Advances in Immunology Volume 6

A D Y A N C E S IN Immunology VOLUME 6 CONTRIBUTORS TO THIS VOLUME WERNERBRAUN FRANK J. DIXON RICHARDW. DWITON ANN ...

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A D Y A N C E S IN

Immunology VOLUME 6

CONTRIBUTORS TO THIS VOLUME WERNERBRAUN FRANK J. DIXON

RICHARDW. DWITON ANN E. GABRIELSEN

PHILIPG. H. CELL

ROBERTA. GOOD Ammw S. KELUS P. J. LACHMANN OTTOJ. PLESCIA .IRTHUR M. SILVEF~STELN JAROSLAV

STERZL

EMILR. UNANUE

ADVANCES I N

Immunology EDITED B Y

J.

F. J. DIXON, JR.

H. H U M P H R E Y

Division of Immunology Nafionol Inrfifute for Medico1 Reseorch Mill Hill London, England

Division of Experimenbl Pathology Scripps Clinic and Research Foundation La lolla, California

ACADEMIC PRESS

V O L U M E 6

1967

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@ N e w York

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ACADEMIC PRESS A Subsldlary of Harcourt

Brace Jovenovlch. Publlshers

London T o r o n t o S y d n e y S e n F r e n a h c o

This is an Academic Press Replica Reprint reproduced directly from the pages of a title for which type, plates, or film no longer exist. Although not up to the standards of the original, this method of reproduction makes it possible to provide copies of books which otherwise would be out of print.

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

WERNER BRAWN, Institute of Microbiology, Rutgers, The State University, New Brunswick, New J e m y (231). FRANKJ. DIXON, Division of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California (1). RICHARDW. DUTTON, Divislrm of Experimental Pathology, Scripps Clinic and Research Foundation, L.u Jolla, California ( 253).

ANN E. GABRIELSEN, Pediatric Research Laboratories of the Variety Club Heart Ho~pitdand the Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota ( 91 ) .

PHILIP G. H. GELL, Department of Experimental Pathology, University of Birmingham, Birmingham, England (461). ROBERTA. GOOD,Pediatric Research Laboratories of the Variety Club Heart Hospital and the Department of Microbiology, Univerdty of Minnesota Medical School, Minneapolis, Minnesota (91). ANDREW S. KELUS,Department of Experimental Pathology, University of Birmingham, Birmingham, England (461).

P. J. LACHMANN, Department of Pathology, University of Cambridge, Cambridge, England (479). OTTOJ. PLESCIA,Institute of Microbiology, Rutgers, The State University, New BrunstL.ick, N e w Jersey (231). ARTHURM. SnvERsmm, The Wilmer Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland (337).

STERZL, Department of Immunology, Institute of Microbiology, CzechoslOuMk Academy of Science, Prague, Czechoslovakia ( 337).

JAROSLAV

EMIL R. UNANUE,*Division of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California ( 1 ) . * Present address: Division of Immunology, National Institute for Medical Research, Mill Hill, London, England. V

This Page Intentionally Left Blank

PREFACE When any serial publication of annual review of progressin a particular subject is inaugurated it is usual-and wise-for judgment of its merits to be reserved. The first volume is likely to be reasonably good and useful, for otherwise the work would not have been started. The second and third volumes, with the benefit of experience, may even be expected to be somewhat better. But, it is asked, will the standard be maintained? Will the field of study be sufficiently prolific of new work, of new ideas, and of new authors to justify annual publication? The critical period, when loss of the initial elan is likely to become evident, is commonly expected to arrive after the first five years. Although Advances in Immunology has established its place with gratifying success, this success may have come too easily because the subject itself has been booming. The appearance of Volume 6 represents, therefore, something of a test, but one whose outcome the editors can face without apprehen-11. There are as yet no signs of dearth of ideas, and we have still to exhaust the friendship of all our colleagues (even though we may put it under temporary strain) by persuading them to write reviews for the benefit of immunologists at large. This volume is the last to be edited by both of us, since John Humphrey, under whose auspices, with W. H. Taliaferro, the publication began six years ago, now retires from editorship. It has been a pleasure to work together. Our task has been much eased by the remarkable cooperativeness of the authors and of the staff of Academic Press. In welcoming Dr. Henry G . Kunkel as the future joint editor we can feel confident, therefore, that he will also find the task rewarding. It may also be permissible for the retiring editor to express his personal conviction that Advances in Immunology will grow even stronger in the future. Volume 6 is rather larger than usual. The first chapter by Emil R. Unanue and Frank J. Dixon reviews the various forms of experimental nephritis, especially from the point of view of understanding the pathogenic mechanisms in terms of immunological events. Owing in large measure to studies undertaken by workers at the Scripps Clinic and Research Foundation, it is now possible to account qualitatively and sometimes even quantitatively for the glomerular changes in two forms of experimental nephritis, namely, those due to nephrotoxic serum and to circulating antigen-antibody complexes. A third form, induced by immunization with renal antigens, has been a cause of controversy and vii

viii

PREFACE

confusion, partly due to expectation that a single and similar mechanism would be operative in different species. Although much still remains to be determined, there appears to be no doubt that in some species, such as the sheep, true autoantibodies against glomerular capillaries are produced and are pathogenic, whereas in other species, such as the rat, the damage is due to complexes of antibody and nonglomerular antigen. The second chapter is by Anne E. Gabrielsen and Robert A. Good and constitutes a monumental review of current knowledge of chemical suppression of adaptive immunity. The authors’ interpretation is based on the hypothesis, enthusiastically expounded by Professor Good, that there are two functionally distinct lymphoid systems-the one thymusdependent and involved in delayed-type hypersensitivity, and the other dependent on the bursa of fabricius (or its analogue in mammals) and involved in antibody production. This approach gives a coherence and rationality to a review which might otherwise be forbidding because of its very scope, for no agents of any promise-not even salicylates-are neglected. Immunosuppressive methods, despite their limitations, have already produced successes in homografting undreamed of even five years ago. Their rational use and development will surely be assisted by this review. Antibodies against nucleic acids were vainly sought for many years, and until about ten years ago it was generally thought that nucleic acids were not immunogenic. As Otto J. Plescia and Werner Braun demonstrate in the third chapter, this opinion was erroneous. Not only can antibodies against DNA be found in various autoimmune diseases, but DNA, RNA, various oligonucleotides, and even mononucleosides can be immunogenic under suitable conditions or when complexed with carriers such as methylated bovine serum albumin. The authors discuss some implications of these exciting findings, on the one hand, for the analysis of structure and function of nucleic acids, and, on the other, for understanding antibody synthesis. In vitro studies of antibody synthesis have great attraction because they offer the hope of isolating and examining separately the variables involved in the interaction of antigen with lymphoid cells which results in an immunological response. Such studies are reviewed in the fourth chapter by Richard W. Dutton, in which he analyzes critically the different experimental conditions employed, the types of data obtained, and their interpretation. So far as established antibody synthesis is concerned, the mechanisms and problems involved appear to be similar to those

PREFACE

ix

involved in synthesis of other proteins. Of more peculiarly immunological interest are attempts to induce and demonstrate specific responses in citron One of the simplest changes to measure is increased DNA synthesis, which Dutton himself has studied extensively, and which is also applicable to stimulation by ccll surface antigens. The phenomenon is clear enough, but despite many ingenious experiments, this review shows how little we yet understand about its mechanism. In the fifth chapter Jaroslav Sterzl and Arthur M. Silverstein have achieved a notable collaboration between Prague and Baltimore in their discussion of developmental aspects of immunity. They review not only the immunological status of fetal and neonatal mammals-to whose study their own contributions are well known-but also various aspects of the ontogeny of the immune response when examined by the most sensitive technique presently available. After arguing inter a2h that the increase of -mmunological capacity with age and the appearance of natural antibodies result solely from specific antigenic stimulation, they develop their brief to include a unitarian concept of cell differentiation and proliferation underlying any form of immunological response. Antibodies specifically directed against the combining groups of other antibodies have been looked for intermittently throughout the past twenty-five years, and have probably not been found. However, in looking for them, antibodies which behave in certain respects as anti-antibodies have often been discovered, and the interpretation of their significance has been confusing. In the sixth chapter Philip Gel1 and Andrew Kelus distinguish four different kinds of such antibody, namely, antiallotype, rheumatoid factor-like, anticomplex, and anticlone. The existence of each kind of antibody poses interesting problems relating to the structure or genetic control of antibodies, and each provides a tool for their investigation. Although the first two kinds are already widely exploited, this brief but lucid review is likely to arouse more interest in the others also. The final chapter by Peter J. Lachmann is about conglutinin and immunoconglutinins. Conglutinin is a remarkable serum protein peculiar to ruminants which interacts with fixed C’3 of all species tested, or with a polysaccharide component of yeasts and some related organisms. Immunoconglutinins are a group of predominantly IgM autoantibodies formed apparently in response to antigenic stimulation by components of an animal’s own fixed complement-commonly C’3, but sometimes C’4. Apart from lending themselves to various elegant tricks for demon-

X

PREFACE

strating fixed complement, these materials pose intriguing problems in relationship to their biological roles. We are confident that this volume contains matter of interest to all immunologists, and wish to thank the authors not only for the care and trouble they took in writing their chapters, but also for having these ready so as to allow the publishers to produce the volume by the scheduled time.

J. H. HUMPHREY F. J. DIXON,JR.

London, England Nouemher, 1966

CONTENTS CONTRIBUTORS..............................................

V

..........................................................

vii

CONTENTS OF PREVIOUS VOLUMES......................................

xv

LIST

OF

PREFACE

Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms

EMILR . UNANUEAND FRANK J. DIXON

I . Introduction ............................................... I1 Nephrotoxic Serum Nephritis .................................. I11. Glomerulonephritis Induced by Antigen-Antibody Complexes ......

.

IV. Nephritis Induced by Immunization with Renal Antigens .......... References .................................................

1 2 42 54 79

Chemical Suppression of Adaptive Immunity

.

ANN E GABRIELSONAND ROBERTA . GOOD

I. Introduction ............................................... 92 I1 Early Experiments on Immunosuppression with Cytotoxic Agents .... 102 I11. Salicylates ................................................. 104

.

IV. Adrenal Steroid Hormones ................................... V. Alkylating Agents .......................................... VI . Folic Acid Antagonists ...................................... VII . Antimetabolites of the Purine Bases ............................ VIII . Analogs of the Pyrimidine Bases .............................. IX . Antibiotics ................................................. X. Plant Alkaloids ............................................. XI . 8-Aminocaproic Acid ........................................ XI1. Acriflavine ................................................. XI11. Ataractic Drugs ............................................ XIV . Methylhydrazine ............................................ XV. p-3-Thienylalanine .......................................... XVI . Penicillamine ............................................... XVII . Hydroxyurea ............................................... XVIII . Antilymphocyte Serum ....................................... XIX. Discussion ................................................ References ................................................

xi

109 125 138

148 168 170 183 187 190 192 194 196 197 198 200 202

205

xii

CONTENTS

Nucleic Acids as Antigens

O n 0 J . PLESCLAAND WERNERBRAUN I . Introduction ................................................ I1. Some Details of Early Studies ................................ I11. Rationale for the Use of a Carrier in Producing Nucleic Acid-Specific Antibodies ...................................... IV. Polynucleotides. Oligonucleotides. and Mononucleosides as Haptens . . V. Ribosomes as Immunogens ................................... VI . Potential Uses of Nucleic Acid-Specific Antibodies . . . . . . . . . . . . . . . VII . Concluding Remarks ........................................ References .................................................

In Vitro Studies

231 233 235 236 243 245 249 250

of Immunological Responses of lymphoid Cells

RICHARDW . DUTTON I. Introduction ............................................... I1. Development of In Vitro Systems for the Study of Antibody Synthesis I11. Mechanism of Antibody Synthesis ............................. IV. Antibody-Forming Cells in Blood and Lymph . . . . . . . . . . . . . . . . . . . V. Initiation of an Immunological Response or the Transfer of Immunity with Cell Extracts ................................. VI. Reactivity of Cells from Delayed Hypersensitive Animals . . . . . . . . . . VII . In Vitro Demonstration of Homograft Reactions . . . . . . . . . . . . . . . . . VIII . Cell Proliferation in the Immunological Response ................ IX. Responses of Lymphoid Cells to Homologous Cellular Antigens . . . . X. Conclusion ................................................ References ................................................

Developmental Aspects

254 255 278 290 292 302

306 314 323 327 328

of Immunity

JAROSLAV STERZLAND ARTHUR M. SILVEFSTEIN I . Introduction ............................................... I1. Methods for Studying Developmental Aspects of Immunity . . . . . . . . I11. Immunological Development ................................. IV. Developmental Stages of Immune Reactions and Their Mutual Relationships ............................................... V . A Unitarian Concept of Immunocytological Mechanisms Based upon the Proliferation and Differentiation of Immunologically Functioning Cells ...................................................... References .................................................

337 338 363 411 429 435

CONTENTS

xiii

Anti-antibodies

PH~LIP G . H . GELL AND ANDREWS . KELUS

I . Introduction ................................................ I1. Anti-antibodies in the Imniunization Process and “Subcomplementarity” ....................................... I11. Immunogenicity of “Altered” IgG ............................. IV. Rheumatoid Factor and Rheumatoid Factor-like Antibodies . . . . . . . . V. Changes in the IgG Molecule Leading to the Production of Rheumatoid Factor-like Antibodies: the Fc Piece ................ VI . Distortion in the Fab Piece of IgG: Anticomplex Antibodies . . . . . . . . VII. “Natural” Anti-antibodies: Agglutinators ....................... VIII . Experimental Anticomplex Antibodies .......................... IX . Molecular Location of Revealed Determinants . . . . . . . . . . . . . . . . . . X. “Anticlone” Antibodies ...................................... XI . Anticlone Antibodies by Heteroimmunization . . . . . . . . . . . . . . . . . . . XI1. Isoimmune Anticlone Antibodies (Idiotypes) .................... XI11. Biological Significance of Anti-antibodies ....................... References .................................................

461 463 465 466 467 468 469 469 471 471 472 473 475 477

Cong lu tin in and Irn rnunoconglutinins

P . J . LACHMANN

I. I1. I11. IV. V. VI .

Introduction ............................................... Nomenclature .............................................. Conglutinin ................................................ The Conglutinogen of Yeast .................................. Immunoconglutinins ......................................... The Reaction of Conglutinin and Immunoconglutinins with Fixed Complement ............................................... VII . Conglutination as a Serological Tool ........................... VIII . The Biological Significance of Conglutination . . . . . . . . . . . . . . . . . . . IX . Summary .................................................. References .................................................

480 481 483 496 498 504 515 516 523 524

AUTHOR INDEX......................................................

529

SUBJECTINDEX ....................................................

566


Con tents of Previous Volumes Volume 1 Transplantation Immunity and Tolerance

M. H A ~ E K A., LENGEROV~, AND T. HRABA Immunological Tolerance of Nonliving Antigens

RICHARDT. SMITH Functions of the Complement System

ABRAHAMG. OSLER In Vifro Studies of the Antibody Response

ABRAMB. STAVITSKY Duration of Immunity in Virus Diseases

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

WILLIAM0. WEIGLE Delayed Hypersensitivity to Simple Protein Antigens

P. G . H. GELLAND B. BENACERRAF The Antigenic Structure of Tumors

P. A. GORER AUTHOR INDEX-SUBJECT INDEX Volume 2 Immunologic Specificity and Molecular Structure

FREDKARUSH Heterogeneity of 7-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

CHARLES G. COCHRANE AND FRANK J. DIXON Phagocytosis

DERRICK ROWLEY Antigen-Antibody Reactions in Helminth ;nfections

E. J. L. SOULSBY xv

xvi

CONTENTS OF PREVIOUS VOLUMES

Embryological Development of Antigens

REED A. FLICKINCER AUTHORINDEX--SUBJE(;T I ~ ~ D E X Volume 3 In Vifro Studies of the Mechanism of Anaphylaxis

K. FRANKA u s m

AND 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 A ~ W 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 IKDEZY-SUBJECT INDEX Volume 4 Ontogeny and Phylogeny of Adaptive Immunity ROBERTA. GOODAND BEN W. PAPERMASTER Cellular Reactions in Infection

EMANUEL S u m AND HANSRUEDY RAMSEIER Ultrastructure of Immunologic Processes JOSEPH

D. FELDMAN

Cell Wall Antigens of Gram-Positive Bacteria M \ C L Y S h'ICCARTY AND STFPHEN

I.

MORSE

Structure and Biological Activity of Immunoglobulins

SYDSEY COIIIW.WD RODNEYR. PORTER Autoan ti bodies and Disease

€3. G, KUNPEL ASD E. X I . TAN

CONTENTS OF PREVIOUS VOLUMES

Effect of Bacteria and Bacterial Products on Antibody Response

J. MUNOZ AUTHORINDEX-SUBJECTINDEX Volume 5 Natural Antibodies and the Immune Response

STEPHENV. BOYDEN Immunological Studies with Synthetic Polypeptides

MICHAELSJILA Experimental Allergic Encephalomyelitis and Autoimmune Disease

PHILIPY. PATERSON The Immunology of Insulin

c. G.POPE

Tissue-Specific Antigens

D. C. DUMONDE AUTHORINDEX-SUBJECTINDEX

xvii


Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mecha nismsl EM11 R. UNANUE2 AND FRANK

J. DIXON

Division o f Experimental Pathology, Scrippr Clinic and Research Foundation, l a Jolla, California

I. Introduction . . . . . . . . . . . . . 11. Nephrotoxic Serum Nephritis . . . . . . . . . A. Introduction . . . . . . . . . . . . B. Clinical History of Nephrotoxic Serum Nephritis in Different Species . . . . C. Pathology of Nephrotoxic Serum Nephritis . . D. Immunology of the Heterologous Phase . . . . . . E. Immunology of the Autologous Phase . . . . . . . F. Nonimmunological Factors Influencing Nephrotoxic Serum Nephritis 111. Glomerulonephritis Induced by Antigen-Antibody Complexes . . . A. Introduction . . . . . . . . . . . . B. Acute Glomerulonephritis . , . . . . . . C. Chronic Glomerulonephritis . . . . . . . . D. Pathogenetic Mechanisms . . . . . . . . E. Other Nephritides Possibly Caused by Immune Complexes . IV. Nephritis Induced by Immunization with Renal Antigens . . . A. Introduction . . . . . . . . . . . B. R a t s . . . . . . . . . . . . . C. Sheep . . . . . . . . . . . . D. Rabbits . . . . . . . . . . . E. Other Experimental Animals . . . . . . . . References . . . . . . . . . . .

.

.

I

1 2 9

1

4 8

11 35 39 42 42 43 47 49 52 54 54 56 63

66 70 79

1. Introduction

Near the turn of the century two important observations were made suggesting an immunological pathogenesis for glomerulonephritis. Lindemann ( 1900) demonstrated the nephrotoxicity of heterologous antikidney sera in experimental animals, and Von Pirquet (1911) clearly related clinical nephritis to the immunological events transpiring in serum sickness. There followed numerous studies which have gone far toward defining the immunopathogenetic mechanisms involved in these two forms of nephritis. On theoretical and experimental grounds it now appears that immunological renal injury can be produced by two means: first, by 'This is publication No. 156 from the Division of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California. Present address: Division of Immunology, National Institute for Medical Research, Mill Hill, London, England, 1

2

EMIL R. UNANUE AND lVL4NK J. DIXON

antibodies capable of reacting with antigens fixed in the kidneys as in the case of antikidney sera, and, second, by circulating antigen-antibody complexes, themselves immunologically unrelated to the kidney, which accumulate in the glomeruli as in the case of serum sickness. The immunization of individuals of several species with heterologous or homologous kidney has also been found to produce nephritis. The pathogenesis of nephritis induced by immunization to kidney is not yet completely described for any species. However, it appears that in each species one or both of the same two pathogenetic mechanisms described above are operative. The relative importance of these two mechanisms in any particular situation depends upon the nature and amount of renal antigen employed and the type of antibody response of the host. This review is organized according to pathogenetic mechanisms as far as is possible. Therefore, it is arranged in three sections: the first deals with nephritide caused by antikidney antibodies, namely the heterologous antikidney antibodies of nephrotoxic serum nephritis, the second with circulating antigen-antibody complex induced nephritis, and the third with nephritis produced by immunization to kidney in which both of the above mechanisms appear to play a role. II. Nephrotoxic Serum Nephritis

A. INTRODUCTION Nephrotoxic serum nephritis (NTN) has been the classic experimental model for the study of glomerular injury induced by antiglomerular antibodies. In this experimental disease the serum donor is immunized with the renal tissue from an animal of a different species; the antiserum from the donor is then injected to an animal of the species from which the renal antigen was taken. A diffuse glomerulonephritis develops which is usually chronic and self-perpetuating, Nephrotoxic serum nephritis has not only served as a means of studying the pathological changes caused by antiglomerular antibodies3 but has also permitted the study of the in vivo dynamics of the interaction of antikidney antibodies with tissue antigens and the characterization of mediators of immunological injury. The terms nephrotoxic serum ( NTS) or nephrotoxic y-globulin (NTGG) will be used to defme antisera or y-gIobuIins containing antibodies to whole kidney, renal cortex, glomeruli, or glomerular basement membranes. No differentiation among these antigens will be made unless specifically stated. The term nephrotoxic antibody (NTAb) will be used to indicate the antibody in either NTS or NTGG. The name of the species donating the antiserum will usually be placed before NTAb, NTS, or NTGG.

EXPERIMENTAL GLOAIERULONEPHRITIS

3

The first description of NTN was given by Lindemann (1900) who induced albuminuria and uremia in rabbits after injection of guinea pig antirabbit NTS. Pearce (1903-1904) studied NTN in dogs with rabbit antidog NTS and determined that the renal cortex could be used as the immunizing antigen. Wilson and Oliver ( 1920) obtained clear evidence that the action of NTS was independent of its hemolysin titer and pointed to the glomeruli as its main target. Masugi (1929, 1933, 1934; Masugi et al., 1935) made a detailed pathological study of NTN in rats and rabbits and noted its similarity to human glomerulonephritis. Masugi (1934) also stressed the features of the chronic stage of NTN and reported experiments in which a nephritis was induced by an antiserum directed against a nonrenal tissue ( Masugi et al., 1935). Further detailed studies were reported by Smadel and collaborators with NTN of the rat. They differentiated the anaphylactic effects of the NTS from their antikidney action ( 1936), reported complete clinical and pathological studies of both the acute and chronic stages (Smadel, 1936, 1937; Smadel and Farr, 1937), pointed out differences in response depending on strain, sex, and diet of rats (Smadel and Farr, 1939; Farr and Smadel, 1939; Smadel and Swift, 1941), and demonstrated the biological protective effect of renal extracts injected prior to NTS (Swift and Smadel, 1937). Of crucial significance were the experiments of Kay (1940, 1942) who showed the importance of the host response to the injected NTS in experiments using duck antirabbit NTS in rabbits. Kay postulated that the heterologous NTAb fixed in the kidney without inducing injury and that the antiheterologous v-globulin response of the host reacting with the kidney-fixed NTAb was the main event responsible for injury. The first studies on the in vivo behavior of NTAb‘s were reported by Sarre and Wirtz (1942) who showed that the duration of circulation of the NTAb was very short despite the development of a chronic nephritis, Next followed a series of papers by Pressman and collaborators, using radioiodinated NTGG, which established the interaction of the antibody with renal and nonrenal tissues and gave evidence of its rapid fixation, its site of interaction in the kidney, and its persistence in the tissues (see Pressman, 1957). These, and many other recent contributions, have permitted a fairly clear understanding of the several pathogenetic mechanisms operating in this disease. Nephrotoxic serum nephritis is best understood by dividing it into two phases which are dependent on different pathogenetic mechanisms. The first, or ‘leterologous” phase is that which results from the interaction of the heterologous NTAb‘s with glomerular antigens. Its effects are usually noted promptly after the injection of the NTS or NTGG. The second, or

4

EMIL R. UNANUE AND FRANK J. DIXON

“autologous” phase develops later and is dependent on the immunological response of the host to the heterologous 7-globulin. The NTAb, after its initial fixation, behaves like an antigen planted in the glomerular capillaries to which the host antibodies to the heterologous ,-globulin fix, causing further injury.

B. CLINICALHISTORY OF NEPHROTOXIC SERUMNEPHRITIS IN DIFFERENT SPECIES Although NTN has been studied mainly in rats, rabbits, and dogs, it has also been reported in mice, monkeys, and sheep. 1. Rats The rat has been the most widely used experimental animal. Nephrotoxic antibodies usually have been obtained from rabbits or ducks and have produced similar clinical effects (Hasson and Seegal, 1954; Stavitsky et al., 1956; Hasson et al., 1957; Hammer and Dixon, 1963; Unanue and Dixon, 1964, 1965a). As noted by many investigators, the clinical severity of NTN was dependent upon the amount of NTS or NTGG administered (Masugi, 1933; Smadel, 1937; Smadel and Farr, 1937; Ehrich et al., 1952; Lippman et al., 1952a, 1954b; Weinreb et al., 1954; Rothenberg et al., 1956; Hasson et al., 1957; Seegal, 1959; Churg et al., 1960; Vogt and Kochem, 1961; Seegal et al., 1962). The amount of antibody capable of fixing to glomeruli was the most critical factor in determining the intensity of the clinical and pathological effects of a given NTS (Unanue and Dixon, 1965a). As far as has been determined, the immediate appearance of nephritis was determined by the quantity and not quality of NTAb (Hasson et al., 1957; Unanue and Dixon, 1964, 1965a). If the amounts of NTAb administered were sufficient to cause injury, the onset of the heterologous phase was immediate with proteinuria and cylindruria within a few hours. The clinical symptoms of the heterologous phase then usually blended with those of the autologous phase which developed after 5 to 7 days. The autologous episode was recognized by the identification of rat 7-globulin fixed in the glomeruli or by the detection of antibodies to the heterologous y-globulins (Ortega and Mellors, 1956; Hammer and Dixon, 1963; Fujimoto et al., 1964; Unanue and Dixon, 1964). Lesser amounts of NTAb induced milder immediate nephritis or none whatsoever. In these cases, symptoms first appeared or increased at the time of development of the autologous phase. In the absence of an autologous phase following an immediate heterologous phase nephritis there was disappearance or reduction of clinical and pathological symptoms (after about 1 week) indicating the importance of a sustained

EXPERIMENTAL GLOMERULONEPHRITIS

5

immunological reaction for continuous injury ( Hammer and Dixon, 1963). As the nephritis progressed, rats developed clinical signs and symptoms of impaired renal function, i.e., elevated blood urea nitrogen ( BUN), decreased urea and creatinine clearance, persistent urinary abnormalities, weight loss, anemia, hypertension, low serum proteins, and hyperlipidemia (Masugi, 1933; Smadel and Farr, 1937; Farr et al., 1942; Seegal and Loeb, 1946; Heymann and Lund, 1951; Heymann and Hackel, 1952; Heymann et al., 1952; Lippman et al., 1952a,b; Weinreb et al., 1954; Seegal and Bevans, 1957). With some NTS, acute anaphylactic symptoms such as generalized pallor, shock, hematuria and sometimes death occurred (Masugi, 1929; Smadel, 1936; Baxter and Goodman, 1956). No correlation existed between the nephritogenic and the anaphylactic potency of an antiserum (Masugi, 1929). Masugi suggested that the anaphylactic symptoms could result from antibodies directed to mesenchymal antigens, and he was able to abolish them by absorption of the antiserum with crude muscle extracts (1929). This assumption is probably true and is supported by experiments which showed that injection of antibodies to collagen induced similar symptoms in rats ( Rothbard and Watson, 1956). In addition to anaphylactic effects, large amounts of NTS could induce death in hours or in a matter of days as a result of either acute renal failure or delayed nonrenal action of the antibody (Lippman and Jacobs, 1952). Among the nonrenal effects of the antibody were the development of acute pulmonary lesions (Weinreb et al., 1954; Laus and Iladler, 1960) presumably induced by interaction of the NTAb's with pulmonary antigens. Clinical symptoms varied depending upon strain, age, weight, sex of the rat, and route of injection of the NTS. Rats of 4 to 6 weeks weighing 50-100 gm., upon injection of a potent NTS, were prone to develop a full-blown nephrotic syndrome with marked edema and ascites plus typical biochemical abnormalities such as hyperlipemia, hypercholesterolemia, and marked proteinuria (Smadel and Fan, 1937; Heymann and Lund, 1951). Rats of larger size developed little, if any, ascites and peripheral edema ( Weinreb et al., 1954). Nephrotoxic serum nephritis has also been induced in neonatal rats by injection of NTS a few hours after birth (Hammer et al., 1963; Calcagno et al., 1963). Smadel and Swift (1941) pointed to the differences in both the acute and chronic stages of NTN among different strains of rats. Rats of Whelan strain were especially susceptible to both acute and chronic nephritis. Rats of Long Evans and Wistar strains developed a slow but progressive nephritis after a remission of several weeks. Though not

6


proven, these differences in the chronic stage could well be related to variations in the immune response to the heterologous globulin among the strains. Differences in acute NTN have also been recognized among Sprague-Dawley, Lewis, and Fisher strains; the latter developed the most severe hetcrologous phase with the highest mortality (E. Unanue and F. J. Dixon, unpublished observations, 1964). In a given strain, adult female rats usually developed a milder chronic nephritis than did males (Smadel and Swift, 1941; Lippman et al., 1954a). However, prepubescent female rats showed a more severe chronic nephritis than prepubescent males (Lippman et al., 1954a). The route of administration of the NTAb was important not only in rats but also in the other experimental species. Following intravenous administration the maximum amount of NTAb fixed in the kidney, and nephritis was the most severe (Lippman et aE., 1954a); after intraperitoneal injection, half as much NTAb fixed in the kidney; after intramuscular injection, even less was kidney fixed (Bale et al., 1955).

2. Rabbits As in the rat, the symptoms of the heterologous phase were dependent on the amount of NTAb fixing to the glomerular loops and not on any qualitative characteristics of the antibody. Much of the work on rabbit NTN has employed duck NTS. In most of these studies, apparently relatively small amounts of NTAb were used so the heterologous phase did not develop and only the autologous phase was seen (i.e., proteinuria appearing from 6 to 10 days after injection, coinciding with the presence of circulating antibodies to duck globulin) which led to the erroneous conclusion that duck antibodies were, by themselves, unable to induce nephritis (Masugi, 1934; Hemprich, 1935; Weiss, 1935; Kay, 1940, 1942; Spuhler et al., 1951b; Simonsen, 1953; Wachstein and Lange, 1958; Lange et al., 1958). However, with large amounts of duck NTAb it has been possible to obtain a full-blown heterologous phase (Halpern et al., 1949a,b; Seegal, 1959; E. Unanue and I?. J. Dixon, unpublished observations, 1964). The amount of NTAb fixing to the glomerulus not only determined the degree of severity of the clinical symptoms but also the time of their appearance. If the amounts of antibody were sufficient, the heterologous phase began immediately with heavy proteinuria and cylindruria. Lesser amounts of antibody induced a mild proteinuria appearing either immediately after injection of NTS, or from 1 to 3 days later. Young rabbits, which make little or no immune response to the heterologous 7-globulin, developed a mild heterologous phase nephritis as late as 6 to 8 days after injection of duck NTS (Moench and Rother, 1956; Erdmann, 1958a,b).

EXPERIMENTAL GLOI\IERULONEPHRITIS

7

The autologous phase usually set in 6-10 days after injection of the NTS and 2-4 days after the appearance of circulating host antibodies to the duck globulin (Kay, 1940, 1942). It was characterized by heavy proteinuria, ranging as high as 2-4 gm. per day, and clinical symptoms of acute renal failure. In the absence of a host immune response there was no autologous phase (Kay, 1940, 1942). In contrast to NTN in the rat, the heterologous phase in the rabbit was usually severe for only a few days after injection and then diminished, making the onset of the autologous phase easily recognizable by the reappearance of severe renal symptoms. Rabbits not showing symptoms during the heterologous phase developed their first recognizable nephritis at the time of onset of the autologous phase. Two types of autologous phases were seen in rabbits. Less than half developed a chronic self-perpetuating nephritis with progressive renal failure which was eventually fatal. However, more commonly, there was a clinical cure or a marked amelioration of the symptoms within several weeks (Masugi, 1934). Either course could be seen in rabbits injected with same amounts of NTS, suggesting that host factors were important in determining the continuation of the renal injury. In young rabbits which did not experience an immune response to the heterologous globulin no chronic nephritis was noted (Erdmann, 1958a,b). It is most probable that to sustain immunological injury in the rabbit, a continuing interaction has to take place in the glomerulus between sizable amounts of fixed NTGG and host antibody. The NTGG is known to disappear fairly rapidly from the rabbit glomerulus (Seegal, 1959) which may account for some of the observed remissions. Experiments with the autologous phase in the rat supports this assumption (Unanue and Dixon, 1965b).

3. Dogs The dog appeared to respond to NTAb's as did the rabbit. Upon injection of the antibody, clinical symptoms developed immediately or after a latent period of several days. As with rabbits, approximately half of the dogs developed a progressive chronic disease and the remainder healed either partially or completely. Although complete clinical and pathological reports are available, little has been written about the immunological aspects of this disease ( Pearcv, 1903-04; Wilson and Oliver, 1920; Fouts et d., 1941; Bevans ct a/., 1955; Seegal et al., 1955; Stickler et al., 1956; hlovat ct al., 1961; Steblny and Leppcr, 19611)). Nephrotoxic serum nephritis has been induced in neonatal pups as early as 8-24 hours after birth (Krakower and Greenspon, 1954; Steblay, 1963b).

8


4. Other Species

Few detailed reports of NTN in mice have appeared ( Gobel-Schmitt, 1950; Eisen and Pressman, 1950; Reid, 1956; Arana et al., 1964; Blair et al., 1965). Mice usually developed clinical symptoms of proteinuria several days after the injection of NTAb, and these were usually part of the autologous phase. There appeared to be little correlation between the amounts of NTAb injected and the time of appearance of proteinuria ( Ciibel-Schmitt, 1950; Unanue et al., 1967). The autologous phase could be severe with heavy proteinuria, ascites, and fatal renal failure, or could develop slowly with remission in many cases. Differences in response to given amounts of NTAb were noted among mice of different strains (Unanue et al., 1967). Nephrotoxic serum nephritis has been induced in monkeys (Michael et al., 1961; Huang et al., 1963), but detailed clinical and immunological studies are unavailable, Preliminary studies have been done in sheep with NTN induced by rabbit NTGG. Sheep appeared to be highIy susceptibIe clinically and pathologically to insult by NTAbs (Lerner and Dixon, 1966b).

C. PATHOLOGY OF NEPHROTOXIC SERUMNEPHRITIS Extensive pathological studies of NTN have been reported in detail. For this information the reader is referred to reports based on light microscopy (Wilson and Oliver, 1920; Masugi, 1934; Smadel, 1937; Tsuji, 1937; Ehrich et al., 1938, 1952; Ehrich, 1937) or electron microscopy (Pie1 et al., 1955; Reid, 1956; Miller and Bohle, 1957; Sakaguchi et al., 1957; Churg et al., 1960; Movat et al., 1961; Feldman et al., 1963; Fujimoto et al., 1964). * The glomerulus, and more particularly the basement membrane, was the site of fixation of the NTAb’s and, consequently, the site of the main pathological changes. Ultrastructurally, the principal lesion was on the luminal side of the basement membrane where irregular deposits of electron-dense material became evident (see Fig. 9). This lesion on the luminal side of the basement membrane produced by antiglomerular antibody contrasts with the lesion observed in the nephritis induced by immune complexes which became evident on the epithelial side of the basement membrane (see Fig. 10) (Dixon et al., 1961). Only rarely were deposits of electron-dense material seen on the epithelial side of the basement membrane in NTN and when seen they were few in number and associated with a severely damaged glomerulus (Movat et al., 1961;

EXPERIMENTAL GLOhlERULONEPHRITIS

9

Feldman et al., 1963). Ultrastructurally, serial studies in dogs ( Movat ct al., 1961), rats (Feldman et al., 1963; Fujimoto et al., 1964), and rabbits (Fujimoto et al., 1964) have disclosed a biphasic type of disease which is in accordance with the two immunological phases of the nephritis. The heterologous phase was characterized by wispy, poorly delineated material applied to the luminal side of the basement membrane and appearing as early as 6-12 hours after injection. The autologous phase was characterized by the appearance of new dense and abundant subendothelial material which progressively increased in amount up to 4 weeks after injection. Also, ultrastructural observations in rats as early as 2 hours after injection have demonstrated a glomerular lesion characterized by accumulation of polymorphonuclear leukocytes (PMN's) . The PMN cytoplasm became attached to the endothelial cell cytoplasm or directly to the basement membrane which at this time presented no visible alterations (see Fig. 8) (Winemiller et a]., 1961; Codlrane et al., 1965). The convoluted tubules and the interstitial tissue also exhibited morphological and biochemical abnormalities ( Fischer and Gruhn, 1957; Wachstein and Lange, 1958, 1960), but these were regarded as largely secondary to the glomerular disease. These changes consisted of areas of cellular hyperplasia with increased number of mitoses, hyaline droplets in the cell cytoplasm, and areas of tubular atrophy-and also loss of some cellular enzymes such as succinic dehydrogenase and cytochrome oxidase. In most situations, NTAb's have not been localized in tubules either by immunohistochemical or radioautographic means ( Mellors d al., 1955b; Ortega and Mellors, 1956; Hiramoto et al., 1959). Exceptions to this rule, however, were newborn rats and adult mice in which some localization of NTAb's along tubular basement membranes had been found (Hammer et al., 1963; Unanue et al., 1967). The cause of the tubular changes is unknown but it might be related to hemodynamic abnormalities in the postglomerular capillaries as a consequence of glomerular disease, Internal hydronephrotic changes induced by plugging of the tubules with protein casts could also be a contributing factor (Wachstein and Lange, 1960). There was marked reabsorption of protein in the tubules in NTN (Goodman and Baxter, 1956b) which might account for functional overloading and produce morphological changes. In this connection, Smadel and Farr reported severe tubular changes (tubules with cellular degeneration and necrosis or with marked cellular hyperplasia) in rats fed a high protein diet as compared to mild or no changes in those fed a basal or low protein diet (Farr and Smadel, 1939; Smadel and Farr, 1939).

10


The glomerular alterations in NTN varied depending on the host species, on the species of origin, and amount of NTAb used, on the presence or absence of an autologous phase, and on the duration of the disease. Certain species had a tendency to develop a particular type of glomerular pathology although variation among individuals did occus. Rabbits, dogs, and especially sheep developed proliferative glomerulonephritis with great frequency. Rats showed an early, prominent exudative response and later tended to develop a mixed pattern of proliferative and membranous glomerulonephritis ( Masugi, 1933; Smadel, 1937; Ehrich et ul., 1952; Heymann and Hackel, 1952; Cochrane et al., 1965). Mice usually had little proliferative response but developed intravascular thrombi and marked mesangial and membranous alterations ( GobelSchmitt, 1950; Reid, 1956; Unanue et al., 1967). Another point worth mentioning is that on many occasions there was a lack of uniformity of the glomerular disease in a single kidney (Masugi, 1934; Ehrich, 1937). Thus, a single histological slide might have some glomeruli with an advanced degree of scarring, others with marked proliferative activity and crescents, and others with very mild lesions. This variation in severity of disease among glomeruli may well be related to variations in the amounts of NTAb originally fixing in different glomeruli or even different parts of the same glomerulus (see Fig. 9). Since NTAb fixes within a few minutes after injection, any inequality in circulation throughout the glomerular capillary bed shortly after injection as a result of spasm (Lippman et al., 1952c; Hartman, 1957) or even normal variation would no doubt cause differences in fixation of NTAb, which could result in different degrees of immediate and subsequent glomerular injury. Pathological abnormalities in NTN were also noted in other tissues besides the kidney. However, these changes were usually secondary to the acute and chronic renal insufficiency and usually correlated with the severity of the nephritic process. Among them were cardiac hypertrophy in relation to blood pressure changes (Lippman et al., 1952b), adrenal hyperplasia (Ehrich et al., 1952; Lippman et al., 1952b), moderate hepatomegaly (Lippman et al., 1952b), moderate testicular atrophy ( Lippman et al., 1952b), and vascular lesions ( sclerosis, medial calcification, arteritis) in vessels of the heart, testes, and brain and in the aorta and its major branches (Smadel, 1937; Hackel and Heymann, 1959; Churg and Sakaguchi, 1965). A possibility that should not be overlooked is that the NTAb’s might injure nonrenal tissues by fixing to their basement membrane antigens, an event which is known to occur in vivo (Pressman et al., 1950b; Bale et al., 1955; Hiramoto et al.,


11

1959). However, there is little evidence to support such a mechanism after the usual intravenous injections of NTAb's. Acute pulmonary lesions have been reported as specifically related to the reaction of the antibodies with lung parenchyma (Laus and Hadler, 1960); however, these lesions were not always present even with antiserum to lung tissue (Triedmann et al., 1962). The scarcity of nonrenal lesions is probably related to the small amounts of antibody fixing to the nonrenal tissues and also to the rapid disappearance of NTAb from these sites (Pressman et al., 195Ob; Triedmann et al., 1962; Unanue and Dixon, 1965a). This assumption is supported by experiments in which lung, skin, and joint lesions have been obtained by the local but not the systemic administration of nephrotoxic or other antitissue antibodies (Tsuji et al., 1940; Read, 1958; Krakower and Greenspon, 1963, 1964a; Teodoru and Volk, 1960). Injection of NTS into pregnant rats resulted in a high incidence of embyronic deaths and, more interestingly, in a significant number of congenital malformations in the surviving offspring (Brent et al., 1961; David et al., 1963; Brent, 1964). Nephrotoxic, as well as antiplacental antibodies, were known to localize in placenta (Pressman and Korngold, 1957; Brent et al., 1961; Boss and Craig, 1963) but rarely induced placental injury (Bevans et al., 1955; Seegal et al., 1955). The relationship between the congenital malformations of the newborn and the immunological insult is unknown. OF THE HETEROLOGOUS PHASE D. IMMUNOLOGY

1 . The Glomerular Antigens The antigenic composition of the glomerulus, and mainly of its basement membrane, has not been clearly elucidated, principally because of the difficulties inherent in isolating the different constituents in pure form. The main antigens of the glomerulus are in its basement membrane (Krakower and Greenspon, 1951) which is antigenically and biochemically related to fibrous and scleroproteins of other tissues. The basement membrane is a complex antigenic structure possessing specific antigenic sites as well as sites which are the same or similar to those in vascular and epithelial basement membranes, stromal reticulum, and also collagen fibers. The glomerular basement membrane and renal reticulum are rich in hydroxyproline (Goodman et al., 1955; Windrum et al., 1955; Bonting et al., 1961; Lazarow and Speidel, 1964; Dische et al., 1965; Kefalides, 1966) and have an amino acid composition similar to collagen. However, they differ from collagen in their higher content of carbohydrate, hexosamine, and lipid (Windrum et al., 1955; Dische et al.,

12


1965; Kefalides, 1966), in their greater resistance to treatment with acids (Windrum et al., 1955) and proteolytic enzymes (Milazzo, 1957), and in their histochemical and ultrastructural characteristics. Histochemically the glomeruIar basement membrane reacts strongly with the periodic acid-Schiff stain indicating a high carbohydrate content, and with silver impregnation stains, in contrast to weak reactions exhibited by collagen (for recent comprehensive review on the histochemistry of connective tissues, see Fullmer, 1965). Ultrastructurally the basement membrane consists of three amorphous layers of varying density, two laminae lucidae and one lamina densa with no evidence of structural periodicity like that exhibited by coIlagen fibers (Rhodin, 1955). No collagen fibers are present in the normal glomeruli of most mammalian and nonmammalian species studied (Fawcett, 1954; Rhodin, 1955; Pak Poy, 1957, 1958, 1959). However, the mesangial cells do appear to have the potentiality to synthesize collagen fibers, for the mesangial areas of diseased glomeruli appear to contain them (Bencosme et al., 1959). One experimental approach which has shown the widespread distribution of glomerular antigens among different tissues has been the induction of a glomerulonephritis upon injection of a heterologous antisera directed to nonglomerular antigens. With this technique, a nephritis clinically and morphologically identical to NTN has been induced upon injection of antisera to most tissues studied. Table I offers a summary of these experiments. Though many of these reports lack the identification of the site of localization of the antibody, it is presumed that it is fixing to antigens of the glomerulus which are shared with the immunizing tissue. Krakower and Greenspon (1951, 1958) used this technique in a quantitative way in order to establish the amounts of “nephrotoxic” antigen contained in various tissues. They immunized with different quantities of a given tissue fraction and established the amounts necessary to induce a potent NTS. These investigators established that the glomerular basement membrane was 40 times more effective in stimulating the formation of NTAb than the glomerular cells (1951). They also established a relationship between the degree of vascularity of a given tissue and its content of nephrotoxic antigens (1958). Potent antisera to avascular tissues, such as cornea, lens, and cartilage, were not able to induce nephritis, presumabIy because of a lack of effective antigen, whereas antisera to poor and well vascularized tissue were able to induce a mild and severe nephritis, respectively. The widespread distribution of antigens similar or identical to those of the glomeruli has also been determined by absorbing a known NTS or NTGG with a given tissue preparation prior to its injection. The elimina-


13

tion or reduction of the nephritogenic potency of the serum was taken as an indication that the NTAb had reacted with the antigens of the tissue used for absorption. With this method it has been determined that among the renal structures, the concentration of nephritogrnic antigcn was greatest in glomeruli (Solomon c t al., 1949; Baxter and Goodman, 1956) in accordance with the experiments reported before. However, substantial amounts of this antigen also were found in renal medulla (Baxter and Goodman, 1956). The presence of shared or cross-reacting antigens between tubules, presumably their basement membranes, and the glomerulus has also been established by three other approaches: (1) in vitro agglutination tests, using antisera to tubular or glomerular basement membranes have shown cross-reactions (Goodman et al., 1955); (2) immunohistochemical methods have shown localization of antiglomerular antibodies to tubular basement membranes (see Table I for references), and ( 3 ) injection of antisera to renal medulla has induced mild nephritis in the recipient animals (see Table I for references). Lung and placenta followed by heart, intestine, liver stroma, and muscle also shared antigens with glomeruli as evidenced by their ability to absorb NTAb‘s in vitro ( Baxter and Goodman, 1956). The precise anatomical localization in the tissues of the glomerular or “nephrotoxic” antigens has been accomplished with immunohistochemical techniques (fluorescent antibody). The in uitro method employed either a fluorescent-labeled or unlabeled antitissue serum which reacted (“stained) with a frozen section of tissue; if the antitissue serum was unlabeled, a fluorescent antisera to the antitissue sera was then applied; the site of interaction of the antitissue antibody with the tissue was examined with the ultraviolet microscope. This technique offered an excellent anatomical localization of the target antigens but was not quantitative. Cruickshank and Hill ( 1953) established that antirat glomerulus and antirat lung antisera stained not only the glomeruli but also the basement membranes of other organs ( gastrointestinal tract, bladder, ovary, breast, thyroid, etc.) and the reticulum framework of spleen, lymph nodes, and thymus, the walls of capillaries and arterioles, sarcolemma, and neurilemma. These authors postulated a common group of antigens among these different structures. Antisera to whole rat kidney showed a second, different type of antibody directed to renal tubular cell cytoplasm (Hill et al., 1953). In vitro agglutination techniques have also shown distinct antigenic differences between tubular cell cytoplasm and renal basement membrane antigens (Goodman et al., 1955). The results of Cruickshank and Hill have been substantiated by other authors (Hiramoto et al., 1959; Scott, 1957, 1959; Boss, 1963b, 1965b). Scott

TABLE I ANTITISSUEANTIBODIES: FIXATION IN THE KIDNEY AND NEPHRITOGENIC PROPERTIES*+ Fixat.ion of antibody in kidney ~

Antigen (used to elicit Recipient of antitissue antibody) antibody

Nephritis (upon intravenous injection)

Kidneya

~~~~~~~

I n vitroc

In vivo Glomeruli*

Tubules*

Glomeruli Tubulesd

Tubular cells

Dog, rabbit, rat (1-6)

Glomeruli or glomerular basement membrane

Dog, rabbit, rat (5, 14, 18-21)

Glomerular cells

Dog (19)

+

Immediate or late onset depending on amounts of antibody injected (5, 6)

-I(7, 8, 9, 10)

+

Usually negative (11-15)

+

+

(13, 16, 17)

Dog, rat, (19, 2426)

c

s sl

+

N.S.

+

N.S.

N.S.

N.S.

+

N.S.

N.S.

N.S.

Immediate or late onset depending on amounts of ant.ibody injected (5)

Immediate or late onset. Usually mild (19, 24-26)

+

m

z

Onset not evaluated (19) Medulla

E

r

Kidney Whole kidney or kidney sediment

M

Renal supernate or soluble extracts

Dog, rat', (19, 24, 27)

+

N.S.

N.S.

N.S.

Immediate or late onset. Usually mild (19, 24, 27)

Extrarenal tissues

Adrenal

Rat (24)

(24)

Brain

Choroid plexus

Collagen

Cartilage

Rabbits, Variable results: rats, (4, none or mild with 24,29,30) late onset in rats (4, 24, 29). Severe with late onset in rabbits (30) Dog (20) Onset not evaluated (20)

+

N.S.

N.S.

N.S.

N.S.

N.S.

+

N.S.

-

+

+

Dog, rat, (20, 31, 32)

Variable results: nephritis of late onset in rats primed with Freund's adjuvant (31). None in dogs (20, 32)

Minimal (33)

Dog, rat,

-

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

(20, 21)

Eye Cornea

Minimal (28)

Dog, rat, (20, 21)

(34)

(34)

(20, 21)

(20)

16 EMIL R. UXANUE AND FRANK J. DIXOX

Lung

Dog, rabbit, rat (20, 24, 30, 32, 42, 43)

+ (24)

(9, 44,45)

N.S.

Minimal (10)

Lymph nodes

R a t (10)

Muscle

Rabbit, rat 24, 30, 33, Always of late onset 38, 46) (30, 33, 46)

+

R a t (24)

-

Pancreas

R a t (40)

(24) N.S.

Placenta

Dog, rat, mouse, (24, 47, 48, 49) (24, 47, 48, 49, 51, 52) Rat, mouse, (4, 52) Thrombi of agglutinated red blood cells in glomeruli (4) R a t (4) Thrombi in glomeruli with hematuria (4)

Ovary

Red blood cells

Serum

+

+

+

+ (11, 14, 43)

+

+

(50)

+

+ (16, 17) N.S.

N.S.

(33) Minimal (10) Minimal (40)

-

-

+

(33, 38)

(33, 35)

N.S.

N.S.

N.S.

N.S.

+

-

(29)

+ (51, 52)

N.S.

-

TABLE I (Continued) -

Fixation of antibody in kidney

In vivo Antigen (used to elicit antitissue antibody) Spleen

Recipient of antibody Rat (24, 33)

Synovium

Rat (53)

Testes

Dog (20)

In vitro"

Nephritis (upon intravenous injection)

Kidney"

Glomeruli

Variable. mild (33) or none (24)

+

+

(9, 33)

N.S.

N.S.

(33) N.S.

+

N.S.

N.S.

Minimal

N.S.

Tubule9

Glomeruli

Tubulesd

Tubular cells

Onset not evaluated (20) umors Murphy lymphosarcoma Testicular

Wagner osteogenic sarcoma Walker carcinoma

Rat (54)

N.S.

Mouse (55, 56) Mouse (57)

(56) N.S.

Rat (58,59)

+ -t Mild, late onset in a minority of rats (59)

Vessels Aorta

N.S.

+

Minimal (57) Minimal (58)

Dog, rabbit, Variable: none in Minimal rat (20, rats and dogs (20, (61) 29) and nephritis 29,60, 61) of late onset in rabbits (60)

N.S.

+

+

+

(56)

(52, 55)

N.S.

N.S.

N.S.

N.S.

4-

N.S. (29)

-

Veiin cava

Dog (20)

+

X.S.

N.S.

9,s.

Onset not evaluated (20) ~~

* Numbers in parentheses indicate the following references (this list is not intended to be complete and only refers to the most pertinent contributions) : 1. Fouts et al. (1941). 2. Seegal et al., (1955). 3. Masugi, (1934). 4. Smadel, (1936). 5. Hasson et al., (1957). 6. Unanue and Dixon, (1965a). 7. Pressman and Keighley, (1948). 8. Pressman, (1949). 9. Korngold and Pressman, (1953). 10. Bale and Spar, (1954). 11. Mellors et al., (1955b). 12. Ortega and Mellors, (1956). 13. Hiramoto et al., (1959). 14. Seegal, (1959). 15. Andres el al., (1962). 16. Cruickshank and Hill, (1953). 17. Hill et al., (1953). 18. Steblay, (1965b) 19. Krakower and Greenspon, (1951). 20. Krakower and Greenspon, (1958). 21. Krakower and Greenspon, (1963).

t N.S.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

Krakower and Greenspon, (1964b). Steblay, (1962b). Baxter and Goodman, (1956). Heymann and Lund, (1948). Heymann et al., (1950). Rothenberg et al., (1956). Spar and Bale, (1954). Seegal, (1958). Spuhler et al., (1951b). Rothbard and Watson, (1959). Markowitz, (1960). Katz and Unanue, (1966). Rothbard and Watson, (1962). Hamori and Olah, (1951). Masugi et al., (1935). Boss, (1965a). Boss, (1965b). Pressman et al., (1951). Anigstein et al., (1954). Sulitzeanu et d.,(1963).

42. Chikamitsu, (1940). 43. Triedmann et al., (1962). 44. Pressman and Eisen, (1950b). 45. Eisen et al., (1950). 46. Tsuji, (1937). 47. Seegal et al., (1955). 48. Seegal and Loeb, (1943). 49. McCaughey, (1955). 50. Pressman and Korngold, (1957). 51. Steblay, (1962b). 52. Pierce et al., (1964). 53. Scott, (1957). 54. Day et al., (1956). 55. Pierce et al., (1963). 56. Kleinsmith et al., (1963). 57. Pressman and Korngold, (1953). 58. Bale el al., (1955). 59. Seegal et al., (1957). 60. Strehler, (1951). 61. Pressman et al., (1953b).

t

M

ZJ

5z 4

9

P

8

Em

50 z

M

!iZJ

=I cn

+

= not studied. or - refer to presence or absence of nephritis (column 3) or to fixation or not of the antibody (columns 4 to 9). Refers to fixation as determined by isotopically labeled antitissue 7-globulin. Localization by fluorescent antibody methods except for reference (15) which employed immunoelectron microscspy and reference (28) which employed autoradiography. Localization by in vitro immunohistochemical method. Refers only to tubular basement membrane or intertubular capillaries. a

w

CD

20

EMIL R. UNANUE AND FRANK J. DJXON

(1957, 1959) compared a fluorescent antiglomerular serum and an antisynovial serum and was able to discriminate certain differences between the two. The antiglomerular serum reacted as described above; the antisynovial serum fixed to reticulum of most organs but not to glomerular basement membrane, epithelial basement membranes, or media of vessels. This author postulated that the antisynovial antibody was fixing to antigens in reticulum not present in the above structures. Pierce and collaborators (Pierce et al., 1963, 1964; Midgley and Pierce, 1963; Kleinsmith et al., 1963) have reported experiments in the mouse using antisera to epithelial basement membrane synthesized by a testicular tumor. Fluorescent antisera to their “neoplastic” epithelial basement membrane when absorbed heavily with splenic reticulum fixed only to basement membranes of epithelial structures. The glomerulus appeared to contain both types of antigens, i.e., those common to epithelial basement membranes and those common to vascular basement membranes and reticulum. Similarly, Krakower and Greenspon (1964b) have obtained in vivo localization in the glomerulus of antisera directed to lens capsule which is an epithelial basement membrane product. This dual antigenic composition of the glomerulus is not unexpected if the embryological development of this structure is considered. Electron-microscopic studies of the fetal human glomerulus have indicated the possibility of two basement membranes early in development; one probably derived from the epithelial cells and the other from the endothelial cells (Kurtz, 1958; Vernier and Birch-Andersen, 1962). Table I shows the studies in which heterologous antibodies to renal and nonrenal tissues have been found to localize in vivo and in uitro to the glomeruli. Collagen and the glomerular basement membrane appear to have certain common antigenic groupings but the nature and extent of this relationship is not clear. Antisera to canine tendon agglutinated or precipitated purified glomerular basement membrane ( Goodman et al., 1955; Markowitz, 1960). Rabbit antirat collagen sera contained complement-fixing antibody to rat kidney and fixed in vivo to the glomerular basement membrane (Watson et al., 19.54; Rothbard and Watson, 1959, 1961, 1962). Immunohistochemically in in vitro conditions, the same antisera reacted with glomerular and vascular basement membranes, reticulum, and collagen. Though the possibility of contamination of the rat collagen antigen with vascular basement membranes is always possible, Watson et al. (1954) have provided evidence for the specificity and purity of their preparation. This antiserum, though fixing to the glomerulus, did not induce nephritis unless rats were previously primed with Freund’s adjuvant (Rothbard and Watson, 1959) and then the nephritis,

EXPERIMENTAL GLOhfERULONEPHRITIS

21

apparently, developed as part of the autologous phase. The inability of anticollagen sera to cause acute nephritis apparently is related to the small quantity of antigen in the glomeruli that can react with anticollagen. The amount is too small to react with a nephritogenic quantity of antibody ( Katz and Unanue, 1966). Experiments with NTAb's, however, have not been clear in showing cross-reactivity between glomerular basement membrane and collagen. Antisera to canine glomerular basement membrane agglutinated sonicated or fragmented canine tendon fibers (Goodman et al., 1955; Markowitz, 1960). However, antirat glomerular or antirat whole kidney sera did not have complement-fixing antibodies to rat collagen (Rothbard and Watson, 1959); similarly, fluorescent antisera against glomerulus or whole kidney did not stain collagen of normal tissue (Cruickshank and Hill, 1953; Hill et al., 1953; Scott, 1957, 1959; Boss, 1963b) or immature collagen fibers during fibrillogenesis (Taylor et aZ.,1961). The suggestion that red blood cells and glomerular basement membranes have shared antigens has little supporting evidence ( Markowitz, 1960, 1962). Immunization with renal or other tissue fractions induced hemagglutinins as well as tissue-specific antibodies. These hemagglutinins presumably were induced because of red cell antigens remaining in the immunizing fraction, since it was impossible to remove all red cells from tissues by perfusion (Pressman, 1949; Unanue and Dixon, 1965a). Absorption of NTS with red blood cells did not affect their nephritogenic potency but reduced the clinical side effects of in vivo agglutination and hemolysis of red blood cells (Wilson and Oliver, 1920; Smadel, 1936; Baxter and Goodman, 1956). Similarly, heterologous antibodies to red blood cells did not fix in the glomeruli (Pierce et al., 1964) and did not cause renal injury except for transitory episodes of proteinuria secondary to their reaction with red blood cells (Smadel, 1936). Tan and Kaplan ( 1963) have reported immunohistochemical studies in which antisera to a crude preparation of mouse ,&globulins reacted with basement membranes and reticulum of normal mouse tissues. In the kidneys the reaction was observed mainly in the axial regions of the glomeruli and in focal areas of tubular basement membranes. Whether this reaction was induced because of the prior fixation of the globulins to the basement membranes as suggested by Pierce et al. (1964) or was, indeed, an indication of shared antigens between the p-globulin and basement membrane has not been ascertained. The nephrotoxic antigens were not entirely species-specific for they exhibited some degree of cross-reactivity with renal and nonrenal tissues of many mammalian species. Nephrotoxic serum nephritis has been in-

22

EMIL €3. UNANUE AND FRANK J. DIXON

duced in mice by injection of antibodies directed to rat kidney (Baxter and Goodman, 1956; Arana et al., 1964) and to human kidney (Arana d at., 1964; Blair et al., 1965); in rabbits by injection of antibodies directed to dog kidney (Simonsen, 1953; Markowitz, 1960) and human glomerular basement membrane ( Steblay, 1965b); in dogs by antibodies directed to rabbit glomerular basement membrane ( Markowitz, 1960) and human glomerular basement membrane ( Markowitz, 1960; Steblay and Lepper, 1961b). This cross-reactivity between species has also been demonstrated by in vitro approaches : i.e., immunohistochemical techniques ( Hill et &., 1953; Boss, 1963a), agglutination ( Milazzo, 1957), cytotoxicity (Liu et &., 1957), and complement fixation (Steblay and Lepper, 1961a). Gery et al. (1965), using isotope-labeled rabbit antirat NTGG, demonstrated cross-reactivity between rat and guinea pig, sheep, dog, and calf kidneys. Some physical and immunochemical properties of the nephrotoxic antigens have been established using either glomerular basement membrane or kidney sediment. The antigens were not readily soluble in water, salt solutions, and lipid solvents; they resisted heating at 60°C. for 30 minutes but were partially inactivated at 100°C. (Eisen and Pressman, 1950; Cole et al., 1951; Baxter and Goodman, 1956; Yagi and Pressman, 1958, 1961). Soluble antigens could be obtained either by repeated washing or sonicating the insoluble material, or simply by letting the insoluble material stand for a few days at 0" to 4°C. (Swift and Smadel, 1937; Krakower and Greenspon, 1951; Greenspon et al., 1952). Proteolytic enzymes, such as trypsin or pepsin, made partially soluble the insoluble material (basement membrane or kidney sediment) (Cole et al., 1951; Greenspon et al., 1952; Goodman and Baxter, 1956a; Milazzo, 1957). Products of tryptic digestion of kidney sediment were able to neutralize NTAb's in vitru and in vim (Cole et al., 1951; Goodman and Baxter, 1956a; Milazzo, 1957; Yagi et al., 1956), but were poorly antigenic, per se (Goodman and Baxter, 1956a). The residue of tryptic digestion was still highly antigenic and also able to neutralize NTAb's in vivo and in vitro (Milazzo, 1957). Yagi et al. (1956) recognized two groups of renal antigens on the basis of their susceptibility to trypsin digestion. One group of antigens was made soluble and could neutralize 75% of the antibodies capable of fixing in uivo. The second group was insoluble and was presumed to contain antigens capable of reacting with the remaining antibodies. Similar groups of antigens were also recognized with respect to their response to trichloroacetic acid treatment (Yagi and Pressman, 1958). The antigens made soluble by proteolytic digestion of kidney or of


23

whole undigested kidney sediment contained at least seven antigenic components which precipitated with NTAb's in gel. Some of these antigens were also present in lung, placenta, heart, and muscle extracts whereas others appeared to be organ-specific ( DeOliveira, 1958; Boss, 1963a,b, 196513; Milgrom et al., 1964).

2. Physicochemical Characterization of the Nephrotoxic Antibodies Most studies of NTAb's have employed rabbit or duck NTS. Most of the antibody of rabbit NTS was contained in the ?,-globulin (Yagi and Pressman, 1961; Fujimoto et al., 1964; Unanue and Dixon, 1!335a). However, some antibody could be detected in the macroglobulin fraction of rabbits bled shortly after immunization with rat kidney (Unanue and Dixon, 1965a). The antibodies of duck NTS were contained in three apparently distinct immunoglobulins and showed certain differences in biological behavior ( Unanue and Dixon, 1965a). Of these three immunoglobulins, one was a rapidly sedimentable, fast migrating y-globulin apparently equivalent to mammalian ylM-globulinand the remaining two were y,-globulins with sedimentation rates of 5.8 and 7.4 (Grey, 1963; Unanue and Dixon, 1965a). 3. In Vivo Specificity of Nephrotoxic Antibodies

The in uivo localization of the NTAb has been determined by two methods. In one a crude globulin fraction or purified y-globulin from the antisera was labeled with an isotope (usually 1311) and injected into animals which were later sacrificed. At sacrifice the organs were perfused with saline to remove blood and their content of radioactivity determined. After correction for contamination by nonantibody 7globulin, the precise amount of NTAb fixed in the kidney was determined (Pressman and Keighley, 1948; Pressman, 1949; Pressman et al., 195Ob, 1953a; Bale and Spar, 1954; Bale et al., 1955). The second method localized the injected NTAb in the tissue by immunohistochemical techniques using a fluorescent antisera to the heterologous 7-globulin (Mellors et al., 195%; Ortega and Mellors, 1956). The isotopic method was quantitative and the more sensitive but did not provide information on anatomical localization unless it was combined with autoradiography ; the immunohistochemical method offered excellent anatomical localization but was not quantitative (Pressman et al., 1958). The NTAb's of a given serum were markedly heterogeneous and differed in their degree of specificity for renal antigens. Some NTAb's

24


fixed preferentially to specific glomerular antigens while others would show widespread distribution in many organs. In vivo localization of l3II-labeled rabbit antirat NTAb took place preferentially in the kidney, but also in adrenal, liver, lung, spleen, ovary, gastrointestinal tract, placenta, and muscle (Pressman, 1949; Pressman and Eisen, 1950a,b; Pressman et al., 1950b, 1953a; Bale and Spar, 1954; Bale et al., 1955; Spar et al., 1956; Pressman and Korngold, 1957; Hiramoto et al., 1958; Powell, 1958; Unanue and Dixon, 1965a). The relative concentrations of NTAb in the kidney and other tissues of the rat varied depending on the time elapsed after injection. Immediately following the injection of NTAb there was a relatively high concentration of antibodies fixed to extrarenal sites; however, the rate of disappearance of the NTAb's from these extrarenal sites was much more rapid than from the kidney, and, consequently, the relative concentration of antibody in the kidney increased with time. The large concentration of NTAb's in the kidney is explained by the fact that all NTS contain some renal specific antibodies. Korngold and Pressman (1953) absorbed rabbit NTGG and other antitissue antibodies with insoluble tissue fractions of different rat organs and later eluted these antibodies by 60°C. heat. The eluted antibodies when injected again into rats showed increased specificity for the organs used to absorb, i.e., the NTGG absorbed by kidney sediment when eluted and injected had higher specificity for the kidney than the whole unabsorbed NTGG. Similar results have been obtained by other investigators (Spar et al., 1956; Powell, 1958). Blau et al. ( 195713) studied the ability of normal and bilaterally nephrectomized rats to remove 1311-labeled rabbit NTGG from the circulation within the first 30 minutes after its injection. From their study they concluded that approximately half of the antibodies were directed to specific renal antigens, and these antibodies remained in the circulation of the nephrectomized rats; the remaining antibodies were directed to antigens common to the kidneys and other organs and were removed from the circulation of the nephrectomized rats. Unanue et al. (1966) studied the NTAb which dissociated from a kidney with NTN after its transplantation to a normal isologous rat and found that these antibodies had a higher degree of renal specificity than the whole preparation of NTGG or than the NTAb dissociated from nonrenal tissues. Whether other physiological factors, such as the great blood flow in the kidneys, the high pressure of blood in the glomerular capillaries, and the fenestrations of the endothelial layer of the glomerulus, play a role in favoring fixation of NTAb's in the kidney is not yet known.


25

4. In Vioo Localization of Nephrotoxic Antibodies within the Kidney Nephrotoxic antibodies have been localized in the renal glomerulus of injected animals by autoradiography after injection of I 3 l I NTGG ( Pressman et al., 1949, 195Oa), by immunohistochemical methods ( Mellors et d., 195513; Ortega and Mellors, 1956; Hiramoto et d.,1959; Seegal, 1959; Seegal et al., 1962; Triedmann et al., 1962; Hammer and Dixon, 1963),and by immunoelectron microscopy ( Andres et al., 1962; Arhelger et al., 1963; Vogt, 1965). The most widely used immunohistochemical method employed fluorescent antisera to the heterologous y-globulin which was applied to renal sections of animals with NTN. A positive reaction was obtained uniformly along capillary walls throughout the entire glomerular tuft shortly after injection of NTS or NTGG and subsequently throughout the course of the disease (see Fig. 1). Heterologous 7-globulins, with no NTAb activity, sometimes could be found in the glomeruli briefly after injection, not in the capillary walls but in irregular, patchy aggregates in the axial or central areas of the glomerulus (Unanue and Dixon, 1964). The minimal amount of NTGG detectable by the fluorescent antibody technique has been determined by staining with fluorescent antisera to the heterologous 7-globulin sections of kidneys from rats injected with known amounts of isotopically labeled NTGG (Pressman et al., 1958; Unanue and Dixon, 1965b). This amount, which varied depending on technical factors, was between 1.5 to 12 pg. of antibody per gram of kidney. Pressman and collaborators (1958) estimated 1.4 X lo-' pg. of NTAb per square millimeter of tissue section as the minimum necessary for localization. Another immunohistochemical method, which has been less used, was to label the NTGG with a fluorescent dye and then follow its distribution in the kidney. However, this procedure showed a loss of 20%of antibody activity and was 4-12 times Iess sensitive than the previously described method (Pressman et al., 1958). The precise anatomic localization of the NTAb's in the kidney has been accomplished by electron microscopy using ferritin-tagged antibody. Andres and collaborators ( 1962) applied ferritin-labeled antisera to the heterologous 7-globulin to sections of kidneys of rats with NTN and observed heavy concentration of ferritin particles aIong the glomerular basement membrane which, as discussed before, is the main site of the antigen. Particles were also found, but in lesser amounts, on the endothelium and on the epithelial foot processes of the glomeruli. Heavy concentrations were also seen in glomerular epithelial cell cytoplasm in an amorphous material lining cysternae of endoplasmic reticulum. Far-

26


quhar et al. (1961) had reported these epithelial cell cysternae before and believed, on morphological grounds, that the amorphous matrix was similar to the basement membrane-a hypothesis which was partially supported by Andre's results. Vogt (1965) used a different approach by injecting NTGG labeled with ferritin to rats and noted specific concentration of the ferritin particles, mainly in the glomerular basement membrane along the luminal side,

5. Rate of Fixation and Exchange of Nephrotoxic Antibodies Heterologous NTAb's have been used to study the in vivo kinetics of tissue antigen-antibody interactions. After injection, the majority of these antibodies rapidly fixed to their tissue antigens and were thereby removed from the circulation. Sarre and Wirtz (1942) clamped one renal pedicle for 15 to 25 minutes following the injection of duck NTS into rabbits. Days later severe renal lesions developed in the undamped kidney as contrasted to mild or no lesions in the clamped one. These authors concluded that most of the antibodies had been removed from the circulation during the period of clamping. Pressman et al. (195Oa) determined the rate of fixation of radioiodinated NTGG in mice. They injected mice with l3II-labeled NTGG and minutes later exsanguinated them; the sera from these bleedings were then injected into other mice which were sacrificed days later and their kidneys assayed for radioactivity. If labeled kidney-fixing antibody was still present in the sera of the first mice when sacrificed, it would localize in the kidney of this second group. In these studies no antibody persisted in the circulation after 18 minutes. Further studies, however, have demonstrated that after the initial period of rapid fixation of the antibodies, there remained a small but detectable amount of antikidney antibody in the circulation for much longer periods. Blau et al. (1957a) detennined two types of localization of rabbit NTAb in rats: one which localized very rapidly disappeared from the circulation with a half-life of 4 minutes and a second which appeared to localize slowly and which had a half-disappearance time from the circulation of 100 minutes. These authors concluded that the two types of localization probably indicated antibodies directed to two different antigenic sites: one present in high concentration and in direct contact with the circulation and the other present in low concentration and somewhat removed from the circulation. They also had evidence which indicated that these two localizations could not be explained by saturation of glomerular antigenic sites by antibody. Unanue and Dixon (1965a) using rabbit and duck NTGG in rats determined the rate of


27

disappearance of the antibody from the circulation and also estimated the rate of accumulation of the antibody in kidney, liver, and lungs. In addition, they quantitated and partially characterized those antibodies persisting in the circulation, The rate of disappearance of the antibody from the circulation was similar to that reported by others; however, the rate of accumulation differed among organs. Kidney showed maximum fixation 1 hour after injection whereas both liver and lung had maximum fixation at 10 minutes after injection followed by a rapid rate of removal. The antibody which was found in the circulation after the first hour apparently consisted largely of low-affinity antibodies. The authors concluded that these antibodies persisting in the circulation were of low avidity and were in a state of equilibrium among extrarenal antigens, the kidney, and a free state in the circulation. Further experiments have demonstrated the interchange of NTAb's among rat tissues which apparently, took place continuously, extending far beyond the first few hours after injection. Seegal (1963) parabiosed normal rats to rats with NTN and days later localized by fluorescent antibody methods the heterologous NTGG in the kidneys of the normal partner. This transfer of NTAb between parabionts occurred to a small degree with all NTS and was a continuous process which involved lowaffinity antibodies derived mainly from extrarenal sites of the nephritic partner (Unanue et al., 1965, 1966). The observed rate of loss of NTAb varied from organ to organ. By transplanting kidneys containing labeled NTAb to isologous rats under several conditions, Unanue et al. (1966) established that the apparent disappearance rate of the NTAb from the kidney depended on two processes. One was the dissociation rate of the kidney-fixed NTAb which was remarkably similar regardless of whether or not complement was fixed or whether or not the host made an antibody response to the NTAb. The second was the rate of association of circulating NTAb with glomerular antigens. These results suggested that an antitissue antibody by the continuous process of association and dissociation might continue to injure the target organ days or weeks after its injection or formation. 6 . Qwtntitatwn of Kidney-Fixing Antibodies

A variety of methods have been used to measure the amount of antibody in NTS or NTGG. Among these were hemagglutination techniques ( Rothenberg et al., 1956), precipitation methods with supernates of renal extracts (Smadel, 1936) or with purified basement membranes ( Markowitz, 1960), complement fixation ( Steblay and Lepper, 1961a), antiglobulin consumption tests ( Steffen and Rossak, 1962), modification

28

EMIL R . UNANUE AND FRANK J. DIXON

of fluorescent antibody methods ( Seegal, 1958; Vogt, 1963)) cytotoxic tests using tissue explants (Lippman et al., 1950) or tissue monolayers (Liu et al., 1957), in vivo fall of hemolytic complement titers ( Stavitsky et al., 1956), and agglutination techniques with basement membranes (Goodman et al., 1955) or glomeruli (Milazzo, 1957). Most of these methods do not measure nephritogenic antibodies exclusively and for the antibodies they do measure, they are semiquantitative at best, affording only a rough correlation between titers of antibodies and nephrotoxic potency. The isotopic method developed by Pressman and collaborators, and described before, has been used to quantitate the in vivo fixation of NTAb's to the glomeruli (Unanue and Dixon, 1965a). Quantitation of NTAb by in vitro and in vivo methods almost always gave higher values for the former. This was because, first, in vitro quantitative methods ( agglutination or precipitation of tissue fractions, etc. ) detected antibodies which were directed to renal fractions that were apparently, inaccessible to circulating antibodies by virtue of their anatomic localization, and thus undetectable by in vivo methods. For example, antirat NTS contained antibodies to tubular cell cytoplasm and tubular basement membranes which were usually never detected in vivo (Hill et d., 1953). Second, there was, in all in vivo determinations, a competition for NTAb's by all the tissues of the host which reduced that amount of antibody able to reach and fix in the kidney. As expected, with precise in vivo determinations of kidney-fixing antibody, a good correlation was found among the amounts of antibody injected, the amount of antibody fixing to the kidney, and the degree of resultant nephritis. No qualitative or quantitative difference was found among the nephritogenic potencies of NTAb in different NTS. In rats no immediate proteinuria was obtained with less than 150 pg. of antibody fixed in the kidneys, moderate proteinuria was obtained with 175 to 200 pg., and maximum proteinuria was obtained with 200 pg. or more. The histopathological picture also varied depending on the amounts of antibody fixed in the kidneys: no lesions appeared with less than 150 pg., a mild proliferative glomerulitis developed with 175 to 200 Jug., and moderate to severe membranous and proliferative glomerulonephritis was seen with 200 pg., or more. These studies emphasized the sensitivity of the immunohistochemical demonstration of glomerular-bound NTGG, which was positive with 2 to 12 pg. of NTAb fixed in both kidneys in contrast with the development of renal injury. This difference in sensitivity in all likelihood explains the reports in the literature where no renal injury was observed in spite of fixation of antibody in the glomeruli detectable by fluorescent antibody


29

methods ( Seegal, 1958; Rothbard and Watson, 1961, 1962). Estimates based on isotope techniques indicated that in order to induce acute proteinuria in the rat there had to be fixation of one antibody molecule for every 20 mpz of capillary filtration surface (Unanue and Dixon, 1965a). Depending on steric factors, this meant coverage of approximately half of the filtration surface by antibody molecules. A similar correlation between amounts of fixed NTAb's and renal injury has also been observed in the rabbit and sheep. However, the amounts of NTAb needed to induce acute injury varied among species. The sheep was highly susceptible to injury and on a dose per unit body weight basis it required 15 times less NTAb than rabbits and rats (Lerner and Dixon, 1966b). In mice receiving rabbit NTAbs, no good correlation existed between amounts of NTAb fixed and proteinuria (Unanue et al., 1967). Similar quantitative information has been obtained using 13*I-labeled duck antirat NTAb. The degree as well as the time of appearance of proteinuria was correlated with the amount of antibody fixed in the kidneys (Hasson et al., 1957; Unanue and Dixon, 1965a). However, the acute lesions induced by duck NTAb differed from the lesions induced by rabbit NTAb. From 150 to 200 fig. of rabbit antibody fixed in a rat's kidneys induced immediate, mild, but persistent proteinuria as compared to an immediate, heavy, but very transitory proteinuria produced by a similar amount of duck antibody. These differences in response are probably related to the difference in mediators activated by the two antibodies. A quantitative difference in nephritogenic potency also has been reported between duck yIM-globulinand the duck 7,-globulins. yIM-globulin appeared to be 60 times more nephritogenic on a molecular basis than 7,-globulin ( Unanue and Dixon, 1965a).

7 . Role of Complement in Glomerulur Injury The fixation of complement to the immune complex of NTAb-glomerular antigen was of importance in that it contributed to the clinical and pathological events noted in acute NTN. a. In Vitro Studies. Most work with complement and NTAb's has been done using rabbit, sheep, or duck NTS. It became evident, as the result of early experimental work, that there was a difference in the degree of complement fixation when mammalian or avian NTS was tested. Izumi (1940) found that duck NTS agglutinated rat kidney sediment without fixation of guinea pig complement. Lange and Wenk (1954) reported experiments in which kidneys were perfused in uitro with fresh rat serum. If rabbit NTS was added to the perfusing system, there was a drop in complement titer of the perfusate; no such drop occurred if duck NTS

30


was added. Quantitative complement-fixation tests have demonstrated little fixation of guinea pig complement by duck NTS and rat kidney antigen (Unanue and Dixon, 1964). The inability of duck NTAb to fix mammalian complement is not peculiar to ducks but is common to most avian antibodies. Rabbit NTAb's and kidney antigens readily fixed complement in vitro; however, there was no correlation between the complement-fixing ability of a NTAb as measured in vitro and its nephrotoxic effect in vivo (Steblay and Lepper, 1961a; Vogt, 1963). b. Serum-Complement Titers. Injection of rabbit NTAb's into rats induced a prompt fall in the serum titer of hemolytic complement which apparently resulted from the fixation of complement to the immune complex of NTAb's and tissue antigens, renal and extrarenal (Pfeiffer et al., 1953; Stavitsky et al., 1954; Lange, 1954; Earle, 1959). Bilaterally nephrectomized rats experienced a drop in complement levels comparable to those seen in intact rats 30 minutes after injection of NTS which apparently reacted with extrarenal antigens (Stavitsky et al., 1954). Complement levels usually remained low for the next 2 to 5 days and then recovered. A significant percentage of rats also experienced a second drop in complement levels, usually correlated with the time of onset of the autologous phase (Earle, 1959; Unanue and Dixon, 1964). The degree of fall in complement levels did not correlate with the severity of the NTN (Stavitsky et al., 1954). Injection of an amount of duck NTS capable of producing immediate NTN produced little or no early fall in serum-complement levels (Stavitsky et al., 1956; Unanue and Dixon, 1964). However, as with rabbit NTS when duck NTS was injected to rats or rabbits, there was a fall in complement levels at the time of the autologous phase response (Ogawa and Sato, 1938; Stavitsky et al., 1956;Lange, 1954; Unanue and Dixon, 1964). In evaluating the relationship between nephritis and serum-complement levels it should be remembered that the proteinuria in severe nephritis may result in a moderate to severe drop in serum hemolytic complement levels in rats in the absence of an immunological reaction (Wilson et al., 1958; Earle, 1959). c. Immunohistochemical Observations. Proof that complement fixation occurs or can occur in the glomerular capillaries in NTN has been obtained by two different kinds of immunohistochemical observations. One used an in vitro fixation of complement (Goldwasser and Shepard, 1958) in which a frozen section of kidney from an animal which was injected with NTS was exposed to fresh heterologous serum and later to a fluorescent antiserum to complement proteins of the heterologous species

EXPERIMENTAL GLOhfERULONEPHRlTIS

31

(Klein and Burkholder, 1959; Burkholder, 1961; Vogt and Kochem, 1961). The complement from the fresh heterologous serum would fix to antigen-antibody complexes and y-globulin aggregates in the tissues and be revealed by the fluorescent anticomplement serum (Lachmann et al., 1962). The second approach was to demonstrate the in uiuo fixation of host complement by staining sections of nephritic kidneys with fluorescent antisera to complement proteins ( Klein and Burkholder, 1959; Hammer and Dixon, 1963). The results of these two methods were usually in agreement; however, in rats given small amounts of rabbit NTS in which there was little to no detectable in viuo fixation of host complement, in vitro fixation has been positive (Unanue and Dixon, 1964). In most of these experiments antisera to a mixture of complement components were used. Klein and Burkholder (1959) developed an antisera containing predominantly antibodies to the fourth component of complement. Antisera containing mainly antibodies to plc-globulin, a protein representing the third component of complement ( MullerEberhard et al., l w ) , have been used in the most recently reported experiments (Unanue and Dixon, 1964, 1965a,b; Cochrane et ul., 1965). Rats injected with rabbit NTS or NTGG and rabbits injected with sheep NTS showed immediate in viuo localization of complement to the glomerular capillary walls in a pattern of distribution identical to that exhibited by the NTAb (Klein and Burkholder, 1959; Hammer and Dixon, 1963; Unanue and Dixon, 1964, 1965a; Cochrane et ul., 1965). Complement was detectable in the glomeruli throughout the heterologous phase, increasing moderately after the initial period of fixation. Rats injected with duck NTAb and having acute proteinuria showed in viuo localization of small amounts of complement as well as most other serum proteins in the glomeruli in an axial or mesangial distribution and not along capillary walls (see Fig. 4) (Unanue and Dixon, 1964). A similar pattern of distribution of complement may be noted in glomeruli of rats exhibiting nonimmunologically induced renal disease (Unanue and Dixon, 1964). No in vitro fixation of complement was seen during the first 3 to 5 days after injection of duck NTS into rats (Vogt and Kochem, 1961; Unanue and Dixon, 1964). d . Mechanism of Action. The role of complement in mediating glomerular injury during the heterologous phase of NTN has been studied by two means, either using treated NTAb's which have little or no ability to fix complement or by decomplementing the host. Rabbit NTGGs have been digested with pepsin (Baxter and Small, 1963; Taranta et al., 1963; Small and Baxter, 1965) or papain (Stelos et al., 1961; Baxter and Small, 1963; Small and Baxter, 1965) to produce

32


divalent or monovalent fragments of y-globulin which retain their antibody specificity but fix little, if any, complement (Taranta and Franklin, 1961; Ishizaka et al., 1962; Ovary and Taranta, 1963; Taranta et al., 1963). Papain-treated rabbit antirat NTGG fixed very transitorily to the rat kidney (Stelos et al., 1961) but did not induce a fall in serum-complement levels nor nephritis even when injected in large doses (Baxter and Small, 1963; Fujimoto et al., 1964; Small and Baxter, 1965). Pepsintreated rabbit antirat NTGG fixed to the kidney also with a shorter halfdisappearance time than that exhibited by the intact, untreated NTGG ( H. Jacot-Guillarmod and F. J. Dixon, unpublished experiments, 1965). Rats injected with pepsin-treated NTGG in doses 2 to 4 times those effective for untreated antibody developed mild transitory proteinuria lasting only 2-3 days (Baxter and Small, 1963; Taranta et al., 1963; Small and Baxter, 1965); their kidneys showed mild renal lesions 24 hours after injection as compared to moderate or severe lesions in the kidneys of rats injected with intact antibody (Taranta et al., 1963). It has been claimed that pepsin-treated divalent fragments fix small amounts of complement (Schur and Becker, 1963), and it was possible to demonstrate small amounts of rat ple globulin in the glomeruli of rats injected with pepsin-treated NTGG (E. R. Unanue, H. Jacot-Guillarmod, and F. J. Dixon, unpublished experiments, 19S6). However, the mild nephritis induced by large amounts of pepsin-treated NTGG was not abolished by in vivo decomplementation of the rats or by reducing their number of circulating PMN’s (E. R. Unanue, H. Jacot-Guillarmod, and F. J. Dixon, unpublished experiments, 1966). Treatment of rabbit NTAb‘s by mercaptoethanol reduced their complement-fixing ability. Such preparations when injected into rats induced proteinuria and renal changes, however, of lesser degree than intact preparations (Fujimoto et al., 1964; Unanue and Dixon, 1964). In order to reduce the host’s complement levels prior to injection of NTGG, injections of antibodies to complement proteins (Kurtz and Donnell, 1962), heat-aggregated human y-globulin ( Hammer and Dixon, 1963; Unanue and Dixon, 1964; Cochrane et al., 1965), soluble antigenantibody complexes ( Hammer and Dixon, 1963), and zymosan particles (Unanue and Dixon, 1964) have been used. With all these methods the drop in serum complement was transitory lasting only a few hours and then returning to near normal levels within 12 to 24 hours after injection (Hammer and Dixon, 1963). The degree of depression of total complement levels as well as of specific components of complement varied with these different agents. Heat-aggregated human y-globulin and antigenantibody complexes produced a 90%depletion of total hemolytic activity


33

of the serum (Hammer and Dixon, 1963; Unanue and Dixon, 1964; Ward and Cochrane, 1965) and a marked depression of the first and fourth components and to a lesser extent the second and third components 15 minutes after injection (Ward and Cochrane, 1965)-observations which correlated with in vitro experiments (Christian, 1 W ) . Injection of zymosan particles depleted mainly the third component of complement (Ward and Cochrane, 1965). Some of these agents induced undesirable side effects in the experimental animals such as hematuria, visceral hemorrhages, and anaphylactoid reactions and care should be taken in order to minimize or avoid them. Rats decomplemented by the above methods and injected with usually effective doses of rabbit NTGG failed to develop immediate proteinuria (Kurtz and DonneU, 1962; Hammer and Dixon, 1963; Unanue and Dixon, 1964; Cochrane et al., 1965). However, large doses of NTAb could induce acute proteinuria in decomplemented rats, usually of lesser degree than in intact rats (Hammer and Dixon, 1963). With usual doses of NTAb, proteinuria appeared with the autologous phase despite the much earlier return of serum complement to near normal values and the early presence of detectable amounts of complement fixed in the glomeruli (Hammer and Dixon, 1963; Unanue and Dixon, 1964). Rats immunologically tolerant to rabbit 7-globulin and decomplemented prior to injection of moderate doses of NTGG never developed proteinuria ( Hammer and Dixon, 1963). Histologically, 24 hours after injection, the kidneys of decomplemented rats injected with rabbit NTGG had milder changes than the kidneys of intact rats injected with rabbit NTGG. Ultrastructurally, however, the degree of basement membrane change was similar (J. D. Feldman, unpublished observations, 1964). It appears that a substantial part of the injury that complement mediates in acute NTN was dependent on its chemotaxis of PMN’s (Cochrane et al., 1965). A few hours after injection of rabbit or sheep NTAb into rats or rabbits, respectively, there was an intense but transitory accumulation of PMN’s in the glomeruli (Ehrich et al., 1952; Heymann and Hackel, 1952; Pie1 et al., 1955; Winemiller et al., 1961; Cochrane et aZ., 1965). The number of PMNs infiltrating the glomeruli was proportional to the amount of NTAb injected and, presumably, to the amount of complement fixing to the kidney as well as to the degree of early proteinuria (Cochrane et al., 1965). In this respect, in vitro experiments have demonstrated that the fixation of the fifth, sixth, and, probably, the seventh components of complement generated a chemotactic factor( s ) for PMNs (Ward et al., 1965). It is reasonable to assume that the fixation of complement in the glomeruli generates this chemo-

34


tactic factor which is, thus, responsible for the accumulation of PMN's. Although the chemotactic factor( s ) in all likelihood does not increase the number of PMNs reaching the glomeruli, it probably acts to detain those passing through. The acute, transitory accumulation of PMN's to the glomeruli of rats and rabbits injected with NTAb's was a necessary step in the development of glomerular injury (Cochrane et al., 1965). Depletion of circulating PMN's in experimental animals prior to injection of moderate amounts of NTAb prevented the development of immediate proteinuria despite the fixation of NTAb and complement. The manner in which the PMN's were involved in the acute glomerular capillary injury is not clear. Ultrastructural studies of the early glomerular lesions showed the PMNs in intimate contact with the basement membrane with frequent displacement of the endothelial cells (Winemiller et d.,1961; Cochrane et d., 1965); no granule lysis or phagocytic vacuoles were noted in the cytoplasm of these PMNs (Cochrane et al., 1965). Polymorphonuclear leukocytes are responsible in the Arthus and Shwartzman reactions for capillary injury ( Stetson, 1951; Humphrey, 1955a,b; Cochrane et al., 1959) possibly through the release of hydrolytic enzymes; whether their mechanism of action in NTN is analogous has yet to be determined. Large doses of NTAb's to rats depleted of PMN's induced acute proteinuria though in lesser amount than when similar doses were injected into non-PMN-depleted rats ( Cochrane et al., 1965). This suggested that non-PMN mediators may also play a role in acute NTN. Whether complement can exert a direct deleterious effect on the glomerular basement membrane similar to its effect on erythrocyte, cellular membranes, or bacterial cell walls is unknown at present. The fact that duck NTAb induced nephritis in rats without detectable participation of either complement or PMN's also indicates that other mediators can cause NTN. Decomplemented rats injected with duck NTS developed immediate proteinuria of the same severity as intact rats (Hammer and Dixon, 1963). Polymorphonuclear leukocytes did not infiltrate the glomeruli after injection of duck NTAb's which correlated with the lack of significant complement fixation (Cochrane et al., 1965). The exact nature of the renal injury induced by these antibodies is not known and several attempts to identify a system of mediators have so far failed. Duck NTAb's appeared to fix to a greater number of antigenic sites in rat glomeruli than rabbit NTAb's (Unanue and Dixon, 1965a), but whether this is of importance in the pathogenesis of the nephritis has not been ascertained. Duck antiprotein antibodies also induced mild Arthus reactions in the skin of rats without apparent fixation of complement (Ward and Cochrane, 1965).


E. IMMUNOLOGY OF

THE

35

AUTOLOGOUS PHASE

1. General Characteristics The autologous phase of NTN was dependent upon the presence of the host’s immune response to the heterologous NTGG (Kay, 1940, 1942; Hammer and Dixon, 1963). During this phase, the NTGG appeared to behave as an antigen planted to the glomerular capillary walls to which the host antibodies fixed (Kay, 1940, 1942). The study of the autologous phase is important because it permits a characterization of capillary injury under conditions where the antibody is produced by the host and thereby approaches an autoimmune situation and where the number of antigenic molecules planted in the capillary wall (NTGG) can be known and controlled. Several days after the injection of NTS or NTGG, circulating antibodies to the heterologous y-globulin were detected in the circulation (Kay, 1940, 1942; Fujimoto et al., 1964). Usually coinciding with the presence of the host’s circulating antibody, the autologous y-globulin, presumably antibody to the NTGG, was detected in the glomeruli by immunohistochemical methods (Fig. 1 ) (Ortega and Mellors, 1956; Seegal, 1959; Hammer and Dixon, 1963; Fujimoto et al., 1964). That the autologous y-globulin was antibody to the NTGG was suggested by several observations: it was localized in a pattern of distribution identical to that of the antigen (the NTGG), its appearance usually coincided with the development of a host immune response, and it did not appear in the glomeruli in the absence of a host response (Ortega and Mellors, 1956; Hammer and Dixon, 1963). Also, homologous antibodies to NTGG passively administered shortly after injection of NTAb promptly localized in the glomerular capillaries in a pattern identical to that of the heterologous and autologous y-globulin (Unanue and Dixon, 1964, 1965b). The autologous 7-globulin could also be detected in the glomerulus days or weeks before renal injury became evident in experiments where the amount of NTAb injected was very low (Unanue and Dixon, 1965b). Radioiodinated rat antirabbit y-globulin, when injected into rats which previously had received NTGG, fixed to the kidney and persisted with a half-disappearance time of 17 days (Unanue and Dixon, 19653,). Autologous 7-globulins sometimes could be detected in the glomerulus in nonimmunological renal disease (Unanue and Dixon, 1964; Okuda et al., 1%5) ; however, in these latter conditions, the distribution was focal, irregular, and mainly in the axial areas of the glomerulus.

36


2. Abolition of the Autologous Phase

The importance of the host immune response in the development or in the continuation of renal injury in animals with NTN has become evident in experiments where this immune response has been prevented. In situations where the injection of NTAb did not produce early disease, the prevention of the host’s response to the heterologous y-globulin avoided the development of nephritis altogether. In the situations where clinical symptoms of the heterologous phase were evident, the suppression of the immune response of the host produced a complete or partial clinical remission, Kay (1940, 1942) prevented nephritis in rabbits which had received duck NTS by X-irradiation prior to administration of the NTS. In his experiments the doses of NTS were too small to produce early disease and all symptoms of renal injury in nonirradiated controls occurred in the autologous phase appearing 2-4 days after the presence of circulating host antibodies to duck serum. Previous immunization to duck serum shortened the period between injections of NTS and proteinuria. Irradiated rabbits did not make circulating antibodies and did not present clinical symptoms of nephritis. However, passive administration of rabbit serum containing antibodies to duck serum to the irradiated rabbits induced clinical symptoms. Similar experiments have been repeated by several authors using drugs that inhibited or suppressed the immune response and with comparable results (Spuhler et al., 1951a; Weisberger et al., 1966). Hammer and Dixon (1963) studied NTN in rats which were immunologically tolerant to rabbit y-globulin and which, therefore, did not have an autologous phase response. These tolerant rats did not exhibit rat 7-globulin in their glomeruli after injection of NTGG and showed complete or partial clinical remission of their heterologous phase nephritis, depending on the dose of NTGG administered. Passive administration of rat antirabbit y-globulin to these tolerant rats again induced severe proteinuria.

3. Mechanism of Renal lnjury The actual mechanism and mediators of renal injury during the autologous phase have not been characterized. Since the glomerular injury in this phase usually developed over a much longer period of time than that of the heterologous phase, short-term experiments in which probable mediators could be eliminated were impossible. Early experiments suggested that the amounts of NTAb reacting with the kidney played an important role in the development of the autologous


37

phase. By clamping one renal pedicle during the time of injection of NTS, renal injury was prevented in the temporarily clamped kidney (Sarre and Wirtz, 1942). In these experiments, renal injury appeared to be entirely dependent on the autologous phase for it first became apparent in the undamped kidney several days after the NTS injection. The prevention of delayed renal injury presumably resulted from the reduced amount of NTAb (now acting as a planted antigen) fixing in the temporarily clamped kidney. In other experiments known amounts of rabbit NTAb themselves incapable of causing nephritis have been injected into rats which were then immunized with normal rabbit y-globulin in adjuvant. It was then possible to observe the renal injury resulting from the reaction of a known number of planted antigenic molecules in the glomeruli with an excess of circulating host antibody (Unanue and Dixon, 1965b). Under these experimental conditions using small doses of NTAb, a latent period was noted between the appearance of circulating and renal, fixed, host antibody and the appearance of proteinuria and histological changes. The duration of this latent period was conditioned by the amount of planted antigen in the glomeruli: it was short with relatively large amounts of planted antigen and long (4-6 weeks) with small amounts of planted antigen. It appears that during this latent period there was a continuous immunological interaction in the glomerular capillaries in which host antibodies (and complement) were associating to and dissociating from the more permanently planted antigens. Glomerular capillary injury in these experiments was induced with as little as 2 pg. of planted NTAb in both kidneys which meant an involvement of less than 1%of the filtration surface by planted antigens. It is presumed, though not established, that complement could be an important mediator of injury in this stage acting as it did during the heterologous insult. Complement fixed to the glomerular capillaries during the autologous phase (Unanue and Dixon, 1964)-a fact which was suggested by the fall in titer of serum hemolytic complement at the time of the host’s immune response (Ogawa and Sato, 1938; Lange, 19%; Stavitsky et al., 1956). This fixation of complement appeared to take place as long as there were circulating host antibodies to the NTGG ( Unanue and Dixon, 1964). However, rabbits and mice congenitally deficient in complement components developed the autologous phase of NTN (Rother et al., 1966; Unanue et al., 1967). Complement-deficient rabbits appeared to lack the sixth, and mice the fifth, components of complement. These results implied that either complement was not altogether essential for the development of injury during this phase or that only a partial fixation of complement components was necessary. No morpho-

38

EMIL R. U N A N U E AND FRANK J. DIXON

logical evidence has been obtained that PMN’s infiltrated the glomeruli during this phase (Cochrane et al., 1965), although a continuous low level of involvement could not be ruled out, The experiments on glomerular injury during the heterologous and autologous phases of NTN indicate two variables of importance in immunologically induced glomerular injury which may apply to all types of immunological capillary injury. These factors, which are clearly interrelated are the number of antigenic sites in the capillary wall and the amount of circulating antibody. If a large number of antigenic sites is present, glomerular injury can be induced immediately provided that there is sufficient amount of antibody to react with these sites. Under these conditions the limiting factor is the amount of antibody present as seen in the heterologous phase with minimal doses of NTAb and plenty of host antigen. However, if the number of antigenic sites is the limiting factor, as in the autologous phase following a small injection of NTAb, glomerular injury may be produced after a long period of antigenantibody interaction in the capillary wall. Under these conditions of antibody excess, the exact amount of antibody would not be expected to correlate with the degree of renaI injury.

4. Other Considerations Some investigators have suggested that the autologous phase is due, not alone to antibodies directed to the heterologous y-globulin but also to autoantibodies directed to host renal antigens altered or liberated because of the heterologous insult (Pfeiffer et al., 1954; Sandritter and Pfeiffer, 1959; Sandritter et al., 1960; Lange et al., 1961; Pfeiffer, 1963). The supporting experiments have employed nephritic and normal rats in parabiosis or cross-circulation and have claimed the development of a slight renal disease in the normal partner. The autoimmune interpretation of these observations has, however, been discounted by the demonstration that the heterologous NTAb crosses from the nephritic to the normal partner and can be demonstrated by immunofluorescence or isotope tracer techniques in the normal partner’s kidneys. Whether or not the “normal” partner develops nephritis depends upon the amount of NTAb transferred, which is, in part, dependent on the amount originally injected into the nephritic partner, and upon the amount of antibody the pair of rats makes to the heterologous y-globulin. Any disease developing in the “normal” partner would presumably be analogous to the autologous phase of NTN with very small amounts of heterologous NTAb in the kidneys. The severity of disease in the “normal” partner

EXPERIMENTAL GLOh4ERULONEI’HRITIS

39

can often be increased by immunizing it with the y-globulin of the species donating the NTAb (Unanue et al., 1965). Other experiments have clearly refuted the autoimmune thesis. The experiments mentioned already in which one renal pedicle was clamped during NTS injection (Sarre and Wirtz, 1942; Spuhler et al., 1951a; Okabayashi et al., 1957; Morrin et al., 1959; Rother and Sarre, 1962; Rother, 1963) were the first to show a normal functioning kidney in an animal with nephritis in the other kidney. A comparable situation occurred if neonatal animals were injected with NTAb; injury occurred only in the juxtamedullary glomeruli functioning at the time of injection whereas the glomeruli of the cortical zone which became functional later in life were spared of disease (Hammer et al., 1963; Steblay, 19631,). Also, no autologous phase developed in rats tolerant to the heterologous y-globulin ( Hammer and Dixon, 1963). Final disproof of the autoimmune thesis has been obtained by transplanting normal isologous kidneys to rats in various stages of NTN. In this situation the transplanted kidney remained normal in the presence of nephritic host kidneys or after their removal ( Unanue et aZ., 1965).

F. NONIMMUNOLOGICAL FACTORS INFLUENCING NEPHROTOXIC SERUMNEPHRITIS 1. Physiological Factors The course of NTN in several species can be influenced favorably or unfavorably by conditions not related directly to the immunological events of the heterologous or autologous phases. Strong experimental evidence discussed above indicated that a continuous antigen-antibody interaction was a necessary requirement for maintenance or progression of NTN. In conditions where this interaction ceased, the nephritis improved ( Hammer and Dixon, 1963). Thus rats immunologically tolerant to the y-globulin of the @jetted NTS had a total or partial clinical remission following the immediate heterologous phase. If rats given a single injection of NTAb developed a poor autologous response and a minimal chronic nephritis (Smadel and Farr, 1939; Lippman et al., 1952a), the nephritis could be made worse by immunization to the heterologous 7-globulin ( Unanue and Dixon, 1965b). After the detectable immunological events of NTN have subsided there may remain a residual, consistent, apparently irreversible, injury manifested by a moderate continuous proteinuria (Hammer and Dixon, 1963). However, conditions that increase the functional demand on such kidneys result in an increase in symptoms of renal failure and in progres-

40

EMIL R . UNANUE AND FRANK J. DIXON

sion of the pathological picture. Farr and Smadel (1939) induced a mild, chronic NTN in Whelan rats; if these rats were given a high protein diet, proteinuria and mortality increased as compared to similar rats fed a low protein diet. The rats fed a high protein diet exhibited progression of the glomerular injury and, more strikingly, a high incidence of severe tubular changes (Smadel and Farr, 1939). Similarly, rabbits with NTN kept on a high salt diet developed a much more severe clinical course than untreated rabbits (Teodoru et al., 1959). On the other hand, mild improvement in NTN has been noted when a nephritic rat was parabiosed to a normal rat or when a normal kidney was transplanted to a nephritic rat ( Unanue et al., 1965). In these situations there was evidence to suggest that a functional sparing of the nephritic kidney was accompanied by some degree of morphological improvement. 2. Blood Coagulation Rats and rabbits injected with very large doses of NTS or NTGG may develop, during the heterologous phase or early autologous phase, focal areas of glomerdar thrombosis in addition to the deposition of antibody and complement in glomerular capillary wall (Masugi, 1934; Smadel, 1937; Ehrich et al., 1952; Weinreb et al., 1954; Pie1 et al., 1955; Wachstein and Lange, 1958; Churg et al., 1960; Vassalli and McCluskey, 1964). The thrombi stained strongly positive with the periodic acidSchiff stain and contained fibrin and 7-globulin (Vassalli and McCluskey, 1964). Ultrastructurally if examined early after their development, the lesion consisted of a focal area of intraluminal thrombosis with typical fibrin strands (Churg et al., 1960; Vassalli and McCluskey, 1964). Similar lesions, however, when examined several days later had an amorphous material, presumably remnants of the thrombus, apparently incorporated in the capillary wall between endothelial cells and basement membrane ( Arhelger et al., 1961; Vassalli and McCluskey, 1964). The thrombotic component of NTN was seen only when very large amounts of antigen and antibody reacted in the glomeruli as in the experiments of Vassalli and McCluskey (1964) who injected antibodies to sheep 7-globulin into rabbits which had received sheep NTAb. Certainly it was inconspicuous or absent in nephritis of usual severity in rats and did not appear to be an essential element in immunological glomerular injury. The exact pathogenesis as well as the sequence of events in the development of this lesion have not been fully determined. An early endothelial lesion caused by the NTAb's might initiate thrombosis by inducing an early platelet clot (Winemiller et al., 1961) or local release of blood coagulation activators as a consequence of the antigen-antibody interaction could precipitate thrombosis. Antigen-antibody reactions can,


41

of course, promote blood coagulation as has been observed in vitro (Robbins and Stetson, 1959) and when such reactions are used to precipitate a generalized Shwartzman reaction (Lee, 1963). Arhelger et al. (1957, 1961) obtained severe glomerular thrombotic lesions by injecting rats with NTAb’s and as little as 0.1 @g.of endotoxin. The rats so injected later developed a very severe membranous nephritis implying that the thrombosis had in some way predisposed to the membranous lesions. In their studies the injection of endotoxin 10 days after the NTGG had no deleterious effect suggesting that the processes capable of causing this lesion were only operative at the height of the immunological reactions in the glomeruli. One difficulty in all coagulation studies is that a positive fluorescent antiserum reaction to fibrin does not necessarily imply an in situ coagulation process; fibrinogen also reacts with a similar antiserum and can also be found in the glomeruli as a result of increased permeability. Also, fibrin is difficult to identify with certainty by ultrastructural studies for it not only can appear as typical fibers with a periodicity of 230 to 250 A. (Hawn and Porter, 1947) but, more commonly, as a granular or filamentous material with no periodicity (Hall, 1949; Still and Boult, 1957; Haust et al., 1965). If intravascular coagulation in severe NTN did contribute to the glomerular injury, the use of anticoagulants might be expected to interfere with the process. Treatment of rabbits with heparin (Kleinerman, 1954) or warfarin sodium (Vassalli and McCluskey, 1964) produced a significant amelioration of the histopathological picture of NTN. The degree of intracapillary proliferation was decreased and the development of epithelial crescent and glomerular sclerosis was largely prevented (Vassalli and McCluskey, 1964). However, the essential damage to the glomerular basement membrane was apparently not prevented for the degree of proteinuria was not changed by the treatment (Vassalli and McCluskey, 1964). If, in addition to fibrin thrombi in the glomerular capillaries, fibrin was deposited in Bowman’s space as part of the inflammation and proteinuria of nephritis, it no doubt would lead to organization, scarring, and obliteration of the space with subsequent crescent formation (see Fig. 7). If anticoagulants prevented fibrin formation in Bowman’s space, they would reduce these undesirable sequelae without necessarily altering the immunological injury to the capillary wall. In other experimental situations not related to NTN, massive glomerular thrombosis appeared to lead to some degree of intracapillary proliferation and glomerular sclerosis (Vassalli et al., 1963). It appears that as a consequence of intravascular thrombosis the glomerulus underwent reparative and proliferative changes similar to the process of organization seen in other vessels which have been thrombosed.

42


3. Pharmacological Factors Many reports have appeared on the effect of drugs or surgical treatments on animals with NTN. Most of these experiments have not carefully determined the effect of such treatments on the basic immunology of this disease and thus must be interpreted with considerable caution. Adrenal cortical steroids have been administered to rats and rabbits with NTN. Rats with NTN if treated with deoxycorticosterone acetate showed a moderate to severe enhancement of the nephritis (Knowlton et al., 1946, 1947; Loeb et al.,1949; Lippman et al.,195413). Hypertension usually developed in these treated rats if they were given a high salt diet; restriction of the salt intake would prevent the hypertension as well as the increased severity of the nephritis (Knowlton et al., 1947). Cortisone or adrenocorticotropin ( ACTH) treatment of rats during the development of NTN did not produce any beneficial effect (Knowlton et al., 1949; Hackel et al., 1950; Ehrich et al., 1952; Heymann et al., 1953; Lippman et al., 1954b; Seegal and Hasson, 1954); moreover, the nephritic process appeared to be enhanced in some instances (Ehrich et ah, 1952; Lippman et al., 1954b; Seegal and Hasson, 1954). Spuhler et al. ( 1951a,b) reported beneficial effects if rabbits with NTN were treated with cortisone and attributed these to an impaired host immune response to the NTS. Bilateral adrenalectomy of rats with NTN has been reported to produce a slight decrease in the degree of proteinuria and histological changes (Ehrich et al., 1952; Heymann et al., 1953; Lippman et aE., 1954b). Hypophysectomized rats injected with NTAb's developed lesser proteinuria than untreated rats but similar degree of histological lesions (Heymann et al., 1953). The effects of testosterone and estradiol treatment of rats with NTN have not been reproducible-one publication reported enhancement of the nephritis ( LeCompte, 1944) whereas another reported a mild protection ( Moench and Vogt, 1958). Treatment of rabbits or rats by antihistaminics prior to injection with NTAb's appeared to have little or no influence on the clinical and pathological nephritis process (Halpern et al., 1949a,b; Winternitz and Hackel, 1951; Hartman, 1957), although one report has noted slight improvement ( Reubi, 1946). Ill. Glomerulonephritis Induced by Antigen-Antibody Complexes

A. INTRODUCTION

Soluble complexes of antigen and antibody can produce a wide variety of tissue lesions, These pathogenic effects of soluble antigen-


43

antibody complexes have been recently reviewed by Weigle ( 1961 ) and Dixon (1962, 1963) and will not be discussed here except in reference to nephritis. Glomerulonephritis developed in the presence of circulating, soluble antigen-antibody complexes. Simultaneously with the development of nephritis, complexes were deposited in the glomerular capillary wall. This type of renal injury was basically different from NTN caused by antiglomerular antibodies in that neither the antigen nor the antibody making up the immune complex showed any relationship to renal antigens. These macromolecular, soluble complexes apparently for nonimmunological reasons localized in and exerted their phlogogenic effects on the glomeruli. Renal lesions caused by circulating immune complexes may be either acute or chronic depending upon the level and persistence of the complexes in the circulation. Acute proliferative glomerulonephritis developed in two experimental models: (1) conventional or “one-shot” serum sickness which was induced by a single large injection of foreign serum protein into animals or humans and appeared as the host made an antibody response and circulating antigen-antibody complexes formed, and ( 2 ) passive serum sickness which was induced by injection of preformed immune complexes into experimental animals. Chronic glomerulonephritis developed as a consequence of repeated smaller injections of foreign serum and, perhaps, some nonserum proteins. In this situation complexes were deposited in the glomeruli gradually over a long period of time and accumulated in very large amounts.

B , ACUTE GLOMERULONEPHRITIS 1. One-Shot Serum Sickness Individuals of most species given a single large injection of heterologous serum or purified serum protein after about 1 week developed acute serum sickness characterized by a variety of lesions including acute glomerulonephritis. The experimental disease has been most completely studied in rabbits (Rich and Gregory, 1943a,b, 1944; Gregory and Rich, 1946; Kawn and Janeway, 1947; Ehrich et al., 1949; Wissler et al., 1949; Germuth, 1953; Germuth et al., 1955, 1957; Dixon et al., 1958) in which the nephritis was characterized by proteinuria, cylindruria, and an elevated BUN which lasted for several days or weeks and then disappeared with complete clinical and morphological cure. The immunopathological events in serum sickness were, in part, surmised from early observations in humans undergoing serum therapy in whom the disease

44


developed at the time circulating antigen was rapidly disappearing from the circulation and just before free antibody appeared (Hamburger and Moro, 1903; Von Pirquet, 1911; Longcope and Rackemann, 1918; Mackenzie and Leake, 1921). It was thought that the interaction of antigen and antibody accounted for the rapid elimination of the former, and, in the process, a toxic material was formed which caused serum sickness. Similar results were obtained with rabbits injected with purified protein antigens ( Hawn and Janeway, 1947; Germuth, 1953). Serum protein antigens injected into rabbits disappeared from the circulation in three different phases: in the first or equilibration phase the antigen level in the serum fell rapidly to one-third to one-half of its initial value as the antigen equilibrated between extravascular and the intravascular fluid compartments; in the second or exponential phase, the protein was eliminated from the circulation slowly at a logarithmic rate as a result of nonimmune catabolism by the host; in the third or immune phase, the protein was rapidly eliminated from the circulation as a result of its combination with antibody formed by the host (Glenny and Hopkins, 1923; Talmage et al., 1951; Dixon et al., 1951, 1953; Germuth, 1953; Janeway, 1953; Dixon, 1954). In order to cause one-shot serum sickness an antigen must be ( 1 ) sufficiently immunogenic to induce a sizable antibody response, ( 2 ) capable of persisting in the circulation until antibody is formed so that circulating complexes will be formed, and (3) given in a dose large enough to allow the formation of considerable amounts of complexes (Moll and Hawn, 1952; Germuth et al., 1957; Germuth and Heptinstall, 1957). Bovine serum albumin had an exponential phase half-life in the circulation of adult rabbits of 5 days. Doses of 0.25 gm. per kilogram of body weight induced glomerulonephritis in 67% of rabbits and the minimum dose required to induce nephritis was 0.13 gm. per kilogram. In contrast, bovine 7-globulin had a half-life of only 2 days and doses of 2 gm. per kilogram were needed to cause a 67%incidence of glomerulonephritis (0.5 gm. per kilogram produced minimal lesions) (Germuth et al., 1957). Antigens that did not persist in the circulation, such as aggregated or heavily substituted proteins, avian serum proteins, hernocyanins, egg albumin, and polysaccharides, never induced serum sickness despite being excellent immunogens. The lesions of serum sickness developed at the time of the immune phase of antigen disappearance soon after the first antibody synthesized by the host combined with antigen to form soluble immune compIexes (Dixon et al., 1958). Circulating complexes of antigen and antibody could be detected during this stage in rabbits injected with bovine


45

albumin (Dixon et al., 1958; Weigle, 1958a; Weigle and Dixon, 1958; Weigle and Deichmiller, 1960). Later, as more antibody was produced, the antigen-antibody complexes became larger and were then rapidly eliminated from the circulation. When all the circulating complexes were eliminated, the disease process rapidly subsided, There was a variation in the incidence and severity of serum sickness nephritis among rabbits, probably related at least in part to a variation in their immune responses. Weigle and Dixon (1958) could not establish a close correlation between levels of detectable immune complexes in the serum of rabbits injected with bovine albumin and the severity of serum sickness as judged by histological changes; however, on the whole, rabbits that made the best antibody responses developed the most severe lesions. Occasionally animals made enough antibody to complex with antigen in the circulation but not sufficient to form large complexes which would be rapidly removed. In such animals a moderate degree of nephritis would develop without a concomitant rapid elimination of complexes or antigen (Weigle and Dixon, 1958). Attempts to potentiate and/or standardize the immune responses of rabbits given foreign serum by previous sensitization with or without adjuvants resulted in a more uniform and severe serum sickness (Germuth et al., 1955; West et aZ., 1960; Rhyne and Germuth, 1961; Kniker and Cochrane, 1965). On the other hand, elimination of the host immune response by X-irradiation or treatment with nitrogen mustard prevented the development of serum sickness (Schwab et al., 1950; Dixon et aE., 1958). The amelioration of the glomerulonephritis induced by treatment with cortisone or ACTH (Rich et al., 1950, 1951; Kobernick and More, 1959) was probably related both to an inhibition of the antibody response and to the antiinflammatory effects of the drugs (Germuth et aZ., 1951a,b). Coincident with the development of nephritis and throughout the course of the disease, antigen, host y-globulin, and host complement presumably in complex form were detected as a fine, granular discontinuous deposit along the glomerular capillary walls by means of the fluorescent antibody technique (see Fig. 2 ) (Dixon et al., 1958; Dixon, 1963; Kniker and Cochrane, 1965). This pattern appeared to be specific for antigen-antibody complexes in the glomeruli and was quite different from the uniform linear deposit of NTAb along the capillary walls in NTN. Occasionally in serum sickness induced by y-globulin antigens the deposition of complexes appeared more continuous and linear, probably because of increased amounts of the reactants and coalescence of the individual granular foci ( Kniker and Cochrane, unpublished observations, 1965). Other host proteins, such as albumin and fibrinogen, were

46


occasionally localized in the glomeruli during this disease but were found in irregular mesangial deposits. By light microscopy, the glomerulonephritis of serum sickness was an acute proliferative process with little visible change in the basement membrane (Rich and Gregory, 1943a; Hawn and Janeway, 1947; Ehrich et al., 1949; Wissler et al., 1949; Germuth, 1953; Rich, 1956). The degree of endothelial cell proliferation could be mild or severe; in the latter cases, the glomeruli became ischemic and occasionally were surrounded by epithelial crescents. Severe cases also exhibited focal areas of necrosis and thrombosis of the glomerular capillaries ( Wissler et al., 1949; Ehrich et al., 1949; Rich, 1956; Germuth et al., 1957). These focal lesions which Rich (1956) compared morphologically to those exhibited by patients with subacute bacterial endocarditis were found by Germuth et al. (1957) to be situated mainly in the glomeruli of the corticomedullary areas. These latter investigators suggested that hemodynamic factors could be responsible for this particular localization. The seventy of the glomerular lesions was presumed to be related to the amount of complexes fixing to the glomeruli on the basis of observations by Germuth et al. (1955, 1957) and Weigle and Dixon (1958) who found a correlation between the early appearance of the immune elimination of antigen and the severity and incidence of lesions. Ultrastructurally the glomerular endothelial cells exhibited marked proliferation and swelling with narrowing and occlusion of the capillary lumens; occasional PMN’s were trapped within the narrowed lumens; and the basement membranes were relatively normal and at the most showed focal areas of thickening (Feldman, 1958; Robertson and More, 1961) The deposits of complexes visualized by 3uorescent antibody techniques were not apparent on electron microscopy, presumably because they were small and of indifferent density.

.

2. Passive Serum Sickness Intravenous injections of soluble antigen-antibody complexes could produce serum sickness in mice and rats which was characterized predominantly by glomerulonephritis and to lesser extent by arteritis (McCluskey and Benacerraf, 1959; McCluskey et al., 1960; Benacerraf et al., 1960; Miller et al., 1960; McCluskey et al., 1962). Soluble complexes made from rabbit, mouse, and chicken antiprotein antibodies and rabbit antihapten antibodies with their homologous antigens produced comparable nephritides. The complexes apparently did not dissociate after their injection in vivo (McCluskey et al., 1960) and when injected repeatedly during a %hour period induced an acute glomerulonephritis which per-


47

sisted for 2 to 3 weeks. In rats, a variable proteinuria and elevated BUN were present from 24 to 72 hours after injection of the complexes (Benacerraf et al., 1960). The incidence of glomerulonephritis was high but the severity varied considerably. This disease was characterized by a conspicuous infiltration of PMN’s in the glomeruli and moderate endothelial proliferation. Fluorescent antibody studies disclosed the presence of these immune complexes in small and large droplets in axial and peripheral areas of the glomeruli (McCluskey et al., 1962; Mellors and Brzosko, 1962; Weiser and Laxson, 1962) ; some of the material visualized in the glomeruli appeared to be intravascular precipitates of antigen and antibody rather than deposits within the capillary wall as occurred in active serum sickness. Cortisone treatment of mice prior to challenge with soluble complexes resulted in a nephritis of diminished severity with fewer number of leukocytes in the glomeruli ( McCluskey et al., 1960).

C. CHRONIC GLOMERULONEPHRITIS Repeated injections of foreign serum proteins in rabbits may cause a chronic nephritis which has been studied morphologically by several investigators ( Longcope, 1913; Vaubel, 1932; Masugi and Sato, 1933, 1934; More and Waugh, 1949; McLean et al., 1951; Hamilton-Paterson and Henderson, 1952; Heptinstall and Germuth, 1957). Dixon and collaborators ( 1961) made a detailed immunopathological study of rabbits injected daily with heterologous serum proteins and established a relationship between the presence of circulating immune complexes and the development of chronic glomerulonephritis. In this experiment, rabbits were injected daily with a heterologous protein in doses of 0.5 to 100 mg.; the amount of protein was varied in relation to the amount of antibody made by the rabbit in an attempt to favor the formation of soluble immune complexes. The antibody responses of the rabbits to the heterologous proteins were of three types. (1) Very large antibody responses which maintained the rabbits in a constant state of antibody excess. The antigen injected in these rabbits was always rapidly removed from the circulation presumably because large immune aggregates formed which were promptly eliminated by phagocytic cells. In these rabbits antigen injections produced brief anaphylactic reactions plus occasional emboli composed of antigen, host y-globulin, and complement. (2) No antibody response which did not provide a basis for immune complex formation. These rabbits showed no untoward effects as a result of the antigen injections. (3) Small to moderate antibody responses which provided enough antibody to allow formation of soluble antigen-antibody

48


complexes in a moderate antigen excess environment. These complexes could be detected in the circulation for hours after each antigen injection. During the first 2 weeks of daily injections of antigen two-thirds of rabbits making a large antibody response developed an acute proliferative glomerulonephritis in all respects similar to that of “one-shot” serum sickness. Supposedly the nephritis developed at the time when the rabbits were beginning to make antibody and were changing from an antigen excess to an antibody excess situation and, consequently, were exposed to circulating complexes. When these rabbits established an excess of antibody, the nephritis subsided. Thereafter a few of these rabbits developed focal proliferation of mesangial cells associated with embolization of glomerular capillaries apparently by antigen and antibody precipitates (Dixon et al., 1961; Andres et al., 1963). Almost all the rabbits which had mild to moderate antibody responses developed chronic membranous glomerulonephritis. Most of these rabbits were in slight antigen excess for 1 to 14 weeks before proteinuria developed, i.e., the dose of heterologous protein that was injected each day neutralized the circulating antibody and formed soluble immune complexes which circulated for at least several hours. The development of chronic glomerulonephritis was unrelated to the type of antigen used, the absolute amount of antigen administered, or the absolute amount of antibody circulating but was related to the ratio of antigen administered and antibody in the host. In many rabbits the development of nephritis could be accelerated or retarded at will by varying the dose of antigen. Clinically the glomerulonephritis was characterized by moderate to severe proteinuria, cylindruria, hypoproteinemia, hyperlipemia, and elevated BUN. By light microscopy, the kidneys exhibited basement membrane thickening with a variable degree of endothelial proliferation and PMN accumulation. In the severely nephritic rabbits, the glomerular changes were more advanced with epithelial crescents and scarring. By fluorescent antibody methods the antigen, host y-globulin, apparently specific antibody, and ,&-globulin were localized in the glomerular capillary walls as dense bead-like deposits along the basement membranes (see Fig. 3) (Dixon et al., 1961; Dixon, 1962, 1963; Andres et at., 1963). A correlation was established between the appearance and amount of these immunological deposits in the glomeruli and the appearance and degree of proteinuria and of basement membrane thickening as seen in the light microscope. These immune complexes seen by the fluorescent antibody technique appeared to correspond with dense amorphous masses of various sizes noted by electron microscopy along the epithelial side of the basement membranes of the


49

nephritic rabbits (see Fig. 10). These masses were believed to be accumulations of immune complexes trapped at the outer edge of the basement membrane or between basement membrane and epithelial cells. Andres et al. (1963) demonstrated ultrastructurally with the use of the ferritin antibody technique, the presence of antigen in these masses. Ultrastructurally, besides these deposits, the basement membranes were variably thickened and/or frayed, and there was fusion of epithelial foot processes and a variable degree of endothelial proliferation. Once the glomerulonephritis had been fully established, cessation of antigen injections caused partial, but not complete, reversal of the clinical and pathological symptoms. The immune complexes noted in the glomeruli at the height of the disease diminished very slowly after cessation of injection but were never seen to disappear completely (Dixon et al., 1961); their rate or removal apparently was uninfluenced by administration of an excess of antigen or steroid therapy (Dixon et al., 1965). It has been calculated that during antigen injections there was approximately 300-800 pg. of antigen in complex form in the kidneys of rabbits with this disease and that the half-disappearance time of the antigen from the kidneys varied from 1 to 2 weeks (Dixon et a]., 1965). Chronic glomerulonephritis also has been induced in rats by chronic administration of human serum albumin (Fennel1 et al., 1965). The human serum albumin together with rat y-globulin were localized in the glomeruli by fluorescent antibody methods. Ultrastructurally lesions were present on the epithelial side of the basement membrane similar to those induced in rabbits and described above.

D. PATHOGENETIC MECHANISMS The mechanisms responsible for the localization and inflammatory effects of immune complexes in the glomeruli are only partially known. Several factors such as size and composition of the complexes, state of the reticuloendothelial system, and systemic or local liberation of vasoactive amines may play a role in the localization of the complexes in the glomeruli. The inflammatory effects of the immune complexes appeared to be produced by their interaction with humoral and cellular factors of the host. The size of the immune complexes was of major importance in their localization within the glomerular wall. Clinical evidence mainly obd tained from experiments in one-shot serum sickness indicated the immune complexes that were formed in antigen excess were the ones that tended to localize in the glomeruli. On the other hand, complexes formed near

50


equivalence or in antibody excess were rapidly eliminated from the circulation and deposited in liver, lung, and spleen (Warren and Dixon, 1948; Dixon and Warren, 1950; Weigle, 1958b; Benacerraf et al., 195913; Walter and Zipper, 1959; Weigle et al., 1960; Weiser and Laxson, 1962). The elimination of these large complexes from the circulation appeared to result largely from phagocytosis by cells of the reticuloendothelial system, PMN’s, eosinophiles, and, perhaps, mononuclear leukocytes ( Cochrane et al., 1959; Sorkin and Boyden, 1959; Patterson et al., 1962; Archer and Hirsch, 1963; Sabesin, 1963; Litt, 1964). Immune complexes competed with carbon particles during clearance from the blood and were localized within the Kuppfer cells of the liver (Benacerraf et al., 1959b; Mellors and Brsozko, 1962). WeigIe ( 1958b) injected immune complexes labeled with radioactive iodine into rabbits and followed their removal from the circulation; part of the complexes were rapidly eliminated and the remainder had a longer exponential rate of removal. The amount of complexes that was rapidly eliminated was greater when the complexes were prepared near equivalence than when they were prepared in antigen excess. Another mechanism for removal of the larger complexes, formed near equivalence, from the circulation was their aggregation, precipitation, and embolization in small blood vessels such as occurred in the lungs of rabbits during anaphylaxis (Dixon, 1953; Germuth and McKinnon, 1957; McKinnon et al., 1957). Once the complexes were eliminated from the circulation they were rapidly catabolized by the host (Dixon and Talmage, 1951; Dixon and Maurer, 1953; Masouredis et al., 1953). This brief circulation of large immune complexes explains the failure of the good antibody formers among the rabbits receiving daily injections of foreign serum protein to develop chronic nephritis. The large complexes formed in these rabbits in an antibody excess environment were removed from the circulation before they had a chance to accumulate in the kidneys (Dixon et al., 1961). Another factor influencing the localization of complexes in the glomeruli was the state of activity or blockade of the reticuloendothelial system. That this could be of importance was suggested by experiments performed by Benacerraf and collaborators ( 1959a ) using colloidal carbon particles which were similar in size to immune complexes (approximately 540 A. ), These authors found localization of carbon in the glomeruli of mice with blocked reticuloendothelial cells but not in the glomeruli of normal mice. Last, the vasoactive amines such as histamine and serotonin which are known to be liberated by antigen-antibody reactions may influence the localization of the complexes. Since the antigens and newly syn-


51

thesized host antibody react in the circulation and interstitial fluids, systemic liberation of these amines would seem likely. Injections of histamine favored the localization of colloidal particles in vessel walls (Biozzi et al., 1948; Jancso, 1955; Alksne, 1959; Majno and Palade, 1961; Majno et al., 1961; Cochrane, 1963a) and particularly in glomeruli ( Benacerraf et al., 1959a). This action of histamine on the capillary walls was not the result of increased phagocytosis by the endothelial cell as was thought earlier; the colloidal material appeared to accumulate in gaps between these cells and against the vascular basement membranes (Majno and Palade, 1961; Peterson and Good, 1962; Cochrane, 1963a). Histamine is known to be released by immune complexes presumably acting on mast cells and platelets (for review of this subject, see Austen and Humphrey, 1963). The acute anaphylactic reactions induced by passive administration of immune complexes were also mediated by histamine for they were prevented by antihistaminics ( McCluskey et al., 1960; Weigle et al., 1960; Cochrane, 1963b). Forman et al. (1949) reported a rise in blood histamine levels and platelets 4 days after injection of horse serum to rabbits. Similar rabbits treated with antihistaminic drugs appeared to develop less proteinuria and milder glomerular changes than untreated rabbits. Kniker and Cochrane (unpublished observations, 1965) have offered strong experimental evidence for the importance of vasoactive amines in the localization of immune complexes during serum sickness in the rabbit. Administration of antihistaminics or serotonin inhibitors and platelet depletion at the time that the rabbits were developing the phase of immune elimination markedly inhihited the deposition of immune complexes in the glomeruli and, consequently, reduced the clinical and pathological symptoms of glomerulonephritis. It seem? logical to assume that the inflammatory effect that the complex exerted on the glomerular capillary wall was not related to their mere passage through the basement membrane and/or to their size but rather to their immunochemical composition and their ability to interact with host complement and perhaps other serum factors and host leukocytes. Particles such as ferritin (Farquhar et al., 1961) or Thorotrast (Farquhar and Palade, 19.59) or very large aggregates of globin, as has been excellently demonstrated by Menefee et al. (1964), traversed the basement membrane without causing residual damage and never accumulated on the epithelial side of the basement membrane. The reasons for the persistence of the complexes along the outer aspect of the basement membranes are unknown but it seems possible that they were trapped there by the epithelial cells as they passed through the basement membrane. It has been reasonably speculated that once the com-

52


plexes reach the epithelial side of the basement membrane they were protected from phagocytosis by circulating cells ( Dixon, 1964). The role that complement might play on this type of glomerulonephritis has not been definitely ascertained. Depletion of serum hemolytic complement took place at the time of immune elimination in one-shot serum sickness (Schwab et al., 1950; Moll and Hawn, 1952; Weigle and Dixon, 1958; Rhyne and Germuth, 1961); however, as expected, there was no strict correlation between amount of fall in complement and the incidence of vascular or renal lesions ( Weigle and Dixon, 1958; Rhyne and Germuth, 1961). Complement proteins, as mentioned before, were found with the immunological reactants in the glomeruli (Dixon, 1963; Kniker and Cochrane, 1965). It was difficult directly to demonstrate the role of complement in acute serum sickness, for the period of injury was too long to allow effective decomplementation of the experimental animal (Rhyne and Germuth, 1961). By analogy with the Arthus reaction, complement would be expected to contribute to the development of this nephritis. In the Arthus reaction the antigen-antibody complex which formed in the vessel wall fixed complement which then served to attract PMNs which were largely responsible for the acute inff ammatory reaction (Cochrane et al., 1959; Ward and Cochrane, 1965). Whether anaphylactogenic antibodies, which do not fix complement such as the 7 S yl-globulins of guinea pigs and mice, form nephritogenic immune complexes has not been ascertained.

E. OTHERNEPHRITIDESPOSSIBLY CAUSED BY IMMUNE COMPLEXES There are no other experimental glomerulonephritides besides the ones considered above in which immune complexes have been definitely established as the etiological agents. However, there is suggestive evidence that complexes could be a causal or contributing factor in several other induced or spontaneous nephritides. Immune complexes may be involved in the pathogenesis of any nephritis induced by repeated administration of antigenic materialsbacterial, viral, or chemical. In this situation, continuous formation of complexes between the injected antigens and host antibody might reasonably lead to renal injury. Glomerulonephritis has been induced by repeated injections of microorganisms: Streptococcus viridans into monkeys (Bell and Clawson, 1931); Hemophilus influenzae into rabbits (Teilum et al., 1951); Proteus mirabilis into mice (Wood and White, 1956), and Escherichiu coli into rabbits (Bailey, 1916; Howes and Pincus, 1963). Killed organisms were employed in all the experiments except in the ones of Bell and Clawson and Bailey. There was little evi-


53

dence to suggest a direct effect of the organisms on the glomeruli, for nephritis developed only after repeated injections over a long period of time. The report of Wood and White (1956) is particularly interesting for these authors were able to detect antigens from the bacterium in the glomeruli by the use of fluorescent antibodies. In the experiment of Howes and Pincus (1963), the rabbits developed an amyloid-like material in the glomeruli and circulating rheumatoid factor. Hotchin and Collins ( 1964) have reported chronic glomerulonephritis in mice chronically infected with lymphocytic choriomeningitis virus. Autologous yglobulin was demonstrated in the glomeruli of the nephritic mice. Though experimental renal lesions have been induced in animals by strains of nephritogenic streptococci, these are probably not related to any immunological disease since the lesions are usually localized to renal tubules (Reed and Matheson, 1954a,b; Kelly and Winn, 1958; Tan et al., 1961; Tan and Kaplan, 1962); induced very rapidly after exposure to the organisms (Sharp, 1960, 1964; Tan d al., 1961), and, apparently, associated with toxic bacterial products (Reed and Matheson, 1954a,b; Kelly and Winn, 1958), such as streptolysin S (Tan et al., 1961; Tan and Kaplan, 1962) which injure tubular cells. Whether immune complexes have an etiological role in experimental amyloidosis cannot be ascertained from the evidence available. Amyloidosis was induced by injection of a wide variety of agents, some of which, such as methylcholanthrene ( Rigdon, 1960) and nitrogen mustard (Teilum, 1954), did not appear to have an immunological basis. Amyloid of experimental animals and humans contained a variable amount of y-globulin, complement, and also of other serum proteins (Vazquez and Dixon, 1956; Lachmann et al., 1962; Schultz et al., 1966) and an unidentified fibrillar component (Cohen and Calkins, 1959; Cohen et al., 1960). Spontaneous or experimental diseases in which antibodies are formed to endogenous circulating material could give rise to circulating complexes and glomerular injury. Mice of the NZB strain were prone to develop spontaneously a Coombs-positive hemolytic anemia, hepatosplenomegaly, and antinuclear antibodies ( Helyer and Howie, 1963; Holmes and Burnett, 1963). Some of the diseased mice also developed a severe chronic membranous glomerulonephritis ( Helyer and Howie, 1963; Aarons, 1964; Howie and Helyer, 1965; Mellors, 1965). The pathogenesis of the whole syndrome is unknown but it is believed to be of an autoimmune nature and has been compared to systemic lupus erythematosus. That complexes might be related to the glomerular disease is suggested by ( a ) the detection of y-globulin in the glomeruli in a granular distribution along peripheral loops ( Aarons, 1964), ( b ) the

33

EXIIL R . UXASL’E A T D FRhh-K J. DIXOS

ultrastructural demonstration of electron-dense material on the epithelial side of the basement membrane, and ( c ) the demonstration by Mellors ( 1965) that nephritic mice developed antibodies which reacted not with normal glomenilar capillarics but with a material deposited in the capillaries and probably derived from the serum of nephritic mice. No definite etiology has been ascribed to the renal lesions of cxperimental or clinical diabetes mellitus; however, there is suggestive evidence for an immune complex mechanism. 7-Globulin and in some instances insulin have been found in the gloinerular capillary walls (Berns ct nl., 1962; Burkholder, 1965): repeated injections of insulin have induced renal diseases in rabbits (Grieble, 1960; Mohos et nl., 1963); circulating complexes of antibody and endogenous insulin have been found in some human diabetics and in rabbits receiving esosenous insulin ( Grodsky, 1964). IV. Nephritis induced by immunization with Renal Antigens

A . I~TRODVCTIOS

Experimental animals may develop antirenal antibodies and glomerunephritis if immunized to heterologous or homologous kidney antigens. Species of experimental animals appeared to differ in their immune responses to various kidney antigens ( glomerular bascment m- mbranes, soluble extracts, etc.) and in the incidence and kinds of nephritis which they developed. Consequently, this type of nephritis will be considered according to the species of animal studied. The actual pathogenetic mechanisms involved in these nephritides have not been totally elucidated although on the basis of recent work it would seem that they vary depending upon species of animal and character of antigen. One of the problems in studying this form of nephritis is that the materials used for immunization of different or even the same species have varied greatly. There has been little effort to standardize, reproduce, or evaluate carefull! the methods for obtaining different kidney fractions or antigens. For purposes of clarity in our discussion we shall use the following terminology to describe the different renal fractions: “whole kidney” referring to kidneys ground and suspended in saline and containing all antigens; “kidne!, extract“ referring to the supernate of a lightly centrifuged ( usually 1500-2000 g) suspension of whole kidney and containing cytoplasmic and nuclear antigens as well as soluble basement membrane antigens; “kidney sediment” referring to sediment from kidney extract preparations and containing glomerular and tubular basement membranes. reticulum from interstitial tissue, and, probably, cell


55

membranes; and “glomerular basement membrane” representing the most purified fraction obtained by the procedure of Krakower and Greenspon ( 1951 ) and containing primarily the insoluble basement membrane antigens. The nephritides induced by immunization with kidney (homologous, heterologous, native, altered, with or without adjuvants ) are apparently related to an antibody elicited by the immunization which at least in some instances is capable of reacting with corresponding autologous antigen. Such a response leading to an antibody capable of reacting with host’s constituents will be considered autoimmune, realizing it may not be initiated by an autoantigen. It is not the purpose of this review to analyze the different mechanisms and hypotheses offered to explain autoimmune disorders and, consequently, only as they pertain to renal injury will they be considered. For a recent review of this subject the reader is referred to the paper of Dumonde (1966). On a theoretical basis an antibody induced by immunization with kidney could cause renal injury by two mechanisms. First, the antibody could react with antigens in the glomerular capillaries injuring the capillaries by a mechanism mimicking that operative in NTN. The degree of nephritis would depend on the titer of antibodies, the duration of the immune response, the number of antigens in the glomeruli which could react with the antibody, the quality of the antibody, and the susceptibility of the particular species of animal to renal injury. Second, the antibody could be directed to antigen in the immunizing material and could injure the glomeruli by forming circulating immune complexes through a mechanism similar to that operating in acute and chronic serum sickness. The antigen in this case could be exogenous and derive from the immunizing injection and/or endogenous and derive from cross-reacting host proteins present in the circulation. In some of the nephritides induced by immunization with kidney the antigens are totally unrelated to the glomeruli and as will be discussed, their pathogenesis is probably related to this second mechanism of injury. The direct participation of sensitized cells in a delayed-type hypersensitivity reaction does not appear to be a likely pathogenetic factor in glomerulonephritis on the basis of the morphological character of the lesions or the results of attempts to transfer any form of the disease with lymphoid cells. In only two types of experiments do lymphoid cells appear to injure the kidney and in neither are the glomeruli involved. First, lymphoid cells are involved as part of the homograft rejection in transplanted kidneys (Simonsen et al., 1953; Porter et al., 1964a,b) reacting in large part in the peritubular capillaries and small arteries and

56

EXIIL R . UA-ANLTE A S D F R A S K J. DIXON

veins (Kountz et al., 1963; Porter et al., 1964a). Second, lymphoid cells from parental strain of rats have been shown to cause tubular injury if injected into the kidneys of F, hybrids (Elkins, 1864, 19M). There are two immunological observations relating to the pathogenesis of experimental and clinical nephritides which have been overinterpreted and have led to unwarranted conclusions. The first is the observation of y-globulin in the glomeruli of nephritic kidneys by the fluorescent antibody method which has frequently been assumed to indicate the fixation of antiglomerular antibodies. In human pathology, for example, yglobulin has been detected in glomeruli of diverse clinical entities such as diabetes, essential hypertension, lupus erythematosus, and poststreptococcal glomerulonephritis and interpreted as antiglomerular antibody. However y-globulin in glomeruli may also represent deposition of nonglomerular antigen-antibody complexes or concentrations of nonspecific 7-globulin in areas of glomerular injury or increased permcability. In most cases the pattern of distribution of y-globulin suggests the mechanism of localization and is an important differential diagnostic feature. The second observation is the detection of antibodies to kidney in the circulation. Antikidney antibodies detected in serum by complement fixation, precipitation, or agglutination are not necessarily the antibodies involved in the glomerular injury. In most cases these antibodies fix poorly in uioo, are related to renal antigens inaccessible to circulating antibody, and, consequently, have little relationship with clinical disease. It is apparent that only a method determining the antibody which localizes in ciao in the glomeruli would give meaningful pathogenetic data. In part this has been accomplished experimentally by labeling the antibodies and observing their behavior in uioo ( Pressman, 1957). B. RATS 1 . General Characteristics

Rats repeatedly immunized with extracts from homologous or heterologous kidney in some instances developed glomeru!onephritis ( Cavelti and Cavelti, 1945a,b,c; Frick, 1950; Heymann et al., 1959; Blozis et al., 1962). Cavelti and Ca\-eiti induced nephritis in rats by multiple injections of kidney extract m i d \\-ith streptococcus. However, repeated efforts to reproduce this result have f a i l c d ( Humphrey, 1933; Xliddlcton et d., 1953). Frick ( 1950) briefly reported the production of acute glomrrulonephritis by intraperitoneal injection of kidney in Freund’s adjuvant containing either Af ycobactcriirm tttberculosis or old tuberculin. On the other hand, Heymann cf 01. ( 1959) reported a well studied, reproducible


57

method for inducing nephritis in rats by immunization with kidney. In this procedure the kidney extracts had to be incorporated in complete Freunds adjuvant containing Mycobacterium tuberculosis and were injected weekly or every 2 weeks intraperitoneally. After six to ten injections (3-5 months), a large proportion of rats became proteinuric with hyperlipemia and hypercholesterolemia, and occasionally elevated BUN (Heymann et al., 1959). The renal disease, once fully developed, was progressive and needed no further immunization with kidney extracts. The immunized rats also developed a sterile granulomatous peritonitis with multiple adhesions between the viscera. The same antigen administered with Hemophilus pertussis as adjuvant by Blozis et al. (1962) produced a quite similar nephritis but without the development of peritonitis.

2. Pathology and Immunohistochemistry This type of glomerulonephritis was a chronic membranous disease with very little proliferation or exudation (Heymann et al., 1962a, 1963). The glomerular basement membranes by light microscopy were thickened and stained strongly with periodic acid-Schiff reagents. Tubular and interstitial changes were also prominent-thickened tubular basement membranes, flattened tubular epithelial cells, and increased interstitial fibrosis. These changes have been considered secondary to the glomerular disease though a primary lesion of the tubules has not been excluded. Ultrastructurally the glomerular basement membranes were thickened and characteristically had numerous electron-dense dcposits on their epithelial side (Hess et al., 1962; Blozis et al., 1962; Feldman, 1963) (see Fig. 11). These electron-dense masses appeared amorphous and were usually irregular in outline, size, and density. They resembled the masses of immune complexes noted in rabbits and rats with glomerulonephritis induced by serum protein antigen-antibody complexes; however, the latter were usually more homogenous and regular in outline. Ultrastructurally, the lesion was identical whether induced by homologous or heterologous kidney extract (J. I. Watson, J. D. Feldman, and F. J. Dixon, unpublished experiments, 1965). Fluorescent antibody studies on nephritic rats demonstrated variable concentrations of host y-globulin and ,8,c-globulin in small, discrete, irregular beads or droplets along the capillary walls in a pattern distinctly different from the uniform linear distribution of 7-globulin seen in NTN (see Fig. 5) (Heymann et al., 1963; Dixon et al., 1965; Okuda et al., 196s; Watson and Dixon, 1966). Uniform linear distribution of these reactants was never seen, even in severe nephritis where larger and

58

EI\IIL R. U S A S U E A S D F R A S K J. DIXON

apparently coalescent masses formed. The same pattern of distribution of 7-globillin and PI,.-globulin existed when homologous or heterologous kidney extracts were used as antigen (Watson and Dixon, 1966). These deposits of immune reactants seen by the fluorescent antibody technique corresponded in size and distribution with the electron-dense masses noted ultrastructurally. 3. Immziiiology a. Tlic Antigen. The antigen( s ) responsible for producing nephritiy in this model were present in greatest concentration in kidney extracts. Partial characterization of the antigen was accomplished by differential ultractntrifugation of homologous kidney extracts. The most effcctivc antigenic fraction appeared to contain primarily cytoplasmic materials ( mitochondrial, nuclear, or microsomal ) , whcrcas both the centrifuged sedimcrit containing basement membrane and the supernate after highspeed ccmtrifugation \%'erethe least antigenic (Hess ct al., 1965; Heymann et nl., 1965; Glassock and \\'atson, 1966). The antigen ( s ) were apparently not organ specific. Immunization of Spraguc-Dawley rats with rat liver extracts occasionally resulted in nephritis ( IIcymann et a/., 1959, 19621, 1963; Holm, 1966). Although rat lung and niusclc were ineffective, human lung, on the other hand. was effective ancl induced nephritis in 25% of rats (Heymann et al., 1963). Tlie antigen( s ) nerc not species specific. Sprague-Dawley rats immunizcd with mt. himian. guinea pig, and rabbit kidney extract developcd a similar incidence of disease (approximately 80%) ( Heymann et a/., 1963). Bovine kidney extract was also equally effective (Hess ct nl., 19651. Le\vis rats immunized with human kidney extracts also developed nephritis but with a lower incidence than when they were immuniwd with kidney extracts from Sprague-Dawley rats (Watson and Dixon, 1966). Changes in the chemical composition of the renal cxtracts a\ produced by coupling it with arsanilic and sulfanilic acid resulted in increased incidence and increased severity of disease ( Watson ancl Iliuon. 1966I . The, amount ok kidnc,! extract administered and the frequency of injections \vcrc important in determining the incidence of diseasc. Doul~liiiqthe closc of adjin ant and antigcn resulted in an increased severity of ncphriti\ ( Hr\miaiin r > t ul., 1959); reducing the amount of antigcn t o ont~-thirdor oiie-fifth of the usual amount resulted in a milder disease ( \\7utwn and Diion, 1966). Increasing the frequency of injectioils from l ~ i m o n t h lto ~ ~ crkly resulted in increawd severity of disease

EXPERIMENTAL GLOlMERULONEFHRITIS

59

(Watson and Dixon, 1966) and, similarly, reducing the number of injections resulted in a marked decrease in incidence and in severity of disease ( Heymann et al., 1959). The kidney extracts were effective in inducing nephritis only if they were incorporated in an adjuvant preparation. The procedure devised by Heymann et al. (1959) incorporated the extract in complete Freund’s adjuvant which contained a strain of hlycobacterium tuberculosis, not Alycobactcririm butyricum ( Heymann et al., l962a), and was administered intraperitoneally. A fourfold increase in the amount of mycobacteria used resulted in an increase in incidence of disease from 70 to 100% (Hcymann et nl., 1962a). Similarly Arlacel A used as an emulsifier produced a higher incidence, and a more clinically severe nephritis than Arlacel C (Watson and Dixon, 1966). The second procedure, devised by Blozis et al. (1962) consisted of injecting intraperitoneally the kidney extract mixed with killed Hemophilus pertussis organisms. The incidence of disease with this procedure was only about 30%.Blozis also reported a preliminary experiment in which nephritis was induced by the same antigenic preparation but using the intravenous route. Differences in incidence and severity of nephritis resulted from the use of homologous and isologous antigens in various strains of rats. The antigen could be autologous and still effective in inducing disease as was shown in experiments in which extracts of autologous kidneys induced disease in Sprague-Dawley rats (Heymann et at., 1959) or extracts of isologous kidney induced mild disease in a small percentage of inbred Lewis rats (Watson and Dixon, 1966). However, where compared, homologous antigens have been more nephritogenic than isologous. Inbred Buffalo rats were resistant to the induction of this disease by immunization with either homologous or heterologous kidney extracts. b. The Antibodies. Rats immunized by the method of Heymann developed precipitating antibodies to proteins from rat renal extracts as revealed by the Ouchterlony method (Hunter et al., 1960). Several bands of precipitation were present, some of which showed reactions of identity with liver extracts. The incidence of rats developing antibodies depended upon the time lapsed after immunization. Maximum response was seen between the third and seventh injections at which time 20% of rats tested had antibodies. There was no correlation between the presence of these antibodies and the presence or severity of renal disease ( Hunter et al., 1960). Hemagglutinating antibodies were also present in the sera of a few rats (Hunter et al., 1960). The ./-globulin of nephritic rats if labeled with I 3 l I and injected into normal isologous rats showed specific kidney localization ( Glassock and

60

EllIIL €3. U N A h W A h 9 FRANK J. DIXON

\\’atson, 1966). This antibody, hovwer, fixed predominantly to nonsedimentable rcnal antigens and had a very short half-disappearance time from the kidneys (18 hours) in contrast to the NTAb which fixed to sedimentable renal antigens and had a very long life in the kidneys (Glassock and Watson, 1966). c. Delayed Hypersensititiity. Nephritic rats usually had negative skin tests to renal extracts (Hunter d al., 1960). Holm (1966) demonstrated an in tiitro cytotoxic effect of lymphoid cells from rats made nephritic by Heymann’s method on rat kidney cells, presumably tubular, grown in tissue culture. This effect was only obtained if the lymphoid cells were harvested at a very critical period after immunization with antigen (8-11 days after a booster injection in nephritic rats); the intensity of the cytotoxic effcct correlated with the intensity of the nephritis. 4. Transfer of Nephritis a. Transfer by Lymphoid Cells. Several attempts have been made to transfer this nephritis from diseased rats to normal rats by means of lymphoid cells. Hess et al. (1962) and Heymann et al. (196213) reported successful transfer of disease using an outbred strain of rats. SpragueDawley rats were made tolerant at birth to spleen cells from a pro. spective donor; the donor was then made nephritic by the method of Heymann; once nephritis developed, the rat was sacrificed, its lymphoid cells harvested and injected to the tolerant rat. A mild nephritis developed in most of the recipients starting 2 weeks after transfer characterized by slight proteinuria, hypercholesterolemia, and slight histological glomerular changes. Pathologically the authors reported that the nephritis was similar in both donor and recipient; however, the published electron micrographs of the transfer recipient’s glomeruli failed to disclose the characteristics deposits on the epithelial side of the basement membrane which characterized the disease in the donor. The most evident histological change in the recipient appeared to be endothelial proliferation which was not an important feature of the original disease. Similarly, though the report mentions the deposition of y-globulin in the recipient’s glomeruli, the published photograph disclosed a faint mesangial distribution of y-globulin and not the typical capillary wall pattern noted in the original nephritis. These differences between the original disease and that observed in the recipient make it doubtful that the results of Hess ct al. (1962) and Heymann et al. (1962b) really represent transfer of the primary glomerulonephritic process. Also, in transfer cxpcriments by parabiosis, as will be detailed below, where the

EXPERI-MENTAL GLOMERULOhXPHRITIS

61

number of transferred cells was many times greater than above, tlic disease was transferred only after a lag period of 4 weeks. Attempts to transfer the disease by lymphoid cell of nephritic inbred rats of Fisher and Lewis strains to normal isologous rats have met with complete failure (Watson and Dixon, 1965; Dixon et al., 1963;; Glassock and Watson, 1966). These attempts were made at different time intervals after immunization, with a variable and supposedly optimal number of cells, and with carefully controlled conditions of cell viability. No clinical or pathological changes developed in the recipients. b. Transfer by Parabwsis. Nephritis was transferred if nephritic rats were united in parabiosis with normal rats (Heymann et al., 196213; Glassock and Watson, 1966). Two approaches have been used in these experiments. In one, normal rats were united in parabiosis without a common peritoneal cavity; nephritis was then induced in one rat by conventional methods. The uninjected partner developed nephritis at the same time or 2-5 weeks after the injected partner (Heymann et al., 1962b). In the second approach an already nephritic rat was united to a normal isologous rat which then developed nephritis a minimum of 4 weeks later (Glassock and Watson, 1966). These experiments did not help explain the pathogenesis of the disease for lymphoid cells, immune complexes, emulsion containing antigen, and free antibody no doubt could circulate from the nephritic to the normal partner. Glassock and Watson (1966) in an attempt to define the mechanisms involved in parabiotic transfer pcrformed short-term parabiosis between nephritic and normal rats. In an 11-day parabiosis, no transfer of disease occurred despite a calculated transfer of 52 total rat blood volumes per day. Also, if a rat made nephritic by long-term parabiosis to a primary nephritic rat was separated and then united to a normal isologous rat, no disease occurred in the latter. These studies suggested that for disease to transfer a prolonged period of parabiosis was necessary; that circulating lymphoid cells alone were not capable of transferring the disease; and that apparently a depot of antigen had to be present in the host for disease to occur.

5. Pathogenetic M e c h a n i m The pathogenetic mechanisms involved in this type of nephritis are not clear. Two explanations given to account for the glomerular disease are ( a ) that it is the product of an autoantibody to glomerular capillaries (Heymann et al., 1963) and ( b ) that it is at least, in part, the result of a circulating antigen-antibody complex lodging in the glomeruli ( Dixon et al., 1965).

62

E3IIL R. UNASUE A S D FRANK J. DIXOX

The thcsis that autoantihodies to glomeruli were involved in thiq disease is based on the folloming observations: ( a ) transfer of the disease from nephritic to normal rats by lymphoid cells and parabiosis, ( b ) presence of 7-globulin in the glomeruli of nephritic rats, ( c ) presence of circulating antirenal antibodies in the circulation of nephritic rats. and ( d ) decrease in the incidence of disease if rats at birth were made immunologically tolerant to kidney extract ( Heymann et al., 1963). Still to be explained by those who support this thesis are the reasons for the prolonged lag period between immunization and the appearance ot glomerular r-globulin, the distribution of immune reactants in the glomeruli, the significance of the deposits seen ultrastructurally on the epithelial side of the basement membrane, and the absence of these pathological and immunohistochemical characteristics with well-known antiglomerular antibodies. The thesis that circulating antigen-antibody complexes contribute to the pathogenesis of this nephritis is based on the following facts: ( a ) In all the transfer studies by isologous lymphoid cells a depot of antigen in adjuvant had to be present in the recipient rats for disease to develop. The experiments of \Vatson and Glassock (1966), using parabiosis also supported this point. ( b ) The demonstration of host y-globulin and PICglobulin in ii beaded pattern along the glomerular capillary wall suggests that they are deposited in complex form and are not manifestations of an antiglomerular antibody. In addition the demonstration of endogenous renal tubular antigen in these immunologic deposits in the glomeruli supports this thesis (Edgington et al., 1966). ( c ) The effective antigen in this nephritis appears to be in cytoplasmic tubular fractions and not in glomerular basement membranes; consequently a large part of the antibody found in the circulation reacts with similar fractions. Though these experiments suggest that immune complexes probably form, circulate, and are then deposited in and injure the glomerulus, the nature of the antigen in such immune complexes is unknown. There is a possibility that the antigen could derive from the depot of adjuvant; however, it is more likely that it is an endogenous tissue protein related to the injected antigen. Further studies on the nature of the antigen, on the amounts of antigen needed for inducing disease, on the immunological composition of the glomerular deposits and of the antibodies inade by the rat should aid in the understanding of the pathogenetic mechanisms. Regardless of the immunological mechanisms involved, a direct role of the adjuvant in the renal injury is recognized. Freunds adjuvant had a dual effect on the kidneys. First it enhanced and sustained any im-

EXPERIMENTAL GLOhlERULONEPHRITIS

63

mune response; if the immune responsc was directed to antigens in or of the glomeruli, the ensuing nephritis would be enhanced or maintained (Rothbard and Watson, 1959; Hammer and Dixon, 1963; Arana et al., 1964; Unanue and Dixon, 1965b ). Second, Freund's adjuvant, particularly when administered by the intraperitoneal route, had an injurious effect on the glomerular capillaries which was evidenced by their increased susceptibility to NTAbs. Watson et al. (1965) primed rats with Freunds adjuvant and then challenged them with minute amounts of NTAb's. They found a 15-20 time increase in the primed rats' susceptibility to acute nephritis which was unrelated to an autologous phase response or to delayed hypersensitivity to the tubercle bacillus. The cause of this increased susceptibility to tissue injury in adjuvant primed rats was unknown but it has also been reported for the thyroid (Roitt et al., 1961) suggesting that is was related to a systemic effect of the adjuvant. Also, Freunds adjuvant alone given intraperitoneally to rats apparently caused some degree of clinical and morphological glomerular abnormalities. Mild proteinuria in a small number of animals (Heymann et al., 1962a) and slight ultrastructural alterations have been observed after its use (Watson et al., 1965). Whether the Hemophilus pertussis in the experiments of Blozis et al. (1962) acted in a manner similar to the Freunds adjuvant is not clear. Hmnophilus pertussis vaccine is known to markedly potentiate hypersensitivity reactions in mice, not only by increasing an antibody response but also by systemic effects on capillary permeability (see review of Munoz, 1964).

C. SHEEP

I. General Characteristics Sheep immunized to heterologous or homologous glomerular basement membrane in complete Freunds adjuvant developed glomerulonephritis ( Steblay, 1962a, 1963a). When antigen was administered every 2 weeks, 30-90 days after beginning of immunization, most sheep developed a severe and usually fatal glomerulonephritis. The disease was characterized clinically by its acuteness and rapidly progressive course which lead to terminal renal failure with azotemia (Steblay, 1962a).

2. Pathology and Immunohistochemisty Detailed sequential histopathological studies have not been reported. Iiowevcr, sheep dying of renal failure exhibited a severe proliferative

64

EhIIL H. U S A S U E AND F R A h X J. DIXON

t y p of gloinerulot~c~phritis with a striking nuin1)er of periglomerular epithelial crt~scents.Tubular and interstitial changes wert. also prominent (Steblay. 196%). Throughout the course of the disease gross hcmaturia ivas ~ommoneven 1)efore ;idvanced glomerular lesions developcd ( Lerner and Dixon, 196%) Ultrastructtirally the glomeruli exhibited moderate to severc' endothelial swelling and proliferation and the basement membranes showed small, focal areas of thickening ( Feldman, 1963). immunohistochemical studies on nephritic sheep have disclosed the presence of host r-globulin and pIc-globulin distributed in a linear pattern along the glomerular capillary and tubular basement membranes. The deposition of y-globulin and pIc-globulin usually preceded the appearance of disease b y several weeks (Lerner and Dixon, 1966a,b).

3. Immunology

n. The Atitigcn. Heterologous (human, dog, rat, and rabbit) and, less effcctivcly, homologous glomerular basement membranes would induce nephritis ( Steblay, 1962aa,196Sa). Human placenta in Freund's adjuvant apparently was ineffective ( Steblay, 1962a); human lung, however, was as effective as human glomerular basement membrane in inducing disease (Rudofsky and Steblay, 1965). The incidence of nephritis with heterologous basement membrane immunization appeared to be virtually 10052, based on a relatively small number of animals observed. The antigens were effective only if incorporated in Freunds complete adjuvant containing Mycobacterium butyriczrm ( Steblay, 196%). The route of administration was not critical: intraderrnal, subcutaneous, and intramuscular routes were all effective ( Steblay, 1962a). Administration of complete Freund's adjuvant without antigen to sheep induced mild renal changes as evidenced by slight proteinuria and occasional glomerular changes ( Steblay, 1962a). b. The Antibodies. Sheep immunized to heterologous kidneys developed antibodies capable of inducing NTN if administered to animals of the same species as the donor of the antigen (Steblay, 1962a). In addition, approximately 0.1% of the y-globulin of sheep with nephritis induced by human glomerular basement membrane localized in vivo in the kidneys of a normal sheep as observed with an "'I label (Lerner and Dixon, 1966a,b ) . By fluorescent antibody the injected y-globulin was localized in the glomerular and tubular basement membranes. The kidneyfixing antibody concentration in the serum increased after bilateral nephrectomy of the nephritic sheep suggesting that the nephritic kidney normally fixed most of the circulating autoantibody (Lerner and Dixon, 1966a,b).


65

4. Trciwsfer of Nephritis Nephritis has been transferred from nephritic sheep to normal sheep by a 4-hour cross-circulation ( Steblay, 1964b). This procedure was effective if cross-circulation was established after bilateral nephrectomy of the nephritic sheep. The recipient sheep developed a nephritis identical pathologically to the nephritis of the donor (Steblay, 1964). Globulin from nephritic sheep containing 180 pg. of kidney-fixing antibody when administered to a unilaterally nephrectomized, otherwise normal lamb produced a heavy proteinuria lasting several days, and a prompt accumulation of PMN's in the glomeruli. Sheep ,-globulin and PIc-globulin could be found localized in the glomerular capillary walls shortly after transfer of the nephritic serum. Molecule for molecule the kidney-fixing antibody in these preparations was about one-fifteenth as nephritogenic as rabbit antisheep kidney-fixing antibody. Absorption of the globulin to be transferred with sheep glomerular basement membrane removed its nephritogenic effect ( Lerner and Dixon, 1966b) . By using large amounts of whole plasma from nephritic sheep, Rudofsky and Steblay (1966) have been able to transfer a progressive disease similar to that in the donor to adult recipients pretreated with Freunds adjuvant. The role of the adjuvant and the possible significance of the spontaneous nephritis of adult sheep (see below) in this transfer of a progressive disease were not clear. Pregnant nephritic sheep did not transfer their disease to the offspring ( Steblay, 1963d). However immunohistochemical studies were not performed on the newborn in order to rule out transfer of small amounts of autoantibodies. 5. Pathogenetic Mechanisms The most clearly defined pathogenetic agent of this disease appears to be an autoantibody capable of reacting with the host's glomerular capillaries. If other factors contributed to this nephritis they have not as yet been identified. Direct proof for the autoantibody has been obtained by the induction of a nephritis in normal lambs injected with globulin of nephritic sheep containing kidney-fixing antibody and the removal of the nephritogenic effect by absorption of the globulin with glomerular basement membrane ( Lerner and Dixon, 1%6a,b). Presumably, though not proven, the autoantibody was directed to antigenic components shared by the immunizing antigen and the sheep glomerulus. The sheep appears to develop this antibody with great ease and also to be highly susceptible to immunological renal injury. Heterologous NTAb's are approximately

66

EXIIL R. U S A S U E AXD FR.4h‘K J. DIXOX

1.5 tiniris inor(’ nepliritogtwic in s1icc.p on a dose for \veight basis than in rabbits and rats (Lerner and Dixon, 1966b). Onc problem lvith experimental renal studies on shccp was the high incideiicr of a spontaneous proliferative glomerulonephritis in adult sheep. Lerner and Dixon ( 1966a) examined sheep older than 1 year from different geographical areas of the United States and Europe and found that approximately 90%of animals had a spontaneous proliferative glomerulonephritis with ?-globulin and p,,.-globulin fixed in the glomeruli, accompanied by a 30%incidence of proteinuria and increased BUN. The etiology and pathogenesis of this disease are not understood but it is extremely important that experimental renal work in sheep be done with full realization of this natural complication and all possible measures taken to control it.

1 . Gcncral Characteristics Rabbits repeatedly immunized with homologous or heterologous kidney fractions containing basement membranes may develop glomerulonephritis ( Xlilgroni ct aE., 1964; Unanue, 1966). Some rabbits immunized to heterologous mammalian kidneys intraperitoneally without adjuvants developed acute transitory episodes of proteinuria beginning several days after initiation of immunization and lasting from 3 to 15 days (L‘nanue, 1966). Of the rabbits immunized to homologous whole kidney, kidney sediment, or glomerular basement membrane in complete Freunds adjuvant 20 to 50% developed nephritis within 50 to 90 days after immunization. Clinically the glomerulonephritis was characterized by proteinuria, cylindruria, and a variable course-rapid progressive renal failure in some rabbits or insiduous chronic disease in others (Unanue, 1966; Vnanue and Dixon, 1967). 2. Pathology and 1mmunohistocliemistl.y The glomerulonephritis was predominantly membranous with a moderate proliferative component and on occasions marked infiltration tty PILIN’s. The severity of the nephritis depended upon the material used for immunization and route of injection. Those rabbits immunized to heterologous kidne!. intraperitoncally and developing acute transitory proteinuria exhibited mild glomerular disease. Ultrastructurally they showed focal areas of endothelial swelling and of basement membrane thickening and swelling and flattening of the podocytes. Those rabbits immunized to homologous kidney fractions in Fremd’s adjuvant dc-


67

veloped a more severe glomerulonephritis. Ultrastructurally they showed moderate endothelial swelling and basement membrane thickening. Focal irregular accumulations of electron-dense material were noted on the epithelial side of the basement membrane. The amount and morphological appearance of this material was variable and apparently poorly related to the time and extent of immunization (Unanue, 1966). Immunohistochemically, rabbits immunized with heterologous kidney regardless of whether or not they had symptoms of renal injury showed localization of y-globulin in their glomerular capillary walls in a linear pattern ( Unanue, 1966). The incidence of rabbits having y-globulin fixed in the glomeruli depended upon the heterologous kidney used for immunization. Practically 100%of rabbits immunized with mammalian kidney and 40-5W of rabbits immunized with avian, or mesonephrictype, kidneys showed y-globulin in their glomeruli. At least part of the y-globulin fixed in the glomeruli appeared to be antibody to glomerular antigens. Material eluted at acid pH from kidneys of rabbits immunized to human and rat kidney reacted with glomeruli of frozen sections of normal rabbit kidney as detected immunohistochemically. Approximately 2@50%of rabbits immunized with homologous kidney, i.e., whole kidney, sediment, or glomerular basement membrane in adjuvant showed localization of y-globulin in their glomerular capillary walls in a linear pattern. Rabbits immunized with whole kidney preparation, kidney extract, or sediment also showed localization of y-globulin in their tubular basement membrane (see Fig. 6 ) (Unanue and Dixon, 1967).

3. Immunology a. The Antigen. Preparations of homologous whole kidney, kidney sediment, or glomerular basement membrane were effective in inducing glomenilonephritis when incorporated in complete Freunds adjuvant. Both intradermal and subcutaneous routes of administration and both Mycobacterium butyricurn and M ycobacterium tuberculosis were effective (Unanue, 1966). No higher incidence of glomerulonephritis was obtained by immunizing with heterologous mammalian kidney sediment although more of the immunized rabbits show 7-globulin fixed in their glomeruli. No glomerulonephritis developed if immunization was done with kidney sediment from species phylogenetically remote such as the duck, frog, or turtle. Immunization with homologous renal extracts did not induce nephritis although some rabbits developed antibodies to tubular basement membranes ( Unanue and Dixon, 1967). Autologous basement membranes likewise, were not effective antigens (Unanue and Dixon, 1967). Heter-

68

EXXIL R. U N A N U E A S D FRANK J. DIXON

ologous rat organ sediments such as muscle and heart were not nephritogenic antigens (Unanue, 1966). b. The Antibodies. Rabbits immunized to heterologous kidney fractions developed antibodies which reacted with the immunizing antigen. Thus, complcment-fixing antibodies were detected on the sera of rabbits immunized to mouse (Furth and Kabat, 1941), dog (Simonsen, 1953), and rat (Asherson and Dumonde, 1963) kidney. In the case of rabbits immunized to rat kidney the antibodies were shown to react with both autologous and homologous renal antigens, to be mainly yz-globulins and to have cross-reactivity with other organs (Asherson and Dumonde, 1963). Also. sera from these rabbits fixed to the cytoplasm of tubular cell as detected iminunohistochemically (Johnson et al., 1963). 7-Globulin from somc' rabbits immunized to rat kidney sediment if Iabeled with '?'I localized in tjico in rabbit kidneys ( Unanue, 1966). Labeled 7-globulin from such rabbits, if previously absorbed with rabbit kidney and then injected to rats, had less fixation in the rat kidneys than unabsorbed preparations ( Gcry et al., 1965; Unanue, 1966). These autoantibodies were directed to those antigenic determinants shared or cross-reacting between the ininiunizing kidncy and that of the host (Unanue and Dixon, 19671, The results of immunization with homologous kidney were variable. Some investigators were not able to detect complement-fixing antibodies to rabbit kidney extracts in sera of rabbits immunized with homologous kidney (Parks et al., 1936-37; Peck and Thomas, 1948). However, others using kidney extract mised with streptococcus or staphylococcus toxins ( Schiventker and Comploier, 1939) or with streptococcal organisms ( Cavelti and Cavelti, 1945a) or autolyzed kidney extract ( Sprunt and Dulaney, 1951) were successful in obtaining complement-fixing antikidney antibodies. These results would suggest that certain alterations of the antigen induced by tosins or autolysis rendered it more antigenic or that the toxins were behaving as an adjuvant. Holton and Schwab (1966) have recently shown that a mucopeptide from the cell wall of streptococcus behaved as an adjuvant to rabbits immunized with bovine albumin. Recently Milgrom et al. (1964) were successful in obtaining antikidney antibodies in rabbits repeatedly immunized to whole kidney in complete Freund's adjuvant. These antibodies were detected by complement fixation and by precipitation in agar. By the latter techniquci these iinsestigators \ \ w e able to detect both organ-specific and nonspecific antibodies. Similar13 , globulin of some rabbits immunized to whole Liclney, kidne! sediment, or glomerular basement membrane in complete Freiiiid'\ adjuvant contained kidney localizing antibodies detwtable in citio by lA1Ilabeling (Unanue, 1966). Not all autoantibodies,


69

however, showed in uivo localizing properties, for some were directed to renal antigens apparently unexposed to circulating blood (Unanue and Dixon, 1967). All experimental work with antitissue antibodies in rabbits should take into consideration the presence of naturally occurring antitissue antibodies. Kidd and Friedewald (1942a,b) detected, by complement fixation, in the sera of most adult rabbits a heat-labile antibody reacting with the rapidly sedimentable material of most homologous organs. The antigen was present in decreasing order in kidney, liver, lung, brain, spleen, and heart and was not species-specific. Rabbits less than 24 days old did not exhibit this naturally occurring autoantibody which was identified most often as a macroglobulin although occasional rabbits exhibited some activity in their y-globulin (Asherson and Dumonde, 1963). Another naturally occurring antibody-like material has been found by Spar et al. (1956). These investigators obtained by acid or heat extraction of kidneys of normal rabbits a material which when labeled with I3lI and reinjected into rabbits or rats localized in their kidneys with a high organ specificity; this material was not present in eluates of other organs. It appeared because of its renal specificity to be different from the Kidd-Friedewald antibody. The biological signscance of these antibodies or their anatomic sites of reaction are unknown.

4 . Transfer Studies y-Globulin containing kidney-fixing antibody from rabbits made nephritic by immunization with kidney sediment or whole kidney when injected into normal rabbits produced a mild and transitory proteinurin in the recipients. The injected y-globulin localized in the glomeruli of the normal recipients which exhibited a mild proliferative glomeruliti9 (Unanue, 1966; Unanue and Dixon, 1967). 5. Pathogenetic Mechanisms Rabbits immunized to heterologous or homologous basement membranes developed autoantibodies which reacted with their own glomeruli. The antibodies were apparently directed to those antigens cross-reactive or shared between the immunizing kidney and that of the host (Unanue and Dixon, 1967). Dumonde (1966) has used the term “crossreactive autoimmunity” to define such phenomena. In the case of the kidney, shared antigens have definitely been established among mammals, and also among mammals and distantly related species such as ducks and frogs (Unanue and Dixon, 1967). There was no strict correlation between the presence of autoantibodies and the development of glomerulonephritis in the immunized rabbits. Two factors were apparently of impor-

70

E\\III. H. USANUE AXD FHANK J. DIXON

tance in the development of glomerulonephritis: ( 1 ) the amount of autoantibody synthesized by the host which in most cases was small and transitory; and ( 2 ) the number of antigenic sites in the host's glomerulus to which the antibody could fix (Unanue and Dixon, 1967). Glomeriilonephritis devcloped when sufficient antibody was formed, provided the host's kidney had enough common antigen to allow the fixation of a nephritogenic amount of antibody (Unanue and Dixon, 1967 ) .

E. OTHEREXPERIMENTAL ANIMALS Steblay { 1963c) successfully induced glomerulonephritis in monkeys by immunizing them with heterologous (human, rabbit, rat, and dog) glomerular basement membrane in Freunds adjuvant containing Mycobactc>rittm hutyricum. The nephritis appeared to be of mild to moderate intensity in the majority of monkeys studied. All showed a latent period between beginning of immunization and disease which varied from 30 to 70 days. The same investigator also reported induction of a severe fulminating nephritis in two goats immunized with human glomerular basemrnt membrane in Freunds adjuvant containing Mycobacterium butyriczim ( \\'illiams and Steblay, 1965). Attempts to induce nephritis in mice (Mardiney and Feldman, 1964), guinea pig\ (Heymann et a]., 1962a), and dogs (Heymann et al., 1962a) by homologous kidney extract in Freunds adjuvant were not successful. Milgrom et al. (1964) were successful in inducing antibodies to autologous or Iiomologous kidneys in guinea pigs immunized with homologous whole kidney. but no nephritis developed in these animals. However, guinea pigs immunized to human glomerular basement membrane and rdt and duck kidney sediment developed fixation of autologous 7-globulin and complement in their glomerular capillary walls (E. R. Unanue and F. J. Dixon, unpublished observations, 1966). Also approximately 30% of the ones immunized to human glomerular basement membrane and rat kidne!, sediment de\reloped glomerulonephritis ( Unanue and Dixon, unpubli5hed observations, 1966). Peters ( 1961 ) reported the induction of glomerulonephritis in guinea pigs immunized to mouse fibroblasts in complete Freund's adjuvant. At the time the animals became nephritic they ediihited dc,layed h!persensitivity reactions and no circulating antibodies to the fibroblasts which suggested to the author that the former rt~ictioncould lw related to the nephritis. .ICKSOWI,EDGWXTS Dr. C11,uIt.s C. Cochr.ine foi cloriatinfi Figs. 2 .tnd 8 ant1 to DI. Joseph D. Feltliiian foi cloii;iting Figs. 9, 10, and 11. \\'(%mi q1.ItetiiI to

FIG. 1. Fluorescence micrograph of a glomerulus from a rat with NTN. Rat was sacrificed 30 days after injection of rabbit NTAb. Section was stained with fluorescent antisera to rat y-globulin. Note the uniform linear distribution of autologous y-globdin along the capillary walls. Inset shown at higher power is from a glomerular capillary wall of similar rat but stained for rabbit y-globulin-a similar linear localization is seen. FIG. 2. Fluorescence micrograph of a glomerulus from a rabbit developing acute serum sickness nephritis after injection of bovine serum albumin. Injected antigen is distributed in a granular pattern along the capillary walls. Similar distribution was noted when sections were stained for rabbit y-globulin and PI,;globulin. 71

72

EMIL R. UNASUE A X 9 FRANK J. DIXON

Frc.. 3. Fluorescence micrograph of a glomerulus from a rabbit with chronic $lomeriilonephritis incluced by repeated injections of bovine serum albumin. Section \vas stained with fluorescent antisera to bovine serum albumin. Bovine albumin, riibl,it y-globulin, and p,c-globulin are localized similarly a5 heavy granular deposits ( axrows ) along the capillary walls. FIG. 4. Fluorescence micrograph of a glomerulus from an NZB mouse developing spontaneous glomerulonephritis. Section is stained with fluorescent antisera to mouse fibrinogen. There is heavy concentration of the material in enlarged and distorted axial (mesangial) areas. The peripheral capillary walls (arrows ) show little concentration of fibrinogen. These peripheral areas contained y-globulin and p,,.-gIohulin in a granular pattern. A similar axial distribution of plasma proteins in the glomeruli may be seen i n many nonimmunologically induced nephritides and is n o t indicative of an immune reaction. 1 7 1 ~ ; . .5. Fluorescence micrograph of ii glornerdus from a Lewis rat made nephritic by immunization with rat kidney. Section is stained with fluorescent antisera to rat y-globulin. Rat y-globulin is distributed along the capillary walls in a fine granular pattern. Inset shows a higher magnification of a capillary wall with numerous discrete deposits (arrows).


73

FIG.6. Fluorescence micrograph of several renal tubules from a rabbit imiiiunized with rabhit kidney. Section is stained with fluorescent antisera to rabbit 7-globulin. Rabbit y-globulin is noted in focal areas ( arron. ) along the tubular hsement rnemhrane. Fir;. 7 . Fluorescence micrograph of a glomerulus from R rat with NTN. Rat u-as sacrificed 60 days after injection of STAb’s. Section is stained with fluorescent antisera to rat fibrinogen. Fibrinogen is concentrated in a large epithelial crescent and in interstitial tissue. The epithelial crescent also contained y-globulin ant1 nlliinnin Init i n lesser amounts. Sote ahsence of fibrinogen in glomerular capillarim. 74


75

FIG.8. Electron micrograph of a glomerdar capillary of a rat with NTN sacrificed 2.5 hours after injection of rabbit NTAb's. A polymorphonuclear leukocyte is in intimate contact with the basement membrane and apparently displacing the endothelium above. BM, basement membrane; E, endothelial cell; Ep, epithelial cell; PMN, polymorphonuclear leukocyte.

76

EMIL R. UKANUE AND FRANK J. DIXON

FIG.9. Electron micrograph of :i glonierular capilhry of a rat with NTN sacrificed :3 days after injcctioii of rabbit NTAb. The basement membrane is thickened and shows an irregular deposit ( D ) along its lumiiial side. An area of norm;tl ;ippe;iring, haseinent mtwbranr is shown at X. Endothelid cytoplasm is swollen. Epithelial foot processes are broad. BhI, basement membrane; D, deposits; En, endothdial cell; Ep, epithelial cell; L, lumen; X, normal basement membrane.

EXPERIMENTAL CLOMERULONEPHRITIS

77

FIG. 10. Electron micrograph of a gloinerular capillary of a rabbit with complex nephritis induced by repeated injections of bovine serum albumin. Dense deposits, which are known to contain antigen, host complement and y-globulin, are noted all along the epithelial side of the basement membrane. The basement membrane is in itself irregularly thickened and frayed in areas. BM, basement membrane; D, deposits; En, endothelial cell; Ep, epithelial cell; Rbc, red blood cell.

Frc. 11. Electron micrograph of the wall of a ghneiiilar capillary from a rat matle nephritic by injection with rat kidney. Dense irregular deposits are noted along the epithelial side of the basement membrane which in itself appears normal. Compare deposits xvith those seen in Fig. 10. The deposited material seen in Fig. 11 is more irregular in texture but otherwise quite similar to that seen in complex induced nephritis in rabbits. Bhl, basement membrane; D, deposits; En, endothelid cell; Ep, epithelial cell; I., lunien.

78


79

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Chemical Suppression of Adaptive Immunity1 ANN E. GAB RIEL SEN^ AND ROBERT A. GOOD^ Pediatric Reseorch Loboratories of the Variety Club Heort Hospital and the Department of Micrabiotog University o f Minnesota Medico1 School, Minneapolis, Minnesota

I. Introduction . . . . . . . . . . . . A. The Immune Mechanism . . . . . . . . . B. Specific Immunological Negativity . . . . . . C. Carcinogenesis, Carcinostasis, Teratogenesis, and Immunosuppres. . . . . . . . . sion 11. Early Experiments on Immunosuppression with Cytotoxic Agents . 111. Salicylates . . . . . . . . . . . . IV. Adrenal Steroid Hormones . . . . . . . . . A. Effects of Adrenal Steroids on Antibody Production . . . B. Effects of Adrenal Steroids on Antibody-Mediated Hypersensitivity C. Effects of Adrenal Steroids on the Complement System . . D. Effects of Adrenal Steroids on Phagocytic Mechanisms . . E. Effects of Adrenal Steroids on Delayed Allergic Responses . F. Effects of Adrenal Steroids in Experimental Autoimmune Disease . . . G. Effects of Adrenal Steroids on Tumor Immunity . H. Effects of Adrenal Steroids on Transplantation Immunity to . . . . . . . . . . Normal Tissues . I. Adrenal Steroids and Homograft Rejection in Man . . . J. Mechanisms of Adrenal Steroid Activity in Immunosuppression V. Allcylating Agents . . . . . . . . . . . A. Effects of Alkylating Agents on Antibody Production . . . B. Effects of Alkylating Agents on Phagocytic Mechanisms . . C. Effects of Alkylating Agents on Inflammation . . . . D. Effects of Alkylating Agents on Delayed Hypersensitivity . . E. Effects of Alkylating Agents on Experimental Autoimmune Disease F. Effects of Alkylating Agents on Transplantation Immunity . C . Conclusion . . . . . . . . . . . VI. Folic Acid Antagonists . . . . . . . . . . A. Effects of Folic Acid Antagonists on Antibody Production . . B. Effects of Aminopterin-Amethopterin on Delayed Hypersensitivity C. Effects of Aminopterin-Amethopterin on Allograft Immunity and . . . . . . . . Graft vs. Host Disease .

. . . .

92 95 99 102 102 104 109 110 114 116 116 117 118 120 121 123 124 125 126 130 131 132 133 134 137 138 139 141 143

‘Original work from our laboratories was supported by grants from the U.S. Public Health Service ( AI-00798, HE-02085, NB-02042, HE-06314, HE-05662), the American Heart Association, and the National Foundation. ’ Research Fellow, Department of Pediatrics, U.S. Public Health Service grant 9T1-AI292. American Legion Memorial Heart Research Professor of Pediatrics and Microbiology. 91

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. GOOD

D . Effects of Amethopterin on Lymphocytic Choriomeningitis . E . Effects of Amethopterin on Experimental Autoimmune Disease F . Clinical Use of Aminopterin and Amethopterin . . . . VII. Antimetabolites of the Purine Bases . . . . . . . A . 8-Azaguanine . . . . . . . . . . . B . 6-Mercaptopurine . . . . . . . . . . C . Azathioprine (Imuran) . . . . . . . . . D . 6-Thioguanine . . . . . . . . . . . VIII . Analogs of the Pyrimidine Bases . . . . . . . . IX . Antibiotics . . . . . . . . . . . . A . Puromycin . . . . . . . . . . . B. Mitomycin C . . . . . . . . . . . C . The Actinomycins . . . . . . . . . . . . . . . . . . D . Chloramphenicol . . E . Azaserine . . . . . . . . . . . X . Plant Alkaloids . . . . . . . . . . . . A . Calchicine . . . . . . . . . . . . B . The Vinca Drugs . . . . . . . . . . XI . e-hinocaproic Acid . . . . . . . . . . XI1. Acriflavine . . . . . . . . . . . . . XI11. Ataractic Drugs . . . . . . . . . . . XIV . hlethylhydrazine . . . . . . . . . . . . . . . . . . . . . . XV . P-3-Thienylalanine XVI . Penicillamine . . . . . . . . . . . . XVII . Hydroxyurea . . . . . . . . . . . . XVIII . Antilymphocyte Serum . . . . . . . . . . XIX . Discussion . . . . . . . . . . . . References . . . . . . . . . . .

14F 146 147 148 148 149 164 167 168 170 171 172 172 178 182 183 183 185 187 190 192 194 196 197 198 200 202 205

I . Introduction

It was not long after the clear delineation of the difference between innate and adaptive immunity. by such scientists as Pasteur (1881). Nuttall ( 1888).P. Ehrlich ( 1892). Behring and Kitasato ( 1890). that the notion of unwanted. misdirected acquired immune responses emerged in Von Pirquet’s (1906) definition of allergy. Since that time the categories of harmful immune responses have been broadened and redefined many times; indeed. one of the major areas of contention among immunologists of the present day concerns the nature and scope of “autoimmunity” as reflected in Werent experimental and clinical diseases. It seems likely to us that viruses. mycoplasma. and even bacterial organisms will ultimately be identified as the initiators of much “autoimmune” disease; however. this does not in any way minimize the role of immunological processes in production and perpetuation of disease. Indeed some of the clearest recent insights into certain kidney diseases have been gained by the analogy to experimental antigen-antibody complex disease ( Dixon

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et al., 1961; Michael et al., 1964b, 1966a). Other very useful experimental models have been the virus-induced hypergammaglobulinemia of Aleutian mink disease (Henson et al., 1962; D. D. Porter et al., 1965), the experimental allergic diseases, particularly encephalitis (reviewed by B. H. Waksman, 1959; Paterson, 1966) and thyroiditis (reviewed by N. R. Rose et al., 1964), and certain of the graft vs. host phenomena (Oliner et al., 1961). Immunosuppression may also be useful in treatment of several types of hypersensitivity, including classic atopic allergy ( Bukantz and Aubuchon, 1957) and anaphylactic phenomena (Nelson et al., 1950) and in modification of the delayed allergic responses which are the basis of both symptomatology and pathology in bacterial, viral, and fungal disease (reviewed by Lawrence, 1956). During the last 10 years another category of “undesirable” immune response has been created by the technical feasibility of organ allotransplantation, particularly in patients with irreversible kidney disease. Often, however, in a consideration of the triumphs and failures of immunosuppression in the transplantation context, we lose sight of the fact that immunosuppression is an everyday reality in clinical medicine and that safer, more effective agents are sorely needed in many instances. Consider a single problem, related to our own experience. During the past 2 years we have found, as have others, that high levels of immunosuppressive agents-in our own hospital a combination of azathioprine and prednisone-have favorably affected the course and prognosis of patients by all earlier criteria considered to have progressive, irreversible renal disease (Michael et al., 1966b). The goals in immunosuppression vary; often the need is for effective short-term treatment in situations in which the destructive immunological process is broadly directed, but will presumably be resolved when the antigen or toxin is removed. In many other contexts, the need is for longterm therapy aimed at the narrow “target area” of specific tissue incompatibility in allo- and xenotransplantation, for example. Most desirable is the kind of specific immunological negativity to skin and organ allografts that can be produced in adult mice by administration of viable cells, disrupted cell preparations, or subcellular fractions from mice of the donor strain, provided the histocompatibility barrier between donor and recipient is not too great (reviewed by Good et al., 1964, 1966). We have known for some time that tolerance can be facilitated by X-irradiation (Main and Prehn, 1955) or immunosuppressive agents. Especially if the leads we have on the importance of the form of the antigen (Dresser, 1962; Dresser and Gowland, 1964; Frei et al., 1965) and on the route and timing of administration ( Battisto and Miller, 1962; Mitchi-

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son, 1964; Nossal, 1966; Kraft et al., 1966) can be translated into clinical terms, specific antigen administration may ultimately permit greatly reduced dosages of immunosuppressants. There is good evidence, both experimental and clinical, that an equilibrium of host and kidney allograft activity is reached in many instances, permitting reduction or discontinuation of immunosuppressive treatment (Pierce et d.,1961; Pierce and Varco, 1962; W. D. Kelly et al., 1965, 1966, unpublished observations; Woodruff, 1964; Starzl et al., 19642,). This is often not classic tolerance, however; dogs bearing functional kidney grafts for long periods will, for example, reject skin from the same donor ( Murray et al., 1964), suggesting that the accommodation of host to graft is mutual. Influence of “immunosuppressive” drugs on the graft cannot entirely be discounted; it has been suggested that cortisone, known to stabilize membranes of certain types, may act in part to protect the graft, perhaps by limiting the tissue-destructive activity of lysosomal enzymes (Billingham et al., 1951; Dempster, 1953, Scothorne, 1956; Weissmann and Thomas, 1964). Our review is necessarily a limited one; we are restricting ourselves to adaptive immune responses: antibody production, responses of delayed hypersensitivity, homograft immunity, and immunological memory, together with their humoral and celiular correlates. Arbitrarily excluded are the suppression of inflammation and allergic phenomena requiring complex effector mechanisms largely active on vessel walls and smooth muscle ( see Brocklehurst, 1962). We are also excluding immunosuppression by irradiation except as a basis for comparison with the activity of chemical agents. Our survey attempts to be selcctive and interpretive rather than exhaustive; an effort has been made to include the experiments or investigations that have provided basic definition of the activity of the agent or agents and to leave the derivative studies for fuller treatment elsewhere. The bias is in the direction of agents that may prove to be useful clinically; however, certain agents or groups of drugs-particularly those with well-defined biochemical activity-will be considered because of the insights they offer into the nature of the immune response, rather than their usefulness pharmacologically. Indeed, one of the paradoxes which will become evident as w e proceed is that the most widely used, most effective immunosuppressants-notably the cortisone group and the purine analogs-are not well charactcrized in terms of the biochemical basis of their effects on neoplastic cells or cr.11~involved in immune responses. Initially, we should like to present a view of the immune mechanism. indicating as ell as we can where it might be vulnerable to pharma-


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cological agents and where it is known to be vulnerable to them. It has often been said that all chemotherapy is selective toxicity; most immunosuppression is not selective, and the price of immunosuppression in terms of gastrointestinal toxicity and hematopoietic depression is often prohibitive. Greater specificity of effect must be sought. Surely, as knowledge of the protein synthetic mechanisms involved in immunological processes increases, a “narrower aim” will be possible, with therapy directed at vulnerable biochemical activities peculiar to the immunologically active cell without damaging vital cell systems. This, of course, does not resolve the issue of the suppression of most normal immunological defenses in the patient in the process of suppression of a single segment. Indeed, in many kidney transplant series, infection has taken a greater toll than graft rejections. The outlook here is perhaps brighter than we might have anticipated earlier; there is every evidence from both experimental and clinical experience that if histocompatibility barriers are minimized and the patient or experimental animal can be carried successfully past the early rejection threats by immunosuppressive agents, other factors favoring the specificity of tolerance to the graft become increasingly dominant so that the drugs can be reduced to minimal levels or discontinued entirely.

A. THEIMMUNE MECHANISM In this section we will focus our attention largely on agents which affect the nucleic acid metabolism of lymphoid cells, the processes that assure the specificity of the response to foreignness in the immediate sense and the persistence of that specific experience in immunological memory. However, there are processes which precede the specific immune response and processes which follow it, both vital to the effectiveness of the total response and both susceptible to pharmacological influence. The afferent arm provides the initial reception and processing of antigen; as we shall see, it is fundamentally dissociated from the cell systems concerned with the specificity of the cellular or humoral response. This phase of the immune response is also susceptible to inhibition by pharmacological means. At the effector end of the immune response, nonspecific mechanisms come into play, notably the complement system; here, too, pharmacological manipulation is possible and may ultimately be practicable at the clinical level. Cellular immunobiology has been an area of controversy for many years, but it is only in the last 15 years that the role of the plasma cell has been well defined, and only in the last year that the relationship of the plasma cell to germinal centers and small lymphocytes has been

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clarified. We have, on other occasions, reviewed the long controversy of the lymphocyte vs. the plasma cell as the antibody-producing cell (Good, 1957; Good et al., 1962). The shift of evidence in favor of the plasma cell came with the experimental work of Kolouch (1938), Bjplrneboe and Gormsen ( 1943), and Fagraeus ( 1M8), supported by clinical evidence in diseases characterized by hypo- or hypergammaglobulinemia ( Bing and Plum, 1937; Good and Campbell, 1950; Good, 1954; Craig et al., 1954). The work of Coons d al. (1955) and other investigators using their immunohistochemical method ( Vazquez, 1962; Mellors and Korngold, 1963) established firmly that most antibody-containing cells are morphologically plasma cells. This has gained further support from electron microscopic studies showing that plasma cells have the protein synthetic apparatus typical of a secretory cell, cellular machinery largely lacking in small lymphocytes (reviewed by J. D. Feldman, 1964)Recent electron microscopic and immunohistochemical data in a variety of contexts have clearly modulated the earlier view of the plasma cell as mi generis; it now seems evident that elements that are clearly lymphocytic by classic criteria may have the protein synthetic machinery and the immunoglobulin content typical of plasma cells (Vazquez, 1964; Zucker-Franklin, 1963; T. N. Harris et d.,1966). The recent work of Cooper and co-workers (1965, 1966a) has added greatly to our understanding of these cellular elements. It had been known that neonatal thymectomy in rodents affected primarily the cellular immune processes and depleted chiefly the small lymphocyte component in the circulation and the tissues (see Good and Gabrielsen, 1964). In chickens, however, it had been difficult to demonstrate such depression following thymectomy in the newly hatched period (Warner et al., 1962; Aspinall et al., 1963; E. J. Yunis et al., unpublished observations, 1963-1964), whereas many investigators had confirmed the original findings of Glick et al. (1956) that extirpation of the bursa of Fabricius of the chicken in early life greatly curtailed the capacity of the maturing animal for circulating antibody production (Mueller et al., 1960; Papermaster et al., 1962; Isakovib et al., 1963). At the time of the studies by Cooper, there was evidence that circulating immunoglobulins were reduced in chickens surgically bursectomized at hatching or hormonally bursectomized by treatment during incubation (P. L. Long and Pierce. 1953; Ortega and Der, 1964; Carey and Warner, 1964); but there, too, the findings were inconsistent. The basis of the experiments by Cooper was the thesis that some of the variability in results might reflect development of the peripheral lymphoid tissues at the time of hatching and that less ambiguous models of immunological dc+icienc!- might be obtained


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by surgical bursectomy or thymectomy on the day of hatching, followed by heavy X-irradiation to deplete the lymphoid cells or their precursors already in the periphery. The results, discussed in detail elsewhere (Cooper et al., 1965, 1966a), showed that the chicken thymus is analogous in function to the mammalian thymus. The thymectomized-irradiated chickens had defective homograft immunity, greatly impaired delayed hypersensitivity to diphtheria toxoid, and a very low level of graft vs. host potential. They had relatively normal immunoglobulin levels, but produced reduced amounts of specific antibody to bovine serum albumin and Brucella abortus organisms. The cellular element most affected was the small lymphocyte; the white pulp of the spleen and other peripheral lymphoid tissues were greatly depleted, and circulating lymphocytes were low. Most remarkable was the agammaglobulinemia produced in the bursectomized-irradiated chickens ( Cooper et al., 1965, 1966a). These animals produce no antibody even when hyperimmunized and they have no immunoglobulins corresponding to human IgM or IgG on immunoelectrophoresis, proteins consistently present in irradiated controls. Their graft vs. host capacity is normal, and they reject skin homografts after only a slight delay. They have normal levels of circulating lymphocytes and normal numbers of small lymphoid cells in the white pulp. The morphology of the spleen and intestinal lymphoid tissues of these chickens was perhaps the most remarkable of all: germinal centers and plasma cells are entirely lacking, section after section, bird after bird. These findings suggest then that there are two lymphoid systems, that these systems are dependent for their ontogenetic development in the periphery on different central lymphoid tissues, and that either one can be defective without greatly affecting the other. That the mammalian lymphoid system is also composed of two major components is suggested by the experiments on neonatal thymectomy in rodents, noted earlier, and by clinical syndromes in which extreme lymphopenia is associated with an extreme defect in thymic development ( Glanzmann and Riniker, 1950; Tobler and Cottier, 1958; Hitzig et al., 1958; Allibone et al., 1964; Nezelof et al., 1964), as well as by the sex-linked recessive agammaglobulinemia noted by Bruton (1952) that parallels the immunological and morphological deficits of bursectomized-irradiated chickens-lack of germinal centers and plasma cells, almost no circulating IgG or IgM, and virtually complete inability to produce antibody to any antigen (reviewed by Good et al., 1962). Children with this disease have relatively normal thymuses (when these have been studied) (Good et al., 1963; Gitlin and Craig, 1963), are abIe to develop responses of the

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delayed allergy type, and usually reject skin homografts though by a slow, abnormal process. Further evidence of the validity of the two-component view of the mammalian lymphoid system has come from a series of experiments on rabbits b y Coopcxr ct al. ( 19661)). Srvcral years ago wc’ (Archer et al., 1963, 1964) noted the morphological similarity of the chicken bursa to the rabbit appendix and sacculus rotundus (intestinal tonsil); more recently, particularly in the findings in man by Cornes (1965) and in our own morphological data on the rabbit, there has been evidence that the Peyer’s patches are also bursa-like morphologically and ontogenetically. Cooper et al. (19fMb) have been able to extirpate large portions of this intestinal tissue in rabbits, irradiate them, and show a selective immunological defect-relatively intact responses of the delayed hypersensitivity type and slightly delayed but complete skin allograft rejection, but significant depression of antibody responses to a range of antigens including potent bacterial preparations. A most intriguing problem, if we accept the two-systems view, is that of antibody production. All antibody production is, of course, eliminated when the germinal centers and plasma cells are eliminated (Cooper et d.,1965, 1966a); however, when the thymus-dependent small lymphocyte development is depressed, the animals are deficient antibody producers, particularly to some antigens, despite normal levels of circulating immunoglobulins and the presence of normal germinal centers and plasma cells in the tissues. This suggests that antibody production may be a two-cell process: that a recognition-information component, at least for some antigens, is provided by the small lymphocytes and their progeny and that instructions for synthesis of the specific antibody protein are by some means transferred to the immunoglobulin production system. Recent electron microscopic studies of the rabbit spleen during an antibody response have suggested that the earliest changes are, indeed, seen among the small lymphoid cells and that blast cells develop, which Movat and Fernando (1965) call immunoblasts. These blasts are ultrastructurally distinct from other large pyroninophilic cells, plasmablasts, which seem to be clearly in the line of descent to plasma cells. Large pyroninophilic blast-type cells have often been equated with plasma cell precursors; but all the present evidence points to distinct and separate lines of this type of cell in the two systems. Our hypothesis, to which we shall return during the review proper, is that formation of the immunoblast, the large pyroninophilic cell characteristic of both the early stages of delayed allergic responses and the early stages of antibody production, is the basic adaptation yielding


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specificity of the effector protein, whether cell-bound or humoral ultimately. These cells yield small specifically sensitized lymphoid cells which probably effect delayed hypersensitivity and the classic allograft rejection. We believe that, depending on the stimulus, these same cell types can confer the elements of specificity on immunoglobulin producing cells of certain kinds. There is much speculation as to what form such a transfer might take: an antibody virus (Smithies, 1965), liquid crystals (Osgood, 1964), or components on the combining sites themselves as polypeptide chains (Burnet, 1964). Any of these seems to be consistent with what is known of the immunoglobulin molecule, including the heterogeneity of certain portions of both the light and heavy chains in the region of the antigen combining sites (Cohen and Porter, 1964; E. C. Franklin, 1966). If this construction is valid, then immunosuppression might be most profitably directed at the earliest phase of the response, particularly before specifically sensitized cells have proliferated. As will be evident later, immune responses are vulnerable at various points, but can be most consistently affected with most inhibitors during the initial “tooling up” following antigenic stimulation. Indeed, with certain inhibitors, the period of unusual susceptibility to inhibition has been narrowed to hours (Berglund, 1962, 1965; Frisch and Davies, 1962a). Many clinical applications call for ‘late” efforts at inhibition of cellular or humoral immune responses; and many experimental efforts with immunosuppressive agents have been addressed to this problem of curtailing the secondary response, abolishing immunological memory, interfering with ongoing allograft rejection, and the like. Thus, while the prototype of the drug-inhibited immunological response is a primary antibody response, often to a relatively weak antigen, the therapeutic challenge clinically is usually more formidable.

B. SPECIFICIMMUNOLOGICAL NEGATIVITY It seems to us that specific immunological negativity is the aim of immunosuppression in many situations-a specific deletion of a narrow zone of reactivity, as contrasted with the wide-spread obliteration of immune responsiveness which occurs in much immunosuppression. This type of immunological lapse has been known for many years. It received renewed interest in 1945 with the classic observations of Owen of fraternal twin cattle chimeras, i.e., two blood types, and was dubbed “tolerance” by Billingham and associates, in 1953, following their artificial creation of mouse chimeras which accepted skin grafts of both strains. Since that time an extraordinary research effort has sought to

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implement the promise of such an approach, particularly in tissue transplantation, but certainly in other contexts as well, and has sought also understanding of the phenomenon itself. Because specific immunological negativitv has been produced in many antigenic situations with the aid of immunosuppressives, as we shall note in later sections, and because this is one of the ways to solve many of the dilemmas of immunosuppression, we believe that a brief review of “tolerance” will provide helpful background. The mechanisms of immunological tolerance are under intense scrutiny in several laboratories, and some very recent observations seem to enhance greatly the possibility that this state may be predictably achievable with minimal hazard to the host ( Mitchison, 1964; Nossal, 1966; Kraft et al., 1966). If we were to consider the classic examples of specific immunological negativity which preceded the initial paper of Billingham and associates (1953), we would cite the suppression of antibody responses in the work of Glenny and Hopkins with horse serum antigen in rabbits (1923), the demonstration of “tolerance” to lymphocytic choriomeningitis virus in mice by Traub (1936), the observation of Owen (1945) in twin cattle mentioned above, immunological paralysis in mice as described by Felton (Felton and Ottinger, 1942; Felton, 1949), and the demonstration of negative adaptation to specific hapten as a consequence of feeding, discovered by Chase (1946). Though disparate in many ways, all were specific negative adaptations to antigenic stimuli. The observations of Owen (1945), the interpretation of Burnet and Fenner (1949), and the experiments of Billingham et al. (1953) all emphasized the role of immunological immaturity in establishment of tolerance. This served for several years to set neonatally acquired tolerance to allografts in mice and rats apart from specific immunological negativity induced in adult life, as in the immunological paralysis described by Felton (Felton and Ottinger, 1942, Felton, 1949) and the protein overloading type of negativity (A. G. Johnson et al., 1955; Dixon and Maurer, 1955). In a series of developments, in our laboratories and elsewhere (reviewed by Good et al., 1964, 1966), it became evident that tolerance to skin allografts may be induced in adult mice with intact cells, disrupted cells, or cell fractions, if sufficient numbers of cells or amounts of antigen are administered. Numbers of cells and amounts of antigen can be appreciably reduced if lymphoid cells are depleted by irradiation, drugs, antilymphocyte sera, or early thymectomy ( Howard ct al., 1962; Medawar, 1963; Monaco et a?.,1966b; Hilgard et al., 1964). They can also be reduced by a variety of other immunosuppressants active as nucleic acid inhibitors rather than as agents merely cytotoxic to lymphoid cells (Medawar, 1963; McKneally et al., 1964).


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How specific immunological negativity is established and maintained is not known. Among the provocative recent observations are the following. In a series of experiments Dresser (1962) and Dresser and Gowland (1964) were able to induce tolerance to serum protein antigens in both mice and rabbits with very small amounts of a soluble fraction of the antigen. Battisto and Miller (1962) achieved a similar result in guinea pigs by administering small amounts of antigen into the portal circulation. Frei et al. (1965) provided a telling confirmation of these results by showing that the soluble nonphagocytized portion of bovine serum albumin was not immunogenic, but would induce tolerance to the whole antigen in rabbits, suggesting that tolerance was induced much more easily if the reticuloendothelial system was by-passed and the soluble portion of the antigen presented directly to the lymphoid cells. Although earlier evidence suggested that a state of tolerance does not modify subsequent processing of that antigen by reticuloendothelial cells (Nossal, 1965), more recent data indicate that this phase of immune function may be affected (Nossal, personal communication, 1966). Some recent observations, from the work of M. Feldman (1966) also suggest that the function of phagocytic cells may be modified in specific immunological negativity. These investigators were able to “break” tolerance with macrophages from syngeneic mice stimulated with the antigen to which the recipient was tolerant. One way of accounting for tolerance that has a good deal of “surface appeal” is that of pseudo-feedback, perhaps by minute amounts of antibody, as suggested by the work of Fitch and Rowley (19%) and Elion et al. (1965). Another is that there may be specific suppression, at the level perhaps of enzymatic controls, that removes an essential component of the particuIar immune response, leaving an otherwise intact immune mechanism. There is a parallel in the work of van Bekkum and Nieuwerkerk (1965) in which the enzyme tryptophan pyrrolase was suppressed by its administration before birth in mice. Mitchison ( 1964), with a serum protein antigen, and Nossal ( 1966), with a Salmonella flagellar antigen, have charted positive and negative adaptation as one dimension and antigen dosage and time as the other two, in a three-dimensional view of the response to a single antigen in a single species. These most remarkable maps tell clearly what has happened, but they do not provide understanding of the basis of tolerance at the cellular level. They show clearly, however, that there are dosage ranges conducive only to positive or negative adaptation when given repeatedly and that at high doses a positive response will ultimately yield to a negative one. Kraft et al. (1966) in their work with acriflavine as an immunosuppressive agent have similarly defined two

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zoiies of antigcm dosage, Ion. and high, piirticularly conducivc to induction of specific immunological negativity. This approach seems to us to be of enormous value and one that should yield additional insights into the mechanisms of specific iinmunological negativity.

C . CARCINOCESESIS, CARCINOSTASIS, TERATOGENESIS, AND IMMUNOsuPPREssIos

Broadly, these four properties are shared by many agents, though emphases differ. One of the types of immunosuppression which we have arbitrarily excluded from this review is that resulting from treatment with chemical carcinogens, such as the methylcholanthrenes (see Rubin, 1964); however, many of the alkylating agents are carcinogenic under some conditions and will be considered as immunosuppressants (see Berenbaum, 1964a). DiPaolo and Kotin (1966) recently reviewed the parallels of carcinogens and teratogens, and the same might easily be done for carcinostatic agents and teratogens. Malmgren et al. (1952a), in one of the early comprehensive analyses of diflerent groups of agents, found that toxic compounds lacking in antitumor properties tended not to suppress hemolysin production to sheep cells in mice; whereas antitumor agents, even in nontoxic dosages, were often immunosuppressive. Immunosuppression might be said, in a very real sense, to be "borrowed from cancer chemotherapy. In most of the sections to follow a brief indication will be given of the antitumor properties of the agents, if any; however, in almost every instance, discussions of mechanisms of the immunosuppressants must be set in the context in which most of this work is done, cancer chemotherapy. I t . Early Experiments on immunosuppression with Cytotoxic Agents

There is a remarkable series of experiments, performed before and during World War I, that warrant mention in even an abridged history of immunosuppression because they were so early and because they anticipated so many of the basic findings of later investigators using other agents. The toxin was benzene, and the initial finding was the effect of suitable doses on hematopoietic tissues and resistance to bacterial infection in rabbits and guinea pigs (Selling, 1911; Duke, 1913; Winternitz and Hirschfelder, 1913; Kline and Winternitz, 1913; W. C. White and Gammon, 1914). Several investigators extended the benzene investigations to assessment of immune responses in treated animalsRusk (1914) to the response of rabbits to sheep cells following benzene injection, Schiff (1915) to sensitization of guinea pigs to sheep serum following adequate dosage of benzene, and Simonds and Jones (1915)


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to the hemolysin response of rabbits to dog red blood cells, as well as their agglutinins and opsonins for typhoid bacilli. In the Schiff experiments dosage was of major importance, a low dose of benzene enhancing the anaphylactic response, the higher dose inhibiting it. Camp and Baumgartner, also in 1915, were concerned with the anti-inflammatory effect of benzene in rabbits and attributed the reduced susceptibility of the animals to bacterial infection to the suppression of inflammation as well as defects of the hematopoietic tissues. In two important papers Hektoen ( 1916a,b) presented extensive experiments on the effects of benzene and toluene in a variety of immunological processes. Repeated injection of benzene greatly reduced production of precipitins and lysins against sheep blood in the rabbit and rat. In dogs he found that small doses of benzene enhanced capacity to produce lysins against goat red blood cells. Hektoen noted no difference in the distribution or elimination of antigen by the methods available in benzene-treated animals and no change in concentrations of antibody already present in the circulation. He concluded, on the basis of these studies, that benzene affected the cellular elements responsible for antibody synthesis, probably leukocytogenic centers, particularly those involved in lymphocyte production, and that its effects were more pronounced in the rabbit than in the rat. Noted also in discussion of these results were effects of benzene on leukemia. Toluene had less immunosuppressive activity than benzene ( Hektoen, 1916b). Of great interest was the finding that although total antibody production was decreased, the antibody which was produced persisted longer in toluene-treated than in normal animals. This effect is very reminiscent of findings in animals treated with 6-mercaptopurine (6-MP) in which there is long persistence of 19 S antibody production, apparently a reflection of the suppression of 7 S antibody production. These results in animals treated with 6-MP and in other experiments have suggested that 19 S antibody production is, in part at least, “shut off by the onset of 7 S synthesis (Sahiar and Schwartz, 1964; Finkelstein and Uhr, 1964; Moller and Wigzell, 1965). Many of the approaches and conclusions of these early studies recur again and again in the history of immunosuppression: the variability of effects from species to species, from antigen to antigen, and from dosage to dosage with any given agent; the correlation of effects on antibody production with specific cell types damaged by the toxin; the finding of enhancement of responsiveness rather than suppression under some conditions of timing and dosage; the long persistence of antibody in the circulation in animals whose response was partially suppressed pharma-

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cologically; and the interrelationships of immunosuppression and carcinostasis and of immunosuppression and anti-inflammatory effects. 111. Salicylotes

Interest in the salicylates as immunosuppresr;ants was evidenced as early as 1922 and has persisted to the present time; indeed, some observations during the last year have aroused renewed interest in these compounds and their effects on specific antibody production and the complement system. Swift (1922) first explored the effects on antibody production of treating rabbits with sodium salicylate, in daily doses of 0.16 to 0.2 gm per kilogram administered by stomach tube. The antigen, Streptococcus uiridans or sheep red blood cells, was administered when salicylate treatment was begun; Swift observed signscant lowering of titers of complement-fixing antibodies, agglutinins, and hemolysins. Since salicylate mixed with the antigen in uitro and then injected reduced the antibody response even further, Swift suggested that salicylates might be affecting antigenicity. Beginning in the late 1920's and continuing up to the mid-l940's, the emphasis in research on salicylates was clinical, particularly their effects on serum sickness (Derick et al., 1928) and rheumatic fever ( Schlesinger and Signy, 1933; Schlesinger, 1938; Perry, 1939, 1941; Coburn and Moore, 1942; Coburn and Kapp, 1943; Rank et al., 1946). In 1928, Derick and colleagues reported that 1 5 9 0 grains of aspirin daily would suppress the arthritis of developing serum sickness, while permitting the expression of the skin manifestations. Their studies of the antibodies to horse serum in salicylate-treated patients showed depression to titers below 1:40, well below the level of 1:400 considered to be necessary for development of the arthritis. Derick and associates attributed the urticaria to an active sensitization of the skin by a mechanism not susceptible to salicylates. The major clinical chapter in the history of salicylates during this period concerned their use in preventing initial attacks and recurrences of rheumatic fever, stemming from the assumption that the disease is a consequence of an immunological response. Schlesinger and Signy ( 1933 ) and Schlesinger ( 1938) reviewed clinical and experimental evidence for this view, and in 1939 Perry presented provocative data which he interpreted as evidence for a prophylactic action of salicylates in rheumatic fever. He found that large doses of aspirin prevented both development of antifibrinolysins and symptoms of rheumatic fever. Analysis of the effects of salicylate treatment in several clinical groups indicated that


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antifibrinolysin titers were reduced, although some of the patients developed rheumatic fever. In another clinical series, Coburn and Moore (1942) treated large numbers of established rheumatic patients with 4-6 gm. of sodium salicylate per day. Although they noted no effect on development of such antistreptococcal antibodies as antistreptolysin 0, they believed that they prevented recurrences of rheumatic fever. Later clinical studies, particularly those of Rantz et al., (1946) indicated that salicylate prophylaxis was not effective in preventing rheumatic fever or suppressing production of antistreptolysin 0 following streptococcal infection. This clinical controversy was not fully resolved; the salicylate dosages in the immunosuppressive range were toxic, and the advent of penicillin therapy offered safer, more dependable means of preventing immune responses to streptococci and both initial attacks and recurrences of rheumatic disease (Rantz et al., 1946; reviewed by Rammelkamp, 1956). During this interval of claims and counterclaims regarding the effects of salicylates on rheumatic fever, a number of studies offered evidence for and against immunosuppressive effects of these agents. Hagebush and Kinsella ( 1930) gave sodium salicylate (0.2 gm.per kilogram in 5% aqueous solution intravenously) to rabbits with hemolytic streptococcal joint infections and reported inhibition of development of delayed allergy without evident toxicity. The effect was a suppressive one, since 2 0 5 0 days after discontinuance of salicylate therapy the rabbits were capable of developing the delayed skin lesions. In 1941, Perry studied the effects of salicylates on antibodies to typhoid in man and noted no effect on antibody levels with salicylate dosages of 30-45 grains per day. Coburn and Kapp ( 1943) noted that salicylates inhibited precipitation of serum antigens by their respective antibodies in the test tube, suggesting that immunological reactions could be suppressed without suppressing antibody synthesis. From 1945 to 1949, several groups investigated the effectiveness of salicylates in serum sickness. Three groups reported a lack of effects, at blood levels as high as 300500 ,pg. per milliliter, on the pathological lesions of serum sickness in rabbits ( W. C. Thomas and Stringfield, 1945; Roberts et al., 1949; Forman et al., 1949). Forman et al. ( 1949) suggested that anticoagulant effects of sodium salicylate observed in their studies might, perhaps in combination with toxic inhibition of antibody production, account for reported effects on hypersensitivity. On the other hand, Sullivan and co-workers ( 1948) prevented allergic arterial lesions in rabbits by subcutaneous administration of large doses of sodium salicylate well in advance of the initial administration of horse serum. They found

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moderate levels of antibody and considered that their treatment prevented union of antigen and tissue cells. Actually, the data presented disclose rather striking reduction of the precipitin titer. Another group of antibody studies, beginning in the mid 1940's, was oriented toward the possibility of suppressing anti-Rh antibodies. Homburger (1946) \\.as able to inhibit antibody production against rhesus red cells by salicyIate treatment in both rabbits and guinea pigs, and later Tourtellotte (19.W) suppressed anti-Rh, but not anti-0, antibodies by salicylate treatment of guinea pigs injected repeatedly with Rh-positive, type 0 human red blood cells. McLennan and colleagues (1947) treated an Rh-negative patient during pregnancy and succeeded in lowering antibody titers, but did not prevent erythroblastosis in her child. Jager (1947) showed further that y-globulin IeveIs were reduced among patients with salicylate levels of 300 pg. per milliliter or more, and Jager and Nickerson (1947) demonstrated that antibody levels to typhoid organisms were reduced in patients achieving this salicylate concentration. A remarkable effect of salicylate on anaphylaxis was documented by Campbell (1948); when given just prior to the provoking injection of antigen, salicylate protects rabbits against anaphylactic shock. It has no effect on histamine shock. Since the animals were sensitized before the drug was given, an effect other than prevention of antibody synthesis must be involved. Smith and Humphrey (1949) were able to suppress the edema of the passive Arthus reaction significantly in both guinea pigs and rabbits by salicyIate treatment. Cortisone has similar protective effects against anaphylaxis and Arthus lesions in some species (see Section IV,B ) ; indeed, studies by several investigators (Pelloja, 1952; Done et at., 1954, 1955) have suggested that the primary effect of salicylate may be attributable to stimulation of the pituitary-adrenal axis out of proportion to the stress of intoxication. Such an effect might account for many of the immunosuppressive effects of salicylates. Of particular interest in the contemporary context are the experiments showing an effect of salicylates on cellular immunity, i.e., an effect on the thyrnus-dependent lymphoid system. One immunopathological model, which will recur in many of the succeeding sections, is experimental allergic encephalomyelitis ( EAE ) , a demyelinating disease induced in many species by administration of central nervous system tissue, usually in Freunds adjuvant. The first use of this model as a test of immunosuppression was in our own experiments with salicylates in guinea pigs. In these experiments, we (Good et al., 1949a,b) found that sodium salicylate in large doses, particularly if administered with p -


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aminobenzoic acid to limit elimination of the salicylate, inhibited development of EAE in guinea pigs. Such treatment delayed onset of symptoms and often prevented them, and greatly reduced mortality from the disease. Similar observations on the rabbit were also recorded in our laboratory (R. A. Good et al., unpublished data, 1 9 4 9 ~ ) Figure . 1 illustrates the dramatic suppression of EAE with salicylates. There was no evidence in

FIG.1. Effects of sodium salicylate on EAE in guinea pigs. Drugs were administered beginning 8 days before injection of the encephalitogenic preparation and continuing for 60 days. It will be seen in the graph that sodium salicylate (Sod. Sal.), 0.2 gm. per kilogram per day, and p-aminobenzoic acid (PABA) alone, 0.5 gm. per kilogram per day, did not significantly affect mortality. The lower curves illustrate the low mortality in two of the treated groups: one treated with 0.3 gm. of salicylate per kilogram per day and the other with 0.2 gm. of salicylate per kilogram per day and 0.5 gm. of PABA per kilogram per day. The inset shows onset of disease rather than mortality in each of the groups, but illustrates the same trend. From Good et al. ( 1949a).

our experiments of a therapeutic effect; the medications seemed effective only when treatment was started before or shortly after sensitizing injections of central nervous tissue were given. Later, Kolb and co-workers ( 1952) reported insignificant effects of salicylate administration on EAE in guinea pigs, but Field and Miller (1961) confirmed our finding of significant protection by administration of sodium salicylate. They were able to influence the disease provided salicylate administration was begun at the time of, or a week after, the encephalitogenic injection, but could

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demonstrate no effect of salicylate therapy before the EAE-inducing injection. That salicylates may inhibit transplantation immunity was indicated in the experiments of Sherman and Friedell (1958) involving xenografting of human malignant melanoma to the hamster. The tumor, which did not grow in the untreated host, in some instances grew after administration of cortisone and even more often after conditioning with sodium salicylate. Much of this work and many additional studies, particularly on allergic phenomena, were reviewed in 1963 by Austen. There are very few published studies of allografting in salicylatetreated animals, and almost none with positive results. Another recent negative result was documented by Floersheim (1964a) who gave up to 200 mg. of sodium salicylate per kilogram to mice without significantly influencing skin allograft survival. There is, however, a positii.e result with skin grafting not yet published in detail (Cramer et al., 1966) in which high doses of aspirin resulted in significant prolongation in rats. Very recent work with salicylates has been most exciting. These have been in vitro studies in large part, and skirt two problems: toxicity, which has been limiting in our own work with certain of the salicylates in vim, and mediation by physiological effects, such as those on adrenal-pituitary function, which may be far more efficiently attained by more direct means. In the work of Ambrose (1965, 1966), to this writing available only in abstract form, a number of salicylates were screened in a test system involving secondary stimulation in vitro with diphtheria toxoid of lymph nodes from rabbits primarily stimulated in uivo. Salicylate at a concentration of about 16 mg./100 ml. arrested antibody production. Compounds substituted at the meta and para positions, rather than at the ortho position, were far less effective than salicylate itself. Compounds more effective than salicylate were also found; gentisic acid, for example, was three to ten times more active in this system than was salicylate itself. Further analysis of the structure of the most active compounds led Ambrose (1966) to attempt reversal of inhibition of antibody synthesis with structurally similar compounds, pyridoxine and pyridoxamine. Both proved to reduce the inhibition of antibody synthesis. As we have noted earlier, relativeIy Iittle work has been done on pharmacological manipulation of the complement system, although this is an essential part of many immune reactions. Manipulation of this system is beyond the scope of our review, since it is a nonspecific com-


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ponent of the effector stage of processes whose specificity is contributed by other cellular and humoral elements. However, in many experimental contexts, pharmacological effects on relatively nonspecific components of immune responses, such as phagocytic mechanisms at the “afferent” end and complement at the “effector” end, need to be considered in defining the effects and potential usefulness of a drug in manipulation of normal or aberrant immunological activity. Briefly, Pickering et al. (1966) have found that sodium salicylate inhibits activity in uitm of total complement and all four classic complement components. The effect is dose- and time-dependent, but differs significantly from the effect of corticosteroids on complement in the fluid phase recently reported by Gewurz et al. (1965). The possibility of in vivo anticomplementary activity has been suggested by preliminary experiments in guinea pigs. These two sets of experimental results and the long history of in uiuo studies in experimental animals and man suggest that intensive study of salicylates and their derivatives-with an eye to clinical application, as well as to analysis of immunological mechanisms-is warranted. Indeed, approaches to the little-understood action of these drugs themselves may also be derived from such analyses.

IV.

Adrenal Steroid Hormones

In many recent reviews of immunosuppression, antimetabolites have been emphasized and scant attention given to the adrenal steroids, although there are several earlier reviews on the effects of these agents on the immune mechanism and its function (Kass and Finland, 1953; Shwartman, 1953; Dougherty, 1952a, 1955; Germuth, 1956; Rose, 1959; McMaster and Franzl, 1961). In part this reflects the fact that adrenal steroids have become a “staple,” as it were, of immunosuppression; in part, perhaps, it reflects the dominance of antimetabolites, particularly 6-MP and its analogs, in the standard transplantation regimen. Whatever the reason, the extraordinary clinical usefulness of these agents, including their major role in control of rejection reactions in recipients of kidney allografts, make a review of their activity especially pertinent. We noted in the Introduction that immunosuppression may affect one or more phases of the immune response, from the initial processing of antigen by the cells of the reticuloendothelial system, through the steps of the adaptive response, involving cell differentiation, essentially a synthesis of “new” macromolecules, and cell proliferation; and the effector stage, either by sensitized cells themselves or by their products, antibody, in the circulation, with or without the participation

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of complement. Perhaps the most remarkahle attribute of the adrenal steroids is that there is good evidence of suppression at each stage, at least in complement-mediated antibody responses. Indeed, in some instances, the failure or inhibition of an immune response, particularly to living celis, may well reflect the effects of adrenal steroids on the antigen as well! Many phases of work on the mechanisms of immunosuppression by cortisone and its analogs are more easily discussed in the context of a particular type of response and then summarized in a brief concluding statement. Several of the more prominent activities of these agents might be mentioned as a preface, however. It has long been known that adrenal steroids inhibit proliferative responses of many types in different tissues (reviewed by B. L. Baker, 1951), including certain types of malignant growth (Heilman and Kendall, 1944)) that they are lympholytic, with a particular affinity for thymic cells (reviewed by Dougherty, 1952a), and that they are antiphlogistic ( Dougherty and Schneebeli, 1950). This is, of course, descriptive; at the biochemical level, the adrenal steroids are little understood but are under intensive investigation. Two phases of this recent work seem to us to be particularly pertinent to immunosuppression: the stabilizing effect of adrenal steroids on cell membranes and on membranes of organelles within cells, particularly lysosomes (reviewed by deDuve, 1964 and by Weissmann and Thomas, 1964), and the capacity of these hormones to induce formation of a variety of adaptive enzymes, many of them of basic importance in a wide range of biological adaptations (reviewed by Nichol and Rosen, 1964). There is widespread sentiment for a single metabolic locus of adrenal steroid activity which would encompass the disparate effects on a variety of tissues; in such an effort, Glenn et al. (1963) proposed inhibition of glucose metabolism as such a common denominator. A. EFFECTS OF ADRENALSTEF~OIDS ON ANTIB~DY PRODUCTION In a group of papers published between 1944 and 1949, Dougherty ct al. ( 1944), Dougherty and White ( 1945), and White and Dougherty ( 1944, 1945 ) characterized lymphocytes as antibody-containing cells which, as a result of adrenal cortical activity or steroid treatment, released y-globulin and preformed antibody into the circulation, in essence effecting an anamnestic response. Later work by Eisen et al. (1947) and Fischel and co-workers (1949) did not support this thesis; indeed, in the experiments of Fischel et al. there was no anamnestic rise in circulating antibody following almost complete lysis of small lymphocytes in the circulation after irradiation. In the early 195O's, as cortisone and adrenocorticotropin (ACTH)


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became increasingly available to investigators, several groups did detailed studies of antibody production in cortisone- and ACTH-treated animals. Fischel (1950) showed that antibody synthesis was regularly prevented or reduced in rabbits if ACTH treatment was begun at the time of initial exposure to antigen, but that anaphylaxis and Arthus reactivity were little affected by ACTH treatment in rabbits sensitized earlier. Bjprrneboe et al. (1951) noted reduced antipneumococcal antibody levels in cortisone- or ACTH-treated rabbits, when treatment was begun at the time of initial stimulation and also when the hormone treatment was begun after multiple immunizations. These investigators noted decreased numbers of plasma cells in these steroid-treated animals. A similar inhibition of plasma cell production was also noted by Teilum et az. (1950). Germuth et al. (Germuth and Ottinger, 1950; Germuth et al., 1951a,b) used both cortisone and ACTH in rabbits to inhibit sensitization with crystalline egg albumin, to prevent or even depress ongoing antibody production, and to suppress the pathology of the Arthus reaction. They were, however, unable to affect the passive Arthus reaction or the tissue response to antigen-antibody complexes or to influence the rate of disappearance of circulating antibody in the passively immunized rabbit. Fischel et al. (1952) provided further evidence of inhibition of rapid production of antibody in the secondary response by cortisone treatment begun at the time of antigen injection. Also in 1952, Craig (1952) analyzed the histology of lymph nodes from antigenically stimulated animals given cortisone and noted effects on mitotic activity in the cortex and the germinal centers. Thus, in an extensive series of experiments, the extraordinary potency of adrenal steroids in high dosage was evident in preventing antibody production and antibody-mediated hypersensitivity and in inhibiting ongoing antibody synthesis. These agents did not, however, foster degradation of antibody or alter appreciably certain mechanisms of immunological injury. During the next 2 or 3 years many studies in a variety of species with a range of antigens under varied experimental conditions documented the immunosuppressant capability of the adrenal steroids (B. N. Halpern et al., 1951, 1952; Malkiel and Hargis, 1952; Dougherty, 1952b; Hayes and Dougherty, 1952; Moeschlin et al., 1952,1953; Fischel, 1953, Whitney and Anigstein, 1953; Hanan and Oyama, 1954; Newsom and Darrach, 1954a,b; Kass et al., 1955). Mountain (1955) used an in uitro system to analyze the effects of steroid hormones on the immune response; indeed, she was one of the first to use such a system as a test of immunosuppressive activity. With

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~onc.t,iitr,itioiisof cortisone o f 32 mg./100 ml., she \\-as able to inhibit ongoing anti-H-agglutinin production by rabbit spleen fragment cultures. A wide range of compounds also proved inhibitory in this systcm. A major observation of this same period was that of Kaliss and associates ( 19Fj6) , who apparently produced immunological tolerance in adult mice with cortisone as an adjunct. Generally they found that the secondary response \vas difficult to suppress with cortisone; however, Fome animals given cortisone during the primary response failed to form antibody not only in the primary response but on subsequent stimulation from 6 to 10 weeks later. A recent series of experiments by Claman and Bronsky (1966) has also demonstrated that a corticosteroid, hydrocortisone in this case, facilitates tolerance development in mice. These investigators produced specific negativity to bovine y-globulin (EGG) in mice that already had circulating antibody to BGG following primary immunization with BCG-bentonite. This was done by repeated administration of a soluble preparation of the antigen in large doses; but it was done more easily when hydrocortisone or radiation treatment was given before the course of antigen injections was begun. One of the most revealing series of experiments on immunosuppression with adrenal steroids has been that of Fagraeus and Berglund, beginning in 1954 and extending to the present time. Initially, they (Fagraeus, 19535”;Berglund, 1952) confirmed depression of antibody to S. tuphi H in cortisone-treated rabbits and rats with appropriate dosage and timing. Later they (Berglund, 1956a,b, 1962, 1965; Berglund and Fagraeus, 1956, 1961; Fagraeus and Berglund, 1961) used the primary hemolysin response to sheep erythrocytes in rats to study the optimum conditions for immunosuppre~sionwith cortisone. When Berglund used 4 mg. of the drug per 100 gm. weight he found that suppression was achieved only if antigen was given after cortisone. Treatment with this dose after antigen administration, even for as long as 7 days, did not atrect the antibody level. Increasing the dose to 8 mg./100 gm. did suppress ongoing antibody production, but it had to be given for a prolonged period. Antigen dosage was also a factor, suppression being more difficult when larger numbers of cells were administered. Berglund and Fagraeus (1956) emphasized the striking similarity of the effects of cortisone and X-irradiation on the primary hemolysin response. This pattern is a recurrent one in efforts to suppress antibody production-the ease of inhibiting the primary response compared to responses to subsequent stimulation and the vulnerability of the earliest phases of the response, the so-called induction period, to pharmacological manipulation. Suppression of on-going antibody synthesis is possible with corti-

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sone and its analogs, as with some of the other immunosuppressive agents; and one of the central questions-of great importance in understanding both the antibody-producing mechanism and the immunopharmacology of the agents themselves-is whether production is not always affected by way of the initiation process rather than by inhibition of synthesis itself. A major insight into the mechanism of corticosteroid suppression of antibody production (see Section IV,J) was offered by a series of experiments by Berglund and Fagraeus (Berglund and Fagraeus, 1956, 1961; Fagraeus and Berglund, 1961). They found that the inhibition of hemolysin production in rats could be reversed by exogenous lymphoid cells, from either spleen or thymus, allogeneic or xenogeneic, but intact. If disrupted cells were given, no restoration was evident (Berglund and Fagraeus, 1961). It will be recalled that Jaroslow and Taliaferro (Jaroslow and Taliaferro, 1956; Jaroslow, 1960; Taliaferro and Jaroslow, 1960) reconstituted antibody production in irradiated animals by similar means, but that the limitations of cell type and cell condition were not nearly as great as in the Berglund-Fagraeus work. Thus, cell fractions were effective, as were yeast cells, HeLa cells, intact mammalian cells, and completely disrupted cell preparations. Pharmacological enhancement of immune responsiveness is, as we have noted earlier, beyond the scope of our review. We noted briefly, in the section on the earliest use of cytotoxic agents to suppress antibody production, that enhancement of susceptibility to anaphylaxis had been noted with low doses of benzene, whereas higher doses were inhibitory ( Schiff, 1915). This has been shown repeatedly with irradiation (Taliaferro et al., 1952; Dixon and McConahey, 1963). Similarly, in experiments by Winter et al. (1962a,b) steroids enhanced or depressed antibody synthesis by rats to Brucella melitensis depending on dosage and timing. Stimulation was greatest with moderate drug dosage several days before antigen administration. Paradoxically, at sufficiently high dosages, timing became less important; thus inhibition was observed when antigen was given several days after termination of high-dose cortisone treatment. These investigators correlated their results with the morphology of the lymphoid tissues, emphasizing the availability or lack of availability of stem cells in sufficient numbers as the crucial variable. Diefenbach et al. (1962) also emphasized the variables of steroid dose and time of treatment in relation to antigenic stimulus. A series of in vitro studies by Ambrose (1964a,b) and Ambrose and Coons (1963a) has also shown dose dependence of corticosteroids in enhancing or inhibiting antibody production. Their system, it will be recalled, involves secondary stimulation with diphtheria toxoid and

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bovine serum albumin (BSA) of lymph node fragments from stimulated rabbits. They showed that small amounts of cortisone, 0.01-10 pg. per milliliter, will substitute for a plasma requirement in the culture and permit or even enhance antibody production. However, when 100 pg. or lo00 pg. is incorporated per milliliter, antibody production is suppressed. Another very interesting in vitro result was reported recently by Strander (1966) who used the Jerne plaque technique for assessing levels of 19 S hemolytic antibody against sheep cells. Puromycin and actinomycin reduced the number of plaques, as did hydrocortisone after 3 hours of exposure to 100 or loo0 pg. per milliliter, the concentrations used by the experiments of Ambrose and Coons and well above clinical levels. Perhaps the most precise work on the effects of adrenal steroids as immunosuppressants in siuo is that of Berglund (1962, 1965) and Borum and Berglund (1964). A model was devised involving stimulation of mice with sheep red blood cells, at varying intervals before and after four hourly injections of prednisolone. The maximum effect was noted in mice whose treatment was begun between 14 and 8 hours before antigen, but some suppression was noted in those treated as early as 18 hours before sheep cell injection or as late as the time of injection. The animals in these experiments remained in good condition. Berglund concluded that the immunogenic tissue of the mouse must be damaged before the thirteenth hour after antigenic stimulation to influence the response. The injury does not need to be sustained; apparently all that is needed is about an hour of damage at a crucial time in the evolution of the antibody response. Berglund (1965) extended these experiments to rats and found that they required larger doses for longer periods for similar effects; but again he was able to delimit sharply the period of susceptibility to the action of prednisolone. The functional appraisal was supplemented with both histological analyses and assays of the proliferative activity of the lymphoid tissues using tritiated thymidine. The autoradiography revealed maximum depression of thymidine uptake 9 hours after discontinuation of steroid treatment, a valley to 13 hours, and progressive increases at 17 and 21 hours. This sequence correlated impressively with the results of the immunological studies. B. EFFECTSOF ADRENALSTEROIDSON ANTIRODY-MEDIATED

HYPERSENS~~~V~~Y In the section on antibody production, we noted briefly that Germuth e t al. (1951b) and Fischel (1950) observed no appreciable effect on


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anaphylactic shock in guinea pigs and rabbits sensitized prior to steroid therapy. On the other hand, Wolfram and Zwemer (1935) had reported inhibition of anaphylactic shock in guinea pigs with the adrenal cortical extract, cortin; and P)rskov (1949) found that anaphylactic mortality in guinea pigs was reduced by administration of 0.8 mg. ACTH 1 5 hours prior to the shocking dose of antigen. In a number of experiments with guinea pigs, adrenal steroids either had no influence on the course of the anaphylactic response (Leger et al., 1948; Arbesman et al., 1951) or reduced the mortality though not preventing the reaction (Simonsen, 1950). Lecomte and Beaumariage (1957), on the other hand, gave hydrocortisone to sensitized rabbits twice, 90 and 30 minutes before the shocking injection, and inhibited the response to a great extent. Humphrey (1951) offered an insight into the disparate results with adrenal steroids in guinea pigs by passive sensitization with small and large amounts of antibody. Here the antibody level was clearly the significant variable, the severity of anaphylaxis being decreased by cortisone in animals given small amounts of antibody, but not in those injected with large amounts. In at least two experiments, cortisone offered some protection against reversed passive anaphylaxis in guinea pigs (Arbesman et al., 1951; Johnstone and Howland, 1957). The Arthus reaction, both passive and active, has also been studied in guinea pigs. Humphrey (1951 ) found that 5 mg. of cortisone per kilogram weight intramuscularly reduced both edema and vascular permeability in the passive and reversed Arthus reactions, Later, Jaques et al. (1959) inhibited the passive Arthus with 10 to 30 mg. of prednisone or prednisolone per kilogram; and Christensen ( 1959) noted diminution of the passive Arthus by daily administration of 10 mg. of corticotropin before the test injection, but found that the response reappeared when treatment stopped. Good and Good (unpublished data, 1952) did a series of experiments on anaphylaxis in rabbits and found little evidence of protection with cortisone doses of 1-25 mg. per kilogram at intervals of 2 days to 4 hours before the shocking injection. In adrenalectomized rabbits, however, considerable protection was afforded by amounts as low as 0.25 mg. per kilogram. Systemic anaphylaxis in mice is particularly susceptible to modification by cortisone and related agents (Kind and Parfentjev, 1951; Solotorovsky and Winsten, 1954; Cameron, 1957). Similar modification was shown by Treadwell and Rasmussen (1961) in conditions of acute and chronic istress and by Malkiel and Hargis (1965) in mice implanted with

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an adrenotropic pituitary tumor. One of the reasons advanced for the particular efficacy of adrenal steroids in anaphylactic responses in mice is the apparent dependence of these responses on lysosomes ( Treadwell, 1965) in the light of the stabilizing effects of corticosteroids on lysosomal membranes ( see deDuve, 1964; Weissmann and Thomas, 1964). In summary, the adrenal steroids can affect antibody-mediated hypersensitivity in two ways: by suppressing production of antibody if administered before or at the time of sensitization and by modifying the local or systemic response in animals actively or passively sensitized before the drug is given. Many variables affect the results of the second type of experiment-species, the agent used, the dosage, the route and time of administration, and the amount of antibody. C. EFFECTS OF ADREXALSTEROIDS ON

COMPLEMENT SYSTEM In the section on salicylates, we noted briefly their effects on the complement system, a nonspecific component of adaptive immune responses. As reviewed recently by Gewurz et al. (1965), in viuo studies of the effects of adrenal steroids on the complement system have been largely inconclusive. The demonstrated in vitro effects have been difficult of interpretation because the criterion of activity has usually been cell lysis, and anticomplementary activity has not been easily separable from the stabilizing effects of corticosteroids on cell and cell organelle membranes. We believe that the recent work in our laboratory (Gewurz et al., 1965) has established that hydrocortisone is anticomplementary in the fluid phase, depressing the activity of both whole complement and all four classic components considered individually. As with most experiments on stabilization of membranes, the cortisone concentrations in the complement experiments were far greater than those attainable in oiuo; and it is clearly not possible to correlate therapeutic effccts with such data. At the same time effects of this type should be kept in mind, particularly in dealing with agents so useful, and yet so enigmatic, as the adrenal steroids. THE

D. EFFECTSOF ADRENALSTEROIDS ON PHAGOCYTIC MECHANISMS The effect of adrenal steroids on phagocytic mechanisms is another influence on the “nonspecific” components of adaptive immunity, in this instance the afferent side or afferent arm of the immune response. The qualifying quotation marks above reflect growing evidence that however impartial a macrophage may be in its initial apprehension and ingestion of exogenous antigens, it probably has a much greater role in processing, or perhaps even creating in part, adequate antigenic

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stimuli to the cell systems which effect specific immune responses than was evident earlier. Thus, it may well prove to be as “adaptive” and as crucial to specificity and adequacy of immune response as the lymphoid cell systems. Without detailing the methods of study and the disparity of results, there are numerous studies showing impairment of clearance of particulate substances in corticosteroid-treated animals ( Cornwell, 1953; Cornwell and Good, 1953; Heller, 1955), although there is also contrary evidence (Martin and Kerby, 1952; Gel1 and Hindc, 1953). Another form of inhibition of phagocytic function by cortisone was noted by Benacerraf et al. (1954). They found that cortisone-treated animals recovered bloodclearing capacity after an intravenous “blocking” dose of carbon much more slowly than did controls. The studies of Lurie et al. (1952) showed that another dimension of reticuloendothelial function is also affected by cortisone. In cortisonetreated rabbits, bacterial antigens were engulfed by macrophages, but tended to pile up in the phagocytic cells and were not as well digested as in control animals. Similar findings were documented by Clawson and Nerenberg (1953) and Kass et al. ( 1953), As has become clear to us in considering clinical immunological deficiencies, ingestion and digestion are dissociable functions of at least the polymorphonuclear leukocytes, and the different experiments with cortisone suggest that cortisone may affect them separately. That cortisone and its analogs in high dosage stabilize both cell membranes and intracellular membranes, such as those of lysosomes, has been noted earlier and will be referred to again. There are a number of ways in which this type of activity might interfere with adaptive immune responses, but one is obvious here-in inhibiting the release of lysosomal enzymes active in digestion of particulate antigens. There is another observation which warrants mention because it is so typical of dosage relationships. Snell (1960) found, using a colloidal chromic phosphate clearance technique, that phagocytosis was enhanced by very small doses of cortisone, hydrocortisone, and corticosterone.

E. EFFECTSOF ADRENALSTEROIDS ON DELAYED ALLERGICRESPONSES There are many inherent difficulties in dealing with inhibition of delayed allergic responses, notably that establishment of sensitivity is not usually separated from expression of sensitivity and that there is no “measurement” of degree of responsiveness. A large group of early studies (D. A. Long and Miles, 1950; S. Harris and Harris, 1950; Sheldon et al., 1950; Derbes et al., 1950; J. B. Long and Favour, 1950)

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showed unmistakably, in patients as well as in experimental animals, that cortisone and ACTH suppressed established delayed sensitivity, most often to tuberculin. There were also instances in which the delayed allergic response was not prevented, although in some cases it was modified (Uehlinger and Siebenmann, 1952; Sonck and Miescher, 1952; Cummings and Hudgins, 1952). Many of these and subsequent investigations of the effects of cortisone on delayed allergic responses have been reviewed by Stoerk (1953), Dougherty (1955), Germuth ( 1956), and Rose (1959). Germuth and Rose, in particular, consider the question of whether there is specificity to this inhibition. It seems to us, largely from the data on suppression of antibody production (see above) and of thymus system-mediated responscs other than delayed allergy per se (see next section) that cortisone administration in high dosage at the time of sensitization or shortly thereafter probably modifies the sensitization process itself. There is a paradox running through the history of immunosuppression with adrenal steroids and also with certain other agents: that clinically effective doses of corticosteroids-in severe eczema and eczematic erythroderma, as noted by Sonck and Miescher (1952), for example-are often not effective doses in experimental suppression of the immune mechanisms presumed to be operating in the disease. Sonck and Miescher, for example, were not able to prevent delayed sensitivity to 2,4dinitrochlorobenzene (DNCB) in guinea pigs despite high doses of cortisone during the period of sensitization and they produced no definite weakening of the response by treatment in animals previously sensitized.

F. EFFECTSOF ADRENALSTEROIDSIN EXPERIIIE~TAL AUTOIMMUNE DISEASE As we noted in Section I11 on salicylates, EAE has been a very useful model in which to test immunosuppressive agents, approaching as it does some of the clinical states in which immunological mechanisms are important. This is now a less ambiguous model of an immunological disease than it was earlier, when the relative roles of humoral and cellular mechanisms were in dispute. Waksman ( 1959) reviewed the evidence for delayed allergic reactions of the tuberculin type as the primary basis of the pathology in this disease. Antibodies are involved, but these seem to be secondary phenomena and at least in rats (Paterson et a?., 1961; Paterson, 1962) seem to have a n anieliorating influence on the dkease procc’ss. hhst telling, in o w view, have 1)cen the observations in thymectomized rats and chickens, in which the disease is clearly

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modified in the presence of an intact immunoglobulin-producing system ( Arnason et al., 1962; Jankovib and ISvaneski, 1963). Cortisone and its analogs have been shown to inhibit EAE in many studies, including those of Moyer et al. (1950) in the guinea pig, Kabat et al. (1952) in the rhesus monkey, and Ferraro and Roizin (1953) in guinea pigs. Ferraro and Roizin noted that the disease developed after treatment was discontinued and interpreted the suppression during therapy as a reffection of both inhibition of inflammation and depression of the immune response. One of the most provocative of these early studies was that of Gammon and Dilworth (1953) in which ACTH was administered when earliest symptoms of EAE appeared and it arrested the progress of the disease. Other investigators noted no beneficial effect of hormone treatment on established disease (Kolb et al., 1952; Ferraro and Roizin, 1953; Condie and Good, unpublished observations, 1956), although the conditions of the experiments by Gammon and Dilworth were not duplicated. However, in several more recent experiments with guinea pigs (Field and Miller, 1961; Finger, 1961; Brandriss et at., 1965b) there was evidence that corticosteroids modify EAE significantly even if their administration is delayed until signs of the disease are present. In none of these instances have the results been as striking as those of Gammon and Dilworth, however. Another model of immunological disease, adjuvant arthritis in rats, has been extensively studied (C. M. Pearson, 1959; C. M. Pearson et al., 1961; C. M. Pearson and Wood, 1963) and appears to involve a delayed allergic mechanism mediated by the thymus-dependent lymphoid cells; indeed, Arnason et al. (1962) noted delayed onset of the disease and decreased severity in neonatally thymectomized rats. The effectiveness of adrenal steroids in inhibiting the process was shown by C. M. Pearson and Wood ( 1959), a finding confirmed and extended by Houssay and Frangione ( 1961) and Newbould (1963). Newbould used paramethasone as the adrenal steroid, but also demonstrated the effectiveness of salicylates, pyrazolidines, and sodium aurothiomalate. Efforts by Spiegelberg and Miescher ( 1963) to suppress experimental immune thyroiditis in guinea pigs involved amethopterin and 6MP, both effective as suppressants. They had no success with hydrocortisone, dexamethasone, or prednisolone in doses of 15 mg. per kilogram per day, as judged by antibody, delayed hypersensitivity, or thyroid lesions. A major model of “autoimmune disease,” originally described by Bielschowsky et al. ( 1959), is the spontaneous Coombs-positive hemolytic anemia of strain NZB/BL mice. Helyer and Howie (1963) noted reduced

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antibody Irvels and clinical improvement in mice with well-advanced disease that had been treated with ACTH; more recently, Casey and Howie (1965) reported similar benefits with betamethasone. In this instance, of course, thc suppression is of an ongoing process, a closer analogy to the clinical context than many of the experiments above in which the effectiveness of steroids was largely limited to the initiation of the immune process. The greater effectiveness of adrenal steroids administered at the time of the immunological stimulus, as in the EAE model in particular, parallels the sharply limited susceptibility of the antibody-producing mechanism to moderate doses of steroids close to the time of administration of antigen. Thus, there seems to be every reason to believe that specific suppression occurs in these instances, although certainly less specific effects play a role. G.

EFFECTS OF A4DREYAL STEROIDS ON

TUMOR I Mhf U N I T Y

i i s we have indicated earlier and will note again in succeeding sections, many carcinostatic agents are immunosuppressants; indeed in many clinical contexts there is a question of whether depression of host defenses does not largely outweigh the limitation of tumor development achieved. Although cortisone and ACTH were shown early to have favorable effects on experimental malignancies ( Heilman and Kendall, 1944; Burchenal et al., 1950) and such agents are used extensively in certain lymphoid malignancies (0. H. Pearson et al., 1949; Sutow et al., 1965; Zuelzer, 1964). there is also an extensive literature on facilitation of tumor survival or metastasis by adrenal steroid administration. Toolan (1953) reviewed her work and that of others on adrenal steroid effects on tumor transplantation and concluded that cortisone served in varied ways to facilitate takes of both allogeneic and even xenogeneic tumors. Later, Kaliss (1955) showed that allogeneic mouse tumors which ordinarily regress show unimpeded growth in mice treated with high doses of cortisone. That metastasis of certain tumors is more frequent in cortisone-treated mice (Pomeroy, 1954; Baserga and Shubik, 1955) has been shown. Iversen and Hjort ( 1958) have documented similar clinical experience. That pretreatment of the host is most effective in fostering take and growth of transplanted tumors, as it is in suppressing other immune responses, was shown by Moore et at. (1960). With Ehrlich ascites tumor even single cells grew progressively after subcutaneous inoculation into cortisone-treated mice. When tumor cells were injected intravenously,


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5000 cells were sufficient to establish metastases compared to a requirement of 2,000,000 cells in untreated hosts. An impressive effect of cortisone on xenografted tumors was noted by Vasiliev and Garin (1961) who also studied the effects in the same mouse-tumor-to-rat model of irradiation, dibenzanthracene, and alkylating agents. Cortisone was the most effective of the agents used, but was effective only when begun on the day of grafting. The facilitation of tumor growth in these animals was temporary, however, and the tumors ultimately regressed. Another perspective on cortisone effects is offered by the experiments of Rosenau and Moon (1962) using an in uitra system of demonstrating the cytolytic effect of sensitized BALB/c mouse lymphocytes on L cell fibroblasts. Cortisone inhibited the destructive action of the lymphoid cells, an inhibition shown to represent an effect on the cytolytic process per se and not on recognition. This is another of the effects of cortisone that might be attributable to stabilization of lysosomal membranes in either the sensitized or target cells (see Section IV,J). In summary, it is well established that cortisone suppresses certain types of tumor immunity, a fact best established for tumor allografts, but often evident with tumor xenografts as well. The effect on metastases has been extraordinary, in some instances reducing the requirement for an effective transplant to a single cell.

H. EFFECTSOF ADRENALSTEROIDS ON TRANSPLANTATION IMMUNITY TO

NORMAL TISSUES

Some of the most dramatic instances of suppression of transplantation immunity by adrenal steroids have been recorded during the last 3 years in clinical renal transplant experience, noted briefly in Section IVJ. There seems to be little doubt that the adrenal steroids “make the difference” in many rejection episodes. As we shall note in more detail later, these agents may well affect the graft as well as the immune response of the host. It is of interest, too, that some of these agents have been much more effective in the human renal allograft context than the myriad of experimental data would have led us to expect. A number of species were used in skin- and kidney-grafting experiments from 1951 to 1956. In rabbits, Billingham et al. (1951) showed that treatment with cortisone prolonged skin allograft survival by a factor of three to four. Morgan (1951) made similar but independent observations in rabbits. Later, Medawar and Sparrow (1956) noted prolongation of skin allograft survivals in A and CBA mice with cortisone acetate, hydrocortisone, and cortisone and with ACTH in depot form.

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Cannon and Longmire (1952) dealt with skin allografts in chickens and found that they were unable to influence survival significantly by ACTH or cortisone administration in adult animals. When they treated newly hatched recipients, a small proportion of which accept skin allografts without pretreatment (6% in this study), however, they increased the proportion of &week survivals to 20%.These results suggest that cortisone fostered development of tolerance to the grafted tissue in the immunologically immature chick. In guinea pigs Sparrow (1953) was able to double survival of skin allografts with cortisone acetate, but the effect was clearly dependent on both dose and route of administration-25 mg. per day subcutaneously was effective, but neither 5 mg. daily subcutaneously nor 20 mg. daily intraperitoneally had any influence. Krohn (1955) noted little effect of either ACTH or cortisone on skin allografts in monkeys. The dog studies are of particular interest in retrospect, because of the major role of this species in the renal allograft work that preceded effective clinical immunosuppression. The experiments of Persky and Jacob in 1951 involved renal allografts and daily administration of ACTH or cortisone; no prolongation was noted. In an unusual experiment involving treatment of the donor, Dempster (1953) noted modification of the rejection lesions, but no significant prolongation of survival of renal allografts in dogs. Really remarkable results in dogs have been achieved very recently by Zukoski et al. (1963, 1965a) and Marchioro et al. (1964), again with allografted kidneys. Zukoski and colleagues used prednisolone and observed moderate prolongation in a number of animals. In a single instance, these investigators noted very long survival, for 749 days in the absence of medication at the time of the second report, the type of tolerance of renal allografts more often achieved in dogs with such agents as 6 M P and azathioprine. Marchioro et al. (1964), on the other hand, used a basic regimen of azathioprine. In eight animals that developed evidence of renal allograft rejection, prednisolone was given, 50-200 mg. per day intramuscularly. Seven of eight responded favorably, a remarkable parallel to the clinical experience, documented particularly by the same Denver group (Starzl et al., 1964a,b) but also by others, as noted in more detail in Section IV,I. Another type of “transplantation immunity” suppressed by adrenal steroids has been documented by Burnet and Warner (1960) and Warner and Burnet (1961). Boyer (1960) had discovered that focal lesions on the chorioallantoic membrane (CAM) occur after injection of allogeneic blood cells into chick embryos. Burnet and Warner found


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that hydrocortisone added to the inoculum of adult cells markedly reduced the number of CAM lesions. A number of other related agents were also effective, though the degree of inhibition varied from compound to compound. This is another instance in which the activity of thymus-dependent lymphoid cells is being inhibited by the drug. Instances of specific immunological negativity induced with the aid of immunosuppressants are of particular interest; and some experiments of Mitchison (1963), originally oriented in another direction, seem to be in that category. Brown Leghorn chickens were given White Leghorn blood, beginning at hatching, and were considered to be tolerant as judged by the rate of elimination of labeled cells. Mitchison gave hydrocortisone, 5 mg. per kilogram, when tolerance was about to be lost according to findings in other experiments, in an effort to "break" tolerance. Instead, at later challenge hydrocortisone-treated birds were still tolerant. Thus cortisone did not facilitate interruption of tolerance, but seemed to prolong a tolerant state otherwise nearly certain to be lost. Although the mechanism is not clear, it may well be that the hydrocortisone fostered tolerance to antigens released as the red blood cells were catabolized. In summary, then, over a 15-year period, the effectiveness of adrenal steroids in prolonging skin and kidney allografts has been demonstrated in a variety of experimental animals. In a few instances, apparent immunological tolerance has been achieved. As with other immune responses discussed earlier, it seemed that cortisone and its analogs were effective only if administered from the time of grafting; however, in the wake of the clinical experience with suppression of renal allograft rejection by these agents, such reversal of rejection was also demonstrated experimentally in dogs whose initial immunosuppression had been achieved with azathioprine.

I. ADRENALSTEROIDSAND HOMOGRAFT REJECTION IN MAN As noted briefly in the preceding section, there is now extensive experience, representing virtually all the major clinical transplantation centers in the United States and in Britain, that moderate to very high doses of adrenal steroids can reverse even ongoing renal transplant rejection (Glassock et al., 1962; Woodruff et al., 1963; Starzl et al., 1964a,b, 1965a; Reemtsma et al., l W a , b ; R. Y. Calne et al., 1963; Nakamoto et al., 1964, 1965; Hume et al., 1963; MacLean et al., 1965; Kelly et al., unpublished observations, 1963-1966). Practice varies; in some groups, including the one at the University of Minnesota, prednisolone or another cortisone analog is given with

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Imuran throughout the early post-transplant period, and the dosage increased when functional indices suggest rejection (Reemtsma et al., l W a , b ; Ylowbray et al., 1W5; W.D. Kelly et al., unpublished observations. 1963-1966; Starzl et at., 1964b; Nakamuto, rt at., 1965). In others, the major reliance is on Imuran, sometimes given with azaserinc, and an adrenal steroid is given only when rejection threatens (Murray et al., 1%; Starzl et nl., 1964b; K. A. Porter et al., 1965; Hume et al., 1963; Clinical Rounds, 1965). Actinomycin is also administered sometimes in small doses during rejection episodes. L

J.

hfECHASIS\lS OF

ADREXALSTEROID ACTIVITY IN

IMMUNOSUPPRESSION

We noted briefly at the beginning of this section the unsettled state of the biochemistry and pharmacology of adrenal steroids. In considering the immunosuppressive activity of these agents, there is no more basic fact, it seems to us, than their predilection for lymphoid cells, particularly small lymphoid cells of the thymus-dependent type. The focus here is perhaps clearer now than it was a few years ago. Gowans (Gowans, 1962; Gowans et al., 1962) emphasized the immunological capabilities of the small lymphoid cells. The work on neonatal thymectomy in a variety of mammalian species (review by Good and Gabrielsen, 1964) and particularly the experiments of Cooper et al. (1966a) on chickens provided strong experimental underpinning for the link of depIetion of small lymphoid cells to defects in “cellular immunity,” including graft rejection, and also deficient antibody production. Recently, the potency of antilymphocyte serum and thoracic duct drainage as immunosuppressive procedures, particularly in conjunction with thymectomy, has been evident (hlonaco et al., 1965b, 1966b; Jeejeebhoy, 1965; McGregor and Go\\-ans, 1963, 1964; Waksman et al., 1961). In interpreting their early work with prolongation of skin-graft survival in cortisone-treated rabbits and mice, Billingham et al. ( 1951) felt that the graft as well as the immune responses of the host are affected by these agents. Stabilization of lysosomes and other membranous structures has been attained €or the most part with unphysiological doses of corticosteroids, and at present we can only speculate that the hormones have an affinity in v i m with certain cell sites or that other ill-defined factors in the in vit-o situation facilitate this type of action by steroids. Direct antienzymatic effects seem to occur in the case of complement components. So far, this inhibition has been shown clearly only in vitro and, again, at very high dosage levels. This activity has thus far been broken down only to the extent of showing that all four classic comple-

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ment components are affected; further studies in terms of the nine “ncw” components are needed. V. Alkylating Agents

Alkylating agents have been used in immune suppression for more than 40 years, Hektoen and Corper‘s (1921) studies with mustard gas being conducted shortly after World War I. It was only after World War 11, however, as mustards were released for general investigation and as their usefulness in treatment of certain lymphomas became known ( Gilman and Philips, 1946), that extensive studies of their immunosuppressive capacities were begun. For several years dozens of alkylating agents were developed for cancer chemotherapy, and many of these were also tested as immunosuppressives. Perhaps the most impressive successes have been made recently with the powerful new agent, cyclophosphamide, as we shall note below. As in Section IV on the corticosteroids, we shall return to the question of mechanism of immunosuppressive action of alkylating agents after considering the experimental data. Many aspects of the activity of alkylating agents are explained by the simple fact that these agents kill cells, but many other aspects are not. As with other agents, the effects are most pronounced in rapidly replicating cells-a type of selectiviky. Under many circumstances, the effects are more pronounced in tumors than in normal tissues, sometimes a reflection of rate of proliferation but not necessarily so. More than many of the immunosuppressive agents, they are carcinogenic and mutagenic, suggesting effects on deoxyribonucleic acid (DNA) particularly; indeed, one of the major recent controversies in this area, not really resolved, is whether alkylation of DNA is the basis of the carcinostatic activity of difunctional alkylating agents. There are many data, however, suggesting that ribonucleic acid ( R N A ) and other proteins in certain tumors may be a target of at least some of the agents. Several investigators have suggested that effects on transport proteins or sulfhydryl groups are crucial. Many insights into these problems, chiefly from the viewpoint of cancer chemotherapy, are offered by the reviews of Wheeler (1962), Karnofsky and Clarkson ( 1963), Emmelot ( 1964), Brookes and Lawley (1964), and Warwick (1963). Indeed, one of the major limitations in considering the mechanism of activity of the mustard and mustardlike drugs as immunosuppressants is our almost complete “carry-over” of data from work in experimental cancer chemotherapy. A word should probably he said initially about the characterization of these agents as “radiomimetic,” a term very popular a few years ago,

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and probably best discarded. None of these agents mimics more than a fraction of the effects of X-irradiation; conversely many agents which have radiationlike activity in certain respects are not of this group.

A. EFFECTS OF ALKYLATING ACEXTSON ANTIBODYPRODUCTION Some of the earliest studies of the effect of alkylating agents on immune processes were those of Hektoen and Corper ( 1921), an outcome of the research on mustard gas during World War I. They used both rabbits and dogs as subjects, sheep or rat blood as antigens, and mustard gas ( “dichlorethylsulphid”) as the suppressing agent and noted an cffect on precipitins in both dogs and rabbits and on hemolysins in dogs, correlated with reduced white counts. The drug was usually administered before or at the time of antigen administration; less effect was noted if the antigen was given first. Hektoen had worked earlier with benzene and toluene (see Section 11) and classed these agents, mustard gas, and irradiation as leukotoxic agents probably suppressing hematopoiesis and antibody production by different mechanisms. As noted briefly above, it was after World War 11, with the background of the effectiveness of the nitrogen mustards in certain lymphoid malignancies, that several groups of investigators studied the effects of these agents on antibody production in different species. Philips and co-workers ( 1947) administered two mustards, tris ( p-chloroethy1)amine and his ( p-chloroethvl ) amine to goats intravenously in doses sufficient to establish chronic leukopenia. The antigen was ricin, and the focus was on the secondary response. The general effect was a delay in the anamnestic response, but some responses were also decreased. Spurr, also in 1947, used rabbits stimulated with typhoid antigen before, at the time of, or after treatment with nitrogen mustard. Antibody production was suppressed except in the group treated after antigen administration. In a definitive study of nitrogen mustards as suppressants of immunological disease, Bukantz et uZ. (1948, 1949) gave rabbits 0.5 mg. of his ( P-chloroethyl ) amine every 3 or 4 days, beginning 2 days before injection of a large dose of horse serum, and prevented serum sickness in the majority of animals. When the animals were treated for 28 days after the first injection of antigen and 14 days after the second injection of antigen, skin-test reactivity, precipitating antibody production, and vascular lesions were all suppressed. Bukantz and associates also correlated serum precipitin level with vascular lesions : rabbits having more than 0.06 mg. of antibody nitrogen per milliliter had \’i15C3L11i1r- Ieqions, whcrcas those having smaller amounts o f antibody did not dcvclop such lesiony.


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Schwab et aZ. (1950) also investigated the effects of nitrogen mustard [methylbis (p-chloroethyl ) amine or tris( p-chloroethyl) amine] on experimental hypersensitivity in the rabbit. They found that treatment just before administration of whole bovine serum, BSA, or BGG prevented formation of circulating antibody and the consequent tissue lesions and also inhibited the consumption of complement by the immune process. The nitrogen mustard treatment seemed to have little effect on antigen clearance. As part of a larger study on reactions of the reticular tissue to antigens, Marshall and White (1950) administered nitrogen mustard to rabbits immunized with typhoid-paratyphoid (TAB) vaccine 3 or 4 weeks earlier. They did not note a significant drop in antibody titer; however, circulating lymphocyte levels fell to 3040%of normal, and destruction of small and medium-sized lymphocytes was prominent. Large lymphoid cells, plasmablasts, and plasma cells seemed to be resistant. This is another affirmation of the susceptibility of thymus-dependent cells to a variety of agents and the relative resistance of the immunoglobulin production system. It also reinforces the concept of the role of small lymphoid cells in the initiation of antibody responses. Suppression by nitrogen mustard [methylbis( p-chloroethyl ) amine] of the primary response to another bacterial antigen (SaZmoneZZu typhi H ) in rabbits was documented by Green (1958a). The maximum effect was noted if mustard treatment preceded administration of antigen; no effect at all was noted if antigen preceded drug administration by 48 hours. Since the animals recovered immune responses with restoration of the damaged tissues, Green suggested that a delay in breakdown of antigen might be a factor in his results. In further experiments (Green, 1958b) degradation of antigen was assessed by processing splenic tissue from control antigen-injected and drug-treated antigen-injected rabbits for injection into other animals. The rabbits receiving control tissue, if stimulated with the antigen, reacted with a primary antibody response. Recipients of spleen cells from experimental rabbits (mustard-treated and antigenically stimulated ) produced a secondary response to the antigen when they were restimulated, provided the interval from stimulation to processing of the tissue did not exceed 48 hours. Green concluded that the immunosuppressive action of mustards may, at least in part, reflect inadequate processing of antigen. Shamaeva and Pankova (1957) suppressed antibody responses of rabbits to sheep red blood cells with a mustard, Novaembichin [chloropropylbis ( 2-chloroethyl) amine], observing again the importance of giving the drug before or at the time of antigenic stimulation to achieve

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suppression. They also noted an enhancing effect of small doses of the drug on antibody production paralleling results with other agents. Legezhinskii ( 1954) also reported such enhancement, hlalmgren e f al. (1952a). in a group of experiments cited in other sections, tested the effects of two alkylating agents on the antibody response to sheep red blood cells in mice. Both triethylcne melamine ( T E M ) and /3-chloroethyl carbamate reduced the hemolysin titer. Some recent experiments have also shown the potency of certain of the mustards as immunosuppressants in mice. Butler and Coons (1964) used thio-TEPA, 6 mg. per kilogram, for 12 days in an effort to inhibit “priming” of mice to diphtheria toxoid; and it proved to be the most effective inhibitor of their entire series. Uracil mustard has also been shown, in recent studies (Buskirk et a!., 1965; Bradley, 1966), to he a powerful inhibitor of response to human 7-globulin and actinophage. Since the late 195O’s, cyclophosphamide has been the alkylating agent most used in efforts to suppress antibody production. Cyclophosphamide is a transport form of nitrogen mustard, 2-[ di( 2-chloroethyI)amino]-loua-3-aza-2-phosphacycIohexane-2-oxide (Arnold et al., 1958a,b) . After degradation of the mdecnle and splitting off of the azaphosphohexane group, largely in the liver, it is presumably active as a nitrogen mustard. The altered tissue distribution is assumed to account for the reduced toxicity of cyclophosphamide as compared to the traditional mustards. Back et nl. (1964) have recently proposed, on the basis of studies of cyclophosphamide toxicity, that it may act as a powerful phosphorylating agmt. Fairley and Simister (1965) have recently edited the proceedings of a symposium on cyclophosphamide held in 1963. Although this collection of papers does not consider the biochemistry or pharmacology of the drug to any extent, it does present a broad range of clinical experience with tumors of various types and a series of short papers on clinical and experimental immunosuppression with the drug. It includes an extensive bibliography. As we shall note here and in subsequent sections, the differences in the immunosuppression efficacy of cyclophosphamide as compared to the typical nitrogen and sulfur mustards are great enough to be consistent with the concept of a qualitative difference in the activitv of the drugs. This “shift” from moderate to powerful immunosuppression is evident in the results of Stender e f al. (1959) who stimulated rats with Bwcellu melitensis and compared the effects of X-irradiation and cycIophosphamide during different phases of the immune response. Cyclophosphamide inhibited the primary response at various stages; indeed, it


129

affected ongoing antibody synthesis after appreciable amounts of antibody were present in the serum. It was more potent in this system, with less toxicity, than irradiation. Maguire and Maibach (1961a) and Maguire et al. (1961) provided another instance of striking inhibition, suppressing entirely anaphylactic sensitization to ovalbumin in guinea pigs. The doses were calculated to depress the leukocyte count to approximately lo00 per cubic millimeter. Such treatment also seemed to produce specific immunological negativity in most of the animals if ovalbumin and cyclophosphamide were administered together. After 3 months, 73%of animals so treated survived a sensitization and challenge with the same antigen. Only 9%of a control group survived. In another series these same investigators (Maibach and Maguire, 1963a) compared cyclophosphamide with other cancer chemotherapeutic agents for capacity to inhibit anaphylactic sensitization to ovalbumin in guinea pigs. Cyclophosphamide was effective; 6-MP, vincaleukoblastine, and actinomycin, in the dosages used, were not. These investigators, on the basis of these and other studies (see Section V,D on delayed sensitivity), emphasized that species is a major variable in experiments with cyclophosphamide and that the guinea pig seems to be the ideal animal in which to observe its potential. It is interesting that an entirely different agent, amethopterin (see Section VI on folic acid antagonists) is also much more effective, within limits of acceptable toxicity, in the guinea pig than in other species. In a series of experiments by Santos and Owens (1962, 1964) a direct comparison of a nitrogen mustard (mechlorethamine ) and cyclophosphamide was made. Rats were stimulated with sheep red blood cells. All the agents, including these two, were given at a dose level of half the LD,,. The timing of antigen administration was varied from 48 hours before, 4 hours after, or 48 hours after the beginning of the 5-day treatment period. Although the nitrogen mustard had no effect, cyclophosphamide not only delayed the antibody response, but suppressed the titers. Of greatest interest is the fact that cyclophosphamide had this effect irrespective of the time of administration of the sheep cells within the range noted. Schneider (1964) compared the response to Brucella melitensis by rats treated with cyclophosphamide and trenimon ( triethyleneaminobenzoquinone) and noted greater inhibition with cyclophosphamide. Another series using rats (Potel, 1965) and Rrucellu antigen has also been reported in which particular attention was given to time of administration of the cyclophosphamide. Smaller repeated doses were found to be more effective than single massive doses. A single dose was an effective suppressant, however, and could be given as early as 8 days

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before antigen or as late as 8 days after with significant effect. The single treatment was most effective when it was given at the time of injection of Brucella. There are two very recent studies that show the powerful suppression by cyclophosphamide of antibody production in mice. B. L. Halpern and associates (1965) used sheep red blood cells and varied the drug dosage (20-70 mg. per kilogram) and the timing (treatment for 5 days before or 5 days after antigenic stimulation). They were able to suppress hemagglutinin production with the lowest dose administered for 5 days after antigen. Pretreatment was effective in reducing the response only in the 70 mg. per kilogram dosage. Finger (1965) studied the effects of cyclophosphamide on antibody production and anaphylactic shock after pertussis immunization. The dosage of 1 mg. given €or 4 days beginning 1 day before or 1 day after injection of the vaccine completely prevented antibody production and anaphylaxis, as did a single dose of 4 mg. given 1 day before or 4 days after antigen. However, if cyclophosphamide injection was delayed until 11 or 14 days after antigen, antibody production was depressed but not eliminated, and anaphylactic reactions occurred though reduced in incidence and severity. Many other investigations of antibody production in animals treated with alkylating agents could be cited; the work mentioned, we believe, indicates that nitrogen mustards are effective in suppressing the primary antibody response if given before it is underway. Toxicity is clearly limiting; this will be seen more graphicaIIy in the transplantation studies cited below. Cyclophosphamide, on the other hand, has been extraordinarily effective in rabbits, guinea pigs, and mice. It has suppressed not only the initial stages of the antibody response, the phase most easily inhibited with most immunosuppressants, but affected ongoing antibody synthesis. It is clearly capable of facilitating development of specific immunological negativity in guinea pigs. Its effectiveness is not limited to weak antigens, but has been demonstrated with soluble, cellular, and bacterial antigens.

B. EFFECTS OF ALKYLATIXC AGENTSON PHACOCYTIC MECHANISMS In the preceding section on antibody production, there were data reported by Green (1958b) suggesting that Salmonella remained undigested, judged by its antigenicity, for relatively long periods in the spleens of nitrogen mustard-treated rabbits, a process completed much sooner in the controls. In the morphological analysis of Marshall and White (1950), however, there seemed to be little damage to reticulo-


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endothelial cells by nitrogen mustard, as compared particularly with the widespread destruction of small lymphocytes. Benacerraf et al. (1954) addressed themselves specifically to effects on phagocytic capacity. They found, using a carbon-clearance technique, in rats treated with the maximum tolerable dose of nitrogen mustard, that granulopectic activity was not inhibited. If a blocking dose of carbon was administered, however, mustard-treated animals were slow to recover normal function, presumably a reflection of the effect of the drug on proliferation of the cells. Similar results were observed with cortisone, as noted earlier.

C. EFFECTS OF ALKYLATING AGENTSON INFLAMMATION Although our concern is chiefly with the adaptive immune processes, a word about the effects of the alkylating agents on inflammation is warranted since this is such a prominent component in certain immune responses. Page and Good (1958) analyzed the effect of nitrogen mustard on the acute inflammatory reaction in rabbits. Earlier they had studied the local inflammatory reaction in a patient with cyclic neutropenia and had shown that the inflammatory response was defective when polymorphonuclear leukocytes were lacking in the circulation. Rather surprisingly, the inflammatory sites were deficient not only in neutrophiles but in lymphocytes which were present in normal numbers in the circulating blood at the time of study. This observation was essentially confirmed in the mustard-treated rabbits whose granulocytes were greatly depleted; hematogenous mononuclears failed to appear in the tissue. If, however, polymorphonuclear leukocytes were injected into the local inflammatory sites of such animals, the mononuclears promptly diapedesed and the inflammation proceeded. This confirmed the dependence of the mononuclear phase of the reaction on the integrity of the preceding phase dominated by the granulocytes. This effect of nitrogen mustard on polymorphonuclear cells might be a factor in such results as those of Green (195813) (see above) where there seemed to be a defect in the initial processing of a bacterial antigen in nitrogen mustard-treated rabbits. Modification of the effector stage of specific immune responses may also occur by means of mustard depletion of granulocytic cells. Humphrey (1955) and Cochrane et al. (1959), for example, showed that polymorphonuclear leukocytes are required for development of the intense inflammation and vessel wall destruction of the Arthus reaction. When they treated rabbits with enough nitrogen mustard to eliminate polymorphs from the circulation, they were able to modify the response

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so that only mild edema occurred rather than the hemorrhagic necrosis characteristic of the response in untreated animals.

D. EFFECTSOF ALKYLATINGAGENTSON DELAYED HYPERSENSITIVITY In Section \’+4on antibody production, we noted some of the experiments of Maguire and Maibach involving interference with antibody production and anaphylactic sensitivity as a criterion of immunosuppression with cyclophosphamide. These investigators did several experiments on guinea pigs already sensitized or to be sensitized to DNCB (Maguire and Maibach, 1961b). In one series, treatment with cyclophosphamide 10 mg. per day was begun 1week before sensitization was attempted, and treatment continued for another week. Parallel experiments were done on guinea pigs treated with actinomycin D. Of the agents used, cyclophosphamide was the most inhibitory. The animals did become sensitive ultimately, but this was delayed for 3 weeks. Two groups of experimcnts were performed in animals sensitized to DNCB first. Zagula and co-workers (1963) induced leukopenia in DNCB-sensitive guinea pigs by irradiation or cyclophosphamide administration and then challenged them with DNCB. Although the cellular response was inhibited markedly on histological examination, the delayed reaction differed little from that of controls. Little difference was observed in the reaction in this model when the circulating white cells were brought as low as 800 per cubic millimeter (Maibach and Maguire, 1963b). A partial insight into the cellular basis of cyclophosphamide inhibition of delayed hypersensitivity is offered by the observations of Turk and Stone ( 1963). They sensitized guinea pigs to oxazolone (2-phenyl4-ethoxymethylene-S-oxazolone ) 2 days after beginning a dosage of cyclophosphamide, 10 mg. daily, but were unable to evoke hypersensitivity on testing. They also showed the effectiveness of methotrexate in similar experiments. Of great interest were the morphological studies. CVhen the local lymph nodes of cyclophosphamide-treated, oxazolonesensitized guinea pigs were examined, a striking inhibition of the initial burst of “large pyroninophilic cells” was noted. This contrasted with the findings in methotrexate-treated animals in which the block seemed to occur later, at the phase of proliferation of small lymphocytes. An oversimplified-but at least partly justified-view of this difference is that in this model cyclophosphamide affected RNA and methotrexate DNA primarily. Turk and Stone cited other results which showed that these drug-treated animals were able to express this type of sensitivity following passive transfer of cells from sensitized donors.

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In a very detailed study, confirming and extending the discovery of hilaguire and Maibach, Salvin and Smith (1964) showed that cyclophosphamide fosters specific immunological negativity in guinea pigs. They used hapten-substituted proteins and showed that treatment with cyclophosphamide not only prevented both delayed allergy and antibody synthesis in these animals, but rendered the animals incapable of responding with either delayed allergy or antibody production upon subsequent stimulus with the specific antigen in question. Thus, with cyclophosphamide there was much less ambiguity about the results of experiments with delayed sensitivity than with the adrenal steroids, for example. In the Maibach-Maguire series it was clear that treatment before and during the sensitization process was inhibitory, but that cyclophosphamide treatment after sensitization, although it modified the response histologically, did not diminish it significantly by the ordinary criteria. The transfer experiments of Turk and Stone showed that capacity for skin reactivity was maintained in cyclophosphamide-treated guinea pigs. Their morphological studies suggested that the block in the response is very early, in the “tooling up” stage involving large pyroninophilic cells and presumaby a dominance of RNA synthesis.

E. EFFECTS OF ALKYLATIXG AGENTSON EXPERIMENTAL AUTOIMMUNEDISEASE In the early 1950’s two groups assessed the effects of nitrogen mustard on EAE, our own group (Good, unpublished observations, 1951) and Kolb et al. (1952). The latter group used guinea pigs and gave 0.1 mg. of nitrogen mustard three times a week, starting 1 week before administration of the encephalitogenic emulsion and continuing for 60 days. Both paralysis and mortality were appreciably reduced. In our own work, later extended by Condie ( Condie and Good, unpublished observations, 1958; and L. W. Hoyer et al., 1960a,b), we used rabbits and gave nitrogen mustard, 0.4 mg. per kilogram, intravenously every 4 days beginning either just before or at the time of injection of the spinal cord-adjuvant preparation. We found that EAE was inhibited while treatment was continued, but that signs of the disease began to appear after a lapse of about 14 days once mustard treatment was stopped. In an extension of these studies we began nitrogen mustard treatment, in similar dosage on a similar schedule, well after the encephalitogenic preparation had been injected. The crucial variable seemed to be the circulating leukocyte levels at the time the disease would ordinarily appear in untreated animals. Thus, even very late treatment was effective, so long as it was given before signs of the disease appeared and in suf-

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ficient dosage to reduce the numbers of white cells drastically. When the leukocyte levels rose again in the absence of further treatment, signs of the disease appeared. This experience, when considered together with experiments on suppression of EAE with other agents, suggests that the "effector" stage of the immune response in EAE and signs of disease are virtually simultaneous and that significant modification of the specific sensitivity of the cells, or, in this case, their elimination in large numbers, postpones the disease by averting the crucial immunological attack. Again, cyclophosphamide has been extremely effective in this experimental model. D. B. Calne and Liebowitz (1963) compared the effects of cyclophosphamide and methotrexate. In doses of 20 mg. per kilogram per day, cyclophosphamide prevented the disease, even if treatment was begun as late as 8 days after the administration of the encephalitogenic emulsion. Methotrexate, 5 mg. per kilogram per day, was equally effective. Some of the most remarkable results with cyclophosphamide in EAE, those of Paterson et al. (1966)in rats, are thus far briefly described only in abstract; EAE is less frequently fatal in rats than in rabbits or guinea pigs, and is generally more manipulable. Paterson has found that cyclophosphamide treatment begun before signs of the disease appear not only prevents the disease during treatment but often forestalls it completely, suggesting that specific immunological negativity to the nervous tissue antigens has occurred, facilitated by the drug.

F. EFFFXXS OF ALKYLATING AGENTSON TRANSPLANTATION IMMUNITY Efforts to manipulate the transplantation barrier by means of alkylating agents divide into pre- and post-cyclophosphamide periods. During the first period most of the investigations with the mustards yielded slight prolongation at the toxicity limit. With cyclophosphamide on the other hand, striking manipulation of the allograft barrier has been attained in several species. The first series of grafting experiments involving alkylating agents known to us are those of R. Baker et al. (1952) who performed renal allografting on dogs and compared the effects of nitrogen mustard, cortisone, and splenectomy, alone and in combination. None of the treatments individually prolonged graft survival significantly, but all seemed to increase it slightly. When they gave mustard and cortisone together, and particularly when splenectomy was added to treatment with both drugs, significant prolongation of kidney grafts was observed. One of the remarkable results achieved with nitrogen mustard was


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long-term survival of skin allografts in Holtzman rats in the experiments of Levinson and Necheles (1956). The dosage was 0.4 mg. per kilogram on the day of transplantation, on days 4, 9, and 14, and on every sixth day thereafter. Survival was frequently prolonged to 40 days, and in some for as long as 115 days; controls rejected the grafts in 8 days. The grafts had good hair growth, and histologica1 examination disclosed good capillary circulation. Drug administration had been stopped in many of these animals; thus it appeared that long-lasting tolerance had been produced. When McQuarrie et al. (1960), in our laboratories, attempted to confirm these results, they found that the Holtzman rats were partially inbred and that allografts often survived for prolonged periods without immunosuppression. When they exchanged skin grafts between lines of rats and in outbred rabbits, these investigators failed to influence graft survival by nitrogen mustard treatment. They concluded that nitrogen mustard did not suppress allograft immunity in rats and rabbits. With the history of the inbreeding of the Holtzman strain in mind, it may well be that Levinson and Necheles were dealing with a weak histocompatibility barrier. Under these circumstances control grafts might well be rejected in a short time, as they are in the case of very minor histoincompatibilities in mice. Thus, the apparent contradiction between the results of McQuarrie and associates and those of Levinson and Necheles may basically be a discrepancy of the antigenic barriers each group sought to manipulate. The findings of Levinson and Necheles may indicate that nitrogen mustard, like so many of the immunosuppressants, has profound effects on grafts across relatively weak histocompatibility barriers, and the experiments of McQuarrie and colleagues may indicate that stronger barriers are little affected by treatment with these agents. Efforts to manipulate graft vs. host activity with immunosuppressives are of great interest because of the potential of bone marrow destruction and replacement in treatment of certain leukemias and aregenerative anemias. Many of these efforts have involved the use of methotrexate or the purine analogs, 6-MP and Imuran, but Cree (1962) did a series of experiments in rabbits using aminochlorambucil. The drug was given only once in a dosage fatal to most animals not given further treatment. Either marrow or fetal hematopoietic tissue cells from donors of a different strain were administered 48 hours later. About half of the animals survived for 30 days, and about a third for 5-6 months. When donors and recipients were outbred Dutch rabbits, six of ten animals survived more than 9 weeks. Some chlorambucil-treated animals that were not grafted survived, but the survival rate with grafts particularly

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those across strain lines, is undoubtedly significant. Some secondary disease was seen, but there seems to have been an extraordinary balance of donor and host immunological competence. Cyclophosphamide has now been used in efforts to suppress allograft rejection in severai species. Jones et al. (1962) reported significant prolongation of skin grafts in rabbits and later (Jones et al., 1963) detailed their observations, including morphological study of the regional lymph nodes and spleens of the cyclophosphamide-treated grafted animals as compared to grafted controls. They started to give the drug at the time of grafting, giving 25 or 30 mg. per kilogram daily up to 38 days or rejection of the graft. Over-all, rejection was significantly delayed, from a mean of 9 days to about 22 days. In individual instances, grafts were retained for as long as 150 days, long after the drug had been discontinued, again an instance of drug facilitation of specific negativity. In morphological studies, these investigators observed suppression in the lymph nodes of changes associated with the rejection process in untreated animals: enlargement of germinal centers, increased mitotic activity, and formation of pyroninophilic reticulum cells. In another very recent study in rabbits, Brody et al. (1965) also found that cyclophosphamide, 25 mg. per kilogram, prolonged allograft survival. This group examined the rejection sites histologically and found that cyclophosphamide almost completely eliminated the invasion of small mononuclear cells. Instead the rejection lesion was featured by endothelial s\velling of vessels, invasion of large mononuclear cells, and intravascular occlusion, proccsses obscured by the intense inflammation occurring in the untreated controls. These investigators suggested that certain features of allograft rejection, ordinarily obscured hy the I'1'gorous small lymphocyte component, were allowed to proceed in the cyclophosphamide-treated animals and permitted a morphological cxpression rarely seen in classic allograft rejection. It is of particular interest to us that this type of rejection lesion is associated most typically with a complement-dependent humoral rejection mechanism observed in certain xenograft and allograft modeIs and in some human transplant rejections (Clark et al., 1964; Gewurz et al., 1966). Many groups of investigators have shown significant prolongation ot skin allografts across both major and minor histocompatibility barriers in inbred mice treated with c~clophosphamide.Rerenbaum ( 1963) used a single injection of 200 mg. per kilogram, but varied the timing in relation to grafting. He found that the greatest prolongation was achieved if the tirug ~ v a sadininistcred shortly after graft placement or tip to 4 days

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after placcment. Some survived for 38-40 days. The combination was F, hybrid to parent strain, the parental strains differing at the H-2 locus. Graft survival was only prolonged slightly by the treatment when grafts were exchangcd between the parent strains. Sutton et al. (1961, 1963) also observed prolongation of graft survival in cyclophosphamide-treated mice. Cyclophosphamide has been tested in many screening series involving weak and strong histocompatibility barriers and has usually been found to prolong skin allograft survival significantly ( Floersheim, 1964a; Amiel et d.,1964a; Fox, 1965; B. L. IIalpern et al., 1965). There has been relatively little success in prolonging allograft survival in dogs with any of the alkylating agents including cyclophosphamide. Montague et al. ( 1962a,b) showed an effect on splenic allografts histologically with nitrogen mustard pretreatment, although survival of the graft was not significantly prolonged. MacPhee and Wright (1964) and MacPhee (1965) gave cyclophosphamide before and after lung transplants. The greatest success was obtained when peripheral white counts were used as criteria of both dosage and time of administration; three of thc animals survived for 21, 30, and 49 days. Many other experiments could be cited; but we believe that the series described offers an insight into the difficulties of manipulating allograft immunity with most of the alkylating agents, except for the relative success with cyclophosphamide, particularly in mice and rabbits.

G. CONCLUSION There seems to be no doubt, from the several histological studies cited, paralleling either inhibition of antibody production or allograft immunity, that both the prohferation and differentiation of cells in adaptive immune processes are profoundly affected by alkylating agents, particularly cyclophosphamide. This seems to be particularly true of “cellular immunity,” i.e., the thymus-dependent system; indeed, in one of the allograft studies there is the suggestion that humoral responses are less inhibited. Although the experimental evidence, chiefly in tumor experiments, has not clarified the issue of alkylation of DNA and RNA, it seems highly probably that the effects observed in immunosuppression reflect effects on nucleic acids. The full potential of cyclophosphamide in clinical disease remains to be assessed. Limited experience in patients with systemic lupus erythematosus and rheumatoid arthritis has been reported by Hill (1965) and Balme (19f35), both cautiously optimistic. Encouraging, too, have been

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thc reports of \Vest ct al. (1965. 1966) of lxnefits to patients with hypocomplementemic glomerulonephritis and lipoid nephrosis. In our own pediatric renal clinic, we have had a little success in treating steroidresistant nephrotic syndrome with cyclophosphamide ( Herdman et at., unpublished observations, 1963-1966) ; however, in dealing with subacute, membranous, and chronic glomerulonephritis, and the nephritis of systemic lupus erythematosus and Goodpasture’s syndrome, we have had much more favorable responses with a combination of azathioprine and prednisone (see Section VI1,C). VI. Folic Acid Antagonists

Aminopterin ( 4-aminopteroylaspartic acid ) and particularly a methylated analog, amethopterin ( 4-amino-N10-methylpteroylglutamic acid) or methotrexate, are potent immunosuppressive agents whose mode of action is as well characterized as almost any drug we shall be considering. Their effectiveness is based on inhibition of dihydrofolate reductase, which in turn blocks conversion of folic acid to tetrahydrofolic acid, a step essential to many crucial biochemical processes in the cell involving reactions of one-carbon units, including ultimately synthesis of DNA, RNA, and purine-containing coenzymes. In terms of inhibition of cell division, they are among the most powerful mitotic inhibitors of the preprophasic type (Truhaut and Deysson, 1964). In an echinoderm embryo model, Karnofsky (1960) found that amethopterin affected the late blastula and early gastrula, an inhibition prevented by thymidine, suggesting that growth is sustained only through the period of availability of preformed DNA precursors, but that the effect of amethopterin on thymidine synthesis in particular soon prevents further development. Many reviews of the activity of aminopterin and amethopterin have appeared, with particular reference to their antitumor activity ( Bertino, 1963; Werkheiser, 1963; Holland, 1961; R. Porter and Wiltshaw, 1962; J. S. OBrien, 1962). Some of the effects of the folic acid antagonists may be attributed to activity other than inhibition of tetrahydrofolic reductase ( Holland, 1961; Vogel et nl., 1964; Condit, 1965). As we shall note in more detail later, these agents have found only very limited application as immunosuppressives in man, owing primarily to their serious toxicity; however, since the early trials by Farber et al. (1948) they have played a major role in treatment of acute leukemias particularly in children and appear often to have been curative in treatment of chorio-epitheliomas in women. They have other therapeutic applications, as in treatment of psoriasis, where small doses have been shown to be very effective.


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A. EFFECTSOF FOLIC ACID ANTAGONISTS ON ANTIBODYPRODUCTION The potential of the folic acid antagonists as immunosuppressants was recognized not long after their introduction as antileukemic agents, particularly in the studies of Little et al. (1950) and in the little recognized series of experiments of Malmgren et al. ( 1952a). In the experiments by Little immunological deficiency, defined in terms of lowered antibody titers to Brucellu abortus, Salmonella typhosa, and Pasteurellu multocida, was produced in chickens by feeding of a pteroylglutamic acid-deficient diet from hatching. The investigators were able to restore antibody responsiveness by administration of pteroylglutamic acid, but not by vitamin B,, itself. Of greatest interest in the present context was the finding that amino-an-fol (4-aminopteroylaspartic acid ) perpetuated the immunological defect when given at the same time as the pteroylglutamic acid, in essence a demonstration of immunological suppression by a folic acid antagonist. The experiments of Malmgren et al. (1952a) have been noted in several other sections. With methotrexate in BALB/c and C3H mice, they observed a dose-related suppression of hemolysin titers to sheep red blood cells over the full range of dosages used, from 0.18 to 1.5mg. per kilogram per day; treatment was given for 5 days beginning at the time of antigen administration. As we shall note in Sections VI,C and E on allograft immunity and immunological disease, there were two major phases of work with methotrexate as an immunosuppressant between these early antibody investigations and the large group of antibody studies beginning in 1960 and 1961. This included the work of Haas et al. (Haas et al., 1957; Potter and Haas, 1959; Haas and Stewart, 1956; Lerner and Haas, 1958) on lymphocytic choriomeningitis in mice and the initial efforts of Uphoff (1958) on suppression of graft vs. host disease in radiation chimeras. Although there had been some unsuccessful efforts to suppress antibody production with amethopterin in rabbits ( Brooke, 1960; Schwartz, 1960), Sterzl (1961) showed total inhibition with aminopterin (0.15 or 0.3 mg./100 gm.) in 3- to 5-day-old rabbits receiving adult rabbit spleen cells mixed with Brucellu suis antigen in vitro. In mice, Nathan et al. (1961) found only borderline suppression of hemagglutinins to sheep red blood cells with amethopterin, 2 mg. per kilogram. As in the cyclophosphamide studies (Section V on alkylating agents) the species of choice in analyses of the potential of amethopterin as an immunosuppressant is the guinea pig. Bertino et al. (1964) noted that the drug does not accumulate in the bone marrow and intestine in the

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guinea pig, probably the most important sites for the lethal effects in other species. In their initial experiments, Friedman et al. ( 1961a,b) administered methotrexate every other day, in a dose of 1.7 mg., to Hartley strain guinea pigs of 250 gm. weight and were able to suppress completely antibody responses to diphtheria toxoid and ovalbumin in adjuvant. They used systemic and cutaneous anaphylaxis as the criteria to evaluate suppression of antibody. In later studies ( Friedman et al., 196%) they also suppressed completely the antibody response to vaccinia virus. Berenbaum ( 1!362a) included amethopterin among the compounds screened in mice as suppressants of the response to a single injection of TAB vaccine and noted significant suppression of antibody levels. The dosage was 10 mg. per kilogram, and nine of the twelve animals survived to the time of bleeding for antibody titers. With the background of folinic acid “rescue” in amethopterin-treated leukemic mice, Berenbaum and Brown (1965), in the same TAB vaccine system, increased the amethopterin dosage up to 50 and 100 mg. per kilogram and administered folinic acid 8 hours later. The decrease in antibody levels was appreciable and very closely paralleled the decreases in unprotected mice. Deaths from the drug were greatly reduced in the folinic acid-treated group. This approach has also been used by Berenbaum (196413) in allografted guinea pigs receiving amethopterin repeatedly in high dosage, also with success (see Section VI,C below). Santos and Owens (1962) tested a number of antimetabolites and alkylating agents in rats immunized with sheep erythrocytes and found that many of the animals receiving 2050%of the LD,, of methotrexate formed no agglutinins for 6 weeks. In extensions of these experiments, these investigators (Santos and Owens, 1969) assessed the role of timing of the first drug dose in relation to administration of the sheep cells. They found that 2448 hours after antigen administration was the most advantageous time to begin the 5-day administration of the drug. Significant depression was not noted if administration was begun after 48 hours. Depression was seen, however, if amethopterin was started as early as 71, hours before antigen; this, of course meant that the last dose was given a day after antigen administration. They related the vulnerable period to the time of maximum mitotic activity in the rat splenic red pulp after administration of sheep cells, as reported by Gunderson et al. (1962). One of the facts that has been repeatedly brought to our attention, in considering clinical and experimental deficiencies in antibody production, including those imposed by immunosuppressive drugs, is the differential


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between light 7 S and hcavy 19 S immunoglobulin. This has been brought out in striking fashion with 6-MP, and Santos and Owens (1966) have recently documented the selective effect of methotrexate on 7 S antibody in rats. In very recent studies on guinea pigs, Brandriss et al. (1965a) were able to suppress antibody to ovalbumin in guinea pigs up to the time of appearance of small amounts of antibody, but not when production was at its height. They were also able to suppress the secondary response if they began the drug at the time of administration of antigen or 48 hours later. On the basis of antibody studies alone, one would classify methotrexate as one of the most effective immunosuppressants available. It is the only agent to our knowledge whose immunosuppressive activity has been dissociated in time from its major toxic manifestations affecting bone marrow and gastric mucosa, so that it is possible to counter the latter with folinic acid while sustaining the immunological injury. Although the major efforts at immunosuppression with amethopterin in the dog have involved allografts of lung, heart, or bone marrow, Thomas et al. (1962a, 1963) studied the response to distemper virus. When antibody production was inhibited in amethopterin-treated animals, the attenuated virus caused a fatal illness.

B. EFFECTS OF AMIKOPTERIN-AMETHOPTEFUX ON DELAYED HYPERSENSITIVITY Remarkable suppression of tuberculin and contact sensitivity has been achieved in guinea pigs with the folic acid antagonists. Prichard and Hayes (1961) studied the effect of aminopterin not only on the hypersensitivity response but on the lesions of the disease itself in tuberculous guinea pigs. They abolished caseation and necrosis in lesions bearing large numbers of bacilli and suppressed entirely the rcsponse to old tuberculin. We noted earlier the suppression of antibody synthesis (Friedman et al. 1961a,b) with methotrexate, using egg albumin and diphtheria toxoid as the antigens. Delayed hypersensitivity was also suppressed, though with more difficulty than the primary antibody response. In later experiments, this group (Friedman et al., 1962a,b) also prevented development of delayed reactivity to vaccinia virus and suppressed established sensitivity to tuberculin, whether actively or passively acquired ( Friedman, 1964). The other major analyses of suppression of cellular, thymus-dependent responses in guinea pigs have been those of Turk (1964) and Turk and

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Stone ( 1963). In different series they used o.uazolone ( 9-phenyl-4-ethoxymethylene-5-oxazolone), picryl chloride, or DNCB as the sensitizers. It was possible in all instances to prevent sensitization by administration of methotrexate, 5 mg. on alternate days, beginning at the time of sensitization or by day 4 after sensitization. If the drug was withheld until day 6, the depression was irregular. Turk and Stone (1963) showed that these suppressed animals could develop delayed sensitivity if transfused with cells from sensitive donors. There is a discrepancy between these results and those of Friedman and Buckler (1963) in which methotrexatetreated guinea pigs did not develop tuberculin sensitivity following passive transfer. This discrepancy seems to have been resolved in terms of the day on which cells were taken from the sensitized donors; in the series by Friedman and Buckler they were taken on day 5, when the developing response was probably still methotrexate-sensitive, whereas in the series by Turk the donor cells were taken later. The histological studies of Turk and Stone (1963) were mentioned in Section V,D on aUcylating agents and delayed hypersensitivity. The dynamics of two cell populations, large pyroninophilic cells and small lymphocytes, were studied by autoradiography and examination of imprints and sections from the regional lymph nodes during the sensitization process. Cyclophosphamide, as we noted earlier, blocked the formation of large pyroninophilic cells, a cell type dominant at 3 to 5 days; but methotrexate acted later in the sequence, chiefly inhibiting the small lymphocyte population presumably derived from the large pyroninophilic cells. Although amethopterin is an inhibitor of both RNA and DNA synthesis, these data suggest that the locus of inhibition in this model is DNA, primarily affecting cell proliferation. The most ambiguous of these findings, it seems to us, is the suppression of established tuberculin reactivity in the work of Friedman. It seems to us that this can only be accounted for in terms of peripheral suppression rather than of central inhibition. There is evidence, in the recent clinical studies of Hersh et al. (1966) that amethopterin treatment, particularly daily intensive therapy, greatly reduces the number of mononuclear cells in “skin window” preparations of inflammatory sites and that even intermittent therapy has significant effects on the inflammatory cycle. Page (1964), on the other hand, noted normal mononuclear infiltrates in inflammatory cycles of aminopterin-treated rabbits, a result perhaps consistent with the negligible effects of the folic acid antagonists on adaptive immune responses in rabbits. In any event, there is at least a suggestion that inflammation can be profoundly affected by these agents in some circumstances.

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c. EFFECTS OF AMINOPTEEUN-A~~E1.HOPTERIN ON ALLOGRAFT IMMUNITY AMJ

GRAFTvs. HOSTDISEASE

As we mentioned earlier, one of the turning points in experimental work on immunosuppression was the experimental work of Uphoff (1958) involving modification of graft vs. host disease in mice. These experiments had been preceded by the work of Haas and co-workers (Haas and Stewart, 1956) on lymphocytic choriomeningitis and the much earlier work of Malmgren and his associates (1952a) on antibody production. The initial experiments (Uphoff, 1958) involved an effort to ameliorate secondary disease in lethally irradiated (C57BL x DBA/2)F, hybrids treated with C57BL bone marrow. Amethopterin was given in a dosage of 1.5 mg. per kilogram at 48-hour intervals for a total of nine treatments, beginning at the time of usual onset of the disease. Four of seven survived at 70 days, compared to one of six controls. Reducing the number of treatments to five or six, either retaining the 1.5 mg. per kilogram dosage or doubling it, also resulted in significant numbers of survivors, though with a higher incidence of overt homologous disease. In extensions of these experiments, Uphoff used the combination of amethopterin administration, in dosages similar to those of the earlier experiments, together with crude antigenic preparations of donor-strain spleen or thymus and spleen (Uphoff, 1961). When tumor tissue was grafted, even an H-2 histocompatibility barrier was overcome in some of the animals. The drug administered alone had relatively little influence on skin graft survival, but did facilitate tumor growth. Several subsequent experiments have confirmed the borderline immunological supprcssion achieved with methotrexate in mice with skin homograft survival as the criterion (Glynn et d.,1963; Floersheim, 1 W a ; Goldin et d., 1962) and the significant facilitation of tumor development in some strain combinations (Humphreys et al., 1961, 1962, 1963; Glynn et al., 1963). Later investigators have also influenced different forms of homologous or secondary disease in mice by administration of amethopterin (Math6 d al., 1962; Lochte and Thomas, 1962; Russell, 1962; Schwartz and Beldotti, 1965). Clearly, the mouse is not the ideal species in which to ascertain the potential of methotrexate; toxicity usually intervenes at dosages and within periods that seriously limit degree of immune suppression achieved. Inbred mice, of course, offer the enormous advantage of welldefined histocompatibility (antigenic) differences and a large body of data on the bridging of barriers to skin and tumor transplantation by administration of cells and subcellular preparations under different con-

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ditions of age and radiological or pharmacological depletion of thtn immune system. Main and Prehn ( 1955) induced tolerance with radiation and lymphoid cells, as did Uphoff with methotrexate and cells in the studies cited earlier. The latter results have been repeatedly confirmed (Woodruff, 1962; Hilgert, 1965). Of more direct relevance to the problem of overcoming histocompatibility barriers in man has been the induction of tolerance in adult mice by disrupted cells or suhcellular fractions facilitated by immunosuppressive agents. Medawar ( 1963) reported synergistic action of cell extracts and amethopterin in prolonging skin homograft survival. Such efforts have been greatly extended with other agents and offer the promise of reducing toxicity by limiting the drug dosages while enhancing the specific suppression by administration of specific antigens. Another means of enhancing immunosuppression while limiting toxicity was used by Bercnbaum and Brown ( 1965) in mice (see Section VI,A) and has also been used by Berenbaum in guinea pigs with skin grafts. In earlier investigations. Berenbaum (1963) had given 9, 12, or 15 mg. of amethopterin per kilogram on alternate days. The 15-mg. dose killed most of the animals, but significant prolongation of skin allograft survival was seen at the 9-mg. dose and particularly at the 12-mg. level. In individual instances grafts survived 85 and 160 days during continued treatment at the 12-gm. level. When dosage was increased to 18 and 24 mg., administered Mondays, Wednesdays, and Fridays, and 18 mg. of folinic acid per kilogram was given 24 hours after each treatment, the proportion of 3- to 6-week survivals was greatly increased (Berenhaurn, 1964b). When treatment was stopped, between 6 and 10 weeks, all the animals ultimately sloughed the grafts, but sometimes after a lapse of another 4 to 6 weeks. Often the rejection was of the indolent type. Herenbaum suggested that further manipulation might improve this experience and that there seemed to be no reason for believing that immunosuppression could not be continued indefinitely in this manner. This is a very promising approach which warrants further investigation in other species. Protection against extreme toxicity without impairment of immunosuppressive efficiency may well be possible with other agents as well. The Cooperstown group has made major efforts in transplantation of bone marrow, skin, lung, and heart in methotrexate-treated dogs. Secondary disease following allogeneic marrow injection in lethally irradiated dogs has been more difficult to manipulate than secondary disease in mice and rats. Thomas ef al. (Thomas and Ferrebee, 1962; Thomas ct a]., 196%; Hager ef a!., 1961) have had some success with the aid

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of methotrexate. In the most successful series, given 1200 to 1600 r of total-body irradiation and given allogeneic marrow 1 to 3 days post irradiation, they gave 5 mg. of amethopterin every other day, beginning 3 to 14 days after irradiation. Eleven of fifteen had marrow “takes,” with an average survival time of 60 days. Two were alive at 300 days. In another series, the amethopterin dose was lower, 1 mg. three times a week, and four of the fifteen animals survived more than 3 months. This group has established that these animals are chimeras. Blumenstock et al. (1961, 1962a,b, 1963, 1964) have documented an extensive series of orthotopic left lung transplants in methotrexate-treated dogs. In some instances this treatment has been combined with either irradiation or treatment with donor antigens. Some very long survivals of functioning grafts wcre observed, e.g., up to 26 months. The transplanted lungs had an average of 50%ventilatory function and 40%of expected oxygen consumption ( Blumenstock, 1963). In a later report (Blumenstock et al., 1964) all the grafts in place 15 months or longer were essentially nonfunctioning. That a long period of relatively normal function of an allografted lung is possible has been suggested by Kahn ( 1963). The immunosuppressive agents were cyclophosphamide and methotrexate; the graft was across breed lines. Hechtman et al. (1962) also reported prolongation of skin graft survival in methotrexate-treated dogs. They (Hechtman et aZ., 1962, 1964; Epstein et al., 1964) have also used this drug in combination with crosscirculation and radiation in an effort to prolong survival of skin and kidney allografts. Prolongation was negligible in all but one series, and these findings are thus far only available in abstract (Epstein et al., 1964). Pairs of dogs were matched for A antigen and cross-circulated, methotrexate-treated, or both. Survival of the skin grafts was significantly increased in the last group, ranging up to 59 days, and averaging 23. Two groups have succeeded in prolonging the survival of allografted hearts in dogs with methotrexate. Williamson et al. (1962) and Reemtsma ct al. (1962) had half of their animals survive more than 10 days and one survived 26 days; the longest survival in an untreated control was 10 days. Blumenstock et al. (1963) had eight of fifty animaIs survive more than 1 day; of these three died early, but the others lived for 12, 17, 18, 32, and 42 days. It has not been possible to carry immunosuppression with methotrexate far enough, in either guinea pigs or dogs, to result in specific immunological negativity, although this should be possible with as potent an agent as this. Perhaps further efforts with the folinic acid “rescue” will permit this. The “rescue” technique should, indeed, be expanded to

146

A L X E. GABRIELSEN AXD ROBERT A. GOOD

other species and other types of grafting situations; although it does not resolve the problem of blanket immunosuppression, with the attendant vulnerability to infection, it has circumvented the problem of lethal toxicity in two species. It may well be that there are other agents whose major toxic manifestations can be dissociated from their immunosuppressive capability in this way.

D. EFFECTSOF AMETHOPTEHIXOX LYMPHOCI'TIC CHORIOMENINGITIS The use of amethopterin in lymphocytic choriomeningitis in mice was one of the earliest instances of immunosuppression with this drug. Beginning in 1956, Haas ef al. (Haas et aZ., 1957; Potter and Haas, 1959; Haas and Stewart, 1956; Lerner and Haas, 1958) demonstrated the suppression of lymphocytic choriomeningitis (LCM ) in mice treated with amethopterin. It will be recalled that one of the earliest descriptions of tolerance to a virus was that of Traub (1936) who found that mice exposed to the LCkI virus in utero grew up as carriers of the virus bearing none of the stigmata of the disease. Rowe (1956) had shown that LCM virus-injected mice were spared if they were given sufficient whole-body irradiation; and it has since been shown that neonatal thymectomy will have similar effects (Levey et al., 1963). In none of these instances is the multiplication of the virus inhibited; the effects are based on the inhibition of the immune reaction. Histological studies (Lerner and Haas, 1958) showed that the disease process was greatly modified, but that extensive inflammatory reactions were present in the spared mice; thus, the suppression was apparently one of degree.

E. EFFECTSOF AMETHOPTERINON EXPERIMENTAL AUTOIMMUNE DISEASE Rrandriss (1963a,b) and Brandriss et al. (1965b) have had extraordinary success in suppressing development of experimentaI alIergic encephalomyelitis with methotrexate in both guinea pigs and rabbits. As in their studies with cortisone, cited earlier, these investigators were able to modify the disease process with amethopterin to some extent even after onset. \*'hen treatment was stopped, most of the animals developed signs of the disease within 3 weeks, although this often seemed to be attenuated in both rabbits and guinea pigs. Even though a number of guinea pigs escaped the disease entirely, it seems doubtful that the disease was actually prevented in a significant number of animals, since the disease rate in control animals is 6045%. Of great significance were the obsen,ations of greatly lowered mortality and clinical improvement in guinea pigs treated after signs of the


147

disease were present. Brandriss (1963b) used a high initial dose of 10 mg. of methotrexate daily for 3 to 6 days, followed by 5 mg. daily for 2 to 3 weeks. The survival rate in the treated group was much higher during the first 2 weeks, 85 vs. 3315. When the drug was stopped, some of the clinically improved animals relapsed. Spiegelberg and Miescher (1963) used both methotrexate and 6-MP in experimental immune thyroiditis in guinea pigs. In this model disease is induced with thyroglobulin or thyroid-extract protein administered with Freund’s adjuvant. In addition to the histological assessment of the thyroid, treated and control animals were also tested for delayed hypersensitivity and circulating antibody. Amethopterin was the more effective of the two agents. In high dosage, the drug suppressed ongoing disease, as did a large dose of 6-MP. Ward et al. (1964) tested several immunosuppressants in adjuvantinduced polyarthritis in rats and found that amethopterin suppressed the disease without altering hypersensitivity to old tuberculin. Thus, in several species, amethopterin has been shown to depress what we would interpret as thymus system-mediated disease.

F. CLINICAL USE OF AMINOPTERIN AND AMETHOPTERIN It seems evident from all the data that have accumulated, documented here only in part, that amethopterin is one of the most potent immunosuppressive agents known. It has been axiomatic in immunosuppression that toxicity and suppressive effectiveness go hand-in-hand; but this is not true temporally in mice and guinea pigs, as so strikingly demonstrated by Berenbaum with folinic acid “rescue” midway between doses of the drug. Lacking, generally, have been reports of specific immunological negativity induced with methotrexate. It seems probable that methotrexate acts largely as a DNA inhibitor in immunosuppression, and the question arises whether this type of agent can facilitate tolerance induction. Clinical use of the folic acid antagonists in diseases with a presumed immunological component has been limited. These agents have, during the last 15 years, become accepted agents in treatment of severe psoriasis (Gubner et al., 1951), an experience recently reviewed by Van Scott et al. (1964). Gubner et al. (1951) have also reported aminopterininduced remissions in rheumatoid arthritis patients, but suggested that its toxicity was limiting. A major trial of amethopterin in rheumatoid arthritis and psoriatic arthritis was that of W. M. O’Brien et al. (1962) and Black et al. (1964). In that series almost all patients showed significant improvement, skin manifestations responding more rapidly than

14s

ANN E. GABRIELSEN AND ROBERT A. GOOD

joint manifestations. Toxicity was a major concern, however, arid there were two deaths, one from a pulmonary embolism and the other a cerebral thrombosis. Wong ( 1966) has recently reviewed his experience with methotrexate in uveal disease. The drug suppressed cyclitis at dosage levels which resukcd in suppression of the mononuclear phase of the inflammatory cycle and inhibition of antibody formation. Vll. Antimetabolites of the Purine Bases

If we wcrc to single out the major transition in immunosuppression as it exists at present, it would be the recognition, signalled chiefly by the experiments of Schwartz and associates (1958) in rabbits, of the potential of the purine analogs, chiefly 6-MP. There were other earlier experiments which showed immunosuppression with other agents of this group, and Sterzl and Holub (1957) had attempted earlier to use 6-MP as an immunosuppressant and failed; but the results of the Boston group were impressive and were, largely through experiments in dogs, “fed into” the biological and technical build-up toward renal allotransplants in man. In this section, we shall deal principally with three of the agents: 8-azaguanine; 6-MP and its imidazole derivative, azathioprine or Imuran; and 6-thioguanine. : I . 8 - h

~ G u A IS hE

S-Amguanine is a classic antimetabolite. It was synthesized in the mid-1940’s (Roblin ct al., 1945) and was shown initially to function as an antagonist of guanine in a number of microorganisms, particularly B. cereus. Later, its incorporation into RNA of viruses (Lasnitzki et al., 1954) and mammalian normal tissue and tumor (Mitchell et al., 1950; Lasnitzki et al., 1954; Matthews, 1958) was demonstrated, the proportion of 8-azaguaninc to guanine being much less in mammalian tissue than in B. ccrezis. There is an extensive literature on enzyme inhibition and enzyme induction b y 8-azaguanine in a variety of systems (reviewed by Elion and Hitchings, 1965). There has been sentiment, on the part of a number of investigators, away from synthesis of ribonucleoproteins as the primary locus of S-azaguanine action to synthesis of other vital cell proteins ( Karnofskv and Clarkson, 1963; Burdge and Mandel, 1962). Surprisingly, at least a partial test of this hypothesis is available in an in tiitro antibody study by Dutton and associates (1958). These investigators started their experiment with the assumption that 8-azaguanine was a nucleic acid inhibitor; however, their studies with 14C-labeled

CHEMICAL SUPPRESSION OF ADAPTIVE Ihf MUh'II'Y

149

leucine showed that inhibition of incorporation of this amino acid into antibody was no more inhibited than incorporation into the insoluble cell proteins, suggesting that in this context 8-azaguanine inhibition was best characterized as suppression of protein synthesis in general. Although a number of studies in the late 1940's and early 1950's demonstrated the antitumor properties of 8-azaguanine in rabbits and mice (Stock et al., 1949, Kidder et al., 1949, 1951; Gellhorn et al., 1950), clinical use has been very limited because of the violence of the gastrointestinal toxic manifestations and the severity of the dermatoses resulting from its administration. In dogs, too, it has been extraordinarily toxic. It is of interest here because it was used effectively in immunosuppression in mice as early as 1952 and is representative of the antimetabolite approach to control of immune processes which was later to be so productive. The early study of 8-azaguanine, in 1952, was that of Malmgren and colleagues who studied both hemolysins and precipitins in mice and rats stimulated with sheep red blood cells. They were unable to affect the response in rats; however in mice, at the highest dosage level, 120 mg. per kilogram, they observed marked inhibition. In a test of several inhibitors in baby rabbit recipients of adult spleen cells mixed with Brucella suis antigen, Sterzl (1961) observed inconsistent inhibition at a dose level of 1.0 mg., and none at 0.5 mg. Butler and Coons (1964) assessed the effect of 8-azaguanine on priming of white mice by diphtheria toxoid; in their system, the efficacy of the drug is tested by whether or not a secondary response occurs on restimulation. They found 8-azaguanine to be one of the most effective of their series of inhibitors; 55%of the animals gave no evidence of priming. When the primary response had not been inhibited and an effort was made to suppress the secondary response, little inhibition was seen with any of the agents used, and none with 8-azaguanine.

B. 6-MEHCAPTOPURINE Thus far, the development in immunosuppression which has been channeled most directly into the clinical transplantation effort has been the appreciation by Schwartz et aZ. (1958) of the potential of 6 M P in inhibiting antibody production and inducing specific immunological negativity to soluble protein antigens. Several branchings of activity occurred, Schwartz et al. launched a most fruitful series of investigations, still in progress, much of it summarized in a reccnt review (Schwartz, 1965); Calne, in England, undertook experiments on dogs with renal allografts; and in our own laboratory we began a series on rabbits involving suppression of the secondary immune response, induc-

150

ALNX E. GABRIELSEN A S D ROBERT A. GOOD

tion of tolerance, inhibition of ongoing antibody production, and prevention of experimental allergic encephalomyelitis, as well as a series seeking to suppress delayed hypersensitivity to tuberculin in guinea pigs; and, with Pierce and Varco, a series on dogs with renal allografts. Many other groups have, of course, used 6-MP and related agents; our selection has been intended to illustrate the range of immunological process and species, offering, as we have tried to do throughout, some indication of what was done in the particular experiment. As in prior sections, we should like to comment briefly here on the mechanism of action in general, to bring in pertinent observations on means of immunosuppression as they occur in discussing results of experiments, and then to focus on mechanism briefly again at the end. 6-hifercaptopurine was introduced in 1953 by Elion et al. as an outcome of a series of systematic investigations of analogs of the purine and pyrimidine bases, in turn derived from consideration of the requirements of bacteria for these bases (reviewed by Hitchings and Elion, 1954). SMercaptopurine apparently can act as a classic antimetabolite, since its inhibition of growth of certain microorganisms can be reversed by any of the natural purines (Elion and Hitchings, 1957); but in other systems its defined activities on enzyme systems and nucleic acid metabolism are myriad and still being expanded (see Karnofsky and Clarkson, 1963; and Elion and Hitchings, 1965). In mammalian systems in particular, there is little evidence that 6-MP is incorporated to any extent into RNA or DNA (Karnofsky and Clarkson, 1963). The antitumor activity of GMP, beginning with the experimental work of Clarke et al. in 1953 has recently been reviewed by Elion and Hitchings (1965). The greatest therapeutic effectiveness in man has been documented in acute lymphatic leukemia in children. One of the paradoxes of immunosuppression has been that the agents with best defined biochemical mechanism are ineffective in doses feasible in the living animal, whereas the agents whose activities are least understood, are those offering most effective suppression. This is nowhere more evident than in considering 6-MP and azathioprine. Balancing the biochemical and pharmacological data on one hand, and the immunosuppressive effects on the other, it seems probable that the primary, but not the sole, effect of 6-hIP on adaptive immune process is on RNA. 1. Effects of 6-nlar.c.uptop~i,.i,ic> otz Antibody Production

One of the earliest iostances of immunosuppression with a purine analog WCI\ recordcd not with 6-MP but with 3,Sdiarninopurine, the earliest of the purine analogs to show activity against experimental tumors


151

(Burchenal et al., 1949; Skipper et al., 1950). This agent was included in the large series tested in mice by Malmgren and associates (1952a), assessing hemolysin titers following administration of sheep cells. Particularly at the highest dose used, 100 mg. per kilogram, suppression of antibody production was significant. During the next 5 years, we note no further work with this agent; but in 1957 Sterzl and Holub proposed that 6-MP might inhibit immune responses by its antimetabolite action. Their own initial efforts showed no effect on immune responses, but the next year Schwartz and coworkers ( 1958) published their startling results-rabbits treated with 6 mg. of 6-MP per kilogram per day intravenously following primary stimulation with soluble protein antigens showed complete suppression of antibody response. Such drug treatment was ineffective if given prior to antigenic stimulation or if withheld until antibody production was at its height. They were also unable to inhibit the secondary response with the dosages used. As remarkable as the initial findings of virtually complete suppression of the primary response with 6-MP was the observation of Schwartz and Dameshek ( 1959) that tolerance, specific immunological negativity to human serum albumin, could be produced in adult rabbits by 6-MP treatment at the time of primary stimulation. We have noted in other sections that most immunosuppressive agents will foster such negativity to a particular antigen under ideal conditions of timing and dosage. In our own laboratories, following the initial demonstration of the effectiveness of 6-MP by Schwartz and associates, we undertook experiments seeking to suppress the secondary and tertiary response. Initially LaPlante and Condie (1960) and LaPlante et al. (1962) confirmed that 6 mg. of 6-MP per kilogram per day, administered after secondary stimulation, did not inhibit the secondary response to bovine serum albumin; however, when the daily dose was increased to 12 or 15 mg., no secondary response was detectable in most of the animals (Tables I and 11). Perhaps the most intriguing aspect of our early investigations was the discovery that a small number of animals given the 15-mg. dose were unresponsive on tertiary stimulation as well, although no additional 6-MP was given. Even though the group was small, we suggested that 6-MP in high dosage was capable of “erasing” immunological memory. Forsen and Condie ( 1963) and Forsen et al. (unpublished observations, 1964) confirmed and extended these experiments in rabbits and were able to show that unresponsiveness to tertiary stimulation is usually observed if the secondary response has been fully suppressed with 6-MP ( Fig. 2 ) . This unresponsiveness is specific immunological negativity, or

152

ANN E. GABRIELSEN AND ROBERT A . GOOD

Relationship between 6 -MP Depressed Secondary Immune Response a n d Tertiary Response

-,.

2" Response

I* Qesgo-se

,5c-

~

cJL_

3' --

Response

6-MP

a

v,

m

&A

BSA

SSA

6 MP

5s

delayed

3-

p2Q3 Days

1

+ 0

-E

5C

Controls 6-MP

\

-jv

BSA

5SA

5SA

Pretreo tment

55

I

6-VP

Ic)

p'

? 0 rJc m a 5c

-

6 MP

1 0 ZCJ 35 40 5 0 60 7 0 80 90 I I

,-.ad ~

1

35.1

BSA

854

Days

FIG. 2. These graphs show titers of circulating anti-BSA in rabbits, in terms of antibody-binding capacity ( ABC-33 in micrograms of nitrogen per milliliter at 0.4 pg. of RSA nitrogen per milliliter). The upper graphs show the lack of detectable antibody, following both secondary and tertiary stimulation, when 6-MP treatment was begun at the time of secondary antigenic stimulation. The middle group of graphs shows a relative depression of the secondary response and no effect on the tertiary response when 6-liP treatment was started 3 days after secondary stimulation with BSA. The bottom graphs show virtually no effect on secondary and tertiary responses when 6-MP was given for 8 days prior to the secondary antigenic stimulation. In all cases 6-XlP was given in a dosage of 18 mg. per kilogram intravenously for 8 days. Each antigenic stimulation consisted of 100 mg. of BSA per kilogram.

153


TABLE I EFFECTSOF 6-MP ON THE SECONDARY RESPONSE TO BOVINESXRUMALBUMIN I N RABBITSASSAYED BY QUANTITATIVE PRECIPITIN METHOD^ Dosageh

I

I1

I11

IV

Controls

0 0 36.1 70.5 61.4 46.4 64.7 91.5

0 0 97.1 30.6 52.7 15.0 21.9

0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 46.9 146.4

0 341.0 75.0 70.4 85.2 69.5 73.8 96.7 80.3 0 77.7 61.1 131.1 87.7

a All determinations were made on day eight serum samples, by the method of Heidelberger and MacPhenon (1943) and are expresed as micrograms of antibody nitrogen per milliliter. The animals had received primary stirnulation with 20 mg. of BSA 30 days before secondary stimulation with the same dosage. All animals treated had responded to primary stimulation. 0 means that a qualitative precipitin test was negative. Dosage values are: I, 6 mg. per kilogram per day for 25 days before and 4 days after secondary stimulation. I I , 6 mg. per kilogram per day for 8 days after secondary stimulation. 111, 12 mg. per kilogram per day for 8 days after secondary stimulation. IV, 15 mg. per kilogram per day for 10 days after secondary stimulation.

tolerance, since the animals are immunologically capable with respect to other antigens. Another principle of dosage and time of administration was clear in these experiments. Toxicity is limiting in many experiments with 6-MP; with such immunological processes as EAE, for example, the animals are spared from the disease as long as treatment is continued, but ultimately succumb to the effects of the drug on other cell systems (see Section VII,B,4). In these experiments with the secondary response, the dosage is very high, but the effect may be achieved by 4 or 5 days of treatment without significant toxicity. The problems are basically different, since tolerance does not develop in the EAE model as it does with BSA. Another type of suppression of antibody production was evident in the experiments of Condie et al. (1961). In these experiments rabbits

154

A S S E. GABRIELSEN AND ROBERT A. GOOD

TABLE I1 RESPOXSESTO BOVINE SERUM Si,ri>mssIo-i OF SECO\I)ARY TERTIARY ALBTIIIS I& H.APBITS TREATED KITH 6-MPa Dosag&*“ I:e*pon.‘ Setxtrttlrtry SWOlitbAr!-

Tert iar? Tertiary Tertiary

1h.T

(‘oritrolb

I

11

0 11

0/12 11/11 1/11 11/11 11/11

0/13 1/13 0/13 11/13 11/13

0/3 0/3 0/3

0

6 12

0/3 1/ a

Respunsea were teated by a qunlitative precipit in method. The secondary response was given 30 dayh after the primarl, and the tertiary 50 days after completioil of the secondary respoiise studies; 6-MP was administered only at the time of secondary stimulatioii. b Data given as tile ratio of number positive to number tested. c Dosage values are: I, 12 mg. per kilogram per day for 8 days after secondary stimulation; 11, 15 mg. per kilogram per day for 10 days after secondary stimulation.

were hyperimmunized by daily injections of BSA for 40 days, with 6-MP beginning day 28 and continuing for 8 days. Even under these conditions, it was possible to suppress antibody synthesis. Although these observations were originally interpreted as indicating suppression of ongoing antibody synthesis, more recent findings suggest that there is constant recruitment of cells, presumably of the thymus-dependent type, under these conditions of daily antigenic stimulus and that it is arrest of participation of these ceIls that results in suppression of antibody production. The argument that 6hIP acts only on the “inductive phase” of antibody production has been advanced particularly by Sterzl (1960, 1963). His experiments were performed on rabbits, adult unimmunized rabbit spleen cells being mixed with BruceIla antigen in uitro and injected into baby rabbits. Antibody production was suppressed with 6-MP begun on the day of cell transfer. If the cells transferred were from antigenically stimulated animals, Sterzl was unable to suppress antibody production with 6-121P. In his 1963 paper, he summarized other evidence in support of the concept of the inductive phase. Since that time it has taken on additional validity in view of the data on the role of small lymphocytes in certain types of antibody responses. In our own experiments (Forsen and Condie, 1963; Forsen et aZ., 1964) evidence was obtained in strong support of this concept. Figure 3 shows that short-term 6-MP treatment started at the time of injection of antigen completely inhibited thc irnmune response to RSA in rabbits. Treatment begun 2 days after


155

initiation of treatment produced lesser, but still significant, suppression, whereas treatment begun 4 days after injection of antigen did not suppress the immune response at all. In further work with their 6-MP induced tolerance model, with BSA as the antigen in rabbits, Schwartz and Dameshek (1963) showed that the phenomenon was dependent upon dosage of antigen, 66 mg. per kilogram in their animals. With lower doses of antigen they were able to suppress the antibody response with 6-MP, but were not able to Second response to BSA 0

Control

x

6-MP (I8mgJkgJdoy for 10 days) on day of stimulation 6-MP (I8mg./kg./day for 10 days) 2 days after stimulation 6-MP (18 mg./kg./day for 10 days) 4 days after stimulation

L-g X

U v) m

. .h- * -

0t

2

100 mg. BSA I. v.

4

6

8

10

Days following second stimulation with BSA

FIG.3. Suppression of the secondary response to BSA by administration of 18 mg. of 6-MP per kilogram for 10 days, beginning either on the day of secondary stimulation or 2 days later. Antibody production was essentially normal if drug treatment was started 4 days after stimulation.

produce tolerance; this is perhaps not a surprising result, since quantity of antigen is a crucial variable in many tolerance models, although many other factors such as the particulateness vs. solubility of the antigen, route of administration, and maturity of lymphoid development of the animal are involved. It may be that the failure of Goh et al. (1961) to induce tolerance in rabbits given 6-MP was related to the dose of antigen; they used a dosage of 60 mg. of either human serum albumin or human y-globulin. M. Feldman et al. (1962) on the other hand were

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A S S E. G.ABRIELSEN A S D ROBERT A . GOOD

successful in inducing tolerance to human serum albumin in 65%of their rabbits, but the antigen dosage was 70 mg. In our own experience with induction of tolerance in rabbits previously immunized with BSA, the dosage of antigen successful in inducing tolerance when given with a short course of 6-MP was approximately 50 mg. per kilogram. In more recent experiments, Okada et al. (1964) also produced a negative adaptation of this type by administering antigens together with 6-MP. 6-Mercaptopurine was an effective suppressant of antibodies to human red cells in chickens ( Orbach-Arbouys and Eyquem, 1961), but ineffective in two experiments involving antibody to serum protein antigens in guinea pigs (Humphrey and Turk, 1961; Genghof and Battisto, 1961). We shall return to some of these questions of species differences later; however. on this specific difference, between rabbit and guinea pig, the work of Abrams and Bentley (1955) may be pertinent. In the rabbit, bone marrow cells have little xanthine oxidase activity; in the guinea pig, however, there is an efficient mechanism for converting 6-MP to thiouric acid which requires xanthine oxidase. All the foregoing studies in rabbits have involved serum protein antigens; however, in two recent studies bacteria or their products have been used with 6-MP. Rubenstein and Wolff (1964) used Salmonella typhosa endotoxin and 6-MP in a dosage of 18 mg. per kilogram for 4 days. Antibody production was suppressed at several levels of antigen; but the immunosuppression did not affect the development of tolerance to the pyrogenic and toxic effects of the endotoxin. The latter is perhaps best explained in light of the work of Kim and Watson (1965) showing that endotoxin tolerance in rabbits is due to formation of 19 S antibodies. Several investigators have shown that 6-MP is much less inhibitory of antibodies of the 19 S class (Sahiar and Schwartz, 1964; Plescia, 1965; Smith and Robbins, 1965; Bore1 et al., 1965). The studies by Smith and Robbins involved administration of Salmonella, 2 x lo9 organisms, and 6-MP dosage of 18 mg. per kilogram. Although the total antibody response was not much reduced, 7 S antibody production was completely suppressed. IVolff and Goodman (1962, 1963) had earlier reported depression of y-globulin in rabbits, but not in guinea pigs, treated with 6-MP in high dosage. Homeostasis of immunoglobulins is a little-understood realm in immunology. One of the effective approaches to it has been analysis of the type of antibody suppressed pharmacologically and what happens to the other types quantitatively. Experiments in mice have also confirmed the immunosuppressive properties of 6-MP; many such experiments will be included in Sections VII,B,3 and 4 on cellular immunity and allograft reactions. There have also


157

been a number of antibody studies. Nathan et al. (1961) and Bieber et al. (1962) used the popular test system of sheep erythrocytes in mice and found 6-MP to be very effective. ButIer (1961) and Butler and Coons (1964) used diphtheria toxoid in mice and assessed effects on two types of response to antigen: “priming” and the secondary response in animals not drug-treated at the time of priming. At the dosage level of 5 mg. per kilogram per day, for 12 days, priming was inhibited in more than 90%of the animals treated with 6-MP, Butler and Coons were unable to influence the secondary response even in doses three times those effective in inhibiting priming; at this level the drug was lethal in 10-12 days. Sterzl (1963) also pursued the question of the susceptibility of the inductive phase of antibody formation in mice to antimetabolites, as well as the problem of suppressing the secondary response as had been done in rabbits. The results seem at first glance to be the reverse of those of Butler and Coons. Sterzl was able to delay antibody production in the secondary response to sheep eryth;ocytes by F-MP treatment, 50-100 mg. per kilogram, and to reduce the titeis attained. He found, however, that a secondary response occurred even. if the primary had been completely suppressed. This difference seems to be a reflection of type of antigen. The experiments of Berenbaum- and associates with immunosuppression in mice have been cited in discussing several groups of agents. With a test system involving administration of TAB vaccine to A2G mice, Berenbaum (1960) and Berenbaum a n d , Brown (1964) noted significant suppression of antibody production with 6-MP in sublethal doses. These authors emphasized that carcinostatic properties of the agents and their immunosuppressive capability tended to be parallel, whereas toxicity per se and immunosuppressive potency were not correlated, a conclusion similar to that of Malmgren et al. (1952a). In general, there have been few assessments of immunosuppressive agents in primates other than man; however, Janssen et al. (1962) studied resistance to intravenously injected variola virus in 6-MP-treated and control rhesus monkeys. Control monkeys survived, whereas animals treated with 6-MP died of the infection. The primary antibody response was either blocked or delayed. It seems to us probable that the defective host resistance in these animals was attributable largely to the suppression of cellular immunity. The major efforts at immunosuppression with 6 M P in dogs have been directed toward successful grafting of allogeneic kidney; however, Mannick et al. (1962), as part of such a study, assessed the circulating antibody response in dogs treated with 6-MP and stimulated with hemo-

158

ASX E. CABRIELSEN AND ROBERT A. GOOD

cyanin and found precipitin formation to be significantly suppressed. The large body of evidence on immunosuppression in man with purine analogs involves azathioprine (Imuran) and will be considered in Section VII,C. However, several groups have studied antibody production in patients treated with 6-MP for a variety of conditions. Levin and co-workers ( 1963) noted suppression of agglutinin and antitoxin production, as well as lowering of 7-globulin levels, in carcinomatous adults under 6- MP treatment. Cancer patients receiving chemotherapy were also studied by Santos et al. (1964). In the group treated with 6-MP they found reduced responses to Pastewella tularensis and typhoid antigen. Our own results, reported in detail by Page et al. (1964), contradict these findings: typhoid agglutination titers, mumps complement fixation titers, and diphtheria antitoxin responses were usually within the normal range during 6-MP therapy, as was the response to actinophage. Our group was a more varied one; the subjects in the actinophage group were children with active rheumatoid arthritis and those studied for responses to typhoid, mumps, and diphtheria antigens had acute lymphatic leukemia in complete hematological remission. It seems probable that the patients in the other groups were already immunologically deficient to some estent before treatment. This tends to be the case in lymphomas and leukemias other than acute lymphatic leukemia (Miller, 19%; Scharff and Uhr, 196.5). It has been shown experimentally that it is easier to inhibit immune responses in animals with neoplasms. In a series of in oitro studies, described in more detail in the section on the pyrimidine analogs, Dutton and Pearce (1962) assessed the effect of 6-MP on the activity of cultured spleen cells from animals stimulated several times with egg albumin and human y-globulin some weeks before and restimulated just before the spleens were taken for culture. Labeled amino acids were included in the medium in some experiments and their incorporation into both antibody and other cell proteins was assessed. Cell viability was also ascertained. 6-Mercaptopurine was an effective inhibitor of antibody synthesis during the time period studied, days 2 and 3 of the culture period, but it also inhibited protein synthesis in general, although there was not much cell death. This set 6-MP apart from the antipyrimidines, some of them well characterized as DNA inhibitors in other systems and apparently acting as effective inhibitors of antibody production by this means in the it1 vifro system. This suggested to the authors that 6-kIP was too blunt an instrument to warrant further use in efforts to dissect the complex sequence of events resulting in formation of circulating antibody.

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159

2. Efects of 6-Mercaptopurine in Delayed Hypersensitivity Suppression of delayed hypersensitivity was achieved with difficulty in the guinea pig, apparently a reflection of the facility with which guinea pigs degrade 6-MP, as noted in Section VII,B,l on antibodies. Salvin and Smith (19f30) used doses up to 75 mg. per kilogram per day intraperitoneally, but were unable to prevent either Arthus or delayed hypersensitivity. In our laboratory, however, J. R. Hoyer et al. (1962) sensitized guinea pigs with BCG (Bacillus Calmette-Gukrin vaccine), and administered 6-MP intramuscularly or intraperitoneally in doses of 50 mg. per kilogram per day beginning on the day of sensitization. Cutaneous testing was done with 0.005 mg. PPD [purified protein derivative (tuberculin)]. As long as treatment was continued the response was suppressed; however, this occurred only with the intramuscular route of administration, perhaps a further reason for the results of Salvin and Smith. Delayed allergy in rabbits was inhibited with 6-MP in the investigations of Borel and Schwartz (1963, 1964). The antigen was bovine y-globulin. Surprisingly 6-MP more readily suppressed this response, with lower drug dosage and a shorter period of treatment, than it did circulating antibody production. Timing was important: If BMP was administered beginning on the third day after sensitization, the animals were unresponsive at challenge at 14 days; if the drug was given on day 6, sensitivity was evident at 14 days but not at 24 days. Borel and Schwartz (1964) emphasized the role of the anti-inflammatory action of 6-MP in some of these results. It is perhaps well to note here briefly the findings of Page et al. (1962) on the powerful antiphlogistic action of 6-MP. In these experiments the inflammatory stimulus was egg white injected subcutaneously along the rabbit’s back; a connective tissue biopsy technique was used to examine the sites during the inflammatory cycle. A period of pretreatment was needed to demonstrate the anti-inflammatory effect, but it could be achieved with doses as low as 3 mg. per kilogram per day if the treatment was continued long enough. Similar profound inhibition by even small doses of 6-MP on the mononuclear component of the acute inflammatory process in man as well suggest that the effects of immunosuppressant drugs on delayed allergic processes may involve both immunosuppressive and anti-inflammatory actions. SMercaptopurine has also been tested as an inhibitor of delayed hypersensitivity in vitru by the assay system of David et a2. (1964). Kaplan and Calabresi (1966) found that 6-MP in concentrations of

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and 10 Jf abrogated bovine 7-globulin inhibition of migration of peritoneal exudate cells from BGG-sensitive guinea pigs.

3. Effects of 6-lllercaptopurine on Allograft Rejection We approached immunosuppression with 6-MP in three ways, as noted earlier: in studies of antibody production, in an experimental autoimmune disease, and in allografting studies, first with skin in rabbits and then with kidneys in dogs. In the experiments of Meeker et aE. (1959, 1960) skin grafts were exchanged between Dutch and New Zealand rabbits; SMP, in a dosage of 6 or 12 mg. per kilogram was administered at grafting. Toxicity was the limiting factor in these results; the animals tolerated 6 mg., but graft survival was not consistently prolongecl. At 12 mg. rejection was consistently delayed. Rejection took place if the drug was discontinued; but the animals died, often with graft intact, if the treatment was continued. Similar observations were made in rabbits by Schwartz and Dameshek (1960). Meeker et al. (1960) extended their experiments to inbred mice, but were unable to affect skin allograft survival significantly. Similar failure to influence skin graft survival in mice was reported by Rubin and Lewis (1961). Ruhin and Lewis (1961) also reported influencing allograft survival in young chickens with 6-MP. They were unable to prolong survival in adult animals; but when 8-day-old chicks were treated, 30% tolerated skin allograft< for a period of months. This is reminiscent of the finding of Cannon and Longmire (1952) in which cortisone in newly hatched chicks greatly increased the proportion of animals developing tolerance of skin allografts. To our knowledge the initial independent successes with 6-MP in prolonging survival of renal aIlografts in dogs were those of R. Y. Calne (1960) and Zukoski et al. (1960). In both instances individual animals retained functioning grafts for more than a month. Regular prolongation of renal homograft survival was also observed by Pierce and Varco. At that stage all indications were that grafts would be rejected if immunosiippression was stopped. However, Pierce et al. (1961) had one animal with a functioning renal graft at 255 days and stopped the drug cntirely. At the time of the 1961 report the period of tolerance had been increased to 400 days, and at the time of the second report (Pierce and Varco, 1962), the kidney was functioning 18 months after placement, almost 10 months after cessation of therapy. There have been selmxl reports of morphological alterations in the lymphoid tissues of allografted animals treated with 6-MP. Zukoski et al.


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(1960) observed depletion of germinal centers in spleen and nodes in dogs. Pierce et al. (1961) noted thinning of the cortices and depletion of the primary follicles in the nodes of dogs. Andre et al. (1962) noted particularly the inhibition of the large basophilic hemocytoblasts in rabbits retaining skin grafts during 6-MP treatment and the appearance of large numbers of these cells as the rejection process began. Since 1961 and 1962 most grafting studies with purine antagonists have involved Imuran (see Section VI1,C). There is, however, one very recent study in rabbits which represents successful application in outbred stock of techniques hitherto applied only in inbred mice. This is the work of Mannick and Southworth (1966) who combined 6-MP treatment, insufficient in itself to prolong graft survival, with administration of large amounts of disrupted cellular material from multiple allogeneic donors, the rationale of the multiple donors being presentation to the recipient of a random assortment of histocompatibility antigens presumably including those present in the skin allograft. Survival of grafts has been trebled in some rabbits. 4. Effects of 6-Mercaptopurine on Experimental Immune Diseases and Graft DS. Host Responses

As we have indicated in other sections, EAE in rabbits has been a most useful model for assessment of immunosuppressive agents; and an effort to modify this disease process was one of our first thoughts as we sought to define the immunosuppressive capability of 6-MP. In the experiments of L. W. Hoyer et al. (1960a,b, 1962) a dosage of 12 mg. per kilogram per day was required to suppress EAE in rabbits. As in the grafting studies this dosage was toxic, and administration could not be continued for more than 18-20 days. When the drug was discontinued, the rabbits developed the disease in 7 to 18 days. In extensions of these experiments, L. W. Hoyer et al. (1962) delayed treatment to day 9 after administration of the adjuvant-central nervous tissue preparation and still observed sparing of the animals treated with 6-MP (Fig. 4). When paralysis was imminent, however, treatment was ineffective. After Field and Miller (1961) found that 6-MP did not inhibit development of EAE in guinea pigs, L. w. Hoyer et al. (1962) reported inhibition of EAE with 6-MP in guinea pigs. As in other experiments with 6-MP in this species, the dosage and route of administration of the drug were crucial. Onset of paralysis was delayed when 150 mg. per kilogram per day was administered intraperitoneally. Even more effective was 50 mg. per kilogram per day injected into the deep muscles of the back. These dosages are in the range of the LD,, in guinea pigs.

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In csperimcnts described in more detail in Section VI on folic acid antagonists, Spiegelberg and Xliescher ( 1963) suppressed experimental immune thyroiditis in guinea pigs with 6-XIP. It has also heen used in experimeutal alicrgic uveitis (\Virostko and Halbert, 1962) and adjuvant arthritis of rats by sevchral groups (KalliomiiLi ct ul., 1964; Ward ct d., 1963).

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FK. 4. Inhibition of EAE in rabbits treated with 6-MP at 12 mg. per kilogram per day, beginning on the day of nervous tissue-adjuvant administration and continuing for 18 days. It will be seen that the incidence of disease in the control animals was a t the 90%level by day 23, at a time when none of the animals treated with 6-MP had the disease. Over the next 10 clays, however, most of the rabbits developed EAE. From L. W. Hoyer et al. (1962).

Graft vs. host disease represents a challenge to immunosuppression, and a number of agents including 6-MP have been used in efforts to modify this process. Some of the major efforts in this direction have involved the folic acid antagonists; however, 6-MP has also been used, principally by Schwartz ( 1962) and Schwartz and Beldotti ( 1965). Their hybrid-parent strain combination was ( C57BL/6 x DBA/2) F, and C57BL/6. Although the earliest results of Schwartz (1962) suggested that a small dosage of 6-MP (10 mg. per kiIogram per day) given before or at the time of administration of the parental cells prolonged survival of the animals, later experiments (Schwartz and Beldotti, 1965) noted no beneficial effects of any of the 6-MP dosages given before, at the time


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of, or after the inoculation of parental cells. In fact, in most instances the disease was accelerated, apparently a reflection of the adverse effects of 6-MP on the host’s own lymphoid tissues also being attacked by the cells. Prednisone and amethopterin were ameliorative, but were effective at different stages of the reaction (see Sections IV and VI on corticosteroids and folic acid antagonists). In Section VI on folic acid antagonists, we noted the great difficulties encountered in seeking to control secondary disease in dogs given allogeneic marrow after heavy X-irradiation. Even in the best of the series of amethopterin-treated marrow-grafted animals, survivors were few. Several groups have tried to control graft vs. host disease in dogs with 6-MP or Imuran. Kauffman et al. (1965), for example, under a range of conditions of irradiation, 6-MP treatment, and cell administration, achieved only temporary “takes.”

5. 6-Mercaptopurine in the Treatment of Diseases Associated with lmmunological Abnormality Following the demonstration by Schwartz et al. (1958) of the effectiveness of 6 M P in suppressing the primary antibody response in rabbits and their subsequent definition of a tolerant state in some of these animals (Schwartz and Dameshek, 1959)) one of the natural transitions for these investigators, clinical as well as experimental, was to a consideration of an application in human disease. Beginning in 1960, they reported some success in treating such diseases as acquired hemolytic anemia (Dameshek and Schwartz, 1960; Schwartz and Dameshek, 1962; review Schwartz, 1963, 1965). In our own hospital, and we believe that this has been general experience, despite a few notable exceptions, the use of 6 M P as a single drug in rheumatoid arthritis and systemic lupus erythcmatosus has not been advantageous. We have had some success with 6-MP in plasma cell hepatitis (Page et al., 1964), experience paralleled by that of Mackay et al. (1964), all the more remarkable because hepatotoxicity is often considered an attribute of 6-MP and related agents, At the time, we considered that 6-MP might be acting as an antiviral agent in this disease. Efforts to suppress rheumatoid arthritis, nephrosis, and lupus erythematosus in children and adults by treatment with 6-MP alone have uniformly failed in our hands. However, using 6-MP in the form of Imuran, together with cortisone or prednisone, has provided remarkable suppression of systemic lupus erythematosus, subacute nephritis, and some forms of chronic nephritis in children and adults (see below). There have, in our view, been few more exciting-and in the long run more useful-recent developments in immunology than that of immunopharmacology which, even now, means largely the immunopharma-

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cology of 6-MP and derivatives, Malmgren and associates recognized early that this group of agents might be useful in suppressing immunity and Sterzl and Holub recognized specifically that 6-MP might be an immunosuppressant; but there is no doubt that the experiments of Schwartz and Dameshek, using the right type of antigen in the right species with an “aggressive” dosage of the drug, set into motion an extraordinary development, both experimental and clinical. There is no need more pressing in clinical allografting today than better immunosuppression; and yet, were it not for the experimental development which we have sketched so briefly, there would be almost no clinical allografting! We made the analogy earlier of corticosteroids and the purine analogs being the most useful immunosuppressives and, at the same time, those least \\.ell defined as to mechanism. 6-Mercaptopurine is less mysterious, certainly, since it is acting largely on the nucleic acids and, perhaps. proteins, of immunologically competent cells-probably only on cells of the thymus-dependent system, There is some contrary evidence, however, and other data interpretable either way, i.e., those on limitation of ongoing antibody production. As we have noted elsewhere, the body of information, including very limited morphological studies of the lymphoid tissues and certain key in citro observations, suggests that the major target is RNA. The fact that ceIlular immune responses, such as EAE and delayed sensitivity, can be affected ‘late,” although this is done with greater difficulty in the case of antibody production, suggests that the RNA of thymus-dependent cells may be subject to interference or inhibition almost to the effector stage. Dissociation of delayed allergy from antibody responses in some instances may reflect the anti-inflammatory effects of the drug on the former, but it may also reflect the relative shielding of antibody-producing cells from 6-MP. It is known that plasma cells are notoriously resistant to irradiation and drugs which more readily affect other lymphoid cells.

C . AZATIIIOPRIXE (Ihiumx) Azathioprine [6-( l-methyl-4-nitro-5-imidazolyl) thiopurine] was introduced in 1961 (Elion et al., 1961; Rundles et al., 1961) and has largely eclipsed 6-MP in clinical use. In particular it is the “backbone” of the various immunosuppressive regimens used in clinical renal allografting. The clearest advantage over 6-MP seems to be a relative sparing of the epithelium of the gut, which reduces gastrointestinal toxicity associated with oral administration of 6-MP. This analog is presumably enzymatically disrupted to the active form in the liver. Elion and Hitchings, in their recent review (1965), cite a series of renal allograft experiments

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in dogs as support for the claim that azathioprine is the superior suppressant, but indicate that no superiority has been demonstrated for the heterocyclic variant in treatment of leukemia. It seems to us that the point of reduced toxicity is well taken, but that the claim of increased immunosuppressive potency has rather narrow support. Under some conditions in certain species, notably the rabbit, the “extra step” in catabolism of azathioprine greatly decreases its effectiveness as an immunosuppressant ( Schwartz and AndrC, 1962, Schwartz, 1963; Gowland et al. 1965); apparently the rabbit cannot split off the imidazole ring effectively. The recent results of Gowland suggest that the observations by Schwartz and AndrC reflected dosage factors in part; but there seems to be good reason to believe that 6-MP can be more efficient than azathioprine in some situations. Of course, it may well be less efficient in some, as has been suggested. In Section IV,I on corticosteroids, we considered briefly the drug combinations in use by the major clinical transplantation groups: the reliance on azathioprine from the very beginning, and the varied practice on the use of prednisolone or some other adrenal steroid, actinomycin, and azaserine. Starzl et al. ( 1964a) started azathioprine before transplantation and believed that this enhanced the chances of good function and survival of the kidney. Some of the most exciting recent experimental grafting work has involved the use of Imuran in dogs with tissues other than kidney, and it is with some of these results that we should like to conclude. The tissues grafted include pulmonary lobes, portions of the gastrointestinal tract, spleen, heart, and-with remarkable successes-liver. Several groups have undertaken extensive series of lung transplants in dogs (Hardy et d . 1963a,b,c, 1964; Barnes and Flax, 1964; Parsa et al., 1964; Richards et al., 1965). Mean survival times in these groups have been about 30 days, although each group has also recorded individual survivals of 2 months or more. As in the methotrexate series of Blumenstock and associates ( Section VI,C ), some of these long-surviving canine lung allografts were not functional. Preston et al. (1965) and Assimacopoulos and Salmon (1965) have observed prolonged survival of jejunum and stomach allografts in azathioprine-treated dogs. In Section VI on folic acid antagonists, we noted some of the prolonged survivals of dogs with heart allografts. Perhaps the most remarkable series is that of Lower et al. (1965a,b) in which azathioprinc therapy and methylprednisolone have been used as immunosuppressives in dogs receiving cardiac allografts. Most successful has been a program of intermittent therapy, concentrated when electrocardiogram

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( ECG 1 voltage suggrsts rcjrction threat. One animal trcxatcd in this manner had survived for morc than 8 months at the time of the report. This achievement is truly remarkable. It may he that the secret lies in the fact that with this model therapy can be initiated very early in the rejection and thus can be most effective. The observation is sufficiently dramatic to provoke additional consideration of intermittent regimens in other models. When a lymphoid tissue is allografted, there is immunological reaction in both directions, and the problem is one of balancing host activity against graft with graft vs. host reaction ( Starzl et al., 196513). Starzl et al. (1965b) have reviewed some of these problems and described the five clinical spleen transplants performed in Denver. This same group (Marchioro et al., 1964) has documented a number of instances of prolonged splenic allograft survivals in azathioprine-treated dogs. Evaluation of splenic allografts presents a problem in both the experimental and clinical contexts, since there is no functional indicator comparable to those available for kidney and liver grafts. Indeed, in the one case of the Denver spleen allograft series in which an index of function, yglobulin production in the agammaglobulinemic recipient, was available, the increased y-globulin proved to be of host not donor type (Starzl et al., 196%). There is great interest in many centers in liver allografting, chiefly in dogs. With azathioprine, often in conjunction with a corticosteroid, prolongations of survivals to a month or more have been achieved by several groups. Some very long survivals have been observed by Starzl et al. (1964c, 196Sa, 1966), including one liver that functioned 20 months after orthotopic transplantation. This is a paradox in many ways, since azathioprine is considered to be a hepatotoxic drug. In commenting editorially on the outlook for clinical liver transplantation, Starzl et al. (1966) cited bleeding problems as a major obstacle to hepatic transplants. Dozens of reports in the past year or two have attested to the range of diseases in which azathioprine is being used, many of them conditions in which 6-MP had been used earlier with benefit to some patients. One of our own programs (Michael et al., 1964a, 1966b) has concentrated on subacute and chronic glomerulonephritis, as well as lupus nephritis, diseases usually considered progressive and untreatable. The regimen is basically that of transplantation therapy, azathioprine and a corticosteroid, usually prednisone. hlerrill ( 1962) used this combination in a similar patient group, although with lower dosages, and noted greatest improvement in patients with lupus erythematosus. The glomerulone-


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phritis patients did not clearly benefit, although it was Merrill's impression that the progressive disease was slowed. Our own group is large, concentrated in the pediatric age group, and many of the patients have now been treated for 2 to 3 years (Michael et al., 1966b). By both functional and histological criteria, not only arrest of the disease process, but at least partial reversal has been noted in patients with lupus nephritis, subacute and chronic glomerulonephritis, so-called membranous glomerulonephritis, and even the Goodpasture's syndrome. The immunological component in some of these diseases has not been completely defined, but our data suggest that whatever the initial insult to the kidney, the ongoing disease process is dominated by inflammatory, immunological and/or proliferative factors susceptible to large doses of two potent immunosuppressive agents. As we noted earlier, West et al. (1965) have also had some success in treating progressive renal disease, resistant to other forms of therapy, with cyclophosphamide. Supplementing the in &To studies of purine analogs cited earlier is a recent experiment by Wilson (1965a,b). In this system inbred rats are immunized by application of skin grafts, sometimes together with spleen cells. The target cells are taken from tumors of the immunizing strain. Counting surviving target cells after specified incubation periods of sensitized lymphoid cells with allogeneic tumor cells offers an assessment of cytocidal action of the lymphoid cells under different conditions. In one series of such incubations Imuran in varied concentration was added to the growth medium. Trypan blue was used to assess lymphoid cell viability. At 1 and 5 pg. per milliliter, Imuran significantly inhibited destruction of target cells; at these concentrations neither lymphoid cells nor target cells were killed by the drug itself. Wilson (196%) concluded that Imuran interfered with an RNA-dependent process in this system. D. 6-THIOGUANINE 6-Thioguanine is a congener of 6-MP, also developed by Elion and Hitchings (1955), and apparently largely equivalent to 6-MP as an experimental antitumor agent ( see Elion and Hitchings, 1%S), although the mechanisms of their action may not be the same. There is, for example, evidence that 6-thioguanine is incorporated into the DNA of tumor cells and that it is incorporated less into resistant than susceptible tumors (LePage and Jones, 1961). Booth et al. (1964) found that thioguanine resulted in a pile-up of DNA in susceptible cells. Thus, although 6-MP and 6-thioguanine have similar activity in many ways, it seems quite possible, from our present understanding of the molecular processes

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involved, that thioguanine functions more directly through formation of fraudulent or abnormal DNA. As has been noted by Philips et uE. (1956), the patterns of toxicity of 6-MP and 6-thioguanine are also quite different. Like 6-h4P, 6-thioguanine was an effective suppressant in mice of antibody responses to sheep red blood cells in the work of Nathan et al. (1961) and to typhoid paratyphoid vaccine in the studies of Berenbaum and Brown ( 1964). The most extensive assessment of the effects of 6thioguanine on antibody production in mice were the experiments of Frisch and Davies (1969a,b) and Frisch et al. (1962). Using human red cells as the antigen, they showed that 6-thioguanine was an effective suppressant of the primary response and also slowed the secondary response. \\'hen single doses were used, the timing was crucial, effectiveness being greatest at about 18-36 hours after administration of antigen (Frisch et al., 1962). This group was also able to inhibit antibody production in rabbits with 6-thioguanine, using sheep red blood cells as the antigen (Frisch and Davies, 1962b). Sterzl (1960) also tested 6thioguanine, 0.1 and 0.0s mg. per 100 gm. body weight, in baby rabbits receiving adult rabbit spleen cells mixed with Brucella suis antigen. Both doses were inhibitory, with considerable toxicity at the higher dose. WoIff and Goodman (1962, 19631, in experiments in rabbits cited in Section VI1,B on &MP, noted depression of y-globulin production following 6-thioguanine treatment. 6-Thioguanine has been relatively little used in grafting studies, a function apparently of the need for continued administration in this type of suppression and of the very rapid cumulative toxic effects of 6-thioguanine (Philips et al., 1956). 6-Thioguanine, like 6-MP and azathioprine, has been used with some success in treating patients with autoimmune hemolytic anemia (Dameshek and Schwartz, 1960; Schwartz and Dameshek, 1962), steroid reFponsive nephrotic syndrome (Goodman et al., 1963), systemic lupus ervthematosus ( Dameshek and Schwartz, 1960; Goodman et al., 1963; Eisen r t nl., 196?), chronic hepatitis, including plasma cell hepatitis (Goodman et al., 1963), and hyperglobulinemic purpura (Weiss et nl., 1963). 6-Thioguanine, Eke 6-MP, seems to have yielded to azathioprine in efforts at clinical immunosuppression of this type. Vlll. Analogs of the Pyrimidine Bases

The analogs of the pyrimidine bases are chiefly important as carcinostatic and virostatic compounds and have generally not been effective in wppressing immunity in cxpcrirnental animal5 in tokrable dosage.


169

They have been used in a number of in uitro studies of antibody production, however, and these are of interest chiefly for the insights they give into the cellular processes involved in secondary antibody response. Earlier, wc: arbitrarily excluded from our review any extensive consideration of potentiation of immunological response; a number of experiments have shown such enhancement with administration of certain antipyrimidines ( Merritt and Johnson, 1962, 1963, 1964). Perhaps the major series of in vitro investigations of antibody production and these agents is that of Dutton et al. (Dutton and Pearce, 1962; Dutton et al., 1959; Dutton, 1960). Cells from secondary stimulated rabbits were put into culture, and the effect of 5-bromodeoxyuridine (BUDR) tested at 2 and 3 days, a period of rapid transition in this in vitro system; BUDR was found to inhibit antibody production in both tests. Thymidine reversed the inhibition at 2 days, suggesting that the drug was inhibiting DNA. In extensions of these experiments, 5-fluorouracil deoxyriboside (FUDR) was used and found to be a less efficient inhibitor than BUDR. Its effect, too, was partly reversed by thymidine. When these agents were used at 3 days rather than at 2 days, much less inhibition was seen. Dutton and Pearce (1962) used the same in vitro system with a variety of agents, including BUDR, 5-iododeoxyuridine ( IUDR), and 6azauridine among others. The donor animals had been stimulated with two antigens, ovalbumin and human y-globulin. The investigators considered cell survival, incorporation of radioactively labeled amino acids into both antibody and other cell proteins, and antibody levels. They found BUDR and IUDR to inhibit antibody production and the effect to be partially reversed with thymidine. These agents did not affect other cell proteins or kill the cells. The action of 6-azauridine, included in the series as a probable RNA inhibitor, was different. It, too, was much more inhibitory at 2 days than at 3; however, its effects could be prevented by cytidine and uridine, but not by thymidine or deoxycytidine. It was similar to RUDR and IUDR in that it did not affect other cell proteins. These “mixed results suggested to the authors that 6-azauridine was acting as a DNA inhibitor, but that it was probably also affecting RNA synthesis as well, a conclusion consistent with the action of 6azauridine in other systems (see Elion and Hitchings, 1965). Calabresi and Turner (1966) reccntly noted that the locus of the pyrimidine block caused by azauridine is fairly close to that caused by methotrexate and that both would result in RNA and DNA inhibition. It is interesting in this connection that Sterzl (1961), testing a large number of antipyrimidines as possible immunosuppressives, found that only Sazauridine inhibited

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antibody production in baby rabbits receiving adult rabbit spleen cells mixed with Brucella siris antigen. It did so irregularly in a moderate dosc, but was uniformly inhibitory in a large toxic dose. In another culture system, involving spleen cells from secondary stimulated rabbits, Kong and Johnson (1963) used BGG as thc antigen and 5-fluorouracil and 5fluoro-3’-deoxyuridine as inhibitors of antibody production. Both compounds were effective, and the inhibition was partly reversed by thymidine. T. F. OBrien (1961) and T. F. OBrien and Coons (1963) used BUDR in their rabbit lymph node culture system. The donors are stimulated with BSA and diphtheria toxoid, the popliteal lymph node fragments prepared for culture, and secondary stimulation provided in uitro. 5-Bromodeoxyuridine inhibited the antibody response if addcd on the second. third, or fourth day; before or after this interval, it had no discernible effect. They concluded that the secondary response in vitro depends on DNA synthesis during this period. L41thoughin z;iz;o results have generally been negative, there are at least two reports of significant inhibition in mice: one by Merritt and Johnson ( 1963), who used bovine y-globulin as the antigen and FUDR or 5-fluorouracil as the suppressing agent for 12 days, and the other by Bieber et al. (1962), who used sheep red blood cells as the antigen and BUDR as the immunosuppressant for 4 days. The latter group also reported synergistic activity of BUDR and 6-MP and argued that combinations of immunosuppressive agents might enhance effectiveness and reduce toxicity. Although combination programs are being used by the transplantation groups, it seems to us that this approach has not been fully tested either experimentally or clinically. IX. Antibiotics

Although even the commonest antibiotics will alter immunological reactivity to some extent, as noted in the recent review of Makinodan et al. { 1965), we shall concern ourselves here with only five agents: puromycin, mitomycin, actinomycins C and D, chloramphenicol, and azaserine. The delineation of the immunosuppressive potential of chloramphenicol-indeed the “new view” of chloramphenicol as it affects mammalian tissues-has been one of the dramatic recent instances of a welI-kno\ili, widely used drug suddenly taking on a whole new role, as it were. Acriflavine, being considered in Section XII, is another of these: a drug developed more than 50 years ago, recently shown to have remarkable effects on the immune mechanism experimentally. Puromycin and the actinomycins are also agents with very well-


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defined actions on very limited segments of the protein synthetic sequence in the cell. They are agents of very limited clinical usefulness, either as antitumor agents or immunosuppressants, because of their inordinate toxicity; but they have been enormously useful in the biochemical analysis of a wide range of cellular processes, including adaptive immunity. The actinomycins, as we have noted in Sections IV,I and VII,C, have been used by several groups as part of the rejection-crisis regimen in transplant patients. Here the doses have been very small; and the evidence that they affect the process significantly has been meager, it seems to us. On occasion, in a desperate situation the administration of a medication may be more therapy for the therapist than for the patient! A. PUROMYCIN Puromycin was isolated from Streptomyces allo-niger in 1952 and originally characterized as an antimicrobial agent (J. N. Porter et al., 1952). Yarmolinsky and de la Haba (1959) noted the structural similarities of this antibotic and the amino acid-bearing end of soluble RNA and suggested that puromycin inhibits the amino acid transfer from soluble RNA to ribosomal protein. Nathans (1964) and Darken (1964) have reviewed many aspects of the activity of puromycin in a variety of systems. Puromycin has not been an effective in viuo inhibitor of cellular processes within toxic limits; but it has yielded important insights into enzymatic and metabolic processes within cells. It has been used in at least three in uitro systems of antibody synthesis. In the work of Smiley et al. (1964) spleens and lymph nodes from rabbits hyperimmunized with BSA were used. The last immunization had been given 3-5 days before the tissues were placed in culture; the incubation period was 6 hours. Under these conditions, puromycin in concentrations as low as lC3and lC4was inhibitory. Ambrose and Coons (1963b) used lymph node fragments from stimulated rabbits and stimulated secondarily in vitro. In an experiment incidental to their definition of the effects of chloramphenicol, they found puromycin to be profoundly inhibitory of ongoing antibody production. Inhibition of hemolytic plaque formation by puromycin in uitro was noted by Ingraham and Bussard ( 1964). Although other inhibitors have had varied effects in these different in vitro models, relating often to the time variable of the antigenic stimulation and the duration of culture, the results with puromycin were remarkably consistent; in the short term or the long, puromycin has been a

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powerful inhibitor of ongoing antibody production, usually without killing the cells.

B. MITOMYCIXC Mitomycin C was isolated from Streptomyces caespitosus ( Wakaki ct nl., 1958) and proved to be active against a number of murine tumors ( Sugiura, 1961). It depolymerizes DNA and inhibits its replication; recent work by Webb et a7. (1962) has established its status as an alkylating agent. The effect on DNA is seen at low concentrations, while RNA aiid protein synthesis continues; at higher concentrations RNA and protein synthesis are also affected (Sekiguchi and Takagi, 1960). Toxicity has generally been limiting in immunosuppression with mitomycin. JaroGkovA (1965) assewed the effect of mitomycin in 4-dayold rabbit recipients of adult rabbit cells mixed with Brucella antigen in fiitro. She also tested the effects on the response to sheep red blood cells in 10-day-old rabbits. With both models mitomycin was not inhi bitory. Otte and Grosjean (1964) used mitomycin in an effort to modify rejection of renal allografts in dogs. Although the mean survival was almost doubled, toxicity, including serious bleeding, was a major problem. Bloom et a?. (1964) and Bloom (19f33) were able to use mitomycin to inhibit the capacity of cells from picryl chloride-sensitized guinea pigs to transfer reactivity. The cells were incubated in concentrations as low as 100 pg. per milliliter and remained viable. When lower concentrations of mitomycin were used, the transfer was modified, but not eliminated; when tested immediately little effect was seen in the response of the recipient, but when tested at 3 days the response was reduced.

C. THEACFIXOMYCINS One of the agents of enormous interest to immunologists and immunochemists has been actinomycin D, an antibiotic whose structure has been cntirely defined and whose primary locus of activity is also well understood. & Indeed, in the background of the recent immunological studies is a vast body of biochemical investigation in bacterial, tumor, embryonic, and other systems in which actinomycin D has been used very incisively as a tool of analysis. In zjiuo, as w7e shall see, actinomycin D is not an immunosuppressant of great consequence. Clinically, actinomycin C has

’ Actinomycin C h a y also been used in immunosuppression studies. It is a mixture three antibiotics produced by Streptomyces chry.~omalltrsand is often referred to as Smamycin (Bayer ) .

fed. 119, 485. O’Brien, T. P. (1961). Federation Proc. u),27.

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DevelopmentaI Aspects of I mmunity JAROSLAV~ T E R Z LAND ARTHUR M. SILVERSTEIN Deparfmenf of Immunology, lnsfifute o f Microbiology, Czechoslovak Academy of Science, Prague, Czechoslovakia, and The W i l m e r Institute, The Johns Hopkinr Universify School of Medicine, Baltimore, Maryland

I. Introduction . . . . . . . . . . . . . 11. Methods for Studying Developmental Aspects of Immunity . . . A. Ontogenetic Models . . . . . . . . . . B. Studies with Isolated Cells . . . . . . . . . 111. Immunological Development . . . . . . . . . A. Development of Mesenchymal Functions . . . . . . B. Development of Immunoglobulins and Other Serum Proteins . . C. Development of Natural Antibodies . . . . . . . D. Ontogenetic Development of Antibody Formation . . . . E. Ontogenetic Development of Specifk Cellular Reactions . . . IV. Developmental Stages of Immune Reactions and Their Mutual Relation. . . . . . . . . . . . . . ships A. Relationship of Phagocytosis to Specific Cellular Reactions . , B. Relationship of Delayed Hypersensitivity to Antibody Formation . C. Dynamics of Antibody Formation . . . . . . . V. A Unitarian Concept of Imrnunocytological Mechanisms Based upon the Proliferation and Differentiation of Immunologically Functioning Cells . References . . . . . . . . . . . . .

337 338 338 357 363 363 370 375 380 409 41 1 412 414 418 429 435

1. Introduction

The past decade has witnessed broad and significant advances in the field of immunology in a number of diverse directions. Among these areas are two-at first glance not very intimately related-that have been of special interest to the authors of the present review: the ontogenetic development of immune responses in the immature animal and the developmental cellular stages of the immune response in the adult animal. The feeling has been growing in the minds of both authors that the cellular mechanisms underlying the several immune responses have not received the full attention that is their due, with respect either to the evolutionary principles underlying the development of all biological systems or to the broad biological rules which govern the proliferative and differentiative activities of cells in any biologically functioning system. 337

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\\'hen preliminary discussions between the present authors disclosed that both, despite wide separation in space, background, and approaches, shared a deep interest and concern in this respect, it was decided to attempt this long-distance collaboration. Having been surprised and impressed by the broad area of agreement between us on both the practical and conceptual level, we have attempted in the present review of the developmental aspects of immunity to discuss the stages of the development of the immune response in order to emphasize the cellular aspects. In this discussion, the greater emphasis has been placed upon ontogenetic rather than phylogenetic studies, the latter having been extensively reviewed in a recent volume of this serial publication (Good and Papermaster, 1964). Finally, rather than engaging in an exhaustive review of the extensive literature in this field, we have attempted to discuss the pertinent data in terms of our personal viewpoints and approaches to each of the areas covered. I t . Methods for Studying Developmental Aspects of Immunity

The approaches employed in the study of the phylogenetic development of specific immune mechanisms have been extensively reviewed elsewhere (Good and Papermaster, 1964) and require no further elaboration here. Investigations along these lines have involved, in the main, only the standard general techniques of immunology, although close attention must be payed in poikilothermic species to the temperature dependence of the active immune response. For an outline of the approaches to the phylogenetic development of phagocytosis, leukocytic function, and the comparative pathology of inflammation, the reader need look no further than the classic studies of Metchnikoff (1893). In this section we shall restrict our attention primarily to some of the more recent methodological developments and their applications to the study of the ontogenesis of the immune response, including both active and passivt. in zjico responses as ~ e l as l pertinent studies involving the use of isolated cells in zjitro.

-4.ON.I.OGESETIC

hfODELS

The marked interest which has developed during the past decade in the immunological capabilities of the immature animal received its main impetus from two directions. So long as the immunologist was oriented toward an instructive theory of antibody formation, he had no compelling reason to be concerned with such questions as the maturation of lymphoid organs and the differentiation of immunologically functioning cells. With the introduction and elaboration of selective theories of antibody forma-

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tion (Jerne, 1955; Burnet, 1959; Talmage, 1959; Lederburg, 1959) and especially with the recognition of the implications of induced immunological tolerance (Burnet, 1957; see also HaHek et al., 1961; Smith, 1961), the manner in which the fetal and neonatal animal develops immunological competcnce and responds to antigcnic stimulus suddenly assumed a new theoretical importance. The second stimulus of interest in the immunological capabilities of the developing fetus came with the recognition that fetal immune responses or the absence thereof might have interesting implications in the pathogenesis of certain congenital disease processes.

1. The Avian Embryo It might appear at first glance that a discussion of the embryonated egg as a research tool for the study of ontogenetic aspects of immunity would be out of place in this presentation, since in most circumstances this developing organism is incapable of producing active immunological responses. But it is precisely because of the general immunological incompetence of the chick embryo (which has permitted virologists for several decades to employ it as a culture medium) that it has served so well as an immunological tool, although Solomon (1961, 1963a,b, 1965, 1966) has suggested that a degree of competence does exist in the developing chick embryo. Owing to its immunological immaturity, the chick embryo has provided some elegant approaches to the study of immunological tolerance and the graft vs. host phenomenon, a few examples of which will be outlined here. A number of investigators have succeeded in inducing partial or complete tolerance of a variety of antigens by injection of the chick embryo (Stevens and co-workers, 1958; Simonsen, 1956; Smith and Thomas, 1956). Although some of these reports involved the induction of tolerance to mammalian erythrocytes, other investigators have reported failure in this respect (Burnet et al., 1950; Brauer et al., 1956; Owen, 1957; HaHek, 1956), while attempts to induce tolerance of influenza virus ( Burnet et al., 1950) or yellow fever virus (Fox and Laemmert, 1947) have also failed. It is important, in assessing data of this type, to bear in mind two aspects of tolerance that have been brought out in some of these studies. It seems clear that, in most cases, the persistence of the tolerant state depends upon the continued presence of antigen in the host; thus Mitchison (1959) and Mitchison and Dresser ( 1%0) demonstrated that repeated injections of foreign erythrocytes in the young chick are necessary to sustain the tolerant state. These investigators demonstrated that if complete elimination of the red

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cclls from thr circulation 11ere permitted, then tolerance would be lost and the nest challenge dose of homologous erythrocytes would result in an immune elimination of the newly administered cells. The timing of the tolerance-inducing injection of antigen into the chick rmbryo is also critical for the induction of tolerance. Thus Simonscn ( 1956) and Smith and Thoinas (1956) demonstrated an inability to induce tolerance in the chick embryo when it was injected either too early or too late in embryonic development. These studies raise the interesting possibility that the differentiating mesenchymal cells are only susceptible to the tolerance-inducing effect of antigen at a critical stage in their maturation. Before this stage the substrate upon which antigen acts to induce tolerance may not exist, whereas after this stage the substrate may be protected from the action of antigen by the developing antibody-forming capabilities of the host. Many interesting data on the induction of tolerance and on the graft vs. host reaction have also been obtained by the technique of experimental embryonic parabiosis, involving the fusion of the chorioallantoic membranes of two embryos ( HaSek, 1953; Lazzarini, 1960). The immunological immaturity of the chick embryo has rendered it especially suitable for studies of the graft vs. host reaction. These have involved, on the one hand, studies of the histology and cytology of the splenonirgaly and runt disease observed in this reaction (Biggs and Paine, 1959; Davies and Doak, 1960); at the same time the system has permitted the development of techniques for the assay of the immunological competence of cell transplants from a variety of organs. Simonsen (1957) was able to show that intravenously injected adult homologous spleen cells were not only involved in the resulting graft vs. host reaction of the bird ~ i t h i n2 weeks of hatching, but were also involved in the formation of antibody against the erythrocytes of the recipient. A more elegant method for the estimation of the immunological competence of donor cells was devised by Simonsen (1962). This involves the deposition of donor cells upon the exposed chorioallantoic membrane o f the embryonated egg. Competence of the donor cells is manifested by the dose-dependent development of plaques on the chorioallantoic membrane, representing the formation of focal graft vs. host responses.

2. Experimental lntrauterine Approaches In response to the increasing awareness that the mammalian fetus is capable of exercising an active immunological response to antigenic utimulus, many new techniques and experimental models have been developed to permit a more precise evaluation of the various aspects of


341

immunogenesis in the fetal animal. The choice of suitable species and appropriate technical approaches to the fetus is governed by a number of criteria which may appropriately be listed. An important requirement for these studies is the relative ease of access to the fetus and the ability to perform the several operative procedures without interruption of pregnancy. Next, it is most desirable to employ a species with an adequately long gestation period, so that one can have sufficient time to investigate the response at different stages of development and, again, sufficient time to follow the development of these responses in the fetus in utero. It is also desirable to employ a species in which the fetus is large enough during the latter half or twothirds of gestation not only to permit such procedures as orthotopic skin grafting,but also to permit one to obtain a large enough yield of serum from the immunized animal for the necessary serological work-up. Finally, there are many instances in which it is desirable to employ a species in which the placentation is such as to prevent the transfer of antigens or immunoglobulins from mother to fetus or from fetus to mother. Thus the use of such ungulates as the sheep or cow, in which this situation obtains, has offered the possibility of studying circulating antibody in the fetal animal that could unequivocally be identified as being of fetal origin. As will be pointed out below, however, certain techniques do exist which allow the use of species with placentas that permit the transfer of antigens or immunoglobulins, while still furnishing a suitable estimate of the contribution of the fetus to the immune response. Perhaps the greatest benefit to be derived from the use of the fetal model in utero lies in the isolation of the developing animal from the wide range of antigenic stimuli to which the normal laboratory animal is continuously subjected in even the best of laboratory environments. The normal mammalian placenta functions so effectively in the protection of the developing fetus that it is the exception rather than the rule that the fetus receives from the maternal circulation any of the wide variety of organisms or other antigenic stimuli with which the mother is continuously involved. Thus the developing fetus can be considered for all practical purposes to be an immunological virgin. In the normal course of events, it presents to the investigator an immature lymphoid system that has not yet responded to extraneous antigenic stimuli prior to experimental intervention. The importance of being able to employ an experimental animal responding immunologically only to those antigens injected by the investigator cannot be overstated when interpreting results..

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a. Immrrnization of the Fetus. To our knowledge the earliest report on the study ot active antibody formation by a fetus in utero was that of Fennestad and Borg-Petersen ( 1957, 1962) These investigators introduced live Leptospira intravaginally in the preimmunized pregnant COW. The organisms subsequently found their way into the fetus, for at birth the infected newborn was found to have circulating antibody specific for this antigen, as well as a tissue plasmacytosis. In some instances, as in the studies of Uhr (1960) on the fetal guinea pig, it has been possible to immunize the fetus directly through the maternal abdomen and uterus prior to birth, permitting an estimate of fetal response on the day of birth. Perhaps the most suitable approach to this problem involves the direct surgical penetration to the fetus. It has become increasingly more evident in recent years that the mammalian utcrus and developing fetus are much more amenable to a \vide variety of surgical procedures without the interruption of pregnancy than had been previously thought. Since the greater part of the work along these lines has been done with sheep and rhesus monkey fetuses, the techniques involved in dealing with these species will be described most fully (Bangham et al., 1960; D. R. Bangham and P. h4. Cotes, personal communication, 19655; Silverstein et a!., 1963a). During the latter half of gestation, the fetal lamb is large enough, and the uterus thin c,nough, so that the animal can be manipulated up against the uterine all and injected intramuscularly, subcutaneously, intraperitoneally, or even intraocularly ( A . M. Silverstein and K. L. Kraner, unpublished observations, 1964 ) directly through the uterine wall. With these large animals, direct intravenous or intradermal injection can be accomplished by making a relatively small incision through the uterus and fetal membranes and bringing out a fetal limb or, alternatively, the fetal head and neck may be brought out and injection made into either the jugular vein or carotid artery. In younger fetuses it is most convenient to bring out of the uterus either the entire front or hind portion of the animal or often the fetus in its entirety, connected then only by the umbilical cord to the intact placenta. In this way the fetus may be injected by any desired routc. It has been possible to immunize extremely small fvtal lambs ( a t 35-40 days of the 150-day gestation period) by floating thr. fetus weighing 2 to 3 gm. out into a bleb of amniotic fluidcontaining amnion forced out through a small incision in the uterus. In

' Scttc, urlclcri i n proof. T h e literature once again discloses how dificiilt it is no\vac\ays to (lo something coniplctc~lyoriginal. Dr. Frank Adler has kindly cnlld o u r attention t o thc tl(.inonstr;~tioti i i i 1904 that tIw lct;il goat iti f i ( c > r oa c t i ~ d yforms hemolytic antibody [Kreidl, and hlandl, L. (1904). Wien. K h . Wochschr. 17, 611-6121.

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this instance the needle is directed through the intact amnion into the fetus. In all instances, the fetus can readily be returned within the uterus. These procedures can be accomplished with a surprisingly low incidence of fetal mortality and can also be repeated at intervals, so that it has been possible in some experiments to exteriorize a single fetus four or five times for the purpose of serial bleeding or biopsy, without interruption of pregnancy. The approach to the fetal rhesus monkey is very similar in almost all respects, although there is a tendency for the uterus to contract if the whole fetus is exteriorized, thus rendering it impossible sometimes to return the fetus to its point of departure. In view of this, and depending upon the requirements of the moment, a fetal arm or leg can be brought out through an incision in the uterus and fetal membranes for immunization or grafting. As with the fetal lamb, several procedures can be effected with no great difficulty and can even be repeated on the same pregnant animal at intervals, although the pregnant monkey is somewhat more susceptible to the induction of abortion than the pregnant sheep. It may be pointed out that the fetal lamb offers at least two distinct advantages over the fetal monkey for this type of study. As was indicated above, the ovine placenta does not permit the passage of immunoglobulins of any description from mother to fetus, whereas in the monkey, at least the 7 S y-globulins are able to cross the placenta. An added advantage of the ovine fetus is provided by the predominance of twin (and not infrequently triplet) pregnancies in this species, whereas in the monkey twinning is exceptional. This sort of surgical approach to the immunization of a fetus in utero has been,applied to several other species. These include the fetal guinea pig (Hieger and Silverstein, 1965) and the fetal dog (Silverstein et al., 1966). There is little reason to believe that similar approaches to the fetuses of other species will not achieve equal success. A number of recent developments permitting approach to the human fetus in utero offer some hope for future immunological studies in this species. Liley ( 1963) has developed techniques permitting transfusion to the human fetus in utero as a means of ameliorating with donor erythrocytes the consequences of fetal involvement in erythroblastosis fetalis. By means of a needle carefully inserted through the maternal abdomen and uterus, amniocentesis can be accomplished, involving the withdrawal of sufficient amniotic fluid to permit a colorimetric estimation of the extent of red cell destruction in the fetus. Having thus established the existence and severity of the erythroblastotic process in the fetus, a similar penetration of the maternal abdomen, uterus, fetal membranes,

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J.\ROSLAV STERZL A S D ARTHUR hi. SILVERSTEIN

and fetal abdomen permits the insertion of a catheter into the fetal peritoneum. Through this catheter sufficient compatible donor blood may be passed as an intraperitoneal transfusion to sustain the fetus through the last critical weeks of gestation. It may be hoped that such transfused fetuses will be examined carefully after birth for possible evidence of the formation of isoantibodies against erythrocyte or leukocyte antigens occurring in the transfused blood. It may be further hoped that advantage will be taken of this technique in the future to incorporate into the intraperitoneal infusion an appropriately chosen innocuous antigen, so that a better estimate may be gained of the range of immunological capabilities possessed by the human fetus. b. Catheterization of the Fetus. In addition to the “one-shot” immunization procedures described above, the introduction of permanent indwelling catheters into fetal animals has proved feasible, permitting a broader approach to some of these problems. In a study of the induction of immunological tolerance in the fetal monkey, Bangham et ul. (1960) implanted catheters into the peritoneal cavity of fetal monkeys. This approach permits the administration of repeated doses of antigen into the fetus without requiring surgical intervention at each injection. It suffers the drawback, however, that although substances can be injected into the fetus at will, blood samples cannot be obtained from the fetus at intervals such as may be required in certain experimental situations. A solution to this problem has been found in the development of techniques permitting the implantation of permanent indwelling catheters into the large vessels in the neck of the fetus. Plastic catheters have been inserted into the proximal jugular vein or carotid artery of the fetal lamb and sutured in place. The catheter is then brought out through the fetal membranes, the uterus, and the maternal abdomen, and is run subcutaneously to the back of the mother where it exits through a small incision in the skin. This approach permits the injection of substances into the fetal circulation and the withdrawal of blood samples at any desired frequency, without requiring repeated surgical intervention. This technique has been employed (Silverstein et al., 1966) in the fetal lamb to permit for the first time a close examination of the immune elimination of antigen from the fetal circulation and an estimate of the earliest antibodies formed in response to antigen administered to the animal in utero. Thus far it has been possible to maintain patency in these catheters for 1 to 4 weeks. It may be anticipated that improvement of technique will permit employment of these catheters over longer periods of time. c. The Fetal Opossum. The marsupial opossum presents an especially interesting system in that the fetus is born “prematurely” after 12 days


345

of gestation, while still in an extremely primitive state of development. The animal finds its way into the maternal pouch, where it attaches to a teat and continues its normal developmental sequence. Thus the primitive opossum is more readily accessible for experimental work than is true of other mammalian fetuses at a similar stage of immaturity. At the stage at which it is born (La Via et al., 1963), the fetus is without a functioning thymus and lymph nodes and is immunologically incompetent: this competence only develops after a period in the pouch. A number of investigators have employed the easy accessibility of the opossum in the pouch to study its morphological development as related to the timing of its ability to form circulating antibody against such antigens as bacteriophage virus (Kalmutz, 1962) and the flagellar antigen of Salmonella (La Via et al., 1963). The latter authors found the earliest antibody response in the newborn opossum on day 8 after birth, corresponding to the time of appearance of lymphocytes in the maturing thymus. d. Homograft Studies in the Fetus. Using surgical approaches similar to those described above, it has been possible to study the development of fetal ability to reject orthotopic skin homografts and to examine the nature of this rejection in the mammalian fetus. The first such studies of intrauterine graft rejection by a fetus were those of Schinckel and Ferguson (1953),who buried skin homografts subcutaneously in fetal lambs and demonstrated their active rejection at a later date. Slight improvements of technique have more recently permitted the orthotopic grafting of skin auto- and homotransplants in the fetal lamb (Silverstein and Prendergast, 1964) and fetal monkey (Silverstein and Kraner, 1965). The ability to perform repeated surgical procedures on these fetal animals has permitted the serial biopsy of grafts applied in utero for the examination of the rapidity of rejection or, alternatively, the demonstration of persisting tolerance of these grafts under certain conditions. In the same way it has been possible to study the response of the fetus to second-set skin homografts. Since the fetal lamb is not normally involved in immunological responses to antigens other than those introduced by the investigator, the morphological study of the fetal response to homografts is very much simplified in its interpretation by the knowledge that the changes in the graft bed and especially in the draining lymph node are due solely to the graft antigens introduced. e . Thymectomy of the Fetus in Utero. The recent implication of the thymus in immunogenesis has provided a fruitful source for numerous experiments in both the newborn and the fetal animal. Employing tech-

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J.\ROSLAV STEHZL A S D ARTHUR 31. SIL.\7ERSTEIS

niques similar to those described above, it has been possible to thymectomize successfully the fetal lamb (Silver3tein and Kraner, 1965) and the fetal rhesus monkey ( A . M. Silverstein and C. J. Parshall, unpublished data, 1965) in trtero without interruption of pregnancy. The effects of thymectomy may be assayed by subsequent immunization and skin grafting of these fetuses. The question of the possible humoral mediation of thymic control functions has also been amenable to investigation, employing the maternal-fetal system as a sort of “tissue chamber” model. On the one hand, Osoba (1965) has demonstrated that the immunological capacity of a female mouse thymectomized at birth is restored at a certain stage of pregnancy, presumably by the development of thymus in the fetal offspring in utero. The obverse of this situation is presented in the studies of Silverstein and Kraner (1965) whereby thymectomy of a fetal lamb at midgestation was shown to be without effect on the development of its immunological capacities, possibly due to the assumption by the maternal thymus of control functions. f . Study of Congenital Infectious Processes. It is quite evident that in certain species, most notably the human, the opportunity for any extensive experimental investigations of developmental aspects of immunity will be severely limited for one reason or another. In this instance it has proved fruitful in the past, and should become increasingly so in the future, to examine instances of natural congenital infectious processes in the fetus of these species. Advantage can be taken of those instances in which a breakdown of normal placental function has permitted the penetration to the fetus of one or another pathogenic organism which establishes residence in the fetal tissues and incites identifiable immunological responses. Even retrospective histopathological studies of such human congenital diseases as syphilis and toxoplasmosis have been fruitful in permitting the identification of plasmacytosis in the affected fetus and thus providing strong presumptive evidence of immunological competence on the part of the developing fetus (Silverstein and Lukes, 1962). On the one hand, such morphological evidence may shed light on the presence and origin of immunologically functioning cells within the fetus ( Silverstein and Lukes, 1962) or in the placenta (Benirschke and Bourne, 1958). On the other hand, a study of the gestational time of development of plasmacytosis in the human fetus may offer the only presently available clues as to the timing of immunogenesis in this species (Sihrerstein, 1962). It may be hoped that fresh tissues from infected human fetuses will occasionally be available to permit the examination by fluorescent


347

antibody techniques and the verification of the specific function of the plasma cells seen in these lesions. Verification of the immunological competence of the human fetus will occasionally be found by the demonstration of circulating antibodies in newborns infected in utero ( Eichenwald and Shinefield, 1963). g. Interfetal Relationships. It may be appropriate to discuss here some interesting recent developments that would appear to hold promise for future productive work in the area of ontogenetic studies. It has been established in the human and other species that identical twins, receiving the same genetic information from their parents, are unable to respond immunologically to the antigens of their siblings. As Owen (1945) has shown, the early exchange of blood between nonidentical twins leads to the development of chimerism and mutual tolerance of one another. It has been possible (K. L. Kraner and A. M. Silverstein, unpublished observations, 1964) to cross experimentally the fetal circulation of twin lambs by means of catheters, therein providing the possibility for the experimental production of parabionts and chimeras in utero. Even more interesting in its impIications are the observations of Anderson and Benirschke (1962) in the nine-banded armadillo. It has been fairly rigidly established that the quadruplets born normally to this animal are originally derived from a single ovum. The demonstration, therefore, that identical siblings in this species would reject one another’s skin grafts is especially provocative. Since it has been established further (Anderson and Benirschke, 1963) that there is no interplacental exchange of blood among the four armadillo fetuses, the interesting question is raised (and demands further investigation) whether identical siblings may not suffer alterations in the genotypy of their histocompatibility antigens after separation from one another.

3. Premature and Neonatal Studies In choosing an experimental model for studies on the development of immune reactions it is necessary to consider the wide variety of factors which may influence the comparison of results obtained with different species. a. Transfer of Antibodies from Mother to Fetus or Newborn. For the first few weeks of life all newborns are equipped with maternal antibodies which protect them during the initial contact with the microorganisms that normally colonize the newborn ( Mossman, 1937; Schubert and Griinberg, 1949; S. G. Cohen, 1950; Brambell et al., 1951; Buxton, 1952). If the transfer of maternal antibodies to the young animal

348

JAROSLAV STERZL AND ARTHUR M. SLLVERSTEIN

is artificially prevented in calves ( Aschaffenberg et al., 1949a,b; Ingram et at., 1956) and piglets (Trnka et al., 1959), they die within a few days, primarily from Eschmichia coli infection (Dunne et al., 1956; Love11 and Rees. 1961) . The presence of passively acquired immuiioglobuli~is and of antibodies to the various antigens used for experimental immunization of neonates makes the estimation of the onset of immunoglobulin and antibody synthesis difficult. Experimental studies can most readily be performed on animals in which the transfer of antibodies between mother and fetus can be prevented. The transfer between mother and fetus depends upon the structure of placenta, as reviewed by Hemmings and Brambell ( 1961) and Amoroso ( 1961). The epitheliochorial placenta (pig and horse) (Table I ) does not allow passage of ./-globulin molecules, and the newborn receives y-globulin only by suckling maternal colostrum. In these newborn piglets, no trace of antibodies transferred from either passively or actively immunized mothers has been found (Sterzl et al., 1960b). The observation of Myers and Segre (1963) that antibody activity could be found in concentrated sera from the newborn was not confirmed using the same antigens and concentration procedures (Sterzl et al., 196%). Traces of ./-globulin were found in the sera of newborn piglets (SterzI et al., 1960a) with properties different from adult y-globulin and without any demonstrable antibody activity. Data were obtained providing evidencc that this y-globulin is actively synthesized by the newborn (Sterzl et al., 1960a,b; Fran&kand Riha, 1964). Similar results were obtained by Kim et al. (1964). In another type of placenta, the syndesmochorial placenta (Table I ) which has five layers separating the mother and fetus (sheep, goat, and cow) the transfer of significant amounts of y-globulin and antibodies has not been observed, although such detailed studies like those with pigs were not performed on these species. Traces of ./-globulin have been detected in precolostral calf serum (Johnson and Pierce, 1959). The electrophoretic patterns of lamb serum before and after transfer of colostrum were studied by Charlwood and Thomson (1948), and yh1globulin formation by the normal fetal lamb in utero was routinely observed ( Silverstein et al., 1963b). In animals with these two types of placenta, however, the most important factor is the transfer of immunoglobulins during the early postnatal period by the ingestion of colostrum. The ability of piglets to absorb immunoglobulin and other macromolecular substances ( nonselec-

TABLE I CLASSIFICATION OF PLACENTATION ACCORDING TO THE INTERVESING TISSUES A N D THE CORRELATION WITH TIMEO F TRANSFER OF IMMUNITY FROM MOTHERTO OFFSPRING^ TJteriiie tissues Type of

placelitation

Animal

-

0 Epitheliochorial

g Syndesmochorial Endotheliochorial Hemoendothelial Hemoendothelial Hemochorial a

IIorse, pig Sheep, goat, cow, ox Cat, dog, ferret Hat, mouse Rabbit, guinea Pig Man, monkey

Fetal tissues

Endothelium

Connective

Epithelium

Trophoblast

Connective

+ + +

+ +

+

+ +

0 0

0 0 0

0 0 0

+ + +

0

0

0

Symbols employed in this b b l e are: 0 = absence;

0

Time of antibody transmission Endothelium Prenatal Postnatal

+b

-Ifb

+b

+b

+ + + + +

+

+

+

0 0

+ ++ (36 hours) +++ (36 hours)

+ ++ (10 days) + ++ (16-20 days) +++ 0 +++ 0

+ = presence; ++ = major contribution; +++ = Bole contribution.

* According to Amoroso (1961) in rodents placentation is hemochorial.

350

JAROSLAV STERZL AND ARTHUR Xi. SILVERSTEIN

tive absorption) persists for 3648 hours after birth (Nordbring and Olsson, 1958; Speer et al., 1959; Lecce et al., 1961; Wellman et al., 1962). It was found that the period of absorption is not limited to a certain critical time period after birth, but that closing of the intestinal barrier is dependent upon the type of food ingested. If newborns are fed with tea only, the intestinal barrier remains permeable. In contrast with this, when newborns are fed with protein food, e.g., cow milk, intestinal absorption of immunoglobulins is prevented (Lecce and Morgan, 1962; Lecce, 1964). The absorption of immunoglobulins was not found to be specifkally selective ( Kaeberle and Segre, 1964; Olsson, 1959b; Payne and hlarsh, 1962). A comparison of the immunoglobulin spectrum of pig serum, pig colostrum, and piglet serum after ingestion of colostrum has shown that the main part of pig colostrum is yG-immunoglobulin.Antibody activity has been detected mainly in the yc and yhI fractions of colostrum, and after ingestion of colostrum the antibody activity of the newborn serum i5 still confined to the yc: and yar fractions (Rejnek et al., 1966). This is in agreement with results of Pierce and Feinstein (1965) on the immunoglobulin composition of bovine colostrum. Differences in the immunoglobulin content ( 7 and ylr) of human and pig colostrum may be due to the fact that in humans r,-globulins pass across the placenta, and thus the y c concentration in colostrum is not essential. On the other hand, the newborn piglet has not received any 7,-globulin in zitero, so that the content of this component in pig colostrum is necessarily high. The endotheliochorial and hemochorial placentas (see, Table I ) permit the transfer of antibodies in utero. The hemoendothelial placenta (hlossman, 1926) with its fine structure is of hemochorial character, possessing throughout gestation a complete layer of trophoblast ( Amoroso, 1961). In the rat and mouse, immunoglobulins and antibodies are transferred before birth by way of the yolk sac (as in the rabbit), the amniotic fluid, and fetal gut ( Brambell and Halliday, 1956). Postnatally, antibodies can be absorbed from milk or immune serum fed to the newborn till the twentieth day of life (Halliday, 1956), a process which can be modified by steroid hormones ( Halliday, 1959). This: absorption capacity is a selective process, since homologous antibodies are resorbed by the mouse four times more effectively than rabbit antibodies (Hemniings and Morris, 1959). The intrauterine transmission of immunoglohulins and antibodies in rabbits was studied earlier (e.g., Mossman, 1937), Iwt not until the studies of Brambell et al. ( 19Fjo) was conclusive evidence obtaincd that antibodies reach the fetal circulation in the rabbit by \vay of the uterine


351

cavity and yolk sac wall and not across the placenta. No antibodies were resorbed from the intestine after birth. Brambell et al. (1952), Batty et a2. (1954), and Hemmings (1957) apparently observed the nonselective transfer of antibodies, since it was shown that the mechanism is one of pinocytosis (Clark, 1959). However, differences have been detected in the transfer of individual papain split products of homologous y-globulin (Brambell et al., 1960). In primates with a hemochorial placenta, all immunoglobulins and antibodies pass exclusively before birth from the maternal to the fetal circulation across the placental membranes. Kuttner and Ratner ( 1923) observed that antibodies cannot be absorbed from colostrum by the newborn infant. Recently the intrauterine transfer of antibodies from mother to fetus has been restudied, especially in connection with the pathogenesis of certain diseases, e.g., erythroblastosis (Wiener and Silverman, 1940), penicillin sensitivity of the newborn resulting from antibody transfer (Epp, 1962), or sensitivity to cow milk (Berger and BiirginWolff, 1962). The transmission of those antibodies which protect the newborn against infectious agents has been especially studied, as in the protection against diphtheria and staphylococcal toxins ( Vignes et al., 1948; D6biis et aL, 1957; Kleinitz and Kiimmel, 1960), streptococci (Murray and Calman, 1953, Vahlquist et at., 1950; Florman et al., 1951), and viral antigens (Szathm6ry and Holik, 1956; Lipton and Steigman, 1957). Although the transmission of antibodies to antigens of gram-negative organisms has been proved (Schubert and Grunberg, 1949), the antibodies transferred have been predominantly of the 7 S type; the sera of newborns contain only slight amounts of 19 S antibodies (Gitlin et at., 1963). This is probably due to the placenta functioning as a molecular sieve. Another type of selective passage of immunoglobulins is found in the formation of colostrum and milk. In spite of the serum origin of these proteins (Askonas et al., 1954; Larson and Gillespie, 1957; Dixon et al., 1961), yA and y,-immunoglobulins predominate, whereas YG immunoglobulin, prevalent in serum, is found only in small quantities in colostrum (Hanson, 1960; Hanson and Johansson, 1962; Rejnek, 1W). It has been demonstrated that not only proteins but also cells can pass across the placenta to the fetus, e.g., erythrocytes (Lee and Vazquez, 1962) or leukocytes (Desai and Creger, 1963). In a series of experiments with nonimmunized newborn mice and rats, antibody-forming cells were detected immediately after birth (one to three were producing cells in los lymphatic cells), which probably are of maternal origin. In other animals, such as rabbits and piglets, antibody-forming cells have never been detected immediately after birth as the result of passage to

352

JAROSLAV STERZL AND ARTHUR hi. SILVERSTEIN

the fetus from the circulation of the mother (Sterzl, unpublished observation, 1965). b. Transfer of Antigens from Mother to Fetus and Vice Versa. In studies in which fetuses are injected with antigen, the possibility must be considered of passage of antigen across the placenta to the mother, resulting in the formation of antibodies by the mother and their transfer to the fetus or newborn. In the experiments of Silverstein et al. (1963a), who immunized sheep fetuses in utero with various antigens, no increase in antibody titer of mother was detected, furnishing evidence against the passage of critical amounts of antigen. Under normal conditions, antigens in the human do not penetrate the placental barrier ( LemBtayer et al., 1948). The existence of such congenital lesions in the human as syphilis, toxoplasmosis, rubella, and cytomegalic inclusion body disease, indicates that breakdown of the placental barrier may occur in some instances, permitting pathogens to pass from mother to fetus. In experiments with pig fetuses immunized in utero 1 month before term with sheep erythrocytes and Brucellu stiis antigen, no increase in antibody levels to these antigens was found in the serum of the mother (Sterzl, 1963a); these experiments provide evidence that the placental barrier in pigs prevents the passage of critical amounts of these antigens. However, in other animal species, e.g., in guinea pigs (Uhr et al., 1963) or in mice (Medearis and Home, 1962), the penetration of phage 4x174 from mother to fetus was observed after intravenous injection, especially when the concentration gradient of the virus particles between mother and fetus was increased. The possible passage of virus particles from mother and fetus indicates that if fetuses are immunized in utero, the passage of virus into the tissues of the mother can be expected which might stimulate maternal antibody formation. The passage of other virus particles from mother to fetus was observed by Burckhardt (1940) and Kulangara and Sellers ( 1959). c. Maturity of Animals at Birth. There are substantial differences in the maturity of individual animal species at the time of birth; e.g., rabbits are physiologically immature, opening their eyes only on day 10, as do newborn mice and rats. On the other hand, the foal, lamb, and piglet at birth satisfy many criteria of maturity (Witschi, 1956). Some correlation was cqerted between the physiological stage of maturity and the capacity to rrspond immunologically ( Sirotinin, 1963; Zdrodowski, 1963). However. it \\‘as recently shown that the development of lymphatic tissue and antibod!. formation precede fiinctional maturation of some of the physiological functions essential for life (Section III,D,2,a). The physio-


353

logical development of target organs, where the secondary manifestations of immune reactions take place, should also be considered. The specific cutaneous inflammatory reaction in newborns is weaker than in adult individuals (Tschertkow, 1929; Sterzl and Hrubeiovl, 1950; Salvin et al., 1962). This could lead to the erroneous conclusion that in newborns the state of delayed hypersensitivity does not develop. d. Intensity of Metabolic Processes in Individual Species. It was found that catabolism, and the serum protein synthesis connected with it, differ in individual species. The half-life found for homologous 1311-labeled albumin was: cows, 20.7 days; humans, 15 days; rabbits, 5.7 days; and mice, 1.2 days. The half-life of homologous y-globulin was: cows, 21.3 days; humans, 13.1 days; rabbits, 4.6-5.7 days; and mice, 1.9 days (Dixon et al., 1953). This factor may condition the different rate of 7-globulin synthesis that results in a different time of onset of antibody formation after the administration of antigen to different species. In chickens or ducklings, rejection of homologous cells and antibody synthesis is substantially more rapid than in rabbits (Sterzl and Trnka, 1959); the half-life of yG-globulin is shorter than is generally true of mammals--1.45 days in the adult hen (Patterson et al., 1962; I v h y i et al., 1964). The rate of serum protein synthesis associated with differences in catabolism varies not only in individual animal species, but also at different developmental stages in the same species. The catabolism of immunoglobulins in the young is generally slower (Deichmiller and Dixon, 1960; Humphrey, 1961; Pace and Dresser, 1961; Ivlnyi et ul., 1964), in all probability related to the lower activity of enzymatic systems degrading these proteins (Robbins et al., 1963). e. Some Species Used for the Study of Development of Immunity. The choice of animal species for studies on the development of antibody formation is determined by the aim of the individual experiment. Examples of immunological studies in some species of laboratory animals are given below. Mice and rats have been used for the induction of tolerance (Howard and Michie, 1962; Thorbecke et al., 1961; Simonsen, 1956) by repeated intravenous injections shortly after birth, either through the orbital branch of the anterior facial vein (Billingham and Brent, 1956) or through the sigmoid sinus (Billingham and Brent, 19%). The resistance to bacterial endotoxin was studied in newborn rats (Miler, 1962). Mice have been used for the study of the development of antibody reaction by transfer of spleen cells from individuals of different ages (Makinodan and Peterson, 1964).

553

JAROSLAV STERZL AND ARTHUR I f . SILVERSTEIN

In rabbits, tlie dcvelopment of thc homograft rcjection rcaction has been studied (Billingham et ul., 1956; Egdahl, 185’7;Porter, 1960; Ivhnyi and Ivinyi, 1961j . Further. this model has been used for the induction of tolerance (Smith and Briclgcs. 1958). for studies 0 1 1 the resistance to bacterial cnclotosins ( Smitli ant1 ‘Tlionm. l%l), and for thc, drtrction of the timc of onset of antibody formation to various antigens. These studics involved either direct immunization of the young ( Freund, 1930; Sterzl and Trnka, 1957a,b; Riha, 1961) or the isolation of spleen cells from the young of different ages and transfer to recipients after stimulation with antigen in citro (Sterzl, 1958a, 196%). Guinea pigs have been used for the study of the development of transplantation immunity ( Egdahl, 1957), for the induction of tolerance by injection of an antigen into fetuses (IVeiss, 1958) or by injection of bovine serum albumin ( B S A ) or human 7-globulin (HGG) into newborns (Humphrey and Turk, 1961), for the study of the onset of antibody formation (Uhr et al., 1%2a), and for studies on sensitivity to bacterial endotoxin ( Uhr, 1962). Piglets reared under conventional conditions ( i.e., those receiving maternal colostrum and developing in association with microbes ) and those reared under sterile conditions without colostrum have been used for studies on the transplantation reaction after birth (Sterzl et al., 1960b), the onset and mechanism of tolerance (Sterzl, 1965b), and the formation of antibodies to various antigens ( Hoerlein, 1957; Olsson, 195%; Sterzi, 1960a,b; Segre and Kaeberie, 1962a,b) . A line of miniature pigs has been developed for laboratory use (Rempel and Dettmers, 1963; Hariiig ct al., 1963). The details of the specific studies are covered more fully elsewhere in the review.

4 . The Germfree Animal Model In studies on the development of immunity, the adequate control of antigenic stimuli is of prime importance. The fetus is generally protected from most or all antigenic stimuli by the placental barrier (if the placenta is not injured or massively infected). Thus the fetus comes into contact with large numbers of antigenic determinants only after birth, with the colonization of the newborn by intestinal flora and after resorption of food antigens. Exposure to these antigens causes a maturation of lymphoid tissue, the “spontaneous” appearance of antibodies of certain types (see Section III,D,2), and an increase in the number of cells able to react to certain antigens, i.c., the development of processes which are not manifested in the absence of antigenic stimuli (Sterzl ct al., 1965a).


355

Rearing animals under sterile conditions helps to eliminate the antigenic stimuli of bacterial flora. A certain reduction of antigenic stimuli may also be attained in special breeds, in which the bacterial contamination is partly eliminated, e.g., in a colony of NCS mice in which a number of common pathogens, including such normal bacteria as Escherichia coli, Pseudomonas, and Proteus vulgaris, have been eliminated ( Dubos and Schaedler, 1960). The techniques of rearing germfree animals have been described in a series of monographs and reviews (e.g., Reyniers, 1956; Symposium on Germfree Animals, 1959; Reyniers et al., 1959; Luckey, 1963; Pollard, 1964). These reviews provide complete information on the origin and development of techniques used for rearing germfree animals and on the character and physiological properties of adult germfree animals. It has been shown, however, that although the lymphoid tissue of germfree animals is underdeveloped ( Miyakawa, 1959) and antibodies against certain bacteria are not present, some antibodies can be detected (Wagner, 1959a; Cohen et al., 1963). In germfree mice, antibodies reacting with bacterial lipopolysaccharides have been found (Landy et al., 1962b). This fact can be explained on the basis of findings that normal food contains killed bacteria and also plant antigens which cross-react with bacterial antigens, either of which may induce antibody formation when resorbed from the gut. Sterile animals without antibodies either passively transferred or produced as a result of immunization by antigens from food can be obtained under the following conditions: (1) for studies on the development of immunity in germfree species, young animals should be used that respond only to significantly higher doses of antigen than adults (Sterzl and Trnka, 1957a); (2) animals fed with nonantigenic diets should be used (Pleasants et al., 1964; Wostmann et al., 1965; TrhvniEek et al., 1966); and ( 3 ) species should be employed in which the transplacental transfer of maternal antibodies does not occur (see Section II,A,3). This will exclude any possible participation in the responses under study by transferred maternal antibodies. A most suitable model for such experiments has been the germfree and colostrumfree sterile piglet (Mandel et al., 1960) fed with nonantigenic diet ( TrhvniEek et al., 1966). Newborn piglets are delivered by hysterectomy in sterile tanks. They are kept in isolators made of rigid plastic material and fed with a diet of either antigenic character (containing components of cow milk) or nonantigenic character, in which the source of nitrogen components is the dialyzate of an enzymatic casein hydrolyzate tested for pyrogenity and antigenic properties. Using both

356

JAROSLAV STERZL AND ARTHUR M. SILVERSTEIN

types of diet, vitamins are administered to the piglets by intramuscular injection of 10,000 I.U. of vitamin D,, 50,000 I.U. of axerophthol, and 5 mg. of menadiol on the first day of life. On the second day after birth Fe-dextran is injected. The dosage of vitamins is repeated on day 10. The technique has been described in detail by TrhvniEek et al. (1966). Similar work on the rearing of germfree piglets was published by Landy and Sandberg (1961), who also used other large animals (Landy c t aZ., 1961) . The germfree animal model is useful in clarification of the role of intestinal flora in the normal physiological status of man and animals. Investigations have been carried out on the significance of normal intestinal flora on tissue metabolism (du Vigneaud et al., 1951), vitamin metabolism (Luckey et al., 1955), the presence of blood-clotting factors (Waaler ct al., 1964), the state and mitotic activity of intestine endothelium (Schmidt, 1962; Lesher et al., 1964), secretion of mucus (Lindstedt et al., 1965), and the presence of bioactive substances in cecal content (Gordon, 1965). Special attention was given in germfree rats to the metabolism of cholic acid (Gustafsson et al., 1960), bilirubin and urobilin (Gustafsson and Lanke, 1960; Gustafsson and Norman, 1962), and liver cholesterol ( Wostmann and Wiech, 1961). Studies with germfree animals support the view that the effect of antibiotics in animal nutrition is due to their action on intestinal bacteria (Quinn, 1956; Eyssen and De Somer, 1963). The role of intestinal flora has also been followed in some pathological states, especially during hemorrhagic shock ( McNulty and Linares, 1960), in the development of inflammation (Brody and Bishop, 1963), and in the induction of arthritis by adjuvants (Pearson et al., 1963). The germfree model is also used for studies of the complex response of animals to pathogenic and nonpathogenic microorganisms either as monocontaminants or in combinations. In germfree piglets, the pathogenic action of certain strains of Escherichia coli was studied by Trnka et al. (1959); germfree mice were used for studies on the fate of attenuated tubercle bacilli Bacillus CaZmtte-Gm’rin (BCG) by Suter and Kirsanow (1962) and the effect of monocontamination by some viruses (Schaffer et al., 1963; Dolowy and Muldoon, 1964). Germfree guinea pigs were employed in studies of the relationship between amoeba and bacteria (Phillips et al., 1955), and germfree chicks in monocontamination studies with several different microorganisms (Phillips et al., 1962). The germfree animal model has also found application in basic immunological studies, especially in the differentiation of the factors of immunity formed spontaneously without antigenic stimulus from those


357

factors which appear or increase after antigenic stimulation. No differences could be found in the phagocytic activity of sterile and conventionally reared animals, if the tests were performed with particles which did not require specific opsonins of an antibody type (Thorbecke and Benacerraf, 1959; Trnka et al., 1959). The level of complement in germfree individuals after birth develops independent of antigenic stimuli (Newton et al., 1960; Sterzl et al., 1962). Bactericidal activity, which results from the action of antibodies and complement on the bacterial cell wall, is reduced in germfree animals (Ikari, 1964). Using germfree animals, it has been shown that bactericidal activity to the S form of gram-negative bacteria does not develop spontaneously, as assumed for properdin, but only as a result of antigenic stimuli by microbial flora after monocontamination of sterile animals ( Sterzl et al., 1962). Germfree animals have also provided experimental approaches to the problem of the synthesis of immunoglobulins of nonantibody character ( Wostmann and Gordon, 1960; Sacquet et al., 1961; Frangk et al., 1961; Sell, 1964; Wostmann et al., 1965), the development and existence of the so-called natural antibodies (see Section 111,B72),and also the dynamics of onset of antibody formation (Wostmann and Olson, 1964; Sterzl et al., 196513). Using the germfree animal model, it has been possible to determine whether substances of a nonimmunoglobulin nature that can imitate antibodies by binding with certain antigens are naturally present in sera and whether complement can interact with certain surfaces (e.g., R forms of bacteria) in the absence of antibodies (Sterzl et al., 1964, 1965a). CELLS B. STUDIESWITH ISOLATED The immunization of animals and detection of their serum antibodies do not disclose which tissues contain the cells able to react with antigen (immunologically competent cells), nor how many of these cells differentiate into antibody-producing cells. Current methods of immunization do not differentiate whether the reduced immune responses observed in embryos and newborns result from the small numbers of competent cells, from the fact that the capacity of these cells to form antibodies has not yet developed fully, or whether inadequacy is conditioned by the immaturity of some other physiological functions which make the reaction of antigen with cells possible. These problems may be approached with two methods involving the use of isolated cells: ( a ) Methods for detection of immunologically competent cells: Isolated cells from nonimmunized animal are mixed with antigen in vitro and then transferred into a suitable environment (cultivation in vivo), where the cells can dif-

358

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fervntiate into aiiti\,od~-produciiig cells. ( 11) hIethods detecting antibody-producing cells. These include, in the main, the cultivation of cells and bits of tissue in tissue culture media in which the antibody content may he estimated serologically or methods detecting antibody production in individual cells. 1. Transfer of Cells to Recipients

This method arose froin earlier work on the transplantation of organ fragments, primarily spleen, which, after transfer to nonimmunized recipients, continue to form antibody. Using this method, the localization and early onset of antibody formation was followed by Deutsch (l899), Ludke ( 1909), Girgolaff ( 1912), Oshikawa ( 1922), Topley ( 1930), Chase ( 1953), Fagraeus and Grabar ( 1953), and Oakley ct al. ( 1954). A new era in the use of this technique was initiated by experiments in which immunologically nonresponsive animals were used as recipients of transferred isolated cells, i.e., X-irradiated animals ( Harris and Harris, 1954; Harris et al., 1954a,b,c), newborn animals (Sterzl, 1955), or animals made tolerant of the antigen used ( Weigle and Dixon, 1959). This approach makes possible the transfer of isolated cells from lymph nodes or spleen after treatment with antigen in ~jitru(Harris and Harris, 1955; Sterzl, 1957). The results of experiments using this method are discussed elsewhere in this review (especially in Section III,C,4); in this chapter, only those factors affecting the procedures will be discussed. The problems connected with antibody formation by isolated cells transferred to recipients were reviewed by Sterzl and Trnka (1959), Harris and Harris (1960), and Cochrane and Dixon (1962). The main source of cells used in transfer experiments has been from lymph nodes and spleen. Antibody formation has also been observed, however, after the transfer of cells from other sources, e.g., thoracic duct ( Holub, 1958), peritoneal exudate ( Dixon et al., 1957), and thymus and Peyer's patches (Stoner and Hale, 1955). After isolation of the cells, viability is estimated with stains such as eosin or trypan blue; these methods have been evaluated in recent studies (Hoskins et al., 1956; Hanks and Wallace, 1958; Black and Berenbaum, 1964). The number of lymphatic cells was counted, and the smallest number of lymphoid cells able to mediate the production of detectable amount of antibody after transfer was found to be from lo6 to 10' cells (Trnka and Sterzl, 1960). Donor cells are transferred to homologous or isologous recipients. The intraperitoneal injection of heterologous cells was shown to result in almost immediate inactivation of their immunological capacities, prob-


359

ably owing to a direct interaction of donor and recipient cell surfaces (Rosenau and Moon, 1961; Granger and Weiser, 1964). This view was supported by successful experiments in which heterologous cells were transferred within diffusion chambers (Urso and Makinodan, 1963; Gengozian, 1964) which protect the cells from direct contact with the cells of the recipient. In most experiments, X-irradiated recipients (450r ) were used. This irradiation lowers the capacity of the animal to form antibodies, especially to small doses of antigen. In experiments by Sterzl (1963b), however, it was shown that this dose of irradiation does not completely abolish the capacity of the recipient to form antibodies. This observation relates to the findings that antibodies formed to Brucella or lipopolysaccharide antigens (Frankk et al., 1962; Robbins et al., 1965) are predominantly 19 S in character; the development of 19 S antibodies is less influenced by X-irradiation than the development of 7 S antibodies ( Smith and Robbins, 1965). Therefore, in other experiments, newborn animals of different species were used-newborn rabbits (Sterzl, 1955; Dixon and Weigle, 1957; Sparck, 1959; Holub, 1958; Harris et al., 1962) and chick embryos and newborns (Trnka, 1957; Mitchison, 1957; Papermaster et al., 1959, 1962). In the newborn as well as the adult rabbit irradiated with 400 r, the development of immune responses by transferred cells was found to be limited by the homotransplantation reaction (Sterzl, 1958a,b; Dixon et al., 1959). The transplantation reaction of the recipient determines in part what type of antigen can be used in cell transfer experiments. Most successful experiments have been performed with normal cells stimulated in vitro with bacterial antigens, where the earliest antibodies can be detected on the fourth or fifth day after the transfer. If cells are mixed with protein antigen and then transferred to recipients, antibody formation does not appear, because before antibodies to protein antigens can be formed (on days 7-10 after the transfer), the donor cells are destroyed by a transplantation reaction. If the cells are protected in diffusion chambers, then the primary response to protein antigens occurs, even after the transfer to newborns (Holub and Riha, 1960). Substantial improvements were obtained when isologous donors and recipients were used, the recipient being lethally irradiated. This approach eliminated the response of the recipient both to donor cells and to the antigen transferred with the cells (Makinodan et al., 1960). Cells, mostly in the form of a free suspension mixed with antigen, have been injected by a variety of routes, including intraperitoneally, intravenously, into the abdominal musculature, subcutaneously, intra-

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JAROSLAV STERZL AND ARTHUR hi. SILVERSTEIN

ocularly, or onto the chorioallantoic membrane (see Cochrane and Dison, 1963). The cultivation of cells in diffusion chambers is useful for studies of the cytological changes in suspensions of transferred cells (Holub, 1958, 1W; Urso and Makinodan, 1963; Capalbo et al., 1964). 2. Tissue and Cell Culture Studies a. Detection of Antibody Formation in Tissue Culture. Tissue culture has not been used in studies on the development of antibody formation in ontogenesis, since during ontogenesis only small numbers of cells form antibody so that the detection of antibodies in suspensions of these cultured cells is difficult. Tissue culture has been used however for the differentiation of developmental stages of antibody formation in adult animals. Up to the present time, the initiation of a primary immune response in tissue culture has not been successful (Parker, 1937; Steiner and A4nker,1956; Sterzl and Rychlikovh, 1958; Sterzl, 195913). The few reports on the stimulation of a primary reaction completely in vitro (Stevens and McKenna, 1958; Fishman, 1959, 1961) have not been generally conkmed. On the other hand, it is evident that antibody formation in citro can be maintained for a certain time by cells that have already started to produce antibodies in vivo (Fagraeus, 1948; Thorbeckc and Keuning, 1953; Sterzl and Rychlikovb, 1958; Stavitsky and Wolf, 1958; Vaughan et al., 1960; La Via et al., 1960a; Elves et al., 1963; Vas ef al., 1964) . Thus, using tissue culture, the induction of antibody formation can be clearly differentiated from the productive phase, during which cultured cells continue to produce antibodies. It was demonstrated ( Sterzl, 1959b) that immunologically competent cells survive in tissue culture, and if they are mixed with antigen and transferred to a newborn animal after culture for 48 hours, they differentiate into antibody-forming cells. These results provide evidence that the conditions of cultivation of lymphatic tissues in citro are not equal to those in vivo. Although the requirements of lymphatic tissue have been studied by Trowell (1955, 19Fj9, 1963), no system permitting the proliferation of lymphatic cells has yet been found. Lymphatic tissue probably requires certain special metabolites, just as with various easily cultivated cells (fibroblasts and tumour cells), unusual nutritional requirements and unusual metabolic acti\ities have been found (Eagle, 1955, 1965). It has also been demonstrated that there is n selectit c utilimtion and liberation of amino acids, cl~nr~~ctc~ristic of clifft.rc,nt m,iminalinn cells line9 ( hfcCart!. I$)@).Cells from I!inphatic tissue rcquire macromolcdar materials in cu1tm.e media. Those macromolecular substances present in serum-albumin


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(Keilovh and Rychlikovh, 1952; Bazeley et al., 1954; Puck, 1958), fetuin, a,-globulin (Marr et al., 1962), cu,-globulin (Michl, 1961, 1965), and a,-lipoprotein ( Rejnek et al., 1965)-may affect directly the cultivated cells (Gwatkin, 1960) or they may play a protective role in cultivation medium, e.g., in binding some toxic materials and products. This view is supported by the fact that some materials with absorption properties, such as Carbowax, can substitute for proteins (PospiSil and Frankk, 1963). Further, in the direct promotion of cell growth, it was found that 0.01-1.0 pLM of hydrocortisone can substitute for serum in the culture medium ( Ambrose, 1964). It is further possible that, in addition to the special nutritional requirements of lymphatic tissues, more dynamic types of cultivation with a continuous supplementation by fresh medium are needed. During the past 10 years, various types of culture chambers have been discussed, e.g., by Steiner and Anker (1956), Trowel1 (1959), and h i s (1962), which make the exchange of medium possible. Methods of cultivation involving permanent aeration and continuous or intermittent exchange of culture medium have been introduced (McLimans et al., 1957; Danes 1957; Earle et al., 1956; Sterzl, 1959b). However, these improvements in cultivation conditions have also not made possible the complete antibody response in uitro. Better results were obtained with cultivation of small pieces of lymphatic tissue than with isolated cells ( Michaelides and Coons, 1963). Until now the cloning of individual lymphatic cells in viuo and in uitm has not been possible. Recently, lymphocytes isolated from a spleen stimulated by phytohemagglutinin and injected into lethally irradiated recipients were found to form colonies in the spleen, probably as a result of cloning of the injected cells (Mekori et al., 1965). The effect of phytohemagglutinin on small lymphocytes in vitro results in blastogenesis (Berman and Stulberg, 1962; MacKinney et al., 1962; McIntyre and Ebaugh, 1962; Carstairs, 1962; Marshall and Roberts, 1963a,b; Elves and Wilkinson, 1963) and the simultaneous transformation and development of many ribosomes (Elves et aZ., 1964). This may offer a potential method for the stimulation in vitro of differentiation by lymphatic cells producing antibodies. This seems to be the basis for the successful experiments of Globerson and Auerbach (1965) on primary stimulation of spleen fragments by sheep red cells in vitro. However, in this instance also, the results should be evaluated critically as possible secondary response. It is known that the secondary response has been realized in vitro, as described by Michaelides and Coons (1963), Michaelides (1957), Ambrose and Coons (1963), Halliday and Gamey (1964), Pospi6iI et al.

3-32

J.\ROSLAV STERZL A S D IRTIIUR hi. SILVERSTEIN

( 19651, Juhasz and Rose ( 19651. Juhasz and Richter ( 1965), and Richardson and Dutton ( 1963). Conventionally reared animals form antibodies against sheep erythrocytes as carly as the fourtecnth day of life, presumably the result of prior contact \\.it11 cross-reacting antigens. Therefore in adult indi\Gluals, cwry furthcr contact \\.it11 this antigen results in a secondary response. b. Dctectioti of Atitibody Formation by Single Cells. These methods serve for the quantitative estimation of the number of cells which have been transformed into producing cells. They enable us to follow the dynamics of the increase in numbers of antibody-forming cells and permit a study of the nature of primary cell induction, the secondary response, and tolerance. Some of these methods have been reviewed by Nossal and Make15 (196%) and Nossal (1962). Many of these techniques are based on the selective adsorption of antigen onto antibodyproducing cells. The fixation of antigen on individual cells is then detected either by fluorescent techniques (Coons et al., 195Fj), or by labeling the antigen and using autoradiography ( Berenbaum, 1958, 1959), or by adsorption of bacterial antigens onto the surface of producing cells (Rciss et ul., 1950; .\loeschlin and Demiral, 1952; Makela and Nossal, 1961) . The detection of antibody-forming cells by adsorption of erythrocytes was described by Zaalberg ( 1964). In other studies, individual cells have been isolated in microdrops, and the production of antibodies examined by taking advantage of their biological activity, e.g., by immobilization of flagella of selected SalnzoiicZZa strains (Nossal and Lederberg, 1958; Nossal, 195%) or by the inactivation of T2 and T5 phages added to the microdrops (Attardi et nl., 1959. 1964). All of these methods arc- limited in their quantitative applicability. In order to determine the ratio of cells producing antibody to those cells not forming antibody, it is necessary to achieve the maximum Ievel of antibody rcsponse, i.e., at least 1%.of the cells should react in the assay systrm, since it is very difficult with these methods to screen large numI x r s of cc.11~.Thus, these methods do not enable us to follow the dynamics of the onset of antibody formation during the early period or in newborns, lvhen only about one cell per 10'; lymphoid cells starts to form antibodies. At the peak of antibody response to the first dose of antigen, only 0.1%of the cells produce antibody. The possibility of detecting antibody formation by individual cells in st~spension (e.g., one ccll per 1 0 lymphatic cells) was provided by the development of the plaque technique in agar (Jerne and Nordin, 1963). This method, ho\ve\rer, suffered from the anticomplementary


363

effects of agar. Therefore agar, with an admixture of DEAE-dextran (Jerne et al., 1963), or agarose ( BernovskA et al., 1963) was used, maintaining culture conditions corresponding to those in standard tissue culture (Sterzl and Mandel, 1964; Sterzl et al., 1965b). A similar method using carboxymethyl cellulose was described by Ingraham (1963) and Ingraham and Bussard (1964). Modifications of this method make it possible to study not only cell morphology, but also the incorporation of I4C- and 3H-labeled thymidine, i.e., to establish whether the cells have differentiated with or without mitosis (Sterzl and Mandel, 1964; Berglund, 1964; Sterzl et al., 1965b). The method has also been used for electron microscopic studies of antibody-forming cells ( Binet and Bussard, 1964; Bussard and Binet, 1965; Fitch et aZ., 1965). 111. Immunological Development

A. DEVELOPMENT OF MESENCHYMAL FUNCTIONS This section may perhaps best be opened with a repetition of the suggestion made in the introduction-that there is much to be learned about the functions and significance of immunological mechanisms by paying close attention to their development in phylogeny and ontogeny. Although our primary interest here will be a discussion of the development of mesenchymal functions and immunological capabilities in the young maturing animal, it may be useful, for the maintenance of a proper perspective, to review briefly some of the more significant aspects of the development of these functions in phylogeny. This will be done in part because of our firm conviction that there is still much useful insight to be derived from a consideration of evolutionary principles in the study of biological processes and that this May be no less true of the immunological mechanisms than of other biological mechanisms. Again, a consideration of the ontogenetic recapitulation of some of these phylogenetic developments may serve to clarify further some of the issues at hand. The authors also feel that there has been some tendency in recent years to lose sight of the generally true adage that natura non facit mltum. Recent suggestions that the plasma cell, lymphocyte, monocyte (?), thymus, lymphoid reaction center, and perhaps lymph node itself may subserve purely immunological functions seem to imply a degree of biological uniqueness for the immunological mechanisms which may not be fully warranted. Although it may be true that some of these cells or tissues are concerned with purely immunological functions, it would be strange indeed if the elaborate immunological apparatus and functions arose fully formed, like Athena from the forehead of Zeus, at some stage

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JAROSLAV STERZL AND ARTHUR M. SLLVEFSTEIN

of evolution between the hagfish and the lamprey. It would not be unreasonable to expect that more primitive precursor forms of immunological response may be discovered in lower animals and/or that future evidence mav show that many of the cellular or molecular structures which we now accept as possessing immunological attributes may prove to have other, presently unrecognized, biological functions.

I . In Phylogeny u. Phacocytosis and Other Cellular hlechunisms. Two centuries of investigations ( reviewed in kletchnikoff, 1893; Wright, 1958) have furnished convincing evidence that the most basic and fundamental mechanism of defense against infection and injury to the tissues is the i t iflaminatory response invoked by the host. The controversy which lasted over many decades concerning the question of whether cellular or humoral mechanisms were most important in acquired immunity was resolved in part by the recognition that both make important contributions to immunity. Since, however, humoral factors acted so frequently as an adjunct to the cellular reactions, Rich and McKee (1934) felt free to conclude that “in the final analysis it is the phagocytes that protect the bodv in acquired immunity,” a viewpoint shared by Taliaferro ( 1949). It is not without interest to recall that the original function of phagocytosis in the most primitive organisms was concerned with the process of nutrition. Thus, in protozoa, the entire cell may ingest and digest a foreign substance. With the continuing differentiation of species, such coelentrates as Hydra developed two layers of cells: an ectoderm and an entoderm lining the stomach cavity wall which alone retained phagocytic function. In this instance the phagocytes are all fixed and apparently function only in nutrition. With the development of a more abundant mesoderm in metazoa such as the sponge, the capacity for phagocytosis was now shared between the fixed entodermal cells (still retaining the nutritional function) and the mobile cells of the mesoderm (now assuming also a role in defense). Again, it is of some interest to recall hletchnikoffs demonstration (1893) that in the starfish larva the “mesodermal” amoeboid celh of the body cavity have budded off from the entodermal invagination. \17ith the continuing improvement in the intracellular and, finally, extracellular digestive processes of the entoderm, the phagocytic mesodermal histiocyte gradually lost significance in the contribution to nutrition. Simultaneously, however, it gained new significance in its ability to deal with pathogens and other foreign matter that had successfully penetrated into the host. These mobile phagocytic tissue cells have survived throughout all higher species, although the relative


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importance of their protective function has diminished with the development of a vascular system containing cells able to accumulate more rapidly at the site of insult in the tissues. In the most primitive vascular system such as is found in annelid worms, the fluid in most forms is devoid of formed elements. In many of the lower invertebrates there occurs only one kind of leukocyte in the circulatory system, containing a few sparse granules. In others (Insecta, Mollusca) one sees the development of two kinds of circulating leukocytes, one granular and the other hyaline, but both mononuclear. These cells possess amoeboid movement and are phagocytic. There are no polymorphonuclear leukocytic forms found in this group. But the evolution of a system of blood-borne phagocytes has involved something more than an increase in the efficiency with which phagocytes can be mobilized. Since mammalian blood contains five types of leukocytes, only three of which are known to be phagocytic, there has undoubtedly occurred a further specialization of leukocytic function along yet other lines. The circulating monocyte and the polymorphonuclear leukocyte are clearly the most competent and effective phagocytes in the circulation, although the basis for their difference in tastes for different microorganisms is less clear. The eosinophile is also phagocytic, but this does not seem to be its most important role. It has been suggested (Speirs, 1958; Speirs et al., 1961) that this cell is actively concerned in the mechanism of antibody formation, but this speculation would seem to demand further proof than has been forthcoming. The essential function of the basophile is also unknown, although its ability to degranulate and liberate pharmacologically active substances is undoubtedly important. As to the lymphocyte-only a few years ago Harris still felt able to refer to the cell as “the perennial shame of pathologists.” Recent evidence (Gowans et al., 1963; Holub et al., 1965) has unequivocally ascribed to the small and medium lymphocyte an important role in the various immunocytological mechanisms, and Burnet (1965) has suggested that, in fact, the immunological function of the lymphocyte may be its unique activity and that all lymphocytes in the body might possess clonal information and immunological competence against one or another antigen for which the lymphocyte waits to respond. Yet it must be said that the obviously nonspecific appearance of large numbers of these cell types at inflammatory sites (McCluskey et al., 1963; Turk and Oort, 1963; Prendergast, 1963) and their ability to differentiate into other nonimmunological mesenchymal forms suggest that they may subserve functions in addition to those demonstrated immunological ones.

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J.AROSLA\' STERZL AND ARTHUR 31. SILVERSTEIN

1). L!yniplwid Tissues. \\'e have alrcady indicated that the increasing specialization of mesodermal functions and structures led in part to the development of an efficient mechanism for the defense of the organism against the processes and consequences of infection by exogenous agents. Accompanying the dcvelopmcwt of more proficient and more highly specidized cell types was the concomitant evolutionary development of supporting tissues and structures, including the various lymphoid organs and the ubiquitous reticuloendothelidl system. Although these tissues and structures may serve other functions as well, it may be reasonable to assign to each of them a logical piace in the slow development of the general mechanisms of defense against infection. The specific mechanisms which occupy the interest ot the immunologist may be merely among the more recent stages of this developmental sequence. If it is granted that, in the main, the two most important mechanisms of defense are phagocytosis and the inflammatory reaction, then it is possible to assign a reasonable and at least teleologically sound sequential order to the development of mesenchymal function. With the appearance of a primitive circulatory system in lower animals, the presence therein of mobile phagocytic cells may be thought of as providing a mobile reserve able to come rapidly to the aid of the slowly migrating tissue histiocytes. iilthough these cells originally derived primarily from the tissue histiocytes themselves and perhaps also from vascular endothelium, more efficient organs soon developed for the mass production of these cells (lymph nodes, spleen, and bone marrow). Not only are these tissues able continuously to turn out large numbers of these mobile cells, but the presence of a sensitive population of primitive and essentially omnipotential cellular elements enables them further to increase their production on demand. It is for this reason that Taliaferro (1949) has called them the mesenchymal reserve. Once again we see in evolution the development of tissues and cells which are able more competently and more rapidly to accomplish a more primitive basic function. With the appearance of this elaborate and widespread system of lymphoid organs, the further development of an efficient central mechanism for the control of lymphoid cell and organ maturation [thymus, bursa of Fabricius, etc. (Good and Papermaster, 1964)] would seem to represent yet one more logical evolutionary step. At each stage of development one can see the elaboration of a more competent mechanism for the development of inflammatory responses and for the mobilization of phagocytic cells. It may not be unreasonable to assume that modem immunology is concerned with the workings of two further mechanisms which have developed to the same end. These


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concern the ability of the cell: (1) to respond rapidly and specifically to a wide variety of substances (antigens) and ( 2) to elaborate specifically structured molecules (antibodies) able to combine with the antigen. In the lymph nodes or spleen, the sensitivity of the more primitive mesenchymal elements (and perhaps of the more mature ones also) permits them to respond rapidly to the antigenic intruder. The resulting burst of proliferative and dserentiative activity results in a rapid outpouring of the cellular and humoral factors which will participate in the defensive reactions. In addition the more mature circulating members of this series (lymphocytes and monocytes), endowed also with this specific immunological reactivity, may function at the site of inflammation to provide, again by proliferation and differentiation, the same specific and nonspecific protective elements at the point where they are most needed. One of the products of the specific response of these cells is the plasma cell, perhaps the most highly differentiated and uniquely committed of all immunologically functioning cells. Both in the lymphoid tissues and at the site of inflammation, this cell constitutes a very efficient factory for the rapid production of specific antibody protein. In its ability to combine specifically with antigen, antibody contributes on at least three levels to the protection of the host. It may act directly to inactivate the intruding pathogen or toxin; perhaps more important, it functions as an opsonin enhancing phagocytosis; and finally, it increases the inflammatory response of the host by its mediation of immediate hypersensitivity mechanisms. It must be pointed out that each of these new mesenchymal developments in evolution has not led to the complete disappearance of the more primitive cellular mechanisms, but rather to a diminution in the relative importance of their role in defense. Thus, although not so important to the mammal as to lower forms, the fixed tissue histiocytes are still present in the body and may still be able to exercise their primitive functions, a point to which we will return below.

2. In Ontageny Knowledge of the ontogenesis of leukocytic function has lagged somewhat behind information on the phylogenetic development of these cellular activities. Bierman (1961)has summarized this information and points out the sequence of development of these cell types in the human fetus. During the early yolk sac stages of hematopoiesis a few myelocytes and histiocytes may be seen in the blood islands. In the human granulocytopoiesis becomes evident during the second month of gestation, while adult lymphocytes do not appear until the third month of fetal life. Be-

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JAROSLAV STERZL AND ARTHUR hl. SILVERSTEIN

the tourth and fifth month monocytes appcar in the spleen and lymph nodes, It is interesting that in ontogeny the polymorphonuclear granulocyte is the first cell type seen in the fetal circulation, whereas in phylogeny this cell type is a rather recent arrival on the scene. During almost all stages of gestation, the developing fetus has been found (Silverstein and Lukes, 1962; A. M. Silverstein, unpublished observation, 1962) to be able quite competently to form foreign body giant cells and granulomata in response to intrafetal infection or injection with adjuvant mixtures. The thymus appears to be the first organ in the maturing fetus to develop its structural maturity and apparently starts very early to exercise its role in the control of other lymphoid development (Miller, 1964; Good and Papermaster, 1964). The organ grows very rapidly during fetal life, reaching its maximum size in relation to the size of the fetus or newborn shortly before or after birth according to the species involved. There then occurs a diminution in relative size and an involution, presumably owing to the termination of its principal activities. It has been shown in a number of species (Good and Gabrielsen, 1964; Defendi and Metcalf, 1964) that thymectomy of the newborn results in a suppression of immunogenesis, whereas thymectomy after immunological competence has been achieved is usually without untoward effects. Whether the thymus exercises its control functions by the seeding of thymocytes to other lymphoid tissues or by some humoral control mechanism is still open to question, although recent evidence using cell-impermeable chambers (Levey et aZ., 1963; Osoba and Miller, 1964) suggests the humoral alternative. These conclusions are further supported by the results of Osoba (1965) who found that the immunological capacity of the female mouse thymectomized at birth was restored shortly after becoming pregnant, presumably owing to the development of fetal thymic function. Again, intrauterine thymectomy of the fetal lamb at midgestation (Silverstein and Kraner, 1965) was found not to impair the immunological capabilities of the developing fetal lamb. The bursa of Fabricius has been shown to share with the thymus a role in immunogenesis of birds (Good and Papermaster, 1964). Chemical or surgical ablation of the bursa in the developing chick appears to interfere with the development of its capacity to form circulating antibodies, although the development of homograft rejection reactions seems to be thymus-dependent (Warner and Szenberg, 19644). Studies of the opossum fetus in the pouch (La Via et al., 1963) have also lent support to the controlling role of the thymus in the development of immunological competence. The former investigators demonstrated t\vecw


369

that only after the first thymocytes appeared in the primitive opossum epithelial thymus was the developing animal able to form circulating antibody. In the absence of antigenic and other stimuli, lymphoid tissues of the developing mammalian fetus mature only slowly ( Silverstein and Lukes, 1962; Silverstein et aZ.,1963b). At birth the normal mammal has only a moderately cellular set of lymphoid tissues in which follicular activity is generally absent and shows only a poor demarcation between cortex and medulla. Only after birth, with the sudden bombardment of the newborn by the variety of stimuli from its new hostile environment, is there a marked acceleration in lymphoid development and the appearance of the more customary gross lymphoid features and plasma cells forming antibodies. Where these environmental stimuli are kept to a minimum, as in germfree animals, maturation of lymphoid organs is also repressed (Thorbecke, 1959). The fetus, however, may be prematurely induced to a precocious maturation of its lymphoid tissues by chance infection with pathogenic organisms (Silverstein and Lukes, 1962) or by the immunization of the fetus in utem with a variety of antigenic stimuli. The application of skin homografts to the fetal animal also results in a similar accelerated maturation of these structures, although in this instance (Silverstein and Prendergast, 1964) plasmacytosis in the draining lymph node is not seen. As mentioned above, the appearance of these highly organized lymphoid tissues is preceded in the fetus by the appearance of a more primitive and more widely distributed mesenchymal system involving tissue histiocytes and perithelial adventitial cells. Even before the development in the fetus of thymus and/or functioning lymph nodes, these cells may be capable of manifesting one or another form of immunological response, a possibility so important for immunological theory that it is certainly deserving of wider investigation. Thus the fetal lamb at 35-40days of gestation is able to form circulating antibodies (of the macroglobulin type), although at this age it has been impossible to identify any functioning, organized lymphoid tissue ( Silverstein and Prendergast, 1966). Along the same lines, it has been suggested (Silverstein and Lukes, 1962) from studies of the histopathology of congenital infectious processes in the human that the fixed primitive mesenchymal cells found around blood vessels may be capable of assuming immunological function and of proliferating and differentiating into plasma cells. The suggestion of Arnason et al. (1964) and B. Waksman (personal communication, 1965) that thymectomy of irradiated animals does not affect their formation of 2-mercaptoethanol-sensitive antibodies as it

370


does their mercaptoethanol-sensitive antibody response implies that the production of at least some antibody species may be free of general lymphoid control mechanisms.

B. DEVELOPMENT OF IhXSfUXOGLOBULIXS A N D OTHER

SERUM PROTEINS

Some aspects of the immunoglobulins, their nomenclature, structure, genetic determination of peptide chains, and heterogeneity have been summarized in a volume edited by J. Sterzl (1965). However, only a small part of the contemporary knowledge of immunoglobulin molecules has been applied to ontogenetic (Fran15k and Riha, 1964) and phylogenetic studies ( Marchalonis and Edelman, 1965). A series of papers has been devoted to the development of protein fractions in phylogenetically lower animals (see the review by Good and Papermaster, 1964; Fish et al., 1965; Pollara et al., 1966; Gewurz et al., 196f3).This section contains data on the development of immunoglobulins and other serum proteins during ontogenesis. The development of individual serum protein fractions in various species has been studied very intensively during the past 15 years, using various microelectrophoretic methods. In most of these studies, only quantitative levels of the serum components are given, without an analysis of whether these proteins result from synthesis by the fetus or newborn alone or whether they originate by passive transfer from the mother ( see Section II,A,3). In chicks, the changes among the protein fractions in the sera of embryos and in egg fluids have been followed by Marshall and Deutsch ( 1950), Brandt et al. (1951), Schechtman and Hoffman ( 1952), Hradec and Lemei ( 1954), Heim and Schechtman (1954), Weller and Schechtman ( 1957), Amin (1961), and Weller and Schechtman (1962). All of these reports indicate that albumin and a-globulin appear first, p-globulin appears later, and only traces of y-globulin are detected shortly after birth, with no evidence that it is synthesized by the hatched chick itself. Further, the characteristics of the individual immunoglobulin fractions have not been firmly established in this species. Recently, the low molecular weight fraction migrating more slowly than yG-globulin has been described as S-globulin ( Richards and Orlans, 1965). In rats, the development of serum factors has been followed by Salganik (1954), Shmerling and Uspenskaya (1955), and Kelleher et aZ. (1963). Again albumin and a- and ,&globulins were detected as the first serum proteins to appear; immunoglobulins, probably of maternal origin, appeared later. The determination of serum protein development


371

in rats is facilitated by detailed studies in the adult animal (Benjamin and Weimer, 1963). Serum from rabbit fetuses (24 days) has been studied by Brambell et al. (1953), who found that the total protein concentration is lower (about 25 mg. per milliliter) than in the adult serum, with the highest proportion consisting of albumin and p-globulin (both fractions about 11 mg. per milliliter). The protein composition of rabbit fetal fluids has been described by Wild (1965). 7-Globulin ( 2 mg. per milliliter) is again probably of maternal origin. Changes in the concentration of plasma proteins of newborn guinea pigs, and especially the increase in the yG fraction with aging, have been described by Wehmeyer (1954). Electrophoretic estimations of serum fractions of human fetuses and newborns have been published in a number of papers (Moore et al., 1949; Ewerbeck and Levens, 1950; Overman et al., 1951; Pfau, 1954; Sohar et al., 1956; Kuhns and Hyland, 1956; Bergstrand, 1956; Hadnagy et al., 1958; du Pan et al., 1959; Levin et al., 1959; Zapp, 1960). In the fetus, the synthesis of albumin and a- and ,&globulins has been described; 7-globulin is selectively transferred from the mother and during the eighth month it reaches the same or a higher concentration than in maternal serum. The electrophoretic estimation of protein fractions reflects only partially, however, the real synthesis at various stages of ontogenetic development, because some serum proteins ( especially immunoglobulins) are passively transmitted from mother to fetus, varying with the animal species under study. In order to distinguish newly synthesized protein, experimental animals were injected with radioactively labeled amino acids. The inability to detect y-globulin synthesis in newborns during the first 10 days of life has been reported (Sterzl et al., 1958). By injecting I4C-labeled amino acids into newborn rabbits, the incorporation into immunoglobulin fractions has been estimated shortly after birth; the injection of nucleoprotein fractions increases the intensity of immunoglobulin synthesis ( HrubeZovA et al., 1959). Similarly, by incorporation of 35S-labeledamino acids into serum proteins, the synthesis of these proteins can be followed in newborn rabbits (Deichmiller and Dixon, 1960). By injecting I4C-labeled lysine into 24-day-old fetuses through the uterine wall, the highest incorporation was found in albumin and a-globulin. However, significant incorporation also took place into the immunoglobulins ( Kulangara and Schjeide, 1962). By combining immunoelectrophoresis and autoradiography, the incorporation of radioactive s5S-labeledmethionine into immunoglobulins during the first day of life has been observed ( Wainer et al., 1963). The second experimental approach to studying protein synthesis in

372


ne\vboriis involves the use of animals in which transmission of inimunoglobulins from mother to fetus does not occur. Immunoglobulin synthesis in the newborn of an agammaglobulinemic mother has been studied ( Good r’t a!., 1960). Experimentally, agamniaglobulincmic newborns can be o1,tained i n species in which the five- or six-layer placenta prevents transmission of immunoglobulins, e.g., in pigs and lambs. By current electrophoretic methods, no 7-globulins have been found in newborn colostrumfree piglets ( Jacobsen and Moustgaard, 1950; Barrick et a?., 1954; Sokol and Mistrik, 1956; Rutquist, 1958; Sterzl et al., 1960a,b). Using electrophoretic detection, 7-globulin can be found only on thc thirtieth day of life. The eluate from the immunoglobulin region was therefore concentrated 100 times; 1 ml. of precolostral piglet serum contained about 20 to 30 pg. of protein that precipitated with anti-pig immunoglobulin serum. Using 35S-labeledmethionine this small amount of protein was found to be synthesized by the newborn. This protein does not appear to be antibody in character, as discussed in detail in Section III,G,2 (Sterzl et a)., 1960a,b). In further experiments ( Frankk et al., 1961), the immunoglobulin fraction was isolated from newborn serum by preparative electrophoresis on a starch block. After separation on DEAE-cellulose, two components were isolated. One component (about 50% of the newborn immunoglobulin), with sedimentation coefficient s20,w= 2.7 does not react with anti-yo serum. The second component (about %), with a sedimentation coefficient of 5.1, reacts with anti-yo antibodies. The 5 S 7-globulin of newborns has been isolated from large amounts of precolostral newborn piglet serum (Frankk and Riha, 1964). Using 14C-labeled amino acids injected into the newborn, incorporation into the neonatal immunoglobulin fraction has been observed, providing evidence that this protein is actively synthesized in the neonate. The subunits, after sulfonation ( oxidative sulfitolysis ) , were separated chromatographically and electrophoretically and in relation to the H and L chains of 7 S 7-globulin. On applying the fingerprint technique, it was found that these subunits were not identical with the corresponding chains of 7 S y-globulin. Both subunit types of the 5 S globulin displayed a certain structural resemblance to the H chains of 7 S 7-globulin, but peptides corresponding to the L chains of 7 S 7-globulin were not present at all (Fran&k and Riha, 1964). The unusual immunoglobulin in newborns may be synthesized by ;1special lymphoid cell clone, because such microglobulin can be detected also in sera from adults (Berggard, 1961; Porath and Ui, 1964). The formation of immunoglobulins in the fetal lamb has been studied by Silverstein et al. (1963b). In their experiments, serum obtained from the cord blood of fetal lambs delivered at various stages of


373

gestation (from 80- to 150-day full-term fetuses) was studied electrophoretically. The presence of fast and slow 7 S immunoglobulin was not found, but detectable amounts of yM-immunoglobulinwas found throughout this period. After injection of antigen in Freund’s adjuvant, appreciable amounts of 7 S yG-immunoglobulin were produced, even in the absence of detectable circulating antibody of any type. This immunoglobulin production, lacking in detectable antibody activity, is discussed in relation to the production of “normal” immunoglobulin in Section III,B,%Supporting data providing evidence for the formation of immunoglobulin molecules without antibody activity have been reported by Humphrey ( 1964) and Rittenberg and Nelson ( 1962). The limiting factor in serum protein synthesis is the total concentration of these proteins. An increase in y-globulin content is accompanied by a fall in albumin. The fall in albumin concentration was explained as a regulative mechanism for the maintenance of colloid-osmotic pressure (Bjorneboe and Schwartz, 1959). Further, the concentration of 7-globulin in serum has a regulatory influence (feedback mechanism) on new synthesis. Injection of exogenous y-globulin into mice increases the rate of catabolism of homologous y-globulin (Fahey and Robinson, 1963); the rate of catabolism in mice having a high level of y-globulin is also increased ( Humphrey and Fahey, 1961) . In contrast to this, germfree mice with subnormal serum 7-globulin concentrations have slower rates of 7-globulin breakdown than normal mice (Sell and Fahey, 1964). Similarly, in newborn rabbits where the synthesis of their own y-globulin is low, the catabolism of this globulin transferred from the mother is slower (Humphrey, 1961; Wiener, 1951b). y-Globulin transferred from the mother also has a specific effect on the initial synthesis of immunoglobulins. If isoantibodies to an allotype of the progeny are transferred in the maternal y-globulin, they then interfere with the ability of the young to exhibit the particular allotype in its serum. At the same time, a compensatory increase in the other allotypes that are controlled by the allelic gene occurs (Dray, 1962). The synthesis of serum proteins is further influenced by mechanisms of general regulation, especially by hormones, e.g., those of the pituitary (Warner et al., 1957; Beardwood et al., 1962) and the adrenal glands (Levin and Leathem, 1941; Snell and Nicol, 1956; Char and Kelley, 1962). Data have recently been obtained on the genetic control of y-globulin synthesis (Oudin, 1956a,b; Grubb, 1956; Dray and Nisonoff, 1965; Franklin et al., 1965) that permit the formulation of general hypotheses (Fudenberg et al., 1963; Smithies, 1963). Studies on various heritable immunoglobulin deficiencies have contributed to a better knowledge of

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genetic regulation of immunoglobulin synthesis (Bruton, 1952; Good and Zak, 1956; Gitlin et aZ., 1959; Barandun, 1959; Good, 1959). The decisive role in the normal development of immunoglobulins is played by antigenic stimuli arising primarily from microflora colonizing the intestinal tract. Such antigenic stimulation results in the initiation and development of antibody formation, as reviewed in Section IV,C. The role of antigen in immunoglobulin formation is apparent from studies on germfree animals, in which only very low levels of immunoglobulins were observed, even when all antigenic stimuli were not completely eliminated (Thorbeckc ct al., 1957; Gustafsson and Laurell, 1958; Wostmann and Gordon, 1958; Grabar et al., 1964; Wostmann et al., 19s.5). A comparison with thc patterns of hemoglobin synthesis is valuable for an understanding of genetic control of immunoglobulin synthesis. Hemoglobin of adult humans can be separated into thrce components, called Hb-A, Hb-A:, and Hb-A, (Kunkel and Wallenius, 1955). The greater part of fetal hemoglobin differs from these and is called Hb-F, as described by Jonxis (1959). Each hemoglobin component is formed from two types of chains. One of them, the @-chain,is common to all three types of hemoglobin ( Hb-A, Hb-A?, and Hb-F), and its synthesis is controlled by one pair of genes. The second chain, either p, 6, or y, is characteristic for each hemoglobin type, and its structure is controlled by an independent gene pair (Fudenberg et al., 1963). Hb-A and Hb-F have been detected in various proportions in the same erythrocyte (Kleihauer et al., 1957; Retke, 1958; Thomas et al., 1960). During the shift from Hb-F to Hb-A synthesis, suppression of the production of one type of polypeptide chain ( y-chain) seems to occur, with the simultaneous initiation of synthesis of the new type of polypeptide chain ( p-chain). characteristic of the new type of hemoglobin (Baglioni, 1963). Data on the synthesis of hemoglobin form the basis for speculation on immunoglobulin synthesis and the shift of 19 S to 7 S antibodies in the same cell line during differentiation (Sterzl et at., 1966a). \'cry little is known about the development of nonimmunoglobulin serum proteins which may participate in immune processes. The presence of natural conglutinin in the sera of precolostral newborn calves can not be detected (Rice and Duhamel, 1957; Turk, 19S9c). Natural factors other than specific antibody and complement, that contribute to opsonic activity of serum (IIoward and Wardlaw, 1958) and the immune adherence activity of normal serum (Turk, 1959a,b) have not been studied from the developmental approach, because they have not yet b c w i sufficiently defined chemically. The development of complement is relatively well known. In com-


375

parison with sera from adults, low levels of complement were detected in sera from newborn humans ( Wasserman and Alberts, 1940). In the fetal lamb, the full set of hemolytically active complement components appears only during the perinatal period (Rice and Silverstein, 1964). Just as there is a rapid increase in albumin synthesis in newborn animals, so also does the level of complement increase shortly after birth, even when newborns are reared under sterile conditions (Sterzl et al., 1962). This does not imply, however, that synthesis of some components of complement is not present during fetal life. Synthesis of PI-C complement component has been detected in fetal guinea pigs, mostly in the mesenteric lymph nodes and the liver. Similarly, the synthesis of pl-C has been found in the fetal human liver (Thorbecke et al., 1965). OF NATURAL ANTIB~DIES C. DEVELOPMENT

The term “natural antibodies” has been employed to describe substances with antibody activity that are detected in small amounts in the sera of animals before a controlled immunization or apparent infection (Wilson and Miles, 1955; Zilber, 1958). This description covers not only antibodies which may be formed spontaneously on the basis of genetic determination and without antigenic stimuli, but also antibodies which are formed as a result of nonovert immunization with natural microflora (Sterzl et al., 1!362), with antigens of food (Berger and Bauer, 1959; Wagner, 1959a,b), or with antigens inhaled, or as a result of inapparent infection (Metzger and Obst, 1959; Brody and Finch, 1960; Eisler and Von Metz, 1961; Cohen et al., 1961) . The latter type of antibody, even if not recognizably induced by a known antigenic stimulus, is nevertheless the product of an acquired active response and cannot be distinguished from antibodies which result from an active response to any other mode of immunization. The term “natural antibodies” should therefore be reserved for those immunoglobulins which may really be formed spontaneously without an antigenic stimulus, but which satisfy the generally accepted criteria of antibody identity. Is there any evidence that natural antibodies, as so defined, really exist? “Natural” isoagglutinins have been considered to be formed spontaneously by genetic determination ( Hirszfeld, 1932; Landsteiner, 1945). However, other data have suggested the intervention of weak, chance antigenic stimuli during early postnatal life. Wiener ( 1951a) critically evaluated the genetic theory of the origin of isoagglutinins and gave indirect evidence that these natural antibodies resulted from antigenic stimuli by bacteria having determinants in common with blood-group antigens. Numerous examples of common tissue and bacterial antigenic

376

JAROSLAV S'IERZL AND ARTHUR XI. SILVERSTEIN

group ha\ c, Iwcw presentvtl. =\nti-.I ailtibodies werc found to be removable from B and 0 sera by staphylococci ( Illchniann-Christ and Nagel, 1951; Coulter, 1962). Pettcnkofer et al. (1960) detected common antigenic properties in humun 1)lood groups and Enterobacteriaceae. Fedoroff and Webh (1962) found that natural cytotoxic ailtibodies in human blood sera react both with mammalian cells and bacteria. Antigenic similarities may be caused by the presence of common heterophile antigens (Jenkin, 1963). The somatic antigen of E . coli 086:B7 was found to havc common properties with blood-group B of human erythrocytes ( Springer, 1956; Springer et al., 1961) . hiore direct evidence for Wiener's concept was presented in a paper bj. Springer et al. (1959) who studied the formation of anti-human blood-group B agglutinins in germfree chicks artificially contaminated with E . coli 086, as compared with conventionally reared control animals. Germfrce animals did not form antibodies reacting with erythrocytes, whereas conventional animals did form agglutinins, although in lower titer than animals contaminated with E . coli strain 086, which contains antigens common to blood-group B of human erythrocytes. In other experiments with germfree animals, Wagner (195910) found that sera of germfree mice agglutinate Micrococcus epiderniidiu at 1:4 to 1:8. This was probably due to the fact that microbes in the food retain enough antigenicity after sterilization to stimulate agglutinin formation. Agglutinins for Escherichiu coli, Paracolobactrum aerogenoides, Proteus uulgaris, Alcaligenes fecalis, Salmoiwlla enteritidis, Pseudomow aerugiima, Streptococcus fecalis, and Lactobacillus species could not be demonstrated in sera of germfree mice. In studies on natural antibodies a synthetic diet was therefore used, reducing the possibility of immunization with antigens in food ( Sterzl et al., 1965a). In the initial experiments using this model (newborn sterile colostrumfree piglets) no antibodies to ubiquitous bacterial, cell, viral, and protein antigens were detected in the immunoglobulins of these animals. Since no antibodies to the various antigens tested were found in concentrated sera of these animals and since some 200,OOO different antibodies could have been detected in an immunoglobulin fraction containing 2000 pg. antibody nitrogen per milliliter (using serological methods detecting 0.01 p g . AbN per milliliter), it was concluded that at least part (if not all) of the neonatal immunoglobulin is not of antibody character (Sterzl et al., 1960a). Therefore the most critical current need is for the development of new techniques for detection of minimal quantities of antibodies (Sterzl and Kostka, 1963b). Antibody molecules may appear initially in quantities below detectable levels or with binding capacities (avidities) so low that they


377

cannot be demonstrated by current serological tests. It should also be pointed out that some “immunological” phenomena need not result from the participation of immune or natural antibodies, although a clear decision is not yet possible in every instance. Thus Landy and Weidanz (1964) felt that they were in fact demonstrating the presence of true antibody in the germfree lamb, using a bactericidal assay system, although Sterzl et al. (1962, 1965a) concluded that the positive bactericidal test is not necessarily a proof of the presence of antibodies. It has been demonstrated that some surfaces (e.g., of erythrocytes treated with tannin or bacteria in R form) are sensitive to the effect of complement even in the absence of antibodies. It is possible, however, that complement fixation to R surfaces (Tachibana, 1965) may be mediated by some other protein present in the serum or that the effect can be heightened by other serum factors, such as lysozyme (Amano et al., 1955, 1956; Muschel et al., 1959). The formation of complexes of some proteins of nonimmunoglobulin character with antigens can imitate antigen-antibody reactions. An example of this is the binding activity of some serum proteins, e.g., an a2-globulin fraction with insulin, which must be distinguished from the binding of insulin in the immunoglobulin region, i.e., with true antibodies (Clausen et al., 1963; Kraml et al., 1964). Natural conglutinin from bovine serum is a protein quite different from immunoglobulin (Lachmann and Richards, 1964; Lachmann and Coombs, 1965) and combines specifically with some polysaccharide antigens, e.g., zymosan ( Leon, 1957; Lachmann, 1962). This reaction can be inhibited with N-acetylD-glucosamine (Leon and Yokohari, 1964). Turk ( 1959a,b) found that in the p-globulin fraction there is a protein with antibody-like activity participating in immune adherence. Adinolfi et al. (1963) found that normal incomplete cold antibody is not of the y-globulin type. The physicochemical properties of this factor are similar to those of the protein described by Pillemer et al. (1954). On the other hand, plant polysaccharides ( Iectins ) which agglutinate erythrocytes specifically have been described, e.g., by Boyd (1960). Natural antibodies have been distinguished from immune antibodies on the basis of binding criteria and physical properties (Doerr, 1949) and are considered to be less specific than antibodies resulting from immunization ( Skarnes and Watson, 1957). This probably results from the fact that “natural” antibodies are not generally elicited by the antigens with which they are subsequently tested. These “natural” antibodies apparently cross-react with chemical groups present in various bacterial and cell antigens, and therefore their specificity appears to be less than

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JAROSLAV STERZL A N D ARTHUR hf. SILVERSTEIN

is true of antibodies formed after active immunization with microbes and then tested with the homologous antigens. However, the specificity of antibodies formed without controlled immunization can be demonstrated by absorption or blocking experiments (Ingallis, 1937; Michael et oZ., 1962). Most naturally occurring antibodies to bacterial antigens have been detected in the more rapidly migrating yxI (Michael and Rosen, 1963). This may perhaps be explained by the recent demonstrations that small doses of antigen result in the formation of predominantly 19 S antibody molecules (Svehag and Mandel, 1962, 1964; Uhr and Finkelstein, 1963), especially when bacterial lipopolysaccharide antigens are employed ( Pike and Schulze, 1964; Weidanz et al., 1964; Robbins et al., 1965). Naturally occurring antibodies also appear to be less resistant to the effects of heating, although this quality is difficult to assess because of its dependence upon the quantity and nature of other proteins present in the test solutions. The thermostability of early antibodies formed after active immunization and antibodies “naturally” present in sera before immunization appears to be similar (Muschel and Toussaint, 1962; Svehag, 1964). However, other data suggest that natural antibodies are more thermolabile than immune antibodies (Michael et al., 1962). Differences in resistance to heat may be explained as the inactivation of a heat-labile cofactor; the binding capacity of antibodies formed early in response to antigens is so low (Sterzl et al., 1965a) that the presence of another serum component ( a serum cofactor sensitive to heating at 56°C. for 30 minutes) is essential for binding. This cofactor does not seem to be identical to the thennolabile component of complement ( Styk, 1965). If the cofactor is inactivated, then the biological activity of the antibody is reduced or disappears, e.g., the ability of the antibody to neutralize virus or phage can be lowered or eliminated (Dulbecco et al., 1956; Muschel and Toussaint, 1962; Pernis et al., 1963b; Sterzl et aZ., 1965a). In studies on the development of antibody response in conventionally reared newborn rabbits, cells forming hemolytic antibodies to sheep erythrocytes can be detected without antigen injection, using plaque technique, as early as day 15 after birth. Further, the number of cells able to respond to an antigen increases by two orders of magnitude during the first month after birth (Table I ) . However, when animals are reared under controlled sterile conditions on nonantigenic diet, no antibody-forming cells are detected during the first month of life, and the number of cells able to react to the corresponding antigen does not change during this period (Sterzl et al., 1965b; Sterzl, 1965b). These data suggest that antibody formation and the “maturation” of immune re-


379

sponses depend upon antigenic stimuli, primarily by antigens of the intestinal flora and food. These observations contrast with the preliminary observations of Weidanz et al. (1964) that the sera of fetal lambs contain a fairly uniform level of bactericidal anti-Salmonella activity throughout at least the latter half of gestation. This activity is 2-mercaptoethanol-sensitive. It sediments as a macroglobulin and can be specifically absorbed from the serum by the antigen in question, thus satisfying most of the criteria for identification as an antibody. Curiously, however, it was found that immunization of the fetus with Salmonella vaccine does not lead to a boosting of the levels of anti-Salmonella activity (see also Silverstein et al., 1963a), suggesting that if it is a natural antibody in the strict sense, then its production may well be by a mechanism other than that which operates in the active antibody response to exogenous antigens. The existence of a natural antibody, spontaneously formed under genetic control, has implications on two levels. The rigorous demonstration of the existence of such antibodies would clarify greatly our understanding of the precise meaning of the term “immunologically competent cell.” It would also lend some support to the suggestions that have been advanced (Jerne, 1955; Eisen and Karush, 1964) that such natural antibody could mediate the action of antigen in its role as a stimulant for antibody formation and perhaps furnish the molecular basis for the distinction of self from nonself and for tolerance. But it must be concluded at present that an adequate proof of the existence of true natural antibodies has not yet been offered. In most instances in which these antibodies seem to exist, the probability of an inapparent antigenic stimulus is high. In the same way, no direct or convinchg evidence has appeared concerning the role of natural antibodies in the mediation of an active antibody response to an antigenic stimulus. In colostrumfree sterile animals, in which antibodies cannot be detected, the immunological phenomena seem to develop without the intervention of “natural” antibody. Further, quantitative experiments provide evidence against the role of “natural” antibodies. Doses of an antigen which should bind all natural antibodies, if present, do not stimulate an experimentally detectable immune response in newborns (Table 11). The immunization effect of antigen is reached only with increasing doses. This would suggest that the mechanism of antigen recognition occurs on the cellular level; very small numbers of immunologically competent cells which are able to react with injected antigen can, on a statistical basis, meet with the critical level of antigen only if the antigen is injected in relatively large quantities.

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JAROSLAV STJZRZL AND ARTHUR XI. SILVERSTEIN

.\yerage iiumher of plaque-forming cells per 1(P IympIi(iid cells

8 Days after ('oiiwiitratioii (Jf Srtw i i i I-ml. (lohe for I,rlrnilry immuiiimtioii 0 0.01% ( 4 X 10' cell-) 0.1% 1'2 X l O 7 rells) 1 % c2 X 1W cells) 10% (2 X 10g cells) Conc.entrated (% X 10'0)

priinary stimulus, developed by coniplement 0 0

:< 9 3-1

16

5 Days after secondary stimulus with 2 X lo9 Srtlc By complement only (19 S) 2 ,583 2,735 14,070 1,788 978 66

B y anti- YG 4complement (7 S) 732 53'2 102,433 7,810 1,445 166

a Rabbits immuriized after liirth I\ ith different doses of sheep red blood cells (Srbc) and revaccinated 3 weeks after the primary immunization.

In summary, most studies on natural antibodies seem to involve adaptive proteins (formed as a result of uncontrolled immunization) which react with antigenic determinants of \.arious organisms, blood cells, animal cells, etc. True natural antibodies, i.e., immunoglobulins arising by genetic determination and without antigen stimulus or by the chance recombination of immunoglobulin chains, have not yet been conclusively demonstrated. Most experimental data suggest that the appearance of these antibodies is preceded by a covert or inapparent contact of immunologically competent cells with antigen.

D. OSTOGESETIC DEVELOPMENT OF ANTIBODYFORMATION Since the phylogenetic aspects involved in the formation of antibody have been so adequately covered elsewhere (Good and Papermaster, 1964), there would seem to be little point in reciting these facts again here. For other surveys on the developmental aspects of antibody formation, the reader is referred to the publications of Egdahl (1953), Sirotinin (1963), Evans and Smith (1963), Smith (1960b), and Ebert and Delanney (1960). We shall restrict this discussion to some of the ontogenetic factors which are involved in the development of immunological responses, together with the variety of molecular and cellular factors which affect our ability to assay these responses. Just as the young of different species vary so greatly in the extent of their anatomical and functional maturation at birth, even so do they


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manifest wide variations in the degree of their immunological maturation. Any consideration of the ontogenetic development of immunological function must therefore first begin with an outline of these two variables: species, and gestational time of antigenic stimulus. Beyond these, some of the other variables which enter into such a discussion involve consideration of the response of the young animal to different types of antigens as well as the effect of varying the dosage of a given antigen. Attention must also be paid to differences in the molecular species of antibodies formed following initial antigenic stimulus and the temporal order of their appearance. The presence or absence of passively received maternal antibodies will have some effect on the initial immunological response of the developing animal, as will the consequences of such treatment as thymectomy and the use of immunosuppressive agents. Some of these variables have unfortunately only been studied incompletely, so that an adequate interpretation of data in some of these areas is not yet possible.

1. Sensitivity of Detection Methods and Nature of Antibodies Involved The ability to detect an antibody response in ontogenetic studies as in other studies demands a consideration of several critical factors which may seriously affect the meaningful comparison of quantitative or qualitative data obtained with different antigens. These factors involve the type of assay system employed, its sensitivity, and its variations in respect to the different types of antibody molecules which may be formed in the test animal. a. T h Character and Sensitivity of the Assay Systems. The earliest detection of antibodies is determined in part by the level of sensitivity of the immune reaction used. When the sensitivity of different serological methods is compared, hyperimmune sera are usually used; these data were reviewed by Grabar ( 1957). Precipitation, requiring 3-20 pg. AbN per milliliter, is the least sensitive reaction. The quantity of antibodies detectable by precipitation in gel (Finger and Kabat, 1958) is in the same range. Using bacterial agglutination, 0.1 pg. AbN per milliliter is detected on the average. By passive hemagglutination with adsorbed proteins or bacterial antigens, about 0.005 pg. AbN per milliliter can be detected. Very similar quantities of antibody have been detected by passive cutaneous anaphylaxis in the guinea pig. Using the hemolytic reaction under standard conditions, about 0.01 p g . AbN per milliliter can be detected, whereas the minimum amount of diphtheria antitoxin demonstrated by neutralization in rabbit skin is 0.003 pg. AbN per milliliter. It should be stressed that the above-mentioned data correspond to

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JhROSLAV STERZL AND ARTHUR hZ. SILVERSTEIN

antibody with high avidity, and therefore the values estimated need not necessarily be valid for detection of small amounts of the “early” antibodies with very low binding affinity. Macroglobulin-type antibodies, in contrast to the later antibodies of the 7 S 7-globulin type with the capacity of neutralizing toxins (Robbins, 1965), are most effective in serological reactions that damage cell surfaces, i.e., in hemolytic and bactericidal ( bacteriolysis ) reactions and in agglutination tests. The effective enhancement of phagocytosis of particles opsonized by this antibody has also been demonstrated (Smith, 1965). The sensitivity of some of these assay procedures can be raised by appropriate modfications of technique. Thus Sterzl and Kostka (1963a) were able to increase the sensitivity of the hemolytic assay by four orders of magnitude by reducing the number of erythrocytes employed and estimating the 50% hemolytic end point microscopically. Very small amounts of antibody are required in the bactericidal reaction which employs small numbers of organisms and the serum of precolostral sterile piglets or calves, which do not contain antibodies to the bacterial antigen used, as a source of complement. In the bactericidal reaction with sera in which antibody content was estimated by precipitation with 0 antigen (antibody to 0 antigen is responsible for bactericidal reaction), le5pg. AbN per milliliter can be detected (Sterzl et al., 1962; Sterzl and Kostka, 196313; Sterzl et aZ., 1964). Landy et al. (1962a) found the bactericidal reaction only slightly less sensitive. The detection of antibodies by opsonization is at about the same level of sensitivity as the bactericidal reaction. After mixing the organisms with antibody and perfusing the opsonized microbes through isolated livers (the number of microbes is estimated by culture), this reaction proved to be more sensitive by four orders of magnitude than the agglutination reaction ( Miler and TlaskalovL, 1W).This arrangement (upsonization in vitm) seems to be the basis of the high sensitivity observed, since in experiments by other authors (Biozzi et al., 1961; Benacerraf and Miescher, 1960), the same amount of antibodies is required both for opsonization and agglutination reactions. The detection of antibodies in virus and phage neutralization reactions is one of the most sensitive methods. For the neutralization of small viruses, the possibility exists that only one molecule of antibody may neutralize one virus particle (Dulbecco c t al., 1956). Silverstein et al. (1963a) calculated that at least 0.001 pg. AbN per milliliter of phageneutralizing antibody can be detected. For serological activity, ‘‘early” antibodies with lo\\, avidity require a fresh serum for their fixation to antigen. In reactions in which the thermolabile component of the serum


383

(complement) acts on the antigenic surfaces after activation (e.g., in hemolytic and bactericidal reactions), it is difficult to study the influence of the thermolabile serum factor on the binding capacity of early antibodies, Some information has been obtained in virus and phage neutralization reactions, “Early” antibodies to encephalomyelitis and poliomyelitis virus (Dulbecco et al., 1956) and the A2 virus of Asian influenza (Styk et al., 1959) require the presence of a thermolabile cofactor, which increases their binding capacity and mediates their detection. For the demonstration of “early” or “natural” antibodies to phages (Pernis et al., 1963b; Toussaint and Muschel, 1962; Muschel and Toussaint, 1962; Sterzl et al., 1965a) the presence of some thermolabile factor [probably distinct from complement (Styk, 1965)] is also necessary. The participation of a cofactor in the neutralizing effect seems to be dependent not only on the character of antibody, but also on the character of the virus particle surface. The detection of early antibodies to T2 phage requires some serum thermolabile cofactor (Pernis et al., 1963b; Sterzl et al., 1965a), but +X phage can be inactivated by the earliest molecules of antibody produced, without the presence of any cofactor (Styk et al., 1964; HBjek and Mandel, 1966). The role of thermolabile cofactor in the detection of “early” antibodies may be the explanation for the frequently demonstrated thermosensitivity of “natural” antibodies ( Toussaint and Muschel, 1962). b. Molecular Species of Antibody and Their Order of Appearance. The discussion above makes it clear that the production of the several types of immunoglobulin antibodies is important not only for an understanding of the basic mechanisms of the immune response, but also for permitting an evaluation of the dependability and sensitivity of the assay methods employed. It has become clear in recent years (Uhr, 1964) that the primary antibody response in the mammalian adult is characterized by the early appearance of 19 S macroglobulin antibodies followed by the formation of 7 S y-globulin antibodies. Smith (1960a,b) demonstrated that antibodies of macroglobulin type (19 s) were formed in the early postnatal period of children. Antibodies of macroglobulin type had previously been demonstrated by Kabat (1939) in horses immunized with pneumococcal polysaccharide. Stelos and Taliaferro ( 1959) observed the change of 19 S antibody to the 7 S type during immunization of rabbits with sheep erythrocytes. The observation by Smith that the first antibodies formed in newborns are of 19 S type was further extended (Smith et aZ., 1960; Smith and Eitzman, 1964) and confirmed by Fink et al. (1961) and Uhr et al. (1962b) in children and by Bauer and Stavitsky ( 1961), Riha ( 1962), Bellanti et al. ( 1963) in neonatal animals.

384


This typical sequence of 19 and then 7 S antibodies appears to be even more marked in the response of the mammalian fetus (Silverstein et ul., 1963a). However, the typical change from “early” 19 S antibodies (with low avidity requiring cofactor and sensitive to 2-mercaptoethanol) to “hyperimmune” 7 S antibodies need not occur. To some antigens (lipopolysaccharides and erythrocytes ) , 19 S antibodies are predominantly formed even after repeated immunization (Fran6k et al., 1962; Dreesman et al., 1965). During immunization, the properties (e.g., avidity) of even a class of immunoglobulins may change gradually (Sterzl et ul., 1965a). Similarly, the character of 7 S antibodies may change during immunization; 7 S antibodies formed in newborn piglets during the secondary response (after the first intrauterine injection into fetuses) are considerably more sensitive to 2-mercaptoethanol ( Sterzl et al., 1 9 6 ) than hyperimmune 7 S antibodies from adult animals. This is in agreement with the finding by Grey (1963) and Adler (1965) of slowly sedimenting and Zmercaptoethanol-sensitive antibodies. Changes in avidity of 7 S antibodies have also been described by Styk (19ss),who found that not only 19 S but also early 7 S antibodies require a cofactor. It is of some interest that although the normal sequence of, first, macroglobulin and then 7 S y-globulin antibodies is observed in the fetal lamb, the formation of antibody activity associated with ya has not been observed in the fetus of this species. Of further interest is the fact that in the ovine the otherwise normally homogeneous 7 S globulin band is separated into two partially cross-reacting immunoelectrophoretic bands (Silverstein et al., 1963b). Although antibody activity in adult sheep can generally be found associated with both the fast and slow 7 S y-globulin components, the initial 7 S y-globulin activity formed by the fetal lamb in utero is composed almost entirely of the more rapidly migrating component. Antibody activity in the slow 7-globulin range appears only very much later after immunization. These and other similar findings disclose additional problems encountered in the study of the development of the antibody response. We are now aware of the existence of, on the one hand, a number of different molecular specics of antibodies produced in response to antigenic stimulus and, on the other hand, of differences in the properties of antibodies. It is still not entirely clear whether the entire spectrum of such characteristics as the binding capacity of an antibody (avidity) or its susceptibility to the action of 2-mercaptoethanol can exist within a given molecular species of antibody, or whether these differences are related to truly distinct classes of antibody proteins.


385

2. Effect of the Nature and Dose of Antigen a. Response to Different Antigens. It is appropriate to begin this discussion with a consideration of the nature of the response of the developing animal to different antigens, since the data obtained in this area will have significant bearing on the discussion of other variables. Because more information has been obtained on the response of the fetal lamb in this respect than that of any other species (Silverstein et al., 1963a, 19M; Silverstein and Kraner, 1965), the significant points will be made in terms of these data. Most recent speculations about the transition during pre- or postnatal development of the mammalian fetus from an immunological “null” state to one of immunological competence have carried the implicit suggestion that this is a single and more or less discrete transition. It therefore came as somewhat of a surprise to observe that the fetal lamb manifests its abilities to form circulating antibodies against different antigens not simultaneously, but rather at significantly different stages of gestation. Although the number of different antigens examined in the fetal and neonatal lamb is not as extensive as might be desired, yet a certain pattern (or rather lack of pattern) can be noted. Between 35 and 41 days of gestation, the fetal lamb is able to form circulating antibodies against bacteriophage +X 174 virus at a time when the formation of antibodies against any of the other antigens employed can not be detected. Only at about 66 days of gestation (of the normal 150-day gestation period in the ovine) does the fetus appear to initiate antibody formation against the protein ferritin, and not until 125 days of gestation can anti-egg albumin antibody formation be observed following immunization of the fetus in utero. At no time during gestation does the fetus appear able to form antibodies against Salmonella typhosa, diphtheria toxoid, or Bacillus Calmette-Gukin (BCG). Even 6 weeks after birth, when these experiments were terminated, antibody formation against these antigens could not be found in the neonatal animal, although adults of this species were found to respond to all but the diphtheria toxoid in a satisfactory manner (Silverstein et at., 1963a). It should be re-emphasized here that these results are not due to differences in the sensitivity of the several serological methods employed, since the tanned cell hemagglutination techniques for the assay of antiegg albumin and anti-ferritin, the bactericidal technique for the assay of anti-Salmonella activity, and the bioassay for diphtheria antitoxin all have roughly the same order of sensitivity. Preliminary data on the antibody response of the fetal monkey in utero are also suggestively similar. It was found (Silverstein and Kraner, 1965) that immunization of the

386


fetal monkev during the last third of gestation with a variety of antigens led to the formation of antibacteriophage +X 174, anti-egg albumin, and anti-ferritin, although, as in the fetal lamb, foriliation of antibody against diphtheria toxoid was not observed. In the majority of earlier papers (Freund, 1930; Buxton, 1954), bacterial antigens were used for the immunization of newborns. Vaccines prepared for immunization with gram-negative bacteria generally contained the prevailing somatic 0 antigen. However, from papers published previously, the earlier formation of antibodies to H antigens during infection or immunization seems to be quite evident (Felix and Olitzki, 1926; Dulaney and Wikle, 1933; Dulaney et al., 1933). The formation of antibodies to H antigens does not depend on their surface localization. The H antigen has extreme immunizing capacity when injected in purified form (Nossal et al., 19f35). Smith ( 196Ob) immunized children between birth and the thirtieth day of life with mixed typhoid vaccine (TAB) and found the formation of H agglutinins 2 weeks after injection in forty-six of fifty-eight children, but formation of 0 agglutinins was found in only one child. Sterzl et al. (1965a), using the bactericidal reaction for the detection of antibodies to 0 antigens, the microhemolytic test for the detection of antibodies to sheep erythrocytes, and the T2 phage neutralization test ( serological reactions of approximately the same sensitivity), found differences in the immunizing capacity of different antigens for newborn piglets immunized after birth. Antibodies to Salmonella paratyphi B were detected in low titers on day 10 only, although earlier antibody formation, on day 5 after immunization, was detected when sheep erythrocytes, T2 phage, or poliomyelitis vaccine was injected. Thesc results are in full agreement with those of Silverstein et al., (1963a). No perceptible pattern has emerged to explain this apparent stepwise maturation of the immunological capabilities of the fetus, in terms of the chemical nature of the antigens employed. A number of possible explanations of this curious situation can be entertained, each of which has interesting theoretical implications. The observed stepwise maturation may represent a sequence of purely immunological events. If the potential to react against antigens is genetically fixed and present in the zygote, then one would expect that during the development and differentiation of lymphatic tissue the immunological potential would probably be distributed uniformly into lymphatic cells, without time differences in the capability to react against various antigens but with differences between individuals and species. However, there is at present no convincing evidence that individuals of various species form antibodies preferentially to antigens which have exerted a maximum selec-


387

tive pressure. Both for nonmammalian vertebrates (Uhr et al., 1962c) and human beings (Uhr et al., 1962b), phage +X 174 was shown to be the most effective antigen for immunization. There is another possibility -that the time differences in response to various antigens result from spontaneous mutations in the population of lymphatic cells during development ( Burnet, 1965). However, the early appearance of antibodies only to certain antigens and the regularity of the timing of response to some and the delay in response to other antigens would seem to argue against the importance of incidental chance mutations. On the other hand, the possibility exists that the apparent stepwise maturation of immunological capabilities is, in fact, a reflection of an essentially nonimmunological developmental sequence. It is possible that immunological potential against all of these antigens exists in appropriate cells at a very early age, but that the ability of the fetus to respond to any one of them must await the development of suitable enzymes capable of appropriately digesting the antigen into a form suitable for use by the immunological apparatus (Levine and Benacerraf, 1964). b. Significance of Antigen Dosage in Testing for the Onset of Immune Responsiueness. The significance of the antigen dose for the onset of antibody formation has been studied in children immunized against diphtheria and tetanus (Edsall, 1955). Sterzl and Trnka ( 1957a,b) studied the influence of the amount of S . paratyphi B antigen on the response of newborn rabbits. The increasing dosage of antigen to the maximum tolerated by the young animals without death from endotoxin effects was found to shorten the period in which the first antibodies were detected and to increase the quantity of antibody formed, If 5-day-old rabbits were injected with 5 x lo@heat-killed S. paratyphi B organisms (0.70 mg. of dry weight), antibodies were detected on days 4 to 5 in some individuals, with the average falling on day 10 after antigen injection. These findings were confirmed and extended by the experimental data of Bellanti et al. (1963). On immunizing newborn rabbits, flagellar agglutinins were found in animals injected between days 7 to 14 after birth. Only eight of forty animals formed agglutinins to somatic antigens during the first month of life. The largest feasible dose of antigen that could be given as a single injection gave an enhanced rather than depressed immunological response. As mentioned above, the first antibodies formed in newborns were of the macroglobulin type. Very similar effects, i.e., the acceleration and increase in total antibody formed, were found by Riha (1961, 1962) after immunization with sheep erythrocytes. Even if a concentrated suspension of erythrocytes was injected, no inhibition of antibody formation was observed using the hemagglutination reaction.

388

JAROSLAV STERZL A S D ARTHUR M. SILVERSTEIN

In contrast, for the hemolysin response, the optimum dose was 1 ml. of 10 to 20% erythrocyte suspension. If a concentrated suspension was injected, a depression of hemolysin formation occurred, showing the influence of the antigen dose and the quality of the antigen. The influence of Brtrcella abortus antigen on rats injected between birth and day 4 of life was studied by Halliday (1964). Sixteen times the standard dose of antigen was injected into some newborns without evidence that this induced an earlier antibody response. Similarly, Uhr et a/. (1%") failed to observe an earlier onset of antibody formation to +X phage after an increase in the quantity of antigen injected. On the contrary, the smaller the amount of antigen injected, the earlier was the antibody response, as studied by the immune elimination of phage from the circulation. This is probably because bacteriophage +X is very similar to soluble protein antigens in its capability to survive in circulation without being cleared by phagocytosis. It is of interest that these investigators observed that 19 S antiphage formation continued only SO long as the antigen remained available to the host. While 7 S antibody usually followed in due course, preparing the animal for a later booster response, reduction of the initial dose of antigen resulted in the formation of only the 19 S antibody, without the persistence of immunological memory for the secondary response. HBjek and Mandel (1966) compared the influence of various doses of (PX and T2 bacteriophages. Striking differences were found in the first detection of antibodies in relation to the doses of the two types of phage employed. The +X 174 virus circulates until immune elimination occurs; increasing doses of this antigen prolong the period before antibody is first detected in the serum. With T2 phage, however, which is eliminated from circulation within 24 hours, the time of the first appearance of antibodies is independent of the dose. However, the total amount of antibody formed both to (PXand T2 phages is greater on days 5 and 9 after immunization with larger doses of antigen. As mentioned above, the first antibodies formed can neutralize bacteriophage +X without the participation of a thermolabile serum component. For the neutralization of T2 phage with early antibodies, however, the presence of thermolabile cofactor seems to be necessary. The negative phase during which neutralizing antibodies are not detected in newborn colostrum-free piglets lasts at least 24-48 hours. Very similar results were obtained by Kim ct al. (1964), who also did not find antibodies in piglets 24 hours after immunization with actinophage MSP8. Summarizing the results on injection of various amounts of antigens, the significance of the nature of the antigen is quite evident. The chem-


389

ical structure of antigens plays an important role-a given quantity of one antigen may stimulate antibody formation, whereas the same amount of another antigen induces tolerance, The fate of antigen in the hostits persistence in the circulation or quick deposition in tissues-is significant for the onset of antibody formation and its detection in the serum. Thus the increase in the dose of antigen to a certain optimal level must be determined individually in relation to the quality of the antigen and the immunological capabilities of the immunized host. c. Significance of Antigen Dosage for Development of Immune lnhibitwn/ToZerance. In adults, the influence of an overdosage of antigen leading to a depression of the immune response has been repeatedly described (Glenny and Hopkins, 1924; Felton and Ottinger, 1942; Chase, 1946; Taliaferro and Taliaferro, 1951; Johnson et al., 1954; Sterzl, 1954; Dixon and Maurer, 1955). In most of the above-mentioned papers, the inhibition of antibody formation in adults was observed only after repeated doses of antigen, following a marked immune response. In young animals immunological unresponsiveness, later called immunological tolerance, was first observed by Owen (1945). It was found that because of the placental anastomoses of blood vessels in bovine twins and the effective exchange of hematopoietic cells between the dizygotic embryos, the mature animals continued to tolerate these transplants, and the red cell population of each twin was therefore composed of a mixture of two distinct cell types. On the basis of the “self-marker” concept (Burnet and Fenner, 1949), a working hypothesis on the nature of this state was formulated. It was assumed that the development of a condition in which an organism does not react to its own components depends upon the appearance of a metabolic function during embryogenesis, which makes the metabolism of “self” Components possible. (“During this phase appropriate intracellular enzyme systems are being adapted to deal effectively with those components which need to be broken down for reintegration into the metabolic activities of the body”-Burnet and Fenner, 1949, p. 102.) It was assumed that if foreign antigens are injected during embryogenesis, e.g., intravenous inoculation of foreign embryonic blood cells in chick embryos, or if foreign microorganisms are present during embryonic life, the state of tolerance would be evoked. Burnet et al. (1950) tried to verify this hypothesis by injecting human erythrocytes, bacteriophage C16, and living influenza virus intravenously or into yolk sac. The capability to respond to the corresponding antigen was estimated after 5-8 weeks. No change in subsequent response of the hatched chick to the corresponding antigen could be demonstrated. The hypothesis of Burnet concerning the onset of tolerance was con-

390

JAROSLAV STERZL AND ARTHUR &I. SILVERSTEIN

firmed by Billingham c>t ul. (1953), who injected large doses of an antigen during embryogcnesis or immediately after birth. By injecting sufficient numbers of living cells, Billingham detected tolerance in the homotransplantation system. HaSek ( 1953) detected inhibition of antibody formation to mutual blood antigens in chicks (or chick and duck) as a result of mutual embryonic exchange of blood during parabiosis. After these basic experiments, a long series of papers and reviews appeared, furnishing evidence of the possibility of inducing tolerance, i.e., the lowering or elimination of the response to antigens in young animals of various species injected with antigen during the early developmental phase when they react only weakly to antigens (Billingham et al., 1954a,b, 1956; Haiek and Hraba, 1955; Billingham and Brent, 1956, 1959; Harris, 1956; Haiek, 1956; Koprowski et al., 1956; Owen, 1956; Nossal, 19%; Mitchison, 1961; Smith, 1961; Cinader and Dubiski, 1962) . Immunological tolerance was considered to be a special category of inhibition, different from other manifestations of unresponsiveness that follow the overdosage of an antigen (Medawar, 1S60). Differences between tolerance and other types of inhibition were seen in the inability of the unresponsive animal to make specific immune response, resulting from the incapability of lymphoid cells of tolerant animals to form antibodies (Smith and Bridges, 1958; Sercarz and Coons, 1959) and in the inability of an animal already sensitive to be made unresponsive. However, it later appeared that the differences between these types of unresponsiveness are not basic and depend on the different reactivity of animals and the character and quantity of the antigen used. Even in adult animals, appropriate doses of antigen and route of administration may mediate the induction of tolerance (Sercarz and Coons, 1962; Ilresser, 1962b). The state of tolerance has been explained on the basis of at least three possibilitirs: ( 1 ) the prevention of the access of antigc’n to the seat of immunological response; ( 4 ) the absorption of antibodies as they are formed ( treadmill phenomenon); and ( 3 ) a failure of response induced in the immunologically competent cells (damage of the crll or intracellular blocking of the antibody-forming mechanism ) . For an understanding of the nature of tolerance it is necessary, however. to ascertain whether at least some cells in the tolerant host do not share in the over-all tolerant state, i.e., whether a certain degree of immunity can be proved during the state of tolerance. Experimental data obtained in the past few years support the concept that immunological tolerance can be explained on the basis of the functions of immunologically competent cclls. Burnet (1957), on the basis of the clonal


391

selection theory, explained acquired tolerance as allergic damage and elimination of the predetermined cell after contact with a corresponding antigen. Other authors (Dresser and Mitchison, 1960) assumed the formation of the tolerant cell at various stages of its differentiation. Medawar (1959) and LengerovA (1962) assumed that the antigen prevents normal maturation of reactive cells at the stem cell stage and that this leads to the state of immunological tolerance (heterogeneous model). On the other hand, some authors (Michie and Howard, 1962), on the basis of experimental data, assume that the onset of tolerance is possible at all stages of cell differentiation, if antigen is present in excess (homogeneous model). This is in agreement with the view that each specific immune inhibition develops on the basis of a pre-existing or developing specific function (Sterzl, 1962a). This conclusion was based on earlier experiments (Sterzl and Trnka, 1957a,b) in which newborn animals were injected shortly after birth with a very high dose of antigen S. paratyphi B, which is quickly deposited from the circulation into the tissues. This dose accelerated and increased the antibody response in the newborn. After 4 months, the newborn were injected with the same antigen, and a significant inhibition of antibody formation was found in comparison with control animals or animals injected shortly after birth with smaller amounts of the antigen. Thus the dynamics of the onset of immunological unresponsiveness found in newborns was of the same character as the development of immune inhibition in adults, which only follows an active immune response (Taliaferro and Taliaferro, 1951; Sterzl, 1954, 1956). It is usually impossible to find antibodies after the injection of large amounts of an antigen into newborns, especially when it persists in the circulation. Therefore all theories concerning immunological tolerance agree that the excessive amount of antigen may induce tolerance directly, without a previous immune response. In recent experiments (Sterzl, 1965b), the number of cells forming hemolytic antibodies to antigenic determinants of sheep erythrocytes was quantitatively estimated. As in the injection of larger amounts of bacterial antigens, it was found that an increase in the amount of sheep erythrocytes injected into newborn rabbits resulted in an increase of antibody-forming cells, detected on the tenth day after repeated large doses of the antigen. No dose was found that induced an immediate inhibition of antibody formation (Tables I1 and 111).Groups of experimental animals injected with various doses of sheep erythrocytes after birth were reinjected 3 weeks later with the same antigen. It was found that the number of cells producing antibodies after the secondary stimulus was significantly lower in those

392

J-4ROSLAV STERZL AND ARTHUR M. SILVERSTEIN

animals stimulated with maximum doses of antigen during the primary reaction, i.e., those animals that produced the maximum of antibodyforming cells during the primary reaction (Tables I1 and 111). T.-IRLE 111 ASTIBODY FORMATIOX IS STERILE PIGLETS" Average number of plaque-forming cells per 108 lymphoid cells

C'oiwentration of Srbc in 10-nil. dose for primary immunizatioii

10 Days after primary

stimulus, developed complenierit

I)

0

0.01% (2 x 107 cells) 0.1% (2 X 108 cells) 1% (2 x 100cells) 10% (2 X 10'0 cells) Conrentrated (2 X 10" cells)

0 0 22 89 389

5 Days after secondary stimulus with 2 X 10s Srhc

By complement only (19 S)

0 0 0 2,640 1,180 0

+

B y anti-70 complement (7

S)

0 0 0 21,600 1,660 800

Piglets immunized after birth xvith different doses of sheep red blood cells (Srbc) and revaccinated 3 weeks after the primary immunization.

On the basis of the results obtained, it is concluded that acquired tolerance may be considered to be the result of a process of terminal exhaustive differentiation : the majority of or perhaps all appropriate immunologically competent cells in the newborn are stimulated with antigen and in the presence of excess antigen they differentiate directly into short-lived antibody-producing cells. In contrast to the effect of high doses of an antigen, small doses stimulate the differentiation of competent cells and proliferation of their descendants; only a small proportion of these cells differentiate into antibody-forming cells, the others providing the cellular basis for the secondary reaction. The terminal exhaustive differentiation hypothesis furnishes an explanation for most of the facts described in studies of tolerance: spontaneous escape from tolerance, dependence on the dose of an antigen (Smith and Bridges, 1958; Thorbecke et at., 1961; Humphrey, 1964), and induction of tolerance even in individuals previously stimulated to an immune response (Dorner and Uhr, 1964). This hypothesis is in good agreement with the observations of Rowley and Fitch (1965), with variations only in the conclusions. These authors also observed antibody-forming cells after inducing tolerance, but assumed that the number of cells appearing


393

resulted from a feedback control on the part of the antibodies circulating in the serum. 3. Antibody Response in Diferent Species

From the extensive literature which has developed on the immunological response of young subjects, the general impression received is that of a reduced (or absent) capacity of the developing young to respond to antigenic stimuli. Among the subjects so studied are chicks (Wolfe and Dilks, 1948; Buxton, 1954; Wolfe et al., 1957; Papermaster and Bradley, 1960; Solomon, 1965; Riha, 1965a), mice (Overman, 19Sa,b; Moulton and Storer, 1962), rats (Halliday, 1957, 1964), guinea pigs (Dettwiler et aE., 1940; Baer and Bowser, 19631, rabbits (Freund, 1930; Baumgartner, 1934; Sterzl and Trnka, 1957a,b; Harris et al., 1961; Riha, 1961, 1963; Bellanti et al., 1963), piglets (Hoerlein, 1957; Olsson, 1959a,b; Sterzl et aZ., 1960a,b, 1962; Segre and Kaeberle, 1962a,b; Kim et al., 1964; Sterzl et aZ., 1965a), and children (Happ, 1920; Di Sant’Agnese, 1949; Peterson and Christie, 1951; Vahlquist and Nordbring, 1952; Osborn et al., 1952; Dancis et al., 1953; Smith, 1960~;Rannon et al., 1960; Lennette et al., 1962). By way of contrast with these data showing the late appearance of a reduced immunological capacity, the fetal lamb is able to form circulating antibodies against at least one antigen, bacteriophage +X 174 virus, at some time between day 35 of gestation (day immunized) and day 41 of gestation (day sampled). It should be emphasized that this by no means sets the lower age limit on the ability of this species to form antibodies. It has not proved technically feasible to immunize and successfully bleed the fetal lamb at a younger age because of the small size of the fetus at this stage of development. In addition, as was suggested above, it may be possible that other antigens would give even stronger immunological responses at this stage of fetal development (about onefourth of the way through gestation). The technique described earlier for the permanent indwelling catheterization of the fetal lamb in utero has permitted an estimate of the disappearance rate of antigen and the sequential appearance of circulating antibody in this mammalian fetus. It was found (Silverstein et a l , 1966) that the fetal animal responded in a manner almost identical to that of the adult. Normal elimination from the circulation of the bacteriophage +X 174 antigen employed in this study was observed over a period of some 41 to 47 hours, followed by a typically accelerated immune elimination phase detectable over the next 24 to 36 hours. Shortly thereafter, circulating macroglobulin antibody could be detected in the fetal serum in continually increasing amounts.

394

TAI70SLAV STERZL A S D ARTHUl3 M. SILVERSTEIN

The findings of Kalmutz (1962), La Via ~t al. (1963), and Rowlands et al. (1964) indicate that the fetal opossum in the pouch is able to form antibodies as early as 5 days after entering the pouch, a time when, like the fetal lamb, it too is extremely immature in many other respects. Opossums younger than this did not form circulating antibody, at Ieast to the flagellar antigen of Salmonella typhi employed in these studies. Little information is available on the earliest time at which the human fetus is able to form circulating antibodies. Information has only been available with respect to the response to antigenic pathogens concerned with certain congenital infectious diseases. Eichenwald and Shinefield (1963) were able to demonstrate the presence of nonmaternal macroglobulin antibodies in the cord blood of newborn infants with toxoplasmosis. These data support the morphological observations of Silverstein and Lukes (1962) that the human fetus infected with such antigenic pathogens as Treponema pallidurn or Toxoplasma may develop significant plasmacytosis in association with the chronic inflammatory lesions of these diseases. In this respect it has been suggested that the appearance of plasmacytosis and the typical lesions of congenital syphilis in the human only after about the fifth or sixth month of gestation may be significant. Since fetuses younger than this have been described with treponemal infestation and yet without the typical lesions of syphilis, it has been speculated (Silverstein, 1962) that this timing may be a reflection of the earliest appearance of fetal immunological competence to this antigen rather than being due to the age at which the placenta first permits passage of the Treponema from mother to fetus. In the case of nonhuman primates, P. M. Cotes et al. (personal communication, 1965) have found that fetal rhesus monkeys are able to form antibody to bovine serum albumin. These same workers also found that the fetal monkey forms antibody to diphtheria toxoid in utero, as determined by fluorescent antibody techniques. This latter observation is of special interest in view of the consistent failure to observe a response to this antigen by the fetal lamb throughout its gestation (Silverstein ct al., 1963a). Silverstein and Kraner (1965) have observed in fetal rhesus and pigtail monkeys the formation of circulating antibody against a variety of antigens during the latter third of gestation. The data on the ability of fetal animals of other species to form circulating antibody are limited and generally have involved study near the end of gestation. Fennestad and Borg-Petersen (1957, 1962) were able to infect a bovine fetus by intravaginal introduction of viable Leptospira. At birth the newborn was shown to have relatively high titers of circulating antibody and a concomitant plasmacytosis.


395

It is difficult to draw any firm conclusions from this disparate mosaic of experimental facts. Without question, there do exist very real differences among the various species with respect to the timing of their earliest abilities to form circulating antibody. This may be either a consequence of the different rates of immunoglobulin synthesis and catabolism in various animal species, or the differences may result from previous contact of some individuals with antigenic groups common to the antigen used. This pre-exposure to antigens may result from an infection of the fetus or a colonization of the newborn by bacteria. The differences in the antigenic compositions of the immunized animal-their relationship to, or difference from, the antigen used for immunization-also influence the intensity of the immune response. It is not improbable, however, that many of the apparent differences in the studies described may be due to the immunization with different antigens in different quantities, the different sensitivity and character of the serological reaction used for the detection of antibodies, and perhaps the suppressive effect of maternally derived antibody upon the active response by the fetus or newborn. 4. Cellular Dynamics of Antibody Formation

The inadequacy of the newborn to form antibodies was previously understood to result from a general immaturity not only of antibodyforming tissues but more predominantly of the general interior milieu of the organism and the tissue metabolism dependent upon it. This hypothesis was supported by the experimental findings of Dixon and Weigle (1957), who suggested that newborn animals are metabolically inadequate. They found that lymph node cells capable of either primary or secondary antibody response following transfer to adult normal Xirradiated recipients make no response when transferred to neonatal homologous recipients. On the basis of these findings, they concluded that the immunological inadequacy of the neonatal animal is related to its internal environment and not necessarily to the lack of cells capable of antibody synthesis. To support this conclusion, Dixon and Weigle (1959) reported that cells from neonatal rabbits are capable of producing considerable amounts of antibody after exposure to antigen in vitro and transfer to X-irradiated adult rabbits. In other laboratories, however ( Sterzl, 1955, 1957, 1958a; Mitchison, 1957; Tmka, 1958; Holub, 1958; Trnka and Riha, 1959; Sterzl and Trnka, 1959; Nossal, 1959; Papermaster et al., 1959, 1962; Harris et al., 1959a, 1962b; Holub and Riha, 1960), newborns were found to be a good culture medium for cells isolated from donors at different stages of their development of immune responses or for cells isolated from non-

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immunized donors treated with antigen in vitro. It was found, in fact, that embryos provide a better culture medium for transferred cells than newborns because of their lower immunological reactivity ( Simonsen, 1957; Trnka and Riha, 1959; Sterzl and Trnka, 1959). Cells transferred even into newborn rabbits are limited in their function by the homotransplantation reaction ( Sterzl, 1958a,b) which is significantly more rapid during the first week of their life than in adults (Najarian and Dixon, 1962). Employing the method of transfer of isolated cells to the newborn recipients, the capacity of cells isolated from the spleen of young rabbits of difFerent ages to form antibodies was studied (Sterzl and Trnka, 1956). When cells were isolated from 5-day-old rabbits, mixed with Brucellu suis antigen in uitro, and transferred to the recipients, no antibody formation was detected. Only rarely was antibody formation found if cells were isolated from older donors. This problem was restudied more quantitatively (Sterzl, 1963b), and it was found that after the transfer of 10 x los spleen cells (from 20-day-old rabbits) mixed with Bruce& suis antigen in vitro to the recipient, specific antibodies were detected. To obtain the same antibody response after the transfer of cells isolated from the adult recipient, it sufficed to transfer 5 x 1W-50 X lo6 spleen cells (Trnka and Sterzl, 1960). There is thus a 100-fold decrease in the ability to form antibodies in a spleen cell suspension from newborns compared to a suspension of cells from adult donors. These results were interpreted to imply that the lesser immunological capacity of newborns is not dependent on the internal environment of the newborns, but that a suspension of lymphatic cells from the newborn contains substantially fewer cells capable of responding to the antigen used. A similar problem, i.e., the development of immune capabilities on the cellular level, was studied by Makinodan and Peterson (1964). They used a model which is more advantageous than the transfer of cells to homologous recipients. They transferred spleen cells isolated from an inbred strain of mice to isologous lethally irradiated recipients (Makinodan et al., 1960). The development of antibody-forming potential was studied in 1- to 126-day-old mice. In contrast to l-week-old mice, the capacity of 40-week-old mice at the time of highest antibody-forming activity increased 600-fold ( Makinodan and Peterson, 1964). Using the technique of transfering isolated cells mixed with an antigen in uitro, the development of the ability to form antibodies can be follo\ved without, however, the ability to count the antibody-forming cells. It cannot be decided in this system whether the higher antibody potential corresponds to a greater number of cells or the same number


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of cells each synthesizing more antibody. For such evaluation, other methods must be used, permitting the detection of antibody formation by individual cells ( Nossal and Makela, 1962b). For quantitative studies, the best approach seems to be the plaque technique, allowing the detection of hemolytic antibodies produced by individual cells in gel, as described by Jerne and Nordin ( 1963). In mouse lymphatic cells (concentration los ) before immunization, about 20-200 cells producing “natural” antibodies to sheep red cells were found (Sterzl and Mandel, 1964). The question then arose as to when the first antibody-forming cells appear in the developing animal. In nonimmunized rabbits, reared under conventional conditions, single cells forming antibodies to sheep erythrocytes were found between days 15 and 20 of life in los spleen cells. In rabbits immunized immediately after birth with 1 ml. of 10% sheep erythrocytes, the first antibodies appear on days 5-7 after immunization; when 10-day-old animals are immunized, antibody formation can be detected within 72 hours after the contact with an antigen (Sterzl et al., 196513). Although these results and those of Makinodan and Peterson (1964), obtained with the transfer of cells to isologous recipients, provide evidence for the maturation or quantitative increase in the immune capacity during development, they do not adequately answer some basic questions. Does the capacity to form antibodies increase spontaneously? Is the increase in antibody formation dependent on an antigenic stimulus? If antigen stimulates the enlargement of the pool of immunologically competent cells, to what degree does the antigen act specifically and to what degree is the lymphoid maturation due to nonspecific irritation? To approach this problem, Sterzl et al. (196%) used as a model system the germfree piglet, reared on either degraded cow milk (Mandel et d.,1960) or a nonantigenic diet containing only amino acids or low molecular weight peptides (TrAvniEek et al., 1966). The study of the development of antibody formation under these conditions showed that before immunization no antibody-forming cells can be detected in the lymphatic tissues, especially during the first month of life. However, during that period the sterile piglets are able to form antibodies competently to a variety of antigens (Sterzl et al., 1965a). If 1-day-old or 30-day-old piglets are immunized, the first antibody-forming cells to sheep erythrocytes appear at the same interval, i.e., within 72 hours after immunization. The number of plaque-forming cells detected at different time periods after the immunization also does not differ substantially during the development of the young animal. Sterile piglets nonspecifically stimulated just after birth by the injection of lipopolysaccharide or

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Freund’s adjuvaiit, \\.hen immunized bvith shcep red cells at the age of 1 month, have the same number of antibody-forming cells as piglets that are not stimulated or immunized at birth. It is concluded that nonspecific stimuli do not increase the number of immunologically competent cells to noncross-reacting antigens. The immunological potential of the germfree animal does not seem to change, as compared with the increasing capacity of those animals reared under the conventional conditions that arc exposed to many cross-reacting antigenic substances during maturation. These results support the assumption that the increase of immunological capacity may be due only to antigenic stimulation. The results of Pernis et al. (1963a) indicate that antigenic stimulation is required for the specific histological changes associated with antibody and immunoglobulin formation in newborn rabbits. In addition, the results of Jerne (1965) provide evidence that the effect of an antigen on the increase in the number of cells participating in the immune reaction is specific. In 10s lymphatic cells from a newborn animal, the number of antibody-forming cells is about 100 (Sterzl et al., 1965b). If it is assumed that the cells concerned with antibody formation are multipotential, then it is difficult to understand why only one cell per 10; would be found at a phase when it could be activated by any given antigen. The process of differentiation of immunologically competent cells may be considered as the restriction of the original multipotential capacity of the cell to a single response, imposed by antigen (Szilard, 1960). Alternately, we may consider that there has occurred a random suppression of the original multipotential capacities of cells such that all the potentials originally present in the zygote find their expression, but in cells each now limited to but a few antigenic specificities.

5 . Inheritance of Antibody-Fonning Capubilitics It has become increasingly more clear in recent years that individual and species variations in the ability to form antibodies of a given specificity depend upon more than the chemical nature of the antigen and the antigenic composition of the host. In addition, the genetically determined potentialities of the individual must be considered. It has been speculated that the genetically fixed potential to form antibodies is realized spontaneously during the development of an individual. This view on the existence of a true “natural” antibody was supported by the group of workers who supposed that isoagglutinin antibodies appeared spontaneously without prior contact with antigen (Hirszfeld, 1928, 1932; Schermer and Kaempffer, 1932). However, according to the contem-


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porary views (Wiener, 1951a), these antibodies are formed under the influence of exogenous, mostly bacterial antigens (Springer et al., 1959), which cross-react with certain blood antigens ( Watkins and Morgan, 1956; Allen and Kabat, 1959) (see Section 111,C). Guyer and Smith (1923) studied the inherited capacity to form antibodies after immunization with typhoid vaccine. In a cross-bred population of rabbits, Sang and Sobey (1954) studied the heritable characteristics of antibody response to tobacco mosaic virus. Differences were found in the quantity of antigen needed for induction of antibody formation and in the quantity and affinities of the antibodies formed. Levine et al. (1963) and Benacerraf (1965) tested the genetic disposition of guinea pigs to form antibodies to protein conjugates of the hapten polyL-lysine. In rabbits, the genetic influence on formation of isoimmune antibodies was studied by Cohen (1960) and the same control over antibody formation to human serum albumin (HSA) was described by Gillert and Eichhorn (1960). Using BSA antigen coupled by the azolinkage to two haptens (sulfanilic acid and p-aminobenzoic acid), Riha and PeShk (Riha, 196513) found that rabbits could be divided into three groups. The two larger groups were rabbits reacting very well or very poorly to both determinant groups. The third group of rabbits reacted well to only one of the two haptenic groups used. In more recent experiments, the genetic transfer of the capacity to react to these antigens has been demonstrated. McDevitt and Sela (1965) studied the response to the artifical antigen poly ( tyrosine, glutamic) -polyalanine-polylysine (TGAL) in Freunds adjuvant in five inbred strains of mice (Humphrey, 1965). The genetically controlled ability to respond to the determinant group TG (tyrosine, glutamic acid) was demonstrated. The results obtained in guinea pigs suggest that the ability to respond to an antigen is transmitted by a single Mendelian dominant gene. The hypothesis was advanced that the ability to respond is determined by the capacity of the individual to degrade the antigen by cellular enzymes. The emphasis on enzymatic degradation resulted from experiments in which copolymers of L-GAT ( glutamic acid-alaninetyrosine) were antigenic in all the guinea pigs and rabbits tested, whereas copolymers of D-GATwith the same relative composition were not antigenic (Gill et al., 1963; Maurer, 1963). However, in recent experiments Gill et al. (1964) succeeded in eliciting antibody formation to a copolymer of D-tyrosine, D-glutamic acid, and D-lysine in rabbits. Although it is clear that genetic factors may determine variations in individual and species antibody responses, the basis for this control is still unclear. Among the possibilities that exist may be listed the follow-

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ing: (11 a control over the antigenic structure of the immunized organism ( Cinader, 1960); ( 2 ) a control over the enzymatic composition of the cells which participate in the degradation of antigen (Campbell, 1957); and (3) the transcription of the structural gene for the formation of specific protein. It is the consequences of the latter possibility that are so interesting for immunological speculation. 6. The Effect of Passive (hlaternu2) Antibody on the Active Response

The various modes of transfer of circulating antibody from mother to offspring have been too well elaborated by Brambell (1958) and Hemmings and Brambell ( 1961) to require further discussion here. Suffice it to say that the mammalian young acquires maternal antibody by one route or another, in some species while still in utero directly across the placenta or via the yolk sac splanchnopleure, whereas the young of other species must await the suckling period when maternal antibody from the colostrum and early milk is able to cross the intestinal wall (see Section II,A,3). There is currently some difference of opinion as to whether or not the presence of passively acquired maternal antibody plays any role in the subsequent primary responses of the newborn to immunization and whether this effect may be to enhance or to suppress antibody formation. The effect of maternal antibodies on the active immune response of the newborn was first studied primarily for practical reasons: to choose an adequate dosage of antigen and time of injection in diphtheria toxoid immunization. The maternal antitoxin was found to inhibit the subsequent antibody response to diphtheria toxoid ( Vahlquist, 1949; Barr et al., 1950). Butler et al. (1954) found that babies with as little as 0.004 units of antitoxin per milliliter produce only one-third the antibody found in individuals with no passively transferred antitoxin. A similar observation was presented by Perkins et al. (1958, 1959) using poliomyelitis vaccination. After injecting the babies with the second dose 10-12 months later, they found that the infants born with high concentrations of maternal antibody responded poorly to the booster injection, indicating that maternal antibody had inhibited the early response to vaccine. The same results have been obtained with experimental animals; guinea pigs passively immunized with diphtheria antitoxin responded with lower antitoxin levels than controls, when actively immunized (Uhr and Baumann, 1961b). The concentration of antitoxin for this effect should be higher than 0.01 Lf per milliliter (Mason et al., 1955).


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Nossal (1957) injected 4-week-old mice with homologous antiserum to an influenza virus and inhibited antibody formation in mice immunized with this antigen. Passively transferred antibodies to red cells have the same depressing effect (Talmage et al., 1956; Neiders et al., 1962; Rowley and Fitch, 1964; Sterzl et al., 1965a). The mechanism of this inhibition has not yet been made clear. In the majority of reports there is the assumption that the immunizing activity of antigen is decreased following absorption by the antibody present ( Moller and Wigzell, 1965). Some evidence has accumulated, however, that the antibody can influence the onset and production of antibodies through a “feedback” mechanism (Uhr and Baumann, 1961b; Finkelstein and Uhr, 1964; Rowley and Fitch, 1964). In contrast with these results, there are several publications by Terres and Wolins ( 1961), Terres and Sorem (1962), and Terres and Stoner ( 1W2) in which injection of antibody-antigen (BSA) complexes in excess antigen caused an accelerated immune response to the antigen. Similar observations were reported by Segre and Kaeberle (1962a,b) who claimed to overcome the immunological deficiency of colostrumdeprived pigs by furnishing antibody through injections of diluted hyperimmune serum or normal serum from older colostrum-deprived pigs. Sterzl et al. (1960a, 1962, 1965a) did not observe that the presence of traces of antibodies was necessary for the onset of antibody formation against different kinds of antigens. Similar results were obtained by Kim et al. (1964) who were unable to detect any traces of antibody in precolostral piglets that gave a very good antibody response. The presence or absence of pre-existing antibody as a necessary condition for the onset of antibody formation is of great theoretical importance Sahli (1920) and Jerne (1955) ascribe the decisive role in the discrimination between self and foreign antigens and the transport of the antigen into the immunologically competent cells to the presence of small amounts of specific antibodies in the serum prior to antigen injection. This view on the importance of the preformed antibodies has been extended in the paper by Eisen and Karush ( 1964), who proposed an explanation of immunological tolerance in these terms. Discussing the possible mechanisms of antibody formation, Edelman (1965), Karush (1965))and Plescia (1965) think that circulating preformed antibodies provide the simplest answer to the question of how the organism recognizes a foreign antigen. Most of these authors postulate that the circulating antibody in question is a “natural” antibody, in the sense that it is formed spontaneously by the host without obligatory pre-exposure to antigen. The studies of Sterzl et al. (196%) on precolostral germfree

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animals argue against the necessary role of pre-existing antibody for the onset of the function of the antibody-forming mechanism.

7. The Role of the Thynitis and Other Lymphoid Structures It has now been well established that the thymus plays an important part in the maturation of immunological capabilities in the developing vertebrate ( Miller, 1964; Good and Gabrielsen, 1964). Thus thymectomy of the immunologically incompetent newborn of several species, such as the mouse and rabbit, results in a severe impairment of immunological development. Immunological function in these thymectomized animals may, however, be established by restoration of the thymus, even within a cell-impermeable chamber (Levey et al., 1963; Osoba and Miller, 1964; Law et al., 1964; Aisenberg and Wilkes, 1965), or by the thymectomized female mouse becoming pregnant and benefiting from the development of a thymus by its offspring (Osoba, 1965). These papers clearly show that the production of humoral factors, such as lymphocytosis-stimulating factor ( LSF) or lymphocytosis-stimulating hormone (LSH), is the important part of thymus function (Metcalf, 1956; Duplan et al., 1962). Similarly a humoral factor in chickens seems to be produced by the bursa of Fabricius (St. Pierre and Ackerman, 1965). As a result of neonatal thymectomy, wasting syndrome in newborn mammals develops ( Aisenberg et al., 1962; Sherman and Dameshek, 1963; McIntire et al., 1964), a condition which could be considered the result of the lack of some growth-stimulating factors. Recent studies, however, showed that the pathogenesis of wasting syndrome is caused mostly by microbes or their products ( Azar, 1964; Brooke, 1964); under germfree conditions, wasting syndrome has not been observed (Wilson et al., 1964). The conclusions to be drawn from studies on thymectomy and antibody formation are somewhat tempered, however, by the observations of Parrott and East (1964) and Humphrey et al. (1964), that the effect of thymectomy in neonatal mice may be variable, depending upon the antigen employed to assay the effect. After the development of full competence by the growing animal and the involution of its thymus, extirpation of this organ appears to be without effect. Should the adult animal be subjected to the effect of sufficient X-irradiation or radiomimetic drug to render it immunoIogically unresponsive, then apparently it turns once again to the thymus for assistance in the renewal of these capabilities. That this is true has been demonstrated (Miller, 1962) by the fact that irradiation plus thymectomy leads to a situation in which the animal can no lonser recover its immunological capabilities. It may not be inappropriate to represent this situation as an immunological reversion


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to the immature fetal state, wherein the developmental sequences may have to repeat in the adult that which occurred once before during fetal life. It would be of great interest to compare the redevelopment of immunological function in the massively irradiated adult with the maturation of the developing fetus, with respect to such variables as, for example, the sequential development of response to different antigens as described earlier. Although it has generally been considered that functioning lymph nodes and spleen are necessary for the formation of antibodies, it is not yet entirely clear whether they constitute a sine qua nun in this respect. Rowlands et al. (1964) have indicated that the fetal opossum in the pouch forms antibody only after the appearance of lymphocytes in the primitive lymph nodes. Preliminary data on the extremely young fetal lamb (Silverstein and Prendergast, 1966), suggest that this fetus may be able to form circulating antibodies even before it possesses organized functioning lymphoid tissue. In addition to the thymus, other lymphoid structures have been found to play a role in immunological maturation of the young animal. In the developing avian, both the thymus and the bursa of Fabricius appear to share these functions (Mueller et al., 1964; Papermaster et al., 1962; Aspinall and Meyer, 1964; Warner and Szenberg, 1964). Removal of both the thymus and the bursa from the developing chick renders it incapable of immunological response. Removal of the bursa alone seems to impair its ability to form circulating antibody, whereas removal of the thymus alone, although this does not affect antibody formation, appears to interfere with the development of delayed hypersensitivities ( Szenberg and Warner, 1962; Warner and Szenberg, 1964). Evidence has also been presented that in the developing rabbit, other gut-associated lymphoid structures may also share in the control of immunological maturation. These include the appendix (Archer et al., 1963; Sutherland et al., 1964) and intestinal tonsil (sacculus rotunda) (Archer et ul., 1963). The role of the thymus on the development of immune reactions may be explained by two alternative hypotheses, which are not mutually exclusive. ( 1 ) From the studies on cellular traffic (Harris et uZ., 1964) it has been suggested that cells from peripheral sources (probably from bone marrow) immigrate into the thymus, where they acquire the character of immunologically competent cells ( ICC ). According to this concept, the thymus functions as a teaching center for the lymphoid cells. Cells which thus acquire immunological competence then emmigrate and colonize the peripheral lymphatic organs. ( 2) Alternate concepts consider the number of ICC in peripheral lymphatic tissue as relatively constant and their origin not directly dependent on the thymus. These

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JAROSLAV STERZL AND ARTHUR hf. SILVERSTEIN

cells (ICC) are activated into cells with proliferative potentiality only after contact with corresponding antigens. The activated cells proliferate under the influence or with the aid of thymus humoral factors. After thymectomy of newborns, the proliferation of activated cells is depressed. This results in a lowering of the response to the injected antigen when compared with nonthymectomized controls, in which activated cells have pro1iferated .

8. Stimuhtion and Inhibition of Antibody Formation a. Stimulation of Antibody Formation. In general, those factors which enhance the formation of antibodies do so by supplying metabolites which are essential to the development of immune reaction, but may be present in limited amounts. On the basis of many studies of antibody formation, it is apparent that the most important factor is the intracellular synthesis of new nucleic acids. After the period of intracellular induction of the mechanism, which probably includes the synthesis of messenger ribonucleic acid (mRNA), the quantitative increase in antibody formation is realized by the mitotic division of activated (primed) cells or cells aheady forming antibodies. In a series of studies, it has been established that oligoribonucleotides can enhance the synthesis of RNA, whereas oligodeoxyribonucleotides stimulate the synthesis of deoxyribonucleic acid (DNA) (see Braun, 1965). It might be expected that the stock of nucleic acid building materials in the newborn is limited, owing to the rapid growth of the organism. Thus, the supplying of nucleic acid products may increase and accelerate the division of antibody-forming cells. In adult animals, an increase of antibody foimation after administration of nucleic acids was observed (Richter, 1952; Merritt and Johnson, 1965). The onset of antibody formation in newborns was also accelerated by the administration of nucleic acid digests in experiments by Braun and Nakano (1965) and Hechtel et al. (1965). Sterzl et aZ. (1959) studied the effect of repeated administration of vitamin B,, to newborns. It had been found that vitamin B,, increases the degree of protein synthesis (Wagle et al., 1958) and is involved in the metabolism of formate, glycine, and the methyl groups of methionine, which are the precursors of the purine and pyrimidine components of nucleic acids ( Wagle and Johnson, 1957). The administration of vitamin B,, to young rabbits and puppies after birth and to pregnant females during the last days of gestation was not found to affect the time of appearance of the first antibodies, as compared with controls. Morphological maturation of the mesenchymal tissues was stimulated, however, by vitamin BIZ,


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and also the resistance to infections produced by Salmonella paratyphi B, Brucella suis, and Bacillus anthracis was enhanced. Administration of 3H- or 14C-labeled thymidine during antibody formation in mice led to a significant increase in the number of cells forming antibodies, as compared with animals injected with antigen alone (J. Sterzl, unpublished data). Since the administration of thymidine influences total mitotic activity [an effect due to its low concentration which limits the process of cell division (Greulich et al., 19Sl)], it may be expected that administration of this substance into newborns would be even more effective than in adults, The administration of ribonuclease resulted in a significant increase of antibody titer after immunization with sheep erythrocytes. This may perhaps be explained by the release of useful breakdown products of nucleic acids as the result of ribonuclease activity ( Sterzl, 1965a). The stimulatory effect of endotoxin is explained on the basis of the release of cell-breakdown products. According to Braun and Kessel (1964) and Merritt and Johnson (1965), the effect of endotoxin on cells is to release nucleic acid products which then participate in the stimulatory effect proper. This stimulatory effect of endotoxin on adult individuals has been described repeatedly with various antigens ( Brandis, 1954; Condie et al.,1955; Johnson et al., 1956; Rowley, 1956a,b; Howard et al., 1958; DAnotone and Fumarola, 1958; Kind and Johnson, 1959; Cutler, 1960; Franzl and McMaster, 1961; Luecke and Sibal, 1962; Rowley and Turner, 1964; Turowski and Chachulska, 1985). We tried to determine whether a similar stimulatory effect of endotoxin may be detected in newborn individuals (Sterzl et al., 1961).Newborn animals were injected with 1 and 5 pg. of endotoxin per 100 gm.of body weight and the stimulatory effect on antibody formation to HSA and Brucella suis antigen was tested. In contrast to the stimulatory effect of endotoxin on antibody formation in adult animals, lipopolysaccharide from Escherichia coli and Salmonella typhi did not stimulate antibody formation in newborn animals against either the protein or the bacterial antigen. The administration of endotoxin to newborns significantly increased their resistance to infection, however. Endotoxin appears to produce only slight morphological changes in the tissue of young animals as compared with adult animals. The negative stimulatory effect of endotoxin on antibody formation in newborns has been explained by the fact that newborn animals, to which endotoxin was administered, were not yet sensitized to this antigen. This explanation is supported by the results of Sterzl (196Od). A group of rabbits was sensitized with 10 mg. HSA and, after 4 weeks, they were

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injected intravenously with 30 pg. HSA simultaneously with a small primary dose of a second antigen (10 pg. ovalbumin) which does not cross-react with HSA. A comparison with controls showed that the primary dose of ovalbumin, which alone does not evoke the formation of detectable levels of antibody, did induce an increased antibody response when injected simultaneously with the antigen producing a secondary reaction. These experiments clearly demonstrate the importance of a previous sensitization to a noncross-reacting antigen which is readministered simultaneously with another primary antigenic stimulus. Similar results were obtained by Boyden and Suter (1952), who found that the injection of ovalbumin together with tuberculin resulted in higher antiovalbumin titers. Similarly, Good et d. (1957) found that the combination of tuberculin with BSA increases antibody formation to the latter antigen, These data were interpreted to indicate that the reaction to tuberculin has an adjuvant effect on the production of antibodies. Our results with endotoxin in newborns (where no stimulatory effect was found in contrast to adult individuals) may be explained on the same basis. It is assumed that very young individuals are not yet sensitized naturally to the endotoxins of intestinal gram-negative bacteria. There is thus no secondary stimulus with endotoxin and, consequently, no release of active substances which might nonspecifically affect the antibody response to other antigens. This hypothesis recently received additional support from the experiments of Dutton and Eady (1964). Secondary contact with an antigen was found to result in increased incorporation of nucleic acid precursors into ceIls, suggesting increased mitotic activity. Dutton showed, however, that the increase in mitotic activity could not be explained by an increase in the number of specific antibody-forming cells only. It is suggested that a biologically active substance is released from cells prepared for the secondary reaction after their contact with a second dose of antigen and that this substance may also stimulate other celIs to mitotic division. b. Inhibition of Antibody Formation by X-Rays. Only sporadic experimental results have been published on the effect of X-irradiation on antibody formation during ontogenesis. The results of experiments on the irradiation of adults and the regularities of inhibition and stimulation ( Clemmensen and Andersen, 1948; Craddock and Laurence, 1948; Xohn, 1951; Taliaferro et a]., 1952; Dixon ct a/., 1952; Dixon and McConahey, 1963) can be applied to the newborn only with difficulty. Newborn irradiated animals were found to he substantially more radiosensitive, although their regenerative recovery was found to be quite rapid. These


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two factors both operate to modify the immune response of newborns after irradiation. In experiments by Sterzl (1960a), the antibody response of about 100 newborn rabbits to Brucella suis antigen was followed: 1- to 3-day-old rabbits were irradiated with a dose of 400r, followed 2 days later by antigen injection. Most animals died after the irradiation-some of them even before the first antibodies could be detected, i.e., between the fifteenth and twentieth day of life. In those animals in which antibody titers could be tested, no differences in antibody levels were found as compared to controls. Very similar results were obtained with a group of newborns irradiated with 300 r and injected with antigen 48 hours later. Most animals survived, and during the period when the first antibodies are detected no significant differences were found between irradiated and control newborns. The simplest explanation of these results would be that in the animals that survived, the lymphoid tissue function regenerated very quickly. The character of the antigen used may affect regeneration. Thus Brucelh suis antigen, with an endotoxin component, accelerated the repair of tissues (Smith et al., 1958; Ainsworth and Hatch, 1958; Miler, 1965). There is, however, an alternative explanation for this phenomenon. The cells participating in the immune process, especially in the formation of antibodies to Brucella (predominantly 19 S antibodies) may be substantially more resistant to irradiation, when the dose which is lethal for newborn rabbits is low (400r). Isolated spleen cells irradiated with 360r, mixed with Brucella antigen in aitro, and then transferred to normal recipients produce antibodies at about the same level as nonirradiated cells (Harris et al., 195913; Sterzl, 1960e). As the irradiation of isolated cells with 100 to 200r significantly reduces and almost inhibits the mitotic division of cells (Puck and Marcus, 1956), it would appear unlikely that all cells stimulated to antibody production must necessarily pass through a mitotic cycle. This may explain why 300r irradiation of newborn rabbits did not affect antibody formation. Further it supports the findings (Sterzl et al., 1965b) that during the primary response, a portion of competent cells differentiate directly into producing cells, without mitosis. c. Inhibition by Antimetabolites of Nucleic Acids. The effect of a series of substances on the antibody responses of adults has been studied (Malkiel and Hargis, 1952; Sterzl and Holub, 1957; Schwartz et al., 1958; Hitchings and Elion, 1963). In the first experiments by Sterzl and Holub (1957) on the effect of Gmercaptopurine (6-MP) on the immune response, adult rabbits immunized with Salmonella paratyphi B antigen

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were used. The effective inhibition of antibody formation in these animals was reached only when ncar-toxic doses were used repeatedly (20 and 50 mg. per kilogram of body weight). Schwartz et ul. (1958) used a protein antigen and obtained very effective suppression with 6-MP. In further experiments, Sterzl ( 1960b) tried to determine whether the reduced effect of 6-MP with the bacterial antigen was a function of the type of antigen used or was perhaps due to the previous immune state of adult animals, since adult individuals respond to bacterial antigens predominantly with a secondary response. Since it was observed that newborns injected with bacterial antigen respond with a primary reaction, the influence of 6-MP on antibody formation was followed in neonatal animals. Bacterial antigens ( Salmonella purutyphi B, Brucellu suis) were injected in various amounts ( 7 x lo9, 7 x lo8 organisms), followed by 6-MP administered in doses of 0.5 and 0.3 mg. per 100 gm. of body weight during the first 5 days after injection of the antigen. In animals treated with 6 M P , the antibody response was significantly lowered or inhibited, as a function of the quantity of the antigen used. When the smaller amount of antigen (7 x lo8 microbes) was injected, even 0.3 mg. of S M P per 100 gm. of body weight resulted in inhibition of antibody formation. In the experiments of Jaroikovh et ul. ( 1966), 10-day-old rabbits were immunized with 1 ml. of a 10% suspension of sheep erythrocytes; 3 hours before immunization, the animals were injected subcutaneously or intraperitoneally with a dose of actinomycin D in the amount of either 5, 10, 20, or 40 pg./lOO gm.of body weight. The highest dose of drug tolerated by the animals was 10 pg. of actinomycin D per 100 gm. of body weight. No differences in antibody titers (tested by hemagglutination) were found between actinomycin-treated animals and controls. In experiments in which actinomycin D at a dose of 10 pg./lOO gm.of body weight was given simultaneously with antigen and then repeatedly at 2-day intervals, or where actinomycin D was injected at different intervals after the administration of antigen, no inhibition was found, not even a t toxic levels of the drug. Similarly, mitomycin C in doses of 50, 100, 200, and 300 pg./loO gm. weight was used without effect on antibody production. These results are in conformance with experiments published earlier (Sterzl, 1961) in which twenty-one different drugs with different mechanisms of action were tested. The test of inhibition was performed with isolated cells mixed with antigen in vitro and transferred to newborn rabbits. These procedures enabled us to differentiate between the action of drugs on intracellular biochemical processes and their effects on the multiplication of cells, which is not manifested in this model. We found


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that only some drugs, such as 6-MP and 6-thioguanine, were very effective, whereas pyrimidine derivatives were relatively ineffective, as confirmed by Nathan et al. (1961). In addition, substances directly affecting mitotic activity (e.g., colchicine) were not found to inhibit the antibody response. It is possible that the concentrations of mitomycin C and actinomycin D were not high enough to affect the cells furnishing the immune response, since their over-all toxicity for young animals is high. The effect of 6-MP, however, provides evidence that the antibody response may be inhibited with the use of a substance which is effectively involved in metabolic processes during the induction of competent cells (Sterzl, 196Oc), and this effect shows that antibody formation can be inhibited without secondary toxic manifestations during a period when 6-MP does not interfere with mitotic division.

E. ONTOGENETIC DEVELOPMENT OF SPECIFICCELLULAR REACITONS In the present discussion we shall include under the term “specific cellular reactions” all those immunological responses to antigen that are mediated by what have variously been termed immunocytes or immunologically competent cells, i.e., those mechanisms in which the specific effect of antigen is to cause the collection and/or proliferation and/or differentiation of cells in the apparent absence of any obligatory contribution by humral antibody. Such mechanisms are taken to include the act of antibody formation itself, delayed hypersensitivity reactions, and the specific rejection of solid tissue homografts. Since the ontogenetic, development of the first of these responses has been discussed extensively above, we shall here restrict ourselves to the latter two forms of reactivity. For the sake of this discussion, we shall assume that circulating antibodies do not mediate delayed hypersensitivity reactions (Karush and Eisen, 1962) or homograft rejection (Stetson, 1963). We shall also assume that no profound difference exists between delayed hypersensitivity of the classic tuberculin type and that formed in response to protein antigens (Raffel and Newel, 1958). We shall return to a discussion of these interesting questions below.

1. Homograft Rejection Response The study of the ontogenesis of the homograft rejection reaction is apparently beset by as wide a range of species variations as was described above in the case of the development of the mechanism of antibody formation. In some species, such as the rat (Rawles, 1955; Medawar and Woodruff, 1958), there appears to exist at birth a broad deficiency in the ability of the still immature animal to reject tissue homografts.

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On the other hand, the human newborn and premature were found able to engage in vigorous homograft rejection (Fowler et al., 1960). Even the newborn rabbit, a species long considered to develop immunological competence at some time after birth, was found to develop homograft immunity (Harris and Harris, 1960). Egdahl ( 1957) found, in fact, that even the fetal rabbit could engage in a vigorous homograft response in utero, an observation supported by the work of Porter (1960) who found that although the fetal rabbit would uniformly develop immunoIogica1 tolerance in response to spleen cells injected in utero prior to day 22 of gestation, treatment after this date resulted not in tolerance, but rather in an apparent rejection of the cell graft. Similar observations on the ability of the newborn piglet to reject tissue homografts were made by Sterzl et al. ( M o b ) , and by Howard and Michie (1962) in the newborn mouse. The development of competence to reject homografts on the part of the chick embryo has been studied by Solomon ( 1963a, 1964). Somewhat more extensive studies of the onset of homograft rejection have been performed on fetal lambs and monkeys. The pioneering work of Schinkel and Ferguson (1953) demonstrated that the fetal lamb in utero was able to reject a homograft as early as day 117 of gestation. In an extension of this work, Silverstein and Prendergast (1964) demonstrated that the fetal lamb is able to reject homografts specificially and rapidly at any time after about day 80 of gestation (about midterm). Grafting at any time prior to this fetal age, however, failed to evoke any detectable immunological response on the part of the fetus. The fetal monkey is apparently able to reject homografts at an even earlier state of gestation than the fetal lamb. D. R. Bangham et al. (personal communication, 1964) reported on the apparent rejection of adult bone marrow cells by the fetal rhesus monkey in utero as early as the ninth week of gestation. More recent work by these authors (P. M. Cotes et al., personal communication, 19%) has shown rejection by the fetal rhesus monkey of maternal and unrelated adult skin grafts applied in utero. The same approach of intrauterine grafting and, later, biopsy was applied to the fetal monkey by Silverstein and Kraner (1965), who observed typical histologica1 rejection of skin homografts by fetal monkeys as early as day 58 of gestation (full term is about 165 days). It is interesting in these instances that the fetus may develop homograft sensitivity to the mother and vice versa, without interfering with the normal course of gestation and the normal peaceful interrelationship between the two parties. Two other factors may be involved in this general process. The first involves the maturation of antigens in the developing young. Billingham and Silvers (1964) demonstrated that skin grafts from the newborn survive significantly longer than adult skin grafts.


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Second, the transplacental passage of maternal cells to the fetus may influence the development of homograft immunity in the fetus and newborn (Najarian and Dixon, 1963; Billingham et al., 1965). It may be worth pointing out that the early hope of demonstrating in the fetal animal a marked divergence between homograft responses and the formation of antibody was not realized. In the fetal lamb, which has been best studied in this respect, homograft rejection capabilities appear after the development of antibody responses to some antigens, but before the development of antibody formation to other antigens (Silverstein et al., 1963a). It would appear that in the stepwise development of immunological capabilities against different antigens, the histocompatability antigens responsible for homograft rejection may only assume a place like all other antigens in the hierarchy to which the fetus step-by-step develops competence.

2. Delayed Hypersensitivity Responses Appreciably less has been learned about the ontogenesis of this group of responses than of the development of homograft rejection or antibody formation capabilities. This is probably due in great measure to the repeated demonstration, first by Freund (1927) and most recently by Sterzl and HrubelovA (1959) and Salvin et al. (1962), that the skin of the newborn is for some apparently nonspecific reason an unsatisfactory site for the elicitation of delayed inflammatory reactions. But the inability to elicit a delayed hypersensitivity skin response does not prove that the animal is incapable of developing the underlying mechanism as demonstrated in the newborn piglet by Sterzl and HrubelOVA (1959) and in the newborn guinea pig by Salvin et al. (1962). In both of these reports it was demonstrated that although the skin test could not be elicited in the sensitized newborn, the delayed hypersensitivity state could be transferred from the newborn to an older normal recipient. The newborn human infant was shown to develop contact hypersensitivity to poison ivy by Straus (1931) and to dinitrofluorobenzene by Uhr et al. (1960). It has been possible under certain circumstances to sensitize the newborn guinea pig in utero and elicit delayed skin responses after birth (Weiss, 1958; Uhr, 1960). IV. Developmental Stages of Immune Reactions and Their Mutual Relationships

Just as one may attempt to trace the development of immunological reactions in phylogeny or ontogeny, so is it possible to attempt a defini-

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tion of the development of immunological processes in terms of the maturation and interrelationship of various cellular functions and the proliferation and differentiation of the cells concerned with these functions. From the time that antigen enters the organism it is affected by, and in its turn affects, various cells of the host, leading either to the initial stimulation or later elicitation of some form of immunological response. Among the various forms of specifw immunocytological response to antigen are the mechanisms of phagocytosis, the delayed hypersensitivity and related homograft rejection mechanisms, and the apparently complex set of cellular responses involved in the inductive and productive phases of antibody formation. It is our purpose here to review these mechanisms briefly and to indicate where possible how these several cellular mechanisms may be interrelated. A. RELATIONSHIF’ OF PHACOCYTOSIS TO SPECIFIC CELLULAR REACTIONS The importance of the mechanism of phagocytosis in the functions

of immunity and the possibility that the phagocytic mechanism may play an active role in the development of the immune response have been recognized for about 80 years (see Metchnikoff, 1893,1903). The various specific and nonspecific aspects of this mechanism have been reviewed most recently by Suter (1956), Hirsch (1959), Elberg (1960), Rowley ( 1962), and Suter and Ramseier ( 1964).

I . Nonspecific Functions of Phugocytosis As discussed earlier, one of the most important mechanisms which operates to protect the host from infection by pathogens is that of phagocytosis. But it soon became apparent that the greater part of the specificity involved in the phagocytic mechanism of defense against infection was not based upon an ability of the phagocytic cell itself to recognize the specific organism, but rather upon the preliminary interaction of specific antibody with antigen. The name “opsonin” was applied by Wright and Douglas (1903, 1904) to describe the ability of circulating antibody to sensitize a foreign particle and render it more palatable to phagocytic cells. Typical of a multitude of such studies were the observations of Lucke et al. (1933), who confirmed that although there was almost no phagocytosis of coliform organisms without antibody, there existed almost a direct relationship between antibody concentration and the degree of subsequent phagocytosis. All of the early concepts of the nature of phagocytic action, therefore, assigned an immunologically nonspecific role to the phagocytic ceII.


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2. Is There a Specific Immune Phagocyte? Despite the preponderance of data suggesting a nonspecific role in immunity for the phagocyte, there occur in the literature repeated reports that phagocytic cells may share in the recognition mechanisms involved in acquired immunity. Lurie (1942) was one of the first to suggest such a role for phagocytes. He allowed phagocytosis of tubercle bacilli by cells of normal and BCG-sensitized rabbits and then transferred the laden phagocytes to the anterior chamber of normal rabbit eyes. He found a marked inhibition of growth, or even death, of organisms which had been phagocytized by cells from immune animals as compared with those of nonimmune animals. Based on these observations, Lurie suggested that “immune phagocytes” might constitute a true basis for cellular immunity. Similar observations on the specificity of phagocytes in Bruw22a-sensitized animals were made by Holland and Pickette ( 1958). A long series of studies of this phenomenon ensued; these have been reviewed recently by Rowley (1962) and by Elberg (1960). The possible existence of an acquired specific property of macrophages to deal with foreign organisms was reported further in the work of Mackaness ( 1962). This investigator studied the response of macrophages from normal and immunized mice to Listeria monocytogenes and concluded that even in the absence of demonstrable protective antibody in the serum the washed phagocytes of the immune animal were far more capable of inactivating the organism than were phagocytes from nonimmune controls. Further studies on the specificity of phagocytosis of antigen in delayed hypersensitivity reactions were reported by Turk (1960) and Kay and Riecke (1963). These investigators found a greater uptake of radioiodinated PPD ( purified protein derivative) by cells from tuberculinsensitized animals than from those of nonsensitized controls. In contrast with these observations, however, McCluskey et al. (1963), in an electron microscopic study of delayed hypersensitivity reactions to the protein ferritin, found that although phagocytic histiocytes were involved at the site of the delayed hypersensitivity lesion, there was no greater degree of ingestion of this foreign antigen by cells at the specific lesion than occurred at the site of a nonspecific lesion in control animals. In is clear, then, that the question of the ability of the phagocytic cell to employ an intrinsic immunological specificity in the exercise of its functions is still unresolved. Critical evaluation of data in this field must, however, take into account two possibilities. The first concerns studies of passive transfer from immune to normal animals, in which it

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has been demonstrated in other areas that specific cellular mechanisms as well as antibody formation may be transferred simultaneously, The second caution centers on the possibility that, granting a possible multipotentiality of mesenchymal cells, immunologically competent cells may pass a portion of their existence as nonspecgc phagocytes, either before or after exercising their specific immunological functions. 3. The Role of Phagocytosis in the Antibody Response

We have mentioned elsewhere in this review the suggestion that phagocytic reticuloendothelial celIs may play an obligatory role in the mechanism of antibody formation by ingesting and degrading antigen into a form suitable for induction by immunologically competent cells. Thus antigenic subunits rather than complete antigen molecules may pass from the phagocytic cell to the appropriate lymphoid cell in order to exercise their function. In this view, the presence or absence of appropriate enzymes capable of degrading one or another antigen would be most important to the ability of the host to respond. The implications of this possibility have been discussed elsewhere. The possibility that phagocytic cells may play an important role in antibody formation has been approached from a somewhat different direction by Fishman and Adler ( 1963) and Fishman et al. ( 1965). These investigators have suggested that macrophages may ingest antigen and translate this immunological information into a group of RNA molecules. They suppose that there is then a transfer of RNA from the macrophage to lymphoid cells, where the RNA can specifically evoke the antibody response. It has been suggested (Fishman et al., 1965) that even macrophages in tiitro can elaborate this form of response when treated with antigen. The interpretation of these data has been questioned by Askonas and modes (1965) who repeated many of these studies, employing antigens heavily labeled with radioactive iodine. These investigators found that the active RNA fractions isolated from macrophages were, in fact, contaminated with radioactive macromolecules and they suggest that the immunological information may not be incorporated into the RNA, but rather that the RNA-antigen complex may serve as a sort of “super antigen.”

B. Rm.iimn’smP OF DEL.\BED HYPERSENSITIVITYTO ANTIBODY FORNATIOX During the period of over 60 years in which immunogenic inflammatory reactions have been studied, an impressive body of evidence has accumulated indicating that delayed hypersensitivities of the tuberculin


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and contact dermatitis types were distinct in mechanism and development from immediate hypersensitivities of the anaphylactic or Arthus type ( Waksman, 1959; Eisen, 1959). This distinction was based originally upon observations of the relationship of these responses to infectious processes, the temporal nature of lesion development, and the histopathology of the reactions. More recent studies on passive transfer with serum or cells from the sensitized donor to a normal recipient, or on the ability of agammaglobulinemics to develop delayed responses but not circulating antibody (Good and Zak, 1956), have served to reinforce these views. A number of investigators were able to demonstrate that tuberculintype hypersensitivities were not restricted to antigens of bacterial origin, but could be elicited by such a bland antigen as egg albumin, when suitably introduced into the experimental animal (Dienes, 1929; Dienes and Schoenheit, 1929; Dienes and Mallory, 1932; Jones and Mote, 1934; Simon and Rackemann, 1934). Only some 20 years later was the next significant step taken by Gell and Hinde (1954), who demonstrated that a progressive immunization reaction in rabbit skin induced by small daily doses of protein antigen leads to a sequence of lesions in which the early stages histologically resemble the delayed-type response, whereas only at later stages are Arthus-type reactions observed. The subsequent development of techniques permitting the induction of delayed hypersensitivity states in guinea pigs, in the absence of a concomitant circulating antibody response, permitted more extensive studies of these delayed hypersensitivity responses. These approaches generally involved intradermal immunization with very small quantities of antigen, either in the form of antigen-antibody complexes (Uhr et al., 1957), small quantities of protein antigen (Salvin, 1958), or proteinhapten conjugates (Benacerraf and Gell, 1959). With the appearance of these techniques, and with the increasing emphasis on the cellular aspects of immunological responses, the emphasis shifted from attempts at comparison of delayed and immediate hypersensitivity skin reactions to the comparison of the delayed hypersensitivity state with the cellular mechanisms involved in the formation of those antibodies responsible for immediate hypersensitivity reactions. The early observations that delayed hypersensitivity to protein antigens was followed soon after by active antibody formation (Salvin, 1958; Salvin and Smith, 1959; Benacerraf and Gell, 1959) prompted renewal of the speculation that delayed hypersensitivity might constitute an “early” or “immature” stage in the cellular mechanism of antibody formation. This suggestion found support in later observations that

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delayed hypersensitive guinea pigs, though not yet forming antibody, were prepared to yield an anamnestic antibody response to the homologous antigen (Salvin and Smith, 1959; Sell and Weigle, 1959; Coe and Salvin, 1964). These observations seem to be in accord with the histological observations of Gell and Hinde (1954), recently extended by Martins and Raffel ( 1964a), that the initial nonspecific mononuclear infiltrate at the inflammatory site of a delayed skin reaction to protein antigens rapidly gives way to a marked plasmacytosis. There are, however, a number of observations which appear to argue against the existence of a close relationship between delayed hypersensitivity phenomena and the cellular basis for antibody formation. The histological picture of the classic tuberculin reaction differs somewhat from that of the delayed reaction to protein antigens (Martins and Raffel, 1964b). Tuberculin reactions do not eventuate in the marked plasmacytosis and antibody formation that is true of delayed sensitivity responses to protein antigens. It has classically been recognized that tuberculin hypersensitivity is very difficult to desensitize (Rich, 1951), again presenting a marked dissimilarity to the delayed hypersensitivity response to protein antigens (Uhr and Pappenheimer, 1958). These and other criteria led Raffel and Newel1 (1958) to suggest that, in fact, tuberculin hypersensitivity and hypersensitivity elicited by bland protein antigens constitute two different mechanisms. The observations of Gell and Benacerraf (1959) offer a possible explanation for some of these differences. Guinea pigs sensitized to native egg albumin will give delayed skin reactions and then an anamnestic antibody response to the native protein, but will give only the delayed response without subsequent antibody formation when challenged with denatured egg albumin. It will be recalled that “old tuberculin” and its derivative PPD possess as their active factor heat-denatured proteins derived from tubercle bacilli. These data suggest that the nature of the antigen in question may play a large part in determining the extent and direction of the immunocytological sequence of events which can be evoked. The demonstration that delayed hypersensitivity may be elicited by hapten-protein conjugates exposed another apparent discrepancy between the mechanism of delayed hypersensitivity and that of antibody action ( Benacerraf and Gell, 1959). These investigators pointed out that whereas circulating anti-hapten antibodies are generally found to be specific for the hapten alone, in delayed hypersensitivity not only the hapten but also the carrier protein is important in defining the antigenic determinant. The general applicability of this carrier protein effect has been confirmed by many investigators (Benacerraf and Levine, 1962; Salvin and Smith, 196Oa; Gell and Silverstein, 1962). The possibility that the


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wrong comparison was again being attempted, however, emerged from the data of Salvin and Smith (lsSOa), who showed that the same antigenic determinant requirements and carrier effect were demonstrable in the anamnestic antibody response as were true in delayed hypersensitivity. Again these data suggest that on the cellular level the antigenic requirements of these two mechanisms are similar. The recurrent suggestion that purified polysaccharides are unable to function in elicitation of delayed hypersensitivity responses, although serving well for the stimulation of antibody formation, has appeared to represent a possible argument against a relationship between these two mechanisms. Borek and Silverstein ( 1963), however, showed that saccharides function effectively as haptens in the delayed system, when conjugated to protein carriers. These authors suggested that during infection the polysaccharide-protein complex might stimulate the immune response, but that the purified polysaccharide alone might not satisfy the necessary and sufficient antigenic requirements for carrier in the elicitation of delayed inflammatory lesions. Reports from a number of laboratories indicate that the proportion of specifically sensitized cells appearing in a delayed hypersensitivity or homograft rejection infiltrate is extremely small if, in fact, any of the cells are truly specific (McCluskey et al., 1963; Turk and Oort, 1963; Prendergast, 1963). Most of the cells involved in the delayed inflammatory ( McCluskey et al., 1963) and homograft rejection (Gowans et al., 1962) infiltrates were shown to have proliferated only very recently, recalling similar observations by Nossal and Makela (1962a) in the case of antibody-forming cells. It will be recalled that passive transfer experiments in both delayed hypersensitivity and antibody production systems involve lymphoid cell suspensions from the same origin, that have often been shown to transfer both capacities simultaneously. Since the precise identification of the cell types capable of transferring the various immunological responses is still unclear, it becomes evident that the presently available data on cell transfer, cellular dynamics, or the specificity of the two responses do not argue conclusively for or against a relationship between delayed hypersensitivity and antibody formation. It has been suggested that a special type of high-affinity antibody rather than specifically sensitized lymphoid cells may mediate delayed hypersensitivity responses ( Karush and Eisen, 1962). The interaction of antigen with low concentrations of the hypothetical high-affinity antibody is suggested to lead to the infiltration of circulating lymphoid cells and induction of their proliferation and differentiation at the site of the delayed reaction. In extension of these ideas, the same authors have also suggested that circulating antibody might serve also to mediate the

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mechanism of antibody formation (Eisen and Karush, 1964). Without arguing here the merits of such proposals, it may suffice to indicate that such theories do not as yet influence the discussion on the relationship of delayed hypersensitivity to antibody formation. Whether truly cellular or humorally mediated, the inechaiiisms underlying these two forms of response may still be comparable.

c. DYSAMICS OF A~TIBODY FORMATION Ontogenetic models are useful for studies on the development of adaptive immunity and on the stages of the antibody response. In the following section, the results obtained in ontogenetic studies are compared with the development of immune response in adults to clarify some of the qualitative and quantitative differences. The basic difference between these two models results from the fact that individuals developing under conventional conditions are subjected to uncontrolled stimulation by a spectrum of antigenic stimuli. Thus, the purposeful administration of antigen into the adult may initiate the immune response at an increased level, depending upon the intensity of previous antigenic contacts. In the present discussion of the proliferative and differentiative functions of cells participating in the immune response, the following notations will be employed. Cells which possess the capacity of responding to antigenic stimulus, although they have not as yet done so, will be referred to as immunologically competent, or X-cells. The effect of antigen on these cells is to convert them to immunologically activated cells, or Y-cells. The antibody-producing cell results from a process of differentiation, and is referred to as the Z-cell [the X, Y, Z, nomenclature for cells is also used by Sercarz and Coons (1962)l.

1 . The Inductive Phase and ''Primary" Response in Adults In reports on the dynamics of antibody formation (e.g., Taliaferro, 1957; Stavitsky, 1961), a time period immediately after immunization is described, during which no detectable level of antibody appears or no change in pre-existing antibody titers is observed. This period, called the inductive phase, was considered to be caused by either quantitative or qualitative considerations ( Sterzl, 1960f). If the inductive phase depended upon quantitative factors alone, then the negative phase (even if antibodies were formed immediately after the antigen injection) would be a consequence of ( 1 ) a minimal production of antibodies at the beginning of the process, ( 2 ) their dilution in body fluids, and ( 3 ) the binding of these antibodies by persisting antigen. This would explain


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the delay during which the level of antibodies builds up in the serum such that they may be detected by serological methods. However, a series of studies provide evidence that a true qualitative difference exists between the inductive and productive phases. There is a different sensitivity of these two phases of the immune response to X-irradiation (Dixon et aZ., 1952; Taliaferro et al., 1952), inhibitors of nucleic acid metabolism (Sterzl, 196Ob,c; La Via et al., 1960b; Jarogkovi et al., 1966), hormones ( Berglund and Fagraeus, 1953; Berglund, 1962, 1965), vitamin deficiency (Axelrod and Pruzansky, 1955), and body temperature ( Jaroslow and Smith, 1964). The view that there is a difference between the inductive and productive phases of antibody formation is supported further by the results obtained in tissue culture experiments. It has thus far been impossible in tissue culture experiments to induce a primary response, although immunologically competent cells survive in vitro. If spleen cells are maintained in culture for 48 hours and then mixed with antigen and transferred to newborns, they differentiate into antibody-forming cells (Sterzl, 1 9 5 9 ~ ) .On the other hand, if lymphoid cells are explanted after the induction of antibody formation in viuo, they continue to produce antibodies in tissue culture. Induction of primary antibody formation in tissue culture was not detectable even after increasing the number of cells cultured or concentrating of the culture medium. These data argue against a quantitative explanation of the inductive phase ( Sterzl, 1960f). Similarly, experiments on the transfer of lymphoid cells stimulated by antigen in uitro support this view. If 50 x lo6 cells are transferred to a recipient, the inductive phase lasts for about 72 hours and cannot be eliminated even if the number of cells transferred is ten to fifty times greater ( Sterzl, 1960a; Makinodan and Albright, 1963). These data suggest the existence of a true inductive phase of antibody formation, during which processes occur that are different from those involved in the productive phase of antibody formation. Studies on antibody formation by isolated cells, especially by plaque techniques, have supplied additional data supporting the conclusion about the real existence of an inductive phase of antibody formation. In adult mice, the dynamics of the increase of antibody-producing cells after immunization was followed by Sterzl and Mandel (1964) and Sterzl et al. (196513). Even before the administration of erythrocyte antigens, an average of 65 antibody-forming cells were detected per los lymphoid cells (minimum 10, maximum 140). Similar individual differences were found in immunized mice 24 hours after antigen injection: again the

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lowest number of antibody-forming cells per 10’ lymphatic cells was 10, although in some animals at this time the number of plaque formers was double the highest values found prior to immunization (377, 315 per 10”. On the average, however, the increase in plaque formers during the first 24 hours was not substantial. These data are in general agreement with the results of Jerne et d.(1963) and Jerne (1%5), who did not detect significant changes in the number of cells during the first 20 hours even after the administration of saturating doses of the antigen. These results also furnish evidence about the varying immunization history of adult individuals. In adult mice the number of X-cells is relatively very low in relation to the number of immunologically activated cells ( Y ) , that had been primed and caused to proliferate by previous uncontrolled antigenic stimuli. Therefore, if the true primary response is to be evoked in the adults, increased quantities of antigen are required to have sufficient chance to stimulate this relatively low number of immunologically competent cells (X). On the contrary, if the pure primary reaction is evoked in newborn animals reared under sterile conditions (see p. 397), no antibody-forming cells can be detected before immunization, and the inductive phase, i.e., the period during which no producing cells are detected, lasts very uniformly for 48 to 72 hours. Thus, sterile piglets have a very low number of X-cells (approximatively 1-10 per lo6 lymphoid cells); an increased dose of antigen is therefore required to make contact with the few X-cells with antigen (see Table 11). The course of the primary reaction depends mainly on the quantity and quality of the antigen injected. Small doses of antigen do not necessarily lead to the completed primary reaction ( X += Y -+Z), i.e., to the appearance of antibody-producing cells. Small doses may stimulate some immunologically competent cells to differentiate into the proliferating activated cells ( Y ) . In this situation, only the initial phase of antibody induction (the priming) is realized. For the completion of the primary reaction {differentiation of Y- to Z-cells) repeated contact of the activated cells with the antigen is necessary. The fact that at least two contacts with antigen are required is suggested by the experiments of Trnka and Sterzl (1960). The negative phase of antibody formation is also connected with the fate of injected antigen, a factor determined in great measure by its chemical and physical characteristics. Recently White ( 1963) and Nossal et al. (1964) demonstrated the localization of antigen in macrophages with characteristic long dendritic branches. Thus, communications may be created in lymphoid follicles between macrophages digesting the antigen and lymphocytes. The importance of the role of the macrophage


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seems to reside in its enzymatic digestion of antigen and in the binding of antigen to nucleic acid, which increases the hmunogenicity of antigenic fragments (Campbell and Garvey, 1963; Askonas and Rhodes, 1965). There are, however, alternate views, not yet fully supported by experimental results, that the macrophage must play an obligatory role in the initial phase of antibody formation. It is suggested that the macrophage not only digests the antigen which penetrates the immunologically competent cell, but perhaps operates directly in the initiation of the immunological process. The synthesis in the macrophage of specific messenger RNA has been assumed; mRNA would transfer to lymphoid cells and initiate the specific synthesis of antibody protein (Fishman et d.,1965). After completion of the induction process, an exponential increase of antibody formation begins (Uhr et al., 1962a; Ingraham, 1964; La Via, 1964). The change in the number of antibody-forming cells is also exponential in character, with a doubling time of about 5 hours. After the maximum is reached, the number of cells decreases rapidly. The halflife of antibody-producing cells was calculated to be 56 hours. However, the rapid disappearance of cells that is observed is due to the technique used. If, after secretion of antibodies by individual cells, the plaques are developed by complement alone, then only cells forming antibodies of the 19 S (yM)type are detected. If the plate is treated with an anti-yo serum at an appropriate dilution before complement is added, then both 19 and 7 S antibodies are detected upon addition of complement. In mice, only 27% of the antibody-forming cells can be detected by the addition of complement alone on day 10. The remaining 73%of the antibody-forming cells are detected only when anti-yG serum is added. On day 14 after immunization, only 8%of the antibody-forming cells can be detected by complement alone; 92%of the cells secreting antibody appear only after the addition of anti-yG serum (Sterzl and Riha, 1965). Similar results were obtained by Dresser and Wortis (1965). To understand the cellular events accompanying the numerical increase in plaque-forming cells, experiments were designed to determine whether all antibody-producing cells result from mitotic division. Mice were injected with I4C-labeled thymidine at various stages of the immune response. The incorporation of thymidine into the DNA of antibody-forming cells was estimated by a combination of the plaque technique and autoradiography (Sterzl et al., 1965b). If thymidine is injected within 24 hours after antigen, then 14C-labeledthymidine was found in only 25%of the antibody-forming cells by autoradiography (detected 72 hours after antigen injection). The greatest proportion of cells appear

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to differentiate without mitosis during the earliest stages of antibody formation. If W-labeled thymidine is administered at the time of highest numerical increase in antibody-forming cells, i.e., within the third or fourth day after immunization, 75% of the antibody-forming cells incorporate thymidine. The total number of cclls producing antibodies is thus a mixture of cells which have differentiated without mitosis and cells derived from an active proliferative process (Sterzl et al., 1966a). Other data are available supporting the view that the induction of antibody formation is not directly and necessarily dependent on the mitotic division of cells. In experiments by Gowans et al. ( 1963), thoracic duct cells employed in the restoration of a hemolysin response were not labeled by tritiated thymidine after a period of 24 hours. In the graft vs. host reaction, tritiated thymidine was injected into lethally irradiated mice after they had received an intravenous dose of small lymphocytes from the rat. No tritiated thymidine was incorporated until the small lymphocytes were transformed into large pyroninophilic cells. During the first 3 days, mitotic activity could not be found in cells mixed with antigen in uitro and transplanted in diffusion chambers (Holub, 1960). Summarizing, the basis of the primary reaction is the stimulation of immunologically competent cells ( X-cells ) which have not previously encountered the antigen. The quantity and quality of the antigen (persistence in organism, fixing on the cells) affect the course of the primary reaction. The reaction may only take the form of the initial phase of antibody induction (the priming)-in the conversion of X-cells into proliferating compartment of Y-cells. This process provides the cellular basis for the secondary reaction. If the antigen of the primary injection is still available to the Y-cells, it stimulates them for differentiation into antibody-forming cells (Z-cells); then the initial reaction includes a part of the secondary response. Only exceptionally is a pure primary response encountered, unaccompanied by the differentiation of Y-cells.

2. The Secondarg Response The preparation of an organism so that it gives an increased immune response is an integral part of the development of the over-all immune response and may be accomplished by inapparent antigenic stimuli. If antigen is administered subsequently, then the second stimulus leads to a response different in quantity and quality from the response to the first antigenic stimulus. The total rate of antibody production increases, and antibodies are formed predominantly of the 7 S type with increased binding activity (Eisen and Siskind, 1964). The nature of this response has been explained differently by various


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authors. Pauling (1940) thought that a change in the chemical environment, i.e., in salt concentration or pH, allowed a more rapid dissociation of antibody from an antigen-modified template and thus increased the rate of antibody production. Jerne (1955), who also assumed that the primary response was conditioned by the level of pre-existing antibodies, thought that the booster response was dependent on the amount of circulating antibody available at the time of the repeated injection. The nature of the secondary reaction was interpreted by Campbell and Garvey (1963) as persistence of partially degraded antigen in the cell and, after a secondary injection of the antigen, the cell was injured, allowing antigenic material to escape and enter adjacent cells. It has also been considered that specific nucleic acids formed in cells after the primary antigenic stimulus may induce antibody formation in other cells. These considerations were supported by experiments in which antibodies were detected in newborns after the transfer of nucleoproteins (Sterzl and Hrubdov6, 1955). In these experiments, however, the binding of antigen and antibody to the nucleoprotein was estimated. It is likely that antibodies which appear after the transfer of the nucleoprotein result from immunization by antigen bound to nucleoprotein (the “super antigen” of Askonas and Rhodes, 1965) or by preformed antibodies released from the nucleoprotein. In the experiments of Sterzl (1!365c), nucleic acids (RNA, DNA) isolated from hyperimmunized animals and injected either directly into recipients or incubated with isolated spleen cells did not induce antibody formation. This observation is at variance with the results of Mannick and Egdahl ( 196Z), Cohen and Parks (1964), and Friedman (1964). A critical evaluation of experiments performed up to this time with various systems indicates that the “transfer of information” by nucleic acids between mammalian cells has not been proved and this fact suggests that the hypothesis is probably not valid. None of the theories mentioned above provides a rcady explanation for quantitative changes during the secondary response, and especially the changes in the quality of the antibodies produced. In general, only the studies on the cellular basis of the secondary response have clarified the situation to any extent. These include observations on the proliferation of cells during the secondary reaction (White et d.,1955), the nature of the secondary response approached by a combination of the methods employing isolated cells and autoradiography ( Nossal and Makela, 1962c; Gowans and McGregor, 1963); studies of cell proliferation shortly after the second administration of an antigen (Baney et al., 1962; Cottier et al., 1964; Sad0 and Makinodan, 1964), and studies of the effect on the proliferative activity of cells of products from sensitized cells following a second administration of antigen (Dutton and Pearce,

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JAROSLAV STF,RZL AND ARTHUR h l . SlLVERSTFXN

1962). It seems to be generally accepted that the basis of the secondary reaction is the antigen-induced multiplication of immunologically activated cells (themselves derived initially from competent cells by the prior effect of antigen). For a better understanding of the nature of the secondary response, it is necessary to discuss in greater detail whether it differs from the primary response only quantitatively (i.e., whether the character of both these reactions is identical and these reactions differ only in the number of cells involved) or whether there is a fundamental difference in the character of the cells which might form the basis of differences between the two responses. The interpretation of the quantitative difference between primary and secondary responses in terms of the number of producing cells has been presented by Uhr et al. (1962a). However, a series of experimental facts provides evidence also for qualitative differences between thc primary and secondary response. If the definition of the primary response is accepted, i.e., that during the primary reaction, immunologically competent cells which have not yet been activated are stimulated by antigen, then the secondary reaction is based on the contact of an antigen with activated (previously primed) and proliferated cells, which were not changed into antibody-producing cells during the primary reaction. This assumption implies that the immune reaction must be viewed as a process involving several stages. A model of a two-stage reaction has been described by Dixon et al. (1952); the first stage, the adaptive, radiosensitive phase involves the activation of the mechanism of antibody response; the second phase, involving the production of antibodies, is radioresistant. Another hypothesis of antibody formation as a two-stage process has been formulated by Pappenheimer et al. (1959), who postulated that priming may be concerned with the development of a stereospecific mechanism on the cell surface that allows cells destined to produce antibody to capture and transport efficiently the specific antigen into the cells. On the basis of studies on the dynamics of the increase in the number of cells forming antibodies by Leduc et al. (1955)) Sercarz and Coons ( 1962), using a fluorescent technique, published a hypothetical two-stage representation of antibody formation. This idea is in full agreement with the findings and conclusions of Sterzl (1962b,c, 1963a). In these papers, the two-stage model was based on the establishment of radioresistant and radiosensitive phases, as well as on the findings that the change of competent ceIls into acti\,ilted cells ( X to Y ) cannot be inhibited by doses of 6-mercaptopurine which do, however, prevent the terminal


425

differentiation into antibody-producing cells ( Y to Z). Since the first step of the inductive phase, the so-called priming, was not inhibited by 6-MP, a normal secondary response could be elicited in these individuals. Very similar results were obtained by Weisberger et al. (1964a,b) who completely inhibited the primary antibody response with chloramphenicol (300 mg. per kilogram daily). In those animals whose primary response had been thus inhibited, however, a typical secondary response to a further dose of antigen was obtained. These results seem to depend also upon the quantity of inhibitor administered; thus Cruchaud and Coons (1964) succeeded in inhibiting the secondary response when a maximum tolerated dose of chloramphenicol (1500 mg. per kilogram daily) was administered during the primary reaction. This indicates that certain inhibitors can eliminate the terminal differentiation step, i.e., the change of Y- to Zcells. However, if a dose of inhibitor is used which also affects the proliferation of Y-cells, then the preparation for a secondary response is eliminated. With appropriate doses of inhibitor, however, activated cells can proliferate and prepare for the secondary response without the manifestations of a primary. As discussed above, the preparation for a secondary reaction can be observed without the completed primary response, i.e., without the detection of antibody-forming cells. Uhr and Baumann ( 1961b) were able to prepare the organism for the secondary reaction by use of diphtheria toxoid-antitoxin precipitates without the development of detectable serum antibody. The injection of an appropriate dose of antigen into pig fetuses (Sterzl et al., 1 W a ) did not elicit detectable antibodies; revaccination of these animals immediately after birth led to a typical secondary response and the formation of 7 S antibodies. Further evidence for the existence of activated Y-cells is their proliferative activity. The number of immunologically competent cells in 108 lymphoid cells does not change in individuals prior to stimulation by antigen; in contrast, the activated cells increase in numbers by proliferation, as shown in a report by Fecsik et al. (1964). The secondary response in mice was found to increase up to the fortieth day after primary immunization. In experiments with various doses of sheep erythrocyte antigen, a gradual increase in the secondary response was established over a period of several weeks after the initial antigen injection (Jilek and Sterzl, 1966). The existence of proliferation of activated Y-cells may be deduced also from X-irradiation experiments in which adaption for the secondary response can be destroyed. The secondary response can be eliminated only in animals which have been irradiated during the fourth to fifth week after the primary stimulus, at a time when adapta-

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JAROSLAV STERZL A S D ARTHUR M. SILVERSTEIN

tion for the secondary response is fully developed (Porter, 1964; Thorbecke et al., 1964). Therefore the findings of Nossal and Makela ( 1 9 6 2 ~ ) that the cell prepared for a secondary response multiplies even before the second contact with antigen are fully acceptable. Further discussion 011 the nature of the sccondary response will be found in Section V of this review, where some of the theoretical aspects of the immunological mechanisms are treated more fulIy. 3. Immunological Unresponsitjeness

It was mentioned earlier (see Section III,D,2,c) that just as immunological inhibition (immunological tolerance) can be induced in newborn animals by one large injection of antigen, the state of immunological unresponsiveness can be induced in adult animals by repeated antigen administration. It was shown that repeated doses of antigen lead initially to the stimulation of antibody formation (Carlifanti, 1951); only a series of repeated doses results in inhibition. Similar dynamics of the antibody response employing long-term injections of rabbits with sheep erythrocytes was described by Taliaferro and Taliaferro (1951, 1962). Hemolysin titers began to rise sooner, reached a higher peak, and then the antibody titers decreased even if antigen was repeatedly injected. An inhibition by bacterial antigen was induced by daily injections of a heat-inactivated suspension of Salmonella paratyphi B ( Sterzl, 1954, 1956). After the first month of antigen administration, the peak of the antibody response was reached ( 1:4096) ; however, when animals were immunized daily for 3 months, the antibody level was minimal. The formation of antibodies ceased in the spleen, as demonstrated by spleen cell transfer. With bacterial antigens not readily metabolized, such as pneumococcal polysaccharide, antibody formation can be inhibited by a single dose that is, however, 100 times higher than the immunizing dose (Felton and Ottinger, 1942; Felton, 1949; Siskind et al., 1963). The induction of immunological unresponsiveness by repeated high doses of protein antigen was described by Glenny and Hopkins (1924), Johnson et al. ( 1954), and Dixon and Maurer (1955). Recently, on the basis of studies of tolerance in newborns, immunological inhibition of adults was induced in a similar manner-by the administration of one large dose of antigen (Sercarz and Coons, 1962; Mitchison, 1962a)b) and also with substantially lower doses (Dresser, 1962b; Mitchison, 1964)In experiments with high doses of protein antigen, antibodies are not detected in the circulation because of the persistence of the antigen. When the method of Farr (1958) is used for the detection of antigenantibody complexes, an immune response elicited by a dose of 20 mg.


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of BSA was found to precede the development of unresponsiveness (Mitchison, 1964). The main difference between the development of immunological inhibition in neonates and adults results from the previous immunization history of adult animals. It has been shown that in adults there is a substantially higher number of cells capable of responding to the antigen administered. In newborns the facile induction of immunological tolerance is dependent upon the small number of cells in relation to the antigen administered (Cohen and Thorbecke, 1963). In the establishment of a similar condition-immunological unresponsiveness-in adults, increased doses of the antigen are therefore required. Taking into consideration the data mentioned above, that an immune response precedes the development of inhibition, the theory of exhaustive differentiation may be applied to the explanation of immunological unresponsiveness. Talmage and Cann ( 1961) postulated that tolerunce is due to a block in the maturation of antibody-forming cells at an early developmental stage, whereas the induction of immunological unresponsiveness in adult animals was thought to be better explained on the basis of a forced maturation (Talmage and Claman, 1964). We assume that the induction of both immunological tolerance in newborns and unresponsiveness in adults do not differ substantially in their mechanisms. The difference seems rather to lie in the character and composition of immunologically reacting cells. In newborns, immunologically competent cells are stimulated for terminal differentiation predominantly during the course of a true primary reaction. In adult animals, on the other hand, the greatest proportion of reacting cells exists already in the form of immunologically activated cells (Y-cells) . The exhaustion even of cells prepared for a secondary response has been described by Sercarz and Coons (1960,1962) and analyzed by the transfer of antibody-forming cells to recipients. They suggested that repeated doses of antigen expend the store of the sensitive cells by stimulating their differentiation along an irreversible pathway leading to antibody formation. The differences between newborn and adult individuals may also be demonstrated in experiments using antigens of different character. If an antigen is used which the immunized animal has not previously encountered (e.g., certain haptenic substances), then the difference between the newborn and adult is not so readily demonstrable. The simple induction of immunological inhibition in adult animals by various chemical substances has been observed repeatedly-the Sulzberger-Chase phenomenon ( Sulzberger, 1929; Chase, 1946; Battisto and Miller, 1962). Pretreatment of adult animals with active haptens, e.g., by feeding,

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JAROSLAV STERZL A S D ARTHUR X I . SILVERSTEXN

prevents the development of contact sensitivity and the formation of antibodies (Chase, 1959). On the contrary, antigens which stimulate the proliferation of cells able to react immunologically upon repeated contact ( e.g., bacterial antigens) can induce immunological tolerance only in large and repeated doses. The induction of immunological unresponsiveness does not depend only upon the relation between the quantity of antigen and the number of cells able to react; the dynamics of the situation are influenced also by the form in which the antigen is administered. If some component of the antigen is able to “irritate” the activated cells toward proliferation, then enough cells will always be available and prepared for terminal differentiation into antibody-forming cells. In such a situation, even repeated doses of antigen will not induce immunological unresponsiveness. An example of this can be found in the contrast between the induction of an immune response and tolerance by the same antigen, depending upon whether it is given incorporated in a Freund’s adjuvant or as a soluble preparation (Dresser, 1962a). The dose of antigen required for the induction of tolerance can be lowered if the “nonstimulatory” antigen is prepared (freed of a11 particulate matter) by centrifugation at u),ooo3O,OOO g for 2 0 3 0 minutes ( Dresser, 1962b). The administration of certain inhibitors ( e.g., 6-MP, amethopterin ) together with an adequate dose of an antigen results in the induction of immunological unresponsiveness in adults ( Uphoff, 1958; Schwartz and Dameshek, 1959, 1963; Robinson and Christian, 1960; Goh et al., 1961; McLaren, 1961). Similarly, the state of unresponsiveness can be induced by the simultaneous administration of an antigen and X-irradiation of animals (Dixon and Maurer, 1955; Michie and Woodruff, 1962; Nachtigal and Feldman, 1963; Rittenberg and Nelson, 1963; Linscott and Weigle, 1964). We may assume that the inhibitors or X-irradiation reduce the bulk of lymphoid tissue and limit the proliferation of immunologically activated cells. This action of the suppressive agents thus facilitates the effect of antigen in causing the differentiation of the remaining cells into the terminal compartment of immunologically producing cells without a proliferative phase, resulting in an easier induction of inhibition. In some instances the termination of immunological inhibition has been observed as a spontaneous escape from tolerance or unresponsiveness (Terres and Hughes, 1959; Thorbecke et al., 1961; Sercarz and Coons, 1962; Sorem and Terres, 1963; Siskind et al., 1963), probably the result of a progressively decreasing level of persisting antigen. The termination of tolerance can be accelerated by inducing cellular proliferation by nonspecific irritants, e.g., Freunds adjuvant ( Maurer et d.,


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1963), or by the injection of cross-reacting antigens ( Weigle, 1962, 1964a,b). The data on immunological unresponsiveness and tolerance are reviewed further in Section V dealing with the theoretical basis of the immune response. V. A Unitarian Concept of lmmunocytological Mechanisms Based upon the Proliferation and Differentiation of immunologically Functioning Cells

Other recent reviews have stressed the variety and importance of the molecular factors involved in immunological responses. In the present survey we have attempted to emphasize the cellular bases of these responses in terms of developmental studies in ontogenesis and the dynamics of antibody formation both in young animals and adults. The underlying and persistent theme of cellular stimulation, proliferation, and differentiation to be found in the previous sections of this review on the sequential steps in antibody formation, on immunological tolerance, and on specific cellular reactions, appears to provide a basis for the formulation of a unitarian working hypothesis for immunocytological reactions. Just as the formulation of the unitarian concept of antibodies almost 50 years ago was creative (Zinsser, 1921; our modern appreciation of the molecular and energetic heterogeneity of antibodies now renders this theory valid only in part ), a unitarian concept of immunocytological reactions based upon contemporary knowledge of cell proliferation and differentiation may play a similar stimulatory role. A further reason for advancing this hypothesis is that the suggested stages in the development of immune responses and their mutual interrelationships may be tested by modem experimental methods. In describing the sequential stages through which the immunologically functioning cell differentiates, we have chosen to employ the noncommittal terms X-, Y-, and Z-cells, as has been done previously (Sercarz and Coons, 1962; Sterzl, 1962c), related directly to the PC, ( =X), PC, ( = Y ) , PI- P , ( = Z ) of Makinodan and Albright ( 1963). With the exception of the Z-cells, the present state of knowledge of cytology does not permit the further identification of these several cell types on morphological grounds. It should also be pointed out that the development of this concept is not affected by the nature of the spec& instructionist or selectionist theory that one might favor, although as Coons (1965) points out, the strict clonal selectionist might take exception to the distinction drawn between the X- and Y-cells. In the following outline, the sequential stages and critical factors in the development of immune responses will be discussed separately.

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JAROSLAV STERZL AND ARTHUR hf. SILVERSTEIN

1. Tlic li~iriiuriologiccrlly Conzpcterrt Cell-Thc

X-Cell

By the term X-cell are denoted those cells which possess the potentialitzj to respond specifically to an antigen, but which have not themselves yet engaged in any specific response. Experimental data suggest that the number of X-cells does not increase in the absence of antigen, but they appear continuously to arise from some more primitive precursor to furnish a constant level of cells, probably owing to some homeostatic mechanism. It is impossible to draw a firm conclusion at present on whether the X-cell has a restricted capacity to respond only to one or a few antigens, or alternatively, whether it is multi- or omnipotential in this respect. Arguing in favor of the former view are data demonstrating that only approximately one cell per 1Cr; lymphoid cells reacts to complex (bacterial and erythrocyte) antigens studied (Sterzl et al., 1965b). On the other hand, the observation of antibody formation in the face of an extreme paucity of lymphoid cells in the immature fetal lamb prior to its development of functioning lymphoid tissue ( Silverstein et al., 1963a) or in the fetaI opossum (La Via et al., 1963) offers a statistical argument in favor of multipotentiality.

2. The Immunologically Actizjated Cell-The Y-Cell The Y-cell arises by the specific action of antigen on the immunologically competent X-cell and is responsible for the persistence of immunological memory. Sufficient data are not yet available on the biochemical and morphological changes associated with the differentiation of X- to Y-cells. Proliferative activity is the specific characteristic of the immunologically activated Y-cell. It is assumed that after antigen effects the X to Y change, proliferation of the Y-cell may occur in the absence of antigen. If antigen persists or is readministered, then the Y-cell is restimulated for further proliferation and differentiation into the antibody-producing cell. This view is supported by experimental data to the effect that very small quantities of antigen may induce the X- to Y-cell change, accompanied by proliferation of Y-cells ( priming or preparation for the secondary response) without effecting a terminal differentiation of the Y-cell into an antibody-forming cell. It may be that the sequence of generations of activated cells ( Y1, Y,. . . . , Y , ) provides the basis for the heterogeneity of antibodies produced; succeeding generations of an activated cell need not necessarily be the precursors of Z-cells all producing qualitatively the same antibody molecules. Thus the nth generation of a given Y-cell may lead to the


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formation of a more highly avid antibody by the Z, cell than will result from the differentiation of an earlier generation of the same Y-cell line. In a somewhat similar manner, during the proliferation of a line of activated cells a shift might occur in a certain generation (e.g., Y, or Y6) which would result in the synthesis of a new immunoglobulin chain, just as occurs in hemoglobin synthesis (see p. 374). This may provide a partial explanation for the change in antibody synthesis from 19 to 7 S which may be realized in a single line of cells.

3. The Antibody-Producing Cell-The Z-Cell The Zcell results from the terminal differentiation of the competent X-cell and the activated Y-cell under the successive influence of antigen. The half-life of antibody-producing cells has been calculated by Vazquez (1964) and Sterzl et al. (1966b) to be between 2 and 3 days. The type of antibody produced by the Z-cell may depend on the type of its progenitor, i.e., which generation of activated Y-cell was stimulated by the antigen (e.g., Y,, Y2, or Yn). The producing cells which appear shortly after the fist stimulus (4 to 5 days after antigen injection) are mostly of the lymphocyte series [small, medium, and large type (Sterzl et al., 1965b)I. The highly productive Z-cell appears to be identifiable morphologically by the appearance of characteristic endoplasmic reticulum, and at least in the later stages of many of these cells by the developing characteristics of the mature Marschalko-type plasma cell.

4. The Significance of Dosage and Character of Antigen in the Stimulation of Immunologically Functioning Cells The dose of an antigen injected determines how many competent X-cells will be activated during the primary response and in what manner they will be affected. Thus, it conditions the preparation for the secondary response, on the one hand, and for the state of immunological inhibition (tolerance) on the other. The amount of antigen employed further determines how many activated Y-cells will be affected during the secondary response and determines the fate of these Y-cells either for proliferation or differentiation into Z-cells. The type of antigen employed is also very important in these respects. Thus, small amounts of easily and rapidly metabolized antigens may act primarily in the preparation for the secondary response, since they will then not be available in sufficient quantity to act further upon the Y-cells produced. In contrast, even smaller amounts of such dif3cultly metabolized antigens as lipopolysaccharides may persist through the X- and Y-cell stages, leading to terminal differentiation into Z-cells without ex-

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terisivc proliferation. This may account for the fact that such antigens lead primarily to 19s antibody formation. The character of the antigen may also influence the way in which it acts upon the Y-cell in the secondary response, as discussed below in connection with delayed hypersensitivity responses. Thus, although certain antigens may be able to induce both proliferation of Y-cells and their subsequent differentiation into antibody-forming Z-cells, other antigens may lack the intrinsic ability to mediate the conversion of Y-cells to Zcells and may therefore stimulate only the proliferative events associated with the secondary response.

5. The Primary Response It is postulated that the primary immune response involves the exclusive action of antigen on the competent X-cells to convert them into activated Y-cells. The quantitative and qualitative character of the primary antigenic stimulus may induce different results. A small dose of antigen injected as a true primary stimulus in the newborn or fetuses may result only in the X + Y transformation, called “priming”; this process provides the basis of the secondary response ( immunological memory). If a sufficient quantity of antigen is available (antigen persisting throughout the X + Y event), the complete reaction-without proliferation in the Y-cell compartment-may be realized (X + Y + Z). Two additional situations involved in the initial injection of antigen into an animaI need be discussed. In the first, the continuing presence of sufficient quantities of antigen allows the antigen to act further on the proliferated Y-cells (e.g., Y1, Y2, . . . ,Y,) and induces their differentiation into antibody-producing cells, either 7 S or the 19 S situations involving immunological memory. Therefore, it is not infrequent that a single administration of antigen will evoke simultaneously the primary and secondary response. In the second exception to this rule, relating to the observations of Uhr et al. (1962a), either the very small amounts of antigen employed or the quality of certain antigens may lead to a shortcircuiting of the Y compartment and the direct differentiation of cells from X to Z, causing the formation of 19s antibody without persisting immunological memory. 6. The Secondary Response

This reaction is characterized by the effect of antigen on the stimulation of cells in the Y compartment to further high proliferative activity and then generally to differentiation into antibody-forming Z-cells. It is probable that the reintroduction of antigen into a sensitized animal will generally result in the secondary response of Y-cells superimposed

DEVELOPMENTAL ASPECTS OF IMMUXIlT

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on a new primary reaction involving a new generation of X-cells. It may be, however, that the pure secondary response (without admixture of a primary response) can occasionally be obtained by the appropriate administration of low doses of antigen into a host with a high population of activated Y-cells.

7. Specific Cellular ( Delayed Hypersensitivity) Reactions It is assumed that these reactions involve the same cell lines and the same cellular developmental sequences as does the mechanism of antibody formation. In the absence of convincing evidence that these responses are accompanied by appreciable direct and specific destruction of cells or by the so-called “invasive destructive reaction,” it must be concluded that the principal immunological component of these responses is the action of antigen on perhaps a few immunologically activated cells leading to their proliferation and differentiation, perhaps with the release of biologically active substances which promote the appearance of still other nonspecifically functioning round cells. Delayed hypersensitivity and related responses have classically presented operational difficulties of interpretation, since ( 1) they have generally been studied in skin test sites which seem to be unrelated to events occurring in the lymphoid system, which has most generally been assigned the important role in immunocytological responses, and ( 2 ) they have usually been considered in terms of the two main prototypes of these reactions, the tuberculin response and contact dermatitis. In spite of this, however, a body of data has accumulated (see pp. 414-418) which suggested to many investigators that there might exist a relationship between these specific cellular responses and the mechanism of antibody formation. Thus, the suggestion recurs in the literature that delayed hypersensitivity might be an “early” or “immature” stage of the sequence of events in antibody formation. In the present treatment we postulate that delayed hypersensitivity skin reactions represent, in fact, secondary response occurring in “ectopic” locations, related directly to the secondary response observed in antibody formation. Sensitization for delayed reactions is assumed to involve the typical primary response of an X- to Y-cell transformation with proliferation of Y-cells. Under certain conditions and with certain antigens, the first exposure may involve also the Y to Z transformation with antibody formation. Under other conditions, and especially where the antigen possesses special characteristics or where an effort is made to suppress experimentally the antibody response by employing very small amounts of antigen or antigen-antibody complexes, the pure primary X to Y transformation is all that occurs. Elicitation of the delayed hy-

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persensitivity lesion in the local skin site then involves the action of antigen on the circulating Y-cells leading to their proliferation in situ. In the case of certain antigens, such as simple proteins, it is well known that the state of delayed sensitivity represents a preparation for a booster antibody response. and in fact the Y to Z transformation and local antibody formation by plasma cells can be seen at the skin test site, thus representing the full secondary booster response. There are certain other antigens, however, which seem incapable of mediating the Y to Z transformation, and therefore only stimulate the secondary response to the extent of inducing the further proliferation of Y-cells. Among these antigens are such denatured proteins as PPD, heated ovalbumin as described by Benacerraf and Gel1 (1959), and perhaps the histocompatibility antigens. The ability of such antigens to act only incompletely may account for the typical round cell response involving Y-cell proliferation without the presence of large numbers of antibody-forming plasma cells representative of the subsequent and perhaps separate Y to Z transformation. The relationship of this approach to that discussed by Pappenheimer et all. (1959) will be apparent.

8. Immunological Inhibition ( Tolerance) In a unitarian concept of immune responses, it is supposed that the inhibition of antibody formation by excessive doses of antigen may be achieved at any stage of cellular differentiation, based either upon a homogeneous or heterogeneous model (see p. 391). On the basis of the experimental data, we assume that at least some types of specific inhibition may result from a terminal exhaustive differentiation of immunologically competent cells. This is based on the finding that increasing doses of antigen do not result in a direct primary inhibition during true primary stimulation. On the contrary, increasing doses of antigen up to the saturation level stimulate all immunologically competent X-cells present in the organism. After a single large dose of antigen into the newborn, about one to ten antibody-producing cells per lofi lymphoid cells can be detected. Under these conditions, antigen activates the competent X-cell and drives it directly to the antibody-forming Z-cell stage, thus short-circuiting the Y-cell compartment. With excessive doses of antigen, the activated Y-cells present may also be driven without proliferation into a terminal differentiation to antibody-forming Zcells. After exhaustion of the Zcell population (half-life of about 2 to 3 days), only small numbers of X-cells will be available for a subsequent stimulation by antigen (probably again the original number of one competent X-cell per loGlymphoid cells, arising from some more primitive precursor). The situation thus described represents, therefore, a state of


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immunological inhibition, in comparison with that present in normal individuals in whom proliferation of Y-cells establishes the “normal” reaction level and persisting immunological memory. Immunological inhibition induced in adult individuals whose capacity to form antibodies may be exhausted by repeated administration of antigen is probably of this same character. The only difference here is that antigen may act upon a significant population of Y-cells. Depending upon the character and amount of antigen employed, antigen may act in some situations to push these cells into terminal differentiation with resulting tolerance, whereas in other situations their proliferation may be specifically stimulated, resulting in a boosted antibody formation.

REFERENCES Adinolfi, M., Daniels, Ch., and Mollison, P. L. (1963). Nature 199, 3891390. Adler, F. L. (1965). J. Immunol. 95, 28-47. Ainis, H. (1962). Nature 194, 197-199. Ainsworth, E. J., and Hatch, M. H. (1958). Radiation Res. 9, 84. Aisenberg, A. C., and Wilkes, B. (1965). Nature 205, 716-717. Aisenberg, A. C., Wilkes, B., and Waksman, B. H. (1962). J. Exptl. Med. 116, 759-772. Allen, P. Z., and Kabat, E. A. (1959). J. Immunol. 82, 340-357. Amano, T., Morioka, T., Seki, Y., Kashiba, S., Fujikawa, K., and Ichikawa, S. (1955). Med. J . Osaka Uniu. 6, 709-716. Amano, T., Morioka, T., Seki, Y., Kashiba, S., Fujikawa, K., and Ichikawa, S. (1956). Med. J. Osaka Uniu. 6, 1023-1026. Ambrose, C. T. (1964). J. Exptl. Med. 119, 1027-1049. Ambrose, C. T., and Coons, A. H. (1963). J. Exptl. Med. 117,1075-1088. Amin, A. (1961). Nature 191, 708. Amoroso, E. C. (1961). Brit. Med. Bull. 17, 81-90. Anderson, J. M., and Benirschke, K. (1962). Ann. N.Y. Acad. Sci. 99, 399-414. Anderson, J. M., and Benirschke, K. (1963). Transplantation 1, 306310. Archer, 0. K., Kelley, W. D., Papermaster, B. W., and Good, R. A. (1963). Federation Proc. 22, 599. Arnason, B. C., de Vaux St. Cyr, C., and Shaffner, J. B. (1964). J. Immunol. 93,915925. Aschaffenberg, R., Bartlett, S., Kon, S. K., Terry, P., Thompson, S. Y., Walker, D. M., Briggs, C., Cotchin, E., and Lovell, R. (1949a). Brit. J. Nutr. 3, 187-196. Aschaffenberg, R., Bartlett, S., Kon, S. K., Walker, D. M., Briggs, C., Cotchin, E., and LoveU, R. (194913). Brit. J. Nutr. 3, 196-204. Askonas, B. A., and Rhodes, J. M. (1965). In “Molecular and Cellular Basis of Antibody Formation” (J. Sterzl et al., eds.), pp. 503-512, Publishing House of Czech. Acad. Sci., Prague (distrib., Academic Press, New York). Askonas, B. A., Campbell, P. N., Humphrey, J. H., and Work, T. S. (1954). Biochem. J. 56, 597-601. Aspinall, R. L., and Meyer, R. K. (1964). In “The Thymus in Immunobiology” (R. A. Good and A. E. Gabrielson, eds.), pp. 376392. Harper (Hoeber), New York.

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Anti-antibodies PHILIP G.

H. GEL1 AND ANDREW S. KELUS

Department of Experimental Pathology, University o f Birmingham, Birmingham, England

I. Introduction . . . . . . . . . . . . . 11. Anti-antibodies in the Immunization Process and “Subcomplementarity” 111. Immunogenicity of “Altered” IgG. . . . . . . . . IV. Rheumatoid Factor and Rheumatoid Factor-like Antibodies . . V. Changes in the IgG Molecule Leading to the Production of Rheumatoid . . . . . . . Factor-like Antibodies: the Fc Piece VI. Distortion in the Fab Piece of IgG: Anticomplex Antibodies . . VII. “Natural” Anti-antibodies: Agglutinators . . . . . . VIII. Experimental Anticomplex Antibodies . . . . . . . IX. Molecular Location of Revealed Determinants . . . . . X. “Anticlone” Antibodies . . . . . . . . . . XI. Anticlone Antibodies by Heteroimrnunization . . . . . XII. Isoimmune Anticlone Antibodies (Idiotypes) . . . . . . XIII. Biological Significance of Anti-antibodies . . . . . . References . . . . . . . . . . . . .

461 463 465 466 467 468 469 469 471 471 472 473 475 477

1. Introduction

It is remarkable how comparatively recently the proteins of the plasma have been differentiated as physicochemical entities, and antibody activity has been shown to be the property of well-defined proteins rather than of the “serum” as a whole. Only for the last decade have the various classes of immunoglobulins and the possibility of interspecific differences as well as intraspecific differences between immunoglobulins of a single class been recognized. At the same time the concept that an animal may react immunologically against its own autogenous antigens has been generally accepted. An anti-antibody in the sense used here means an antibody which will react as such with an Ig molecule1 because that molecule is an antibody, not just because that molecule is a y-globulin. This does not imply that the antibody-combining site of that molecule is necessarily the locus of interaction, which has never been definitively shown. The word ‘We employ the terminology recently proposed (Nomenclature for Human Immunoglobulins, 1964 ) of immunoglobulins or their components, using the blanket symbol Ig when no particular class of immunoglobulin is referred to; though most of the work discussed in detail refers to “ordinary 7 S y-globulin,” IgG. 461

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PHILIP G . H. GELL AND AXDREW S. KELUS

“becausc,”in fact, covers three quite diffcrent situiiti~l~s. The first is

when anti-antibodies are elicitable because the antibody has reacted with antigen beforehand (in a way characteristic, as far as one can judge, only of antibodies) so as to expose, or to form de nouo, structures which, though potentially present in any Ig molecule. are not immunogenic in the intact molecule. The second is when anti-antibodies are elicitable because the antibody-combining site itself, in the intact molecule, is immunogenic and is involved in the interaction. The third is when there is an absolute correlation between the presence of a particular specific cornbining site and an immunogenic site elsewhere on the molecule. A fourth situation, strictly excluded by this definition, is when the antibodies all come from a highly restricted subclass of, say, IgG, but where there are molecules in this subclass capable of reacting with the presumed antiantibody yet which do not appear to possess antibody activity themselves. These may, of course, be antibodies of low or negligible affinity for the antigen but which are, in fact, present because of and produced by the original induction stimulus; or they may be quite unrelated to it. The earlier speculations and experiments on anti-antibodies were along the lines (1) whether heterologous antisera could be raised against antibodies as such (as opposed to the “normal” proteins of the serum) and ( 2 ) whether the change in immunoglobulin thought to be “induced” by antigen might itself be recognized as foreign by the animal in which it occurred and whether this might play a part in the immunization process. Pioneer work along these lines is discussed in that off-beat but stimulating book “Immuno-catalysis” by Sevag ( 1945). The earliest studies by Ehrlich, Bordet, Moreschi, and others were influenced by the idea that antibody was a substance arising de novo, unrelated in any way to the pre-existing proteins of the serum. Thus antisera that neutralized thc activities of antibodies ( Iysins, agglutinins) were termed “anti-antibodies.” By the time of the paper by Eagle (1930), antibodies were recognized as members of a class of serum protein. Smith and Marrack (1930) were the first to state clearly that, since antitoxin, when precipitated by a precipitin, still combines with toxin, different groups on the antitoxin molecules must be involved in the two reactions-a point confirmed by the studies of Eagle (1936). Treffers and Heidelberger (1941) in a study of horse antisera concluded that “the groupings responsible for the antibody function constitute either a small part of the total protein molecule or else are non-antigenic.” Other work on these lines (e.g., Ando et al., 1938; Northrop, 1942) is difficult to interpret, presumably owing to lack of knowledge at the time of the macroglobulin

ANTI-ANTIBODIES

4.63

(IgM) nature of some horse antibodies and of their possible nonreactivity or cross-reactivity with antisera to IgG. A simple-minded attempt to demonstrate anti-antibodies is, therefore, likely to encounter a number of difficulties. If heteroimmunization is used, ( 1 ) the bulk of the antibody will be species-specific and ( 2 ) antibodies specific to a class or even a subclass of Ig molecules may be confused with specific anti-antibodies. If isoimmunization is used, antibodies may be produced specific for genetic differences on IgG (or other proteins) not common to donor and recipient, i.e., to allotypic determinants. Since the bulk of our discussion here will be concerned with rabbit antibodies, and since the genetic situation with rabbit IgG allotypes (Dray et al., 1962) is fairly simple and well worked out, it is advantageous to outline at this stage the relevant data on this-not because antiallotypic antibodies are anti-antibodies in any sense used here, but because it is necessary to be aware of the allotypic situation whenever intraspecies cross-immunizations are used ( see review, Kelus and Gell, 1967). At present there are two well-defined loci, a and b, giving rise to allotypic determinants. The a-locus determinants are located on the heavy chain (Asl, 2, 3); the b-locus determinants (As4, 5, 6 ), on the light chain, and possibly also in the heavy chain (Feinstein et al., 1963). There is evidence for at least one other locus for IgG, and one for the heavy chain of IgM (Kelus and Gell, 1965). Quite extensive breeding data from both Europe and America for laboratory rabbits have not given any indication of further alleles at either the a or b locus. The blocus determinants are quite strongly immunogenic, although the use of some kind of adjuvant is usually needed to raise strongly precipitating antisera; the a-locus determinants are much less so, and adjuvants are always needed. A proportion of IgG molecules (10-20%) seems to lack determinants from one or the other locus. II. Anti-antibodies in the Immunization Process and “Subcomplementarity”

Before we consider more recent experiments on the direct elicitation of anti-antibodies, the work of Najjar (1963) and his colleagues may be considered. To this group must go the credit of having persistently drawn attention to the possible role of molecular distortions in the immune process, although the rather elaborate theoretical structure which they have erected upon their experiments is not acceptable to many workers today. Najjar considers that “when the first antibody” [to whose process of formation we shall return in a moment] “reacts in the antigenic site, both antibody and antigen are shown to be altered simul-

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taneousl!,. Antibodies are then formed against these sites of altered configuration. This process continues as further sites appear on the antigen and reacting antibody” (Najjar and Robinson, 1959). Thus the antibodies formed in the later stages of immunization would be to novel determinants not present in the “native” antigen, a proportion of them, indeed, to determinants not on antigen but on antibody. “After the third injection [of a native protein antigen] over 80% of antibody protein reacted with [an antigen-antibody complex made at equivalence] . . . leaving behind the remainder which reacted with antigen only” (Najjar and Fisher, 1955). The authors go on to advance as supporting evidence the work discussed below of Milgrom and Dubiski and others on rheumatoid factor-like anti-antibodies. Although it is difficult to accept either the authors’ experiments, or their supporting evidence, as being only, or even most readily, explainable in terms of their theory, this does not imply that the theory, first put forward at a time when autoantibodies were still hardly respectable, does not contain much truth. It is not quite clear whether the distortion of the antibody is thought of as being antigen-specific, which is hard to envisage in physicochemical terms, or a nonspecific result of antigen-antibody interaction, in which case the later stages of immunization should lack all specificity, since antibodies developed against one complex would react with all other complexes. Such antibodies are, indeed, sometimes demonstrable ( see below) but are produced only in special circumstances and not apparently as a general rule. Failure to eliminate allotypic differences between rabbits may make some experiments hard to interpret (Najjar and Fisher, 1955), though not those in which autostimulation is thought to have occurred (Harshman et aZ., 1963); however, application of newer knowledge of Ig structure and the use of the digerential power of gel-diffusion techniques to demonstrate antibodies of different specificities can bring a greater precision to such experiments. Najjar and co-workers go on to postulate that precipitation, in fact, occurs only when the “fit” of antigen and antibody is not exact (“subcomplementary”), so leading to some distortion. “Qualitatively there could then be three types of immune globulin synthesized in response to an antigen stimulus. The first type would seem to be those that are sufEciently sub-complementary to the antigen site to undergo configurational alterations, i.e. antibodies detectable by the usual immunochemical techniques.” [These are considered to arise because antibody is synthesized in relation to antigenic fragments, as discussed by Campbell (1957) and others, as well as to native antigen with its normal tertiary structure.] “To the second type belong the globulins that are comple-

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mentary to the antigen site and, therefore, not detectable under ordinary conditions. The third type falls between these two and includes a spectrum of molecules with varying degrees of sub-complementarity.” Though this theory can be used to explain certain types of cross-reactivity, the basic supposition that immunological precipitation is due, not to “complementary” antigen-antibody interaction leading to lattice formation, but solely to the uncovering of hydrophobic groups as a result of subcomplementary molecular distortion, would seem to ignore the whole history of immunochemistry from Landsteiner and Marrack onward. More recent studies of what conditions do, in fact, lead to molecular distortion, and in what parts of the molecule, have made the situation much clearer. Ill. lmmunogenicity of “Altered” IgG

This work entails five different kinds of experiments: ( a ) experiments with red cells or bacteria coated with antibody and injected into other animals of the same species (isoimmunization) or of a different species (heteroimmunization ) ; ( b) experiments in which antigen-antibody complexes are made in various ratios and used for immunizing other animals of the same species; ( c ) experiments in which IgG is altered in uitro and reinjected into the donor animal; ( d ) the demonstration in normal rabbit sera of IgM antibodies against rabbit IgG antibody which has reacted with antigen; and ( e ) strong immunization with any antigen may be found to give rise to antibodies reacting with Ig of one sort or another. By such experiments, four evidently different kinds of antibodies have been demonstrated, singly or in combination. 1. Antiallotypic antibodies, when isoimmunization by methods ( a ) or ( b ) has been used-such methods led, in fact, to the original demonstrations of IgG allotypes by Oudin (1956). Even where the known allotypes are identical in donor and recipient, a new allotype may be uncovered, the characteristic of this situation being that the preimmunization serum of the donor and of certain other rabbits will react with the antibody elicited. These antiallotypic antibodies need not be considered further here. 2. Rheumatoid factor-like antibodies [methods ( a ) , ( b ) , ( c ) , and ( e ) ]-these characteristically cross-react with Ig of species other than the donor. 3. Anticomplex antibodies [methods ( a ) and ( b ) ; allotypic differences being eliminated]- these can be shown to react with any antigenantibody complexes made with the Ig of the same species or with antibody-coated cells, but not with unreacted antibody. It is an open question

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whether “antigen-specific” anticomplex antibodies also occur. The ‘hatural” antibodies ( d ) fall also into this group. 4. “Anticlone” antibody [methods ( a ) and (b)]-these will react only with the actual antibody used as immunogen, not with preimmunization serum from the same animal-the antigen need not be in the form of a complex. Immunoconglutinin ( Coombs and Coombs, 1953)-although not an anti-antibody under our definition is an additional type of antibody, which is likely to be detectable if looked for with an appropriate test system and which, if unsuspected, may lead to misleading conclusions. However, it has not been recorded as precipitating antibody. It is evidently directed against activated autogenous complement, and occurs regularly as an accompaniment of any fairly intense immunization. Its properties have recently been fully reviewed (Coombs et al., 1961). IV. Rheumatoid Factor and Rheumatoid Factor-like Antibodies

Whether one considers rheumatoid factor ( RF) , as arising in rheumatoid arthritis and some related diseases in man, to be relevant to this discussion depends upon one’s opinion as to its origin. There seems little doubt that it is an IgM antibody induced by “damaged IgG, and it is possible, but not proved, that this damage arises because the IgG has reacted, as antibody, with some extrinsic, e.g., bacterial, antigen, and therefore the RF is brought within our definition of an anti-antibody. This hypothesis was put forward by Dubiski (1958). The extensive chemical and experimental work on human RF has been reviewed elsewhere (e.g., Glynn, 1963). Williams and Kunkel (1965) in a discussion of antiantibodies refer to a number of antibodies of this nature occurring in rheumatoid arthritis sera. Similarly the identification of the Gm and InV allotypes of man by means of normal ( SNagg) sera and rheumatoid (RF, Ragg) sera, is outside the compass of this review. We shall, therefore, not consider further the spectrum of antibodies in human RF sera except when showing analogies with those experimentally produced. Milgrom and Witebsky ( 19sO) showed that autoimmunization of rabbits with their own IgG precipitated with ammonium sulfate or alum (and presumably mildly denatured), in Freunds complete adjuvant, gave rise to precipitating and complement-fixing antibodies which reacted very much better with human than with rabbit IgG. McCluskey et al. (1%2), in a controlled study of various methods of denaturation, recorded the appearance of antibodies of this type. These results can be taken as possible models for the production of anti-antibody.

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The rheumatoid factor-like substance (RFLS) of Abruzzo and Christian (1961), arising from long immunization of rabbits with formalin-killed bacteria, showed somewhat different properties. This was a macroglobulin which reacted with immune complexes, made both with human and rabbit antisera, and with cells coated with heat-aggregated rabbit and human IgG. Cross-absorbtion showed that the rabbit IgG was the homologous, and human IgG the cross-reacting antigen; in these properties it shows an analogy with human RF. Aho and Wager (1961) and Williams and Kunkel (1963) described similar antibodies in rabbits both of 19 and 7s type, after immunization with various protein antigens. In man, RF-like antibodies may be demonstrable in many chronic diseases (as quoted by Abruzzo and Christian, 1961) and even transiently after prophylactic immunization with tetanus or diphtheria toxoid ( Svec and Dingle, 1965). V. Changes in the IgG Molecule leading to the Production of Rheumatoid Factor-like Antibodies: the Fc Piece

There is evidence that the precipitation of RF with heated IgG is the result of changes in the Fc piece of the latter (Henney and Stanworth, 1965b), involving S-S bond rupture and some aggregation. The Fc part of the molecule is also involved in complement fixation and in the irritant properties of antigen-antibody complexes ( Ishizaka and Ishizaka, 1964 ) . The RF-like antibodies induced by Milgrom and Witebsky (1960) and McCluskey et al. (1962) were evidently produced in response to Fc piece changes. This sort of change evidently gives rise to a determinant which is both autoimmunogenic and widely cross-reacting-as with other types of cross-reacting system, heated IgG from some species, e.g., rabbit, reacts very much better with human R F than does that from others, e.g., horse ( Henney and Stanworth, 1965a). Antigen-antibody combination may also give rise to similar Fc piece changes: it is as a result of some such process that the RF-like antibodies of Abruzzo and Christian are likely to have been provoked. Antigen-antibody complexes and reacted antibody on the surface of cells (as in the Rose-Waaler test) will for the same reason react with human RF. The changes induced by antigen-antibody union, however, are more complex than those induced by heat “denaturation.” (An unfortunate word to use in this connection, in that it often implies an “all-or-none” irreversible reaction, whereas the changes which we are considering are progressive, and, in the early stages at least, reversible.) In the first place, those changes occurring when antibody reacts with a soluble antigen are

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critically dependent upon the antigen (Ag)-antibody ( Ab) ratio of the complex formed, being maximal in moderate antigen excess, where the ratio is Ags Ab,. and probably minimal both at the gross antigen excess ratio ( Ag, Ab, Ag,,) and at equivalence. Reaction of antibody with a cell, e.g. ;in erythrocyte, will produce some distortional changes in antibody apparently independently of the ratio of the reactants, and even with soluble antigens there is some evidence that the larger the antigen the wider the range of ratios at which distortion occurs (Henney and Stanworth, 1966). The distortion of antibody on union at a critical ratio is reflected in increase in free -SH (Robert and Grabar, 1957) and in optical rotation (Ishizaka and Campbell, 1959). That the latter was due to changes in antibody rather than antigen was indicated by the fact that the increase in optical rotation was still demonstrable when an optically inactive antigen was used. Distortion of antigen does, however, sometimes occur, most elegantly shown in studies with enzyme-antienzyme systems, e.g., those of Pollock ( 1964) with antipenicillinases where clear-cut changes in substrate specificity and kinetics were demonstrable, as well as those investigated by Najjar (1963).

+

VI.

Distortion in the Fob Piece of IgG: Anticomplex Antibodies

Thus both physical treatment, and union with antigen, will produce alterations of the IgG (antibody) molecule in its Fc piece. In the latter situation there is evidence, however, that other and, perhaps, mere significant changes occur at the Fab end of the molecule. These changes are recognizable by altered immunogenicity, but antisera elicited by the altered molecules still react more strongly with homologous than with heterologous IgG. This is in contrast with the cross-reactivity of the Fc piece changes. The anti-antibodies so produced have the property of reacting with homologous antibody as it exists in a complex at antigen excess or attached to a cell, but not with heated IgG or with heterospecific IgG complexes. They are demonstrable, as discussed below, in a proportion of normal rabbit sera under special conditions, or as a result of experimental immunization with complexes formed in uitro. No unequivocal demonstration that this kind of anti-antibody arises as a result of true spontaneous autostimulation has apparently appeared, though the production of immunoconglutinin (Coombs and Coombs, 1953) provides an analogy. One might expect a course of repeated large injections of antigen, as in the experimental production of “serum sickness nephritis,” to provoke

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their appearance, but Boyns (1966) during experiments of this type has hitherto failed to find any antibodies of this nature. VII. “Natural” Anti-antibodies: Agglutinators

The “anti-antibodies” described by Milgrom and Dubiski ( 1957) were later identified by Dubiski et al. (1958) as probably antiallotypic antibodies. However, Milgrom ( 1962) described antibodies ( “agglutinators”) in a proportion of normal rabbits, which in a subsequent communication (Fudenberg et al., 1963) were found to be macroglobulins capable of reacting with antigen-antibody complexes made from antibody digested with papain and therefore lacking the Fc piece, as well as with rabbit red cells sensitized with rabbit isoagglutinin. Cohen and Tissot ( 1965) analyzing the latter situation, showed that only certain isoagglutinins could, on reacting with the red cells, reveal a site capable of combining with the agglutinator, and there was some evidence from family data that the “agglutinator trait” was under genetic control. Although it was not possible to decide whether the agglutinator resulted from specific stimulation or not, it would seem reasonable to refer to it as a “natural” antibody, by analogy with other “natural” isoagglutinins. VIII. Experimental Anticomplex Antibodies

Antisera were raised by isoimmunization against preformed soluble complexes made in vitro by Leskowitz (1960), but he concluded that the antibodies demonstrated were probably against allotypic determinants rather than against complexes as such. Henney et al. (1965) immunized rabbits with preformed bovine serum albumin ( BSA )-anti-B.S.A. complexes, the known allotypes of donor and recipient being identical, and analyzed the resulting antisera by gel diffusion. In this study no RF-like antibodies were produced, though antibodies were produced against the antigen contained in the complexes used as immunogen. There was no evidence of the production of antiallotypic or other antibodies reacting with the free antibody used to make the immunogenic complex, but true anticomplex (anti-cpx) antibodies could be demonstrated. By this we mean antibodies that will react on agar diffusion with complexes made between other rabbit antibodies and their respective antigens. The reaction of anti-cpx antisera with the immunizing complex ( BSA-anti-BSA) and with BSA alone was more complicated (see Fig. l ) , since the anti-cpx antiserum contained in addition much anti-BSA antibody-as one would expect when immunization was carried out with BSA-antibody complexes in antigen excess. Therefore, when the anti-cpx antiserum was diffused in agar against BSA,

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complexes were formed which then reacted in situ with the anti-cpx antibodies present in the same serum. However, the expected strong spur was formed when complexes made between another antigen and its antiserum were reacted in an adjacent cup. Further information was obtained as to the location in the IgG molecule of the new “determinant,” produced on complexing, in that, though no reaction was obtained between the anti-cpx serum and samples of fresh IgG nor with several samples of heated IgG capable of reacting with R F (via changes in the Fc piece), a definite reaction occurred

FIG.1. Diagram to illustrate the reactions of anticomplex antiserum. Note spur between homologous and heterologous systems; both anti-BSA and anticomplex antibodies pile up in this line. (BSA = bovine serum albumin.)

with the separated Fab pieces obtained by papain digestion and with a (heavy; 7 ) A-chain preparation made by the method of Fleischman et al. ( 1962). It was tentatively concluded that the determinants revealed by this study were in the Fab region of the heavy chain, contiguous to the antibody-combining site, revealed and made immunogenic as a result of the distortion produced by reaction with antigen. Two samples out of thirty of stored IgG, in which some aggregation had occurred, also showed the presence of this same determinant reacting with the anti-cpx serum. Thus though attempts to expose this Fab determinant experimentally by physical means were unsuccessful, one may suppose its appearance not to be absolutely confined to antigen-antibody reactions, but producihlc also 11y other forms of degradation, of unknown nature. A notable characteristic of the anti-cpx serum u7as that it reacted most strongly on gel diffusion with complexes in that ratio at which

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maximal distortion of the molecule occurred as judged by optical rotation and -SH group increases. Similar studies with ferritin (Henney and Stanworth, 1966) showed an essentially similar picture, except that in this case much RF-like antibody was elicited, and complexes made with this larger antigen molecule reacted over a larger range of AgAb ratios. (Here also an additional precipitation line appeared in the anti-cpx-ferritin diffusion pattern, which could possibly be due to antibodies against distortions on the antigen. ) Since, however, even antibody-BSA complexes are reactive with RF sera, one may assume that changes in the Fc piece normally occur coincidentally with changes in the Fab piece, though the former may not always elicit specific antibodies. IX. Molecular Location of Revealed Determinants

From the foregoing experiments it is clear that the distortion of the IgG molecule, either by physical means or as a result of its reaction with antigen, can lead to the appearance of immunogenic determinants, as postulated by Najjar. Different situations arise, however, depending upon whether these determinants are in the Fc or the Fab part of the molecule. Broadly we may say that the antibodies we have described as RF-like are directed against Fc determinants, whereas those described as anticomplex are against Fab determinants. If this general interpretation is correct, it suggests that the determinants on complexes are not created by the process of distortion of the chains, but merely revealed. If, as suggested by electron microscopy (Feinstein and Rowe, 1965), the two halves of the IgG molecule, each containing one antibody-active site, may be forced apart on reacting with antigen, it would not be surprising that areas on the H ( 7 ) chain, normally concealed within the molecule, should be exposed. That these should be potentially immunogenic implies that the normal self-tolerance to autogenous IgG extends to the whole molecule but not to its fragments. X. “Anticlone” Antibodies

The last type of anti-antibody is of a kind quite different from those which we have discussed hitherto, and its lucid description is a matter of some difficulty, owing to the complexity of the system, though in essence it is simple enough. Some preliminary discussion of the use of the word “clone” is, therefore, justified. It is now a commonplace that each myeloma protein, of man or of mouse, possesses an individual specificity which distinguishes it from all other myeloma proteins, and from the “normal” Ig of the same animal

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PHlLIP G . H. GELL AND ANDREW S . KELUS

(Kunkel, 1965). This specificity is quite independent of the specificity that determines light or heavy chain class, but resides in some other part of the H chain. These proteins are often referred to as “monoclonal”-an assumption based, perhaps, upon Burnet’s original postulate of a marked tendency to somatic mutation of Ig-producing cells, and hence on the possibility that any IgG molecule in the serum may be different in detail from all other IgG molecules. Burnet postulated that such differences arise by somatic mutation. The very word, therefore, implies a quite complicated and still controversial theory of Ig biosynthesis, which was devised by Burnet primarily to explain heterogeneity at the antibodycombining site, though without excluding it at other points on the molecule. Nevertheless the concept of a “clone” of cells is a helpful one to indicate the exceptionally limited specificity of certain determinants both on myeloma cells and on the types of antibody pinpointed by the anti-antibodies which we shall describe. As long as one is aware of the danger of allowing a word to direct the track of one’s thinking, and continuously alert to prevent its doing so, it is perhaps safe to use it. By an anticlone antibody w7e mean, therefore, an anti-antibody that will react with another antibody of defined specificity only, and not with normal IgG or other antibodies or complexes from that animal. There has been a gradation both in specificity and in geographical site of action in the types of antibody we have been describing, from the Fc end of the molecule to the Fab end in the region of the combining site- “anticlone” antibodies appear to be directed either to the combining site itself or to a part of the molecule specifically codetermined with it, though not necessarily determined by it. XI. Anticlone Antibodies by Heteroirnmunization

One would expect that immunization of one species with the antigenantibody complexes of another would produce antispecies IgG antibodies, perhaps some RF-like and anti-cpx antibodies, and possibly some antibodies to a limited class of heavy-chain IgG, and stop at that. The situation following the injection of human Ab-Ag complexes into rabbits was analyzed by McDuffie et al. (1958) and they showed that some antibody was only removable by the specific immunogen. Subsequently Kunkel et al. (1963) described rabbit antisera against isolated human antibodies which after full absorption with normal IgG reacted specifically with the immunogen, i.e., the human antibodies had individual specificity. The Fab piece of the molecule contained the reacting site.

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Under the conditions of the experiment it was not possible to demonstrate the absence of the reactant from preimmunization serum. XII. Isoimrnune Anticlone Antibodies (Idiotypes)

An anticlone antibody arising from isoimmunization was described independently by Oudin and Michel (1963) and Gell and Kelus ( 1964a), which arose out of routine attempts to identify new allotypes in rabbits. This kind of antibody has been called “idiotypic” by Oudin (1966). The normal way to raise antiallotypic sera is to take a donor animal of say As1/4 (genotype l,la/4,4b), and immunize it with a bacterial suspension (e.g., Proteus vulgaris). The antibody so raised is coated on a suspension of the bacterium, the coated suspension washed, injected into the recipient animal of allotype say AslJ5 (or any animal having As1 but not As4). Antiallotypic antibodies are then elicited in the recipient specsc for As4. The process of coating onto a bacterium both gives a painless way of separating the immunoglobulin of the donor, and adds (when Profeus vulgaris is used) a marked adjuvant effect presumably due to the endotoxin of Proteus vulgaris intimately associated with the antigen. Much the same effect can be achieved by making equivalence precipitates with antibody from a donor animal with any antigen, say BSA, and injecting the precipitate in Freund’s complete adjuvant (FCA ) (Oudin, 1956). The results to be discussed arose from using this process of immunization between animals of identical allotypes. We shall use for descriptive purposes our own notation and experiments, though the general methods and conclusions are similar to those of Oudin and Michel. Should there be undetected differences between say As4 determinants in various strains of rabbits, or “new” allotypes on other Ig classes, such methods should demonstrate them-indeed, in one such experiment a new allotype on macroglobulin (Msl) was identified (Kelus and Gell, 1965). But a large proportion of the immunized animals produced antibodies (R/a) which would react with the donor antiserum (D/a) only, and not at all with (1) preimmunization donor serum (D/o), ( 2 ) normal serum from progenitors or progeny of the donor, ( 3 ) antibody subsequently or simultaneously raised in the donor to other antigens, and ( 4 ) sera from over fifty other rabbits immunized against the same antigen as that used to immunize the donor (see Fig. 2). On the other hand, anti-antibodies of this type were elicited against the antibody of one donor (D/a) in several other animals; these all gave a reaction of identity on gel diffusion against D/a indicating that a single substance was being identified. Evidence was presented that this

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PHILIP G . H. GFLL Ah?) ANDREW S. KELUS

substance was reactive as antibody against an antigen of Proteus vulgaris and that all this antibody and only this antibody was the “antigen” reacting with R/a. Its electrophoretic mobility was shown to be restricted in the slow region of the IgG range. In a subsequent communication, serum from another donor was analyzed (Gell and Kelus, 196413) which contained a number of antibodies to various constituents of Proteus vulgaris-independent anti-antibodies corresponding to two and possibly three of these multiple components were seen. In the work in progress we have ourselves raised twelve D/a-R/a systems in pairs of homo-

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FIG. 2. Diagram to illustrate the reactions of anticlone antibodies. Pr-Proteus extract; RJa-antiserum of recipient 1 immunized against Dda; &/a-antiserum of recipient 2 immunized against Dda; Dda-donor 1 : anti-Proteus uulgoris antibody; D d o d o n o r 1: preimmunization serum; Ddb-donor 1: long postimmunization 2: anti-Proteus serum (after immunization with another antigen); Dda-donor antibody. (Note: In this diagram R,/a is shown as absorbed with Proteus bacilli so that no reaction occurs against Proteus extract.

allotypic rabbits: none of these D/a determinants were cross-reacting (Kelus and Gell, 1966). If the interpretation of these experiments is correct it would appear that rabbits can recognize individual determinants correlated with and wholly specific to particular antibodies in individual antisera. These determinants are not characteristic of antibodies from different rabbits against the same immunogen, as one might expect if the combining site to, say, a particular Proterls uulgaris component had just one mandatory amino acid composition and arrangement. On this ground we were hesitant in regarding the anti-antibody as being directed against the combining site itself, since one would suppose that all structures, defined

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by a pattern ot atomic radii and potential H bonds, which when functioning as antibody could be complementary to the combining site of a Proteus antigen, would when functioning as antigens provoke the same or at least a cross-reacting antibody. The simplest explanation would seem to be that all the cells producing the D/a antibody are derived from a single progenitor cell which has undergone at least two somatic mutations, one, on the Burnet hypothesis, to shape the antibody-combining site, another, quite independent one to shape the determinant recognized by the anti-antibody. The antibodycombining site may still be nonimmunogenic either from its position on the molecule or because it is entirely dependent on a labile tertiary structure of the Fab piece, which is not preserved in a macrophageprocessed fragment. The correlation of the immunogenic D/a determinants with a particular antibody specificity would then be a purely coincidental effect of random mutations in a single progenitor cell. If this is the case a particular type of D/a determinant will not be under genetic control. Clearly the decision as to whether the determinant on D/a recognized by this kind of anti-antibody is determined by or merely randomly correlated with the combining site will depend upon further chemical analysis. In the noninbred rabbits which we have used, there does not appear to be any simple genetic law controlling the production of D/a determinant in antibody: since among thirty progeny of 2 bucks producing D/a determinants (recognizable by R/a anti-antibodies), no reacting D/a determinants were demonstrable in the progeny sera before or after successful immunization with Proteus vulgaris ( Kelus and Gell, 1966). Nevertheless, the interpretation in terms of random somatic mutation and cloning may be oversimplified or wrong. An alternative hypothesis, in terms of multiple genes and their alleles capable of contributing elements in the structure of Ig (in particular of the Fd and possibly light chains ), among which antigen would “select” those put together in just the right way, is possible and easier to reconcile with the clear demonstration of genetic control of the capacity to respond to certain synthetic antigens ( Levine and Benacerraf, 1964; McDevitt and Sela, 1965). XIII. Biological Significance of Anti-antibodies

Autoantibodies to changes in the reacted antibody molecule, such as those of Abruzzo and Christian, may well play a part in potentiating immune elimination of antigen-especially when the antigen is weakly immunogenic and antibody production against it is poor, so that small

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antigen-excess complexes occur and are cleared rather inefficiently. A similar role has been postulated for immunoconglutinin (Coombs et uZ. 1961) . Whether autoanticlone antibodies can be formed is a much more complex problem. In our own recent work two animals were injected with their own antibody under conditions regularly effective in raising antiantibodies by isoimmunization, without any autoanti-antibodies being produced; but much more experimentation would be necessary to prove that this would never occur. Much speculation has been devoted to the question as to whether and how the body protects itself against (somatic) mutant cells in order to preserve its identity. It is hard to believe that it can regularly react to “private” determinants on its own antibodies, not so much because the process would be self-destructive as that it would lead to an infinite regress of anti-antibody production. If self-tolerance is to be invoked to explain this, then it must exist or be induced specifically to the possible private determinants on any single one of its possible antibodies-sinee the determinants are potentially immunogenic as shown by isoimmunization. Burnet’s clonal elimination theory could give a mechanism for this, but it would entail the elimination of a number of clones equal to the number of possible antibodies. If an antibody site is uniquely associated with a recognizable mutant sequence, then this number would be equal to the number of possible mutations associated with and determining the antibody character of the IgG molecule. Since all possible mutant sequences in this part of the molecule might crop up as antibodies they would all have to be rendered nonimmunogenic. UnIess the amino acid composition at or near this Combining site is very similar in all possible antibodies-which would contradict the demand for a wide range of heterogeneity there-one would expect this wholesale elimination of clones to eliminate response to a wide range of potential antigens. Clonal elimination, that is to say, would have to cope not merely with “autologous I g G but with all possible somatic mutants of IgG associated with the combining site. Alternatively one may admit that antibodies are potentially immunogenic in the host, but that their immunogenicity is very low. Hence thc infinite regress is cut short at an early stage-except, perhaps, in systemic lupus erythematosus, in which such high levels of IgG occur-and one is approaching a position very close to that taken by Najjar, as discussed above. In general it may be said that work on anticlone antibodies, with their uniquely limited specificity, could throw light on many unsolved problems in the analysis of the genesis and mechanism of the immune response.

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REFERENCES Abruzzo, J. L., and Christian, C. L. (1961). J. Ex&. Med. 114, 791. Aho, K., and Wager, 0. (1961). Ann. Med. Exptl. Fenniae (Helsinki) 39, 79. Ando, K., Takeda, S., and Ilamano, M. (1938). J. Immunol. 34, 303. Boyns, A. R. (1966). Pb.D. Thesis, Birmingham University. Campbell, D. H. (1957). Blood 12, 589. Cohen, C., and Tissot, R. G. (1965). J. Immunol. 95, 273. Coombs, A. M., and Coombs, R. R. A. (1953). J. H y g . 51, 509. Coomhs, R. R. A., Coombs, A. M., and Ingram, D. G. (1961). “The Serdogy of Conglutination and its Relation to Disease.” Blackwell, Oxford. Dray, S., Dubiski, S., Kelus, A., Lennox, E. S., and Oudin, J. (1962). Nature 195, 785. Dubiski, S. (1958). Folia Biol. (Cracow) 6, 47. Dubiski, S., Dudziak, Z., and Skalba, D. ( 1958). Proc. 7th Intern. Congr. Microbial., 1958, Almquist & Wiksell, Uppsala, p. 201. Eagle, H. (1930). J. Immunol. 18, 393. Eagle, H. (1936). J . Immunol. 30, 339. Feinstein, A., and Rowe, A. J. (1965). Nature 205, 147. Feinstein, A., Gell, P. G. H., and Kelus, A. S. (1963). Nature 200, 853. Fleischman, J. B., Pain, R. H., and Porter, R. R. (1962). Arch. Biochem. Biophys. Suppl. 1, 174. Fudenberg, H. H., Goodman, J., and Milgrom, F. (1963). Transfusion 3, 422. Gell, P. G. H., and Kdus, A. S. (1964a). Nature 201, 687. Gell, P. G. H., and Kelus, A. S. (1964b). “Molecular and Cellular Basis of Antibody Formation” (J. Sterzl et aZ. eds.), p. 201. Publishing House of Czech. Acad. Sci., Prague (distr., Academic Press, New York). Glynn, L. E. (1963). In “Clinical Aspects of Immunology” (P. G . H. Cell and R. R. A. Coombs, eds.), p. 593. Blackwdl, Oxford. Harshman, S., Robinson, J. P., and Najjar, V. A. (1963). Ann. N.Y. Acad. Sci. 103, 688. Henney, C. S., and Stanworth, D. R. (1965a). In “Protides of Biological Fluids,” (H. Peeters, ed.), p. 155. Elsevier, Amsterdam. Henney, C. S., and Stanworth, D. R. (1965b). Immunology 9, 139. Henney, C. S., and Stanworth, D. R. (1966). Nature 210, 1071. Henney, C. S., Stanworth, D. R., and Gell, P. G. H. (1965). Nature 205, 1079. Ishizaka, K., and Campbell, D. H. (1959). J . Immunol. 83, 116. Ishizaka, K., and Ishizaka, T. (1964). J. Immunol. 93, 59. Kelus, A. S., and Gell, P. G. H. (1965). Nature 206, 313. Kelus, A. S., and Gell, P. G. H. (1966). In preparation. Kelus, A. S., and Gell, P. G. H. (1967). Progr. Allergy 11 (in press). Kunkel, H. G., (1965). Harvey Lect. Ser. 59, 219. Kunkel, H. G., Mannik, M., and Williams, R. C. (1963). Science 140, 1218. Leskowitz, S. (1960). J. Immunol. 85, 56. Levine, B. B., and Benacerraf, B. (1964). J . Erptl. Aled. 120, 955. i’vIcCluskey, R. T., Miller, F., and Benacerraf, B. (1962). J. Erptl. Med. 115, 253. McDevitt, H. 0. and Seln, W., (1965). J. Exptl. Med. 122, 517. McDuffie, F. C., Kabat, E. A., Allen, P. Z., and Williams, C. A., Jr. (1958). J. Immunol. 81, 48.

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hlilgrom, F. ( 1962). Vox Sanguinis 7, 545. Milgrom, F., and Dubiski, S. (1957). Nuture 179, 1351. Milgrom, F., and Witebsky, E. (1960). J. Am. Med. Assuc. 174, 56. Najjar, V. A. (1963). Physiol. Rev. 43, 243. Najjar, V. A., and Fisher, J. (1955). Science 122, 1272. Najjar, V, A,, and Robinson, J. P. (1959). in “Immunity and Virus Infection” (V. A. Najjar, ed.), p. 71. Wiley, New York. Nomenclature for Human Immunoglobulins. ( 1964). Bull. Wmld Health Organ. 30, 447. Northrop, J. H. (1942). J . Gen. Physiol. 25, 465. Oudin, J. (19513). Compt. Rend. Acud. Sci. 242, 2489, 2606. Oudin, J. (1966). Proc. Roy. SOC. B (in press). Oudin, J., and hfichel, M. (1963). Compt. Rend. Acad. Sci. 257, 805. Pollock, 51. R. (1964). Immunology 7, 707. Robert, B., and Grabar, P. (1957). Ann. Inst. Pasteur 92, 56. Sevag, hl. C. ( 1945). “Immuno-Catalysis.” Thomas, Springfield, Illinois. Smith, F. C., and blarrack, J. R. (1930). Brit. J . Exptl. Puthol. 11, 494. Svec, K. H., and Dingle, J. H. (1965). Arthritis Rheumut. 8, 524. Treffers, H. P., and Heidelberger, M. (1941). J. Erptl. Med. 73, 125, 293. Williams, R. C., Jr., and Kunkel, H. G. (1963). Proc. Soc. Exptl. Biol. 112, 554. Williams, R C., Jr., and Kunkel, H. G. (1965). Ann. N.Y. Acad. Sci. 124, 860.

Conglutinin and lmmunoconglutinins P. J . LACHMANN Deporfmenf of Pofhology. University of Cambridge. Combridge. Englond

. . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . I1. Nomenclature A. Complement . . . . . . . . . . . . B. Conglutinin and Immunoconglutinins . . . . . . I11. Conglutinin . . . . . . . . . . . . . A. Isolation of Conglutinin from Bovine Serum . . . . . B. Properties of Bovine Conglutinin . . . . . . . C . The Reactivity of Conglutinin . . . . . . . . D . The Distribution of Conglutinin among Different Spccies . . E. Analogy with Other Serum Factors . . . . . . . . . . . . . . . . IV. The Conglutinogen of Yeast A. Conglutinogen in Yeast Cell Walls . . . . . . . B . Conglutinogen in the “Microsomal Fraction” of Yeast . . . C . Chemical Stability of Yeast Conglutinogen . . . . . D . Anticomplementary Activity of Yeast Conglutinogen . . . E . Antigenicity of Yeast Conglutinogen . . . . . . . . . . . . . . . . . . V. Immunoconglutinins A . The Stimulation of Immunoconglutinins . . . . . . B. The Nature of Immunoconglutinins . . . . . . . C . The Isolation of Immunoconglutinins . . . . . . D . Analogy with Rheumatoid Factors . . . . . . . VI . The Reaction of Conglutinin and Immunoconglutinins with Fixed Com. . . . . . . . . . . . . plement . A . The Reaction Sequence in Complement Fixation . . . . B. “Resuspension” and Sedimentation Pattern Techniques for Measuring Conglutination . . . . . . . . . . C . The Specificity of Conglutinin and Immunoconglutinins toward the Components of Fixed Complement . . . . . . . D . The Preparation and Properties of Conglutinable Intermediates from Different Species . . . . . . . . . . E . The Heterogeneity of the Reactants in Fixed C‘3 . . . . F. Inhibition of Conglutination by Sugars . . . . . . C . The Chemical Stability of Conglutinogen and Immunoconglutino. . . . . . . . gens in Fixed Complement H . The Liberation of Conglutinogen and Immunoconglutinogens from a C‘3 Fraction of Human Serum . . . . . . . . . . . . VII . Conglutination as a Serological Tool . . . . . . VIII . The Bidogical Significance of Conglutination . A . Mechanisms That May Be Involved . . . . . . . 479

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B. The Variations of Conglutinin and Immunoconglutinin Titers in Disease

IX. Summary References

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Introduction

The phenomenon of conglutination came to light during the controversy between the Ehrlich and the Bordet schools on the nature of complement action, when the powerful clumping by bovine serum of erythrocytes which had reacted with antibody and complement was &st observed (Bordet and Gay, 1906). The component in bovine serum giving rise to the phenomenon was given the name conglutinin by Bordet and Streng (1909). During this early period there was considerable controversy about the existence and nature of conglutination and its distinction from other agglutination reactions. At this distance of time it is no longer easy to understand why this controversy should have survived Streng’s development of a conglutinating complement-fixation test ( Streng, 1910, 1911), but echoes of it appear in the literature for another 50 years (Maltaner and Johnston, 1921a,b; Eagle, 1930; Gyorffy, 1932a,b, 1933, 1934; Osler, 1961). The resemblance between conglutinin and an anticomplement antibody led Streng (1930) and Wartiowaara (1932) to produce substances with similar activities by immunizing rabbits with “alexinated bacteria (or erythrocytes) i.e., ones that had reacted with antibody and complement. To these artificially stimulated substances producing conglutination the name of immunoconglutinin was given-with the inference that conglutinin was a naturally occurring antibody of similar specificity. It should be pointed out that Streng’s immunoconglutinin would produce conglutination of bacteria distinct from those used in the immunization, alexinated with a different species of complement. They were not simply antibodies to foreign serum proteins. The phenomenon was reinvestigated extensively by Coombs and his collaborators and the results obtained in his laboratory with a review of other relevant work were published as a monograph (Coomhs et al., 1961). For this reason no extensive review of the literature before 1961 is to be attempted here. These workers showed that the reaction of conglutinin and immunoconglutinins \\as against fixccl complement only and that the preseiice of free complement components in solution did not inhillit the reaction, and suggested on this basis that antigenic sites hidden in complement

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molecules became exposed when complement was fixed and that it was with these newly exposed determinants that conglutinin reacted (Coombs, 1947). This is, to the best of my knowledge, the first formulation of the concept of “hidden determinants” whose validity has been widely confirmed since-and not only in relation to fixed complement. They also pointed out that immunoconglutinins could be stimulated not only by the injection of material previously alexinated with heterologous complement ( a process to which they gave the name “heterostimulation”) but also by injection of bacteria alone ( a process to which the name “autostimulation” was given ) . This observation had originally been made by Wartiowaara (1932). In the latter case the presumption is that it is the animal’s own complement fixed on the bacteria (after antibody has been formed) which is the antigen, and Coombs and Coombs ( 1953) proposed that autostimulated immunoconglutinin should be regarded as an autoantibody. Subsequently (Coombs et al., 1961), when evidence had been obtained that immunoconglutinin raised the resistance of an animal to certain infections, this concept was extended to that of a “physiogenic autoantibody” thus providing one example supported by experimental evidence for the idea that autoantibodies may have a useful role (Grabar, 1963). The growing knowledge of the nature of the complement system and the improved immunochemical techniques becoming available in recent years have made it possible to study the conglutination phenomenon in greater detail in regard both to the properties of the naturally occurring conglutinin and of the various types of immunoconglutinin and to the specificity of their interaction with fixed complement. The picture of conglutination-as a manifestation of complement activity, as a biologically important autoantibody system, and as a serological tool-that is growing up in the light of this newer information forms the subject of this review. II.

Nomenclature

A. COMPLEMENT The nomenclature of the complement system is at the present time in a confused state, particularly with respect to the “classical C’3” components, where the terms used by those working with guinea pig complement are different from those used by those working with human complement. Although there is no absolute proof that the factors in these

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two complements are identical, the terminology to be used throughout this review will be that used by Miiller-Eberhard and his collaborators (Polley and Miiller-Eberhard, 1967) for human complement: E, erythrocyte, the antigen (the term S for “antigenic site” is a frequently used alternative) ; ,4,antibody; E-4, erythrocyte-antibody complex or “sensitized red cell”; C’, complement; C’EDTA, and C’H62, etc., complement reagents, the suffix describing the treatment to which fresh serum as source of C‘ has been exposed. Thus in the example given C’EDTA is whole serum in 0.01hl EDTA, a reagent which acts as source of all the “classical C’3” components; and C’H62 is whole serum heated at 62°C. for 20 minutes ( a t pH 8.5) which, in t h e case of guinea pig complement, serves as a source of C’3 alone of the “classical C’3” components (Klein and Wellensiek, 1965). R1, R2, R3, and R4, complement reagents specifically depleted in one particular component, the suffix describing the absent complement component. In practice, the content of complement components in the commonly used R reagents is less straightforward than their designation would suggest and also varies from species to species. It would, in fact, be more satisfactory, for example, to call ammonia (or hydrazine)-treated serum C’NH, (or C”H,NH,) than to call it R4, but the latter name is so well established that it will be used. 1. Complement Components These components are listed in the order of their interaction with EA: C’1 is made up of three subcomponents (C’lq, C’lr, C’ls) but occurs in fresh serum as a single macromolecular component (Naff et al. lW)-C’lq is equivalent to the 11 S component or C’O of human complement; C’1 is the proesterase; C’4 is identified with P,,-globulin in human serum ( Miiller-Eberhard and Biro, 1963); C’2; “classical C 3 ” is the complement component acting in the absence of divalent cations and producing lysis of EAC’142. It is made up of (at least) the following six subcomponents: ( 1) C’3, also called C‘3a (Klein and Wellensiek, 19%) and C’3c (Linscott and Nishioka, 1963); this is the hydrazine-sensitive moiety of classical C’3 and has been identified with plC-globulin in human serum ( Miiller-Eberhard, 1961) (2-4). Three further components, C’5, C‘6, and C’7, are required to convert EAC’1423 to the “heat stable” alexinated intermediate. These appear to correspond to C’3b and C’33p of Klein and IVellensiek (1965) and to C‘3b, C’3e, and C‘3f of Nelson et aE. (1966). C’5 has been identified with @,,-globulin in human serum (Nilsson and Miiller-Eberhard, 1965). (5-6) Two components, C’8 and C’9 are needed to complete the hemolytic sequence. These appear to


483

correspond to C’3c and C’3d of Wellensiek and Klein (1965) and C’3a and C’3d of Nelson et al. (1966). 2. Alexinated Intermediates

These are designated in the form EAC’ suffixed with the fixed components, e.g., EAC’142. The further suffix “a” is often used to denote that complement component is fixed in an activated form, e.g., EAC’la42a for EAC’142. At the present time this tends to cause confusion with subcomponents such as C’3a. B. CONGLUTININ AND IMMUNOCONGLUTININS Conglutinin and immunoconglutinins are abbreviated K and I-Ks, respectively; I-K refers to “autostimulated I-K except where otherwise stated. Although not strictly logical in terms of English spelling these abbreviations have the merit of avoiding confusion with complement and-in respect to I-K anyway-can claim a respectable lineage deriving from the original work of Streng (1930). In discussing the antigens, or more properly the “reactants,” with which conglutinin and immunoconglutinin react, the terms conglutinogen ( abbreviated K-gen) and immunoconglutinogens ( abbreviated I-K-gens ) (Lachmann and Coombs, 1965) are used. The term “conglutination” is used to describe the specific clumping of alexinated material whether this is brought about by K or I-K. 111.

Conglutinin

The conglutinating activity normally found in cattle serum is now known to stem not from the presence of naturally occurring antibody to determinants in fixed complement but from the presence of an antigenically specific protein with unusual physicochemical properties which is unrelated to the immunoglobulins and reacts with its, predominantly polysaccharide, “antigen”-the “cong1utinogen”-through the mediation of calcium ions (Lachmann, 1962; Lachmann and Coombs, 1965). On this basis an analogy with C-reactive protein has been drawn, since this factor too is an antigenically specific protein reacting with a polysaccharide “antigen” through the mediation of calcium ions. Conglutinin does not, however, share the property of C-reactive protein of being an “acute phase reactant.” CONGLUTININ FROM BOVINE SERUhl For a protein that is present in serum at rather low concentration (in the region of 50 pg per milliliter), conglutinin is relatively easy to obtain A.

ISOLATION OF

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in fairly pure form in that the isolation procedure can be based primarily on its specific reactivity ( Lachmann, 1962). The reactivity employed, which is discussed fully in a subsequent section, is not that with fixed complement but that with the yeast cell wall preparation-zymosan. This reaction requires the presence of no other serum components but is mediated by calcium ions. Thus, if zymosan is reacted with bovine serum, well washed, and then eluted with ethylenediaminetetraacetate ( EDTA ) , the eluate contains the conglutinin in a greatly enriched form. This may then be harvested by precipitation as a euglobulin. The bovine serum is heat-inactivated before use. This prevents fixation of complement components on the zymosan with subsequent elution of C’1 on treatment with EDTA. It would also destroy any antibodies of the type described by Pernis et al. (1963) in baby rabbits which are calcium-dependent, zymosan-absorbable, and hcat-labile. Yokohari and Leon (1965) reported the isolation of calciumdependent antibodies (both Ighif and IgG) from bovine serum by EDTA elution of polysaccharide-antipolysaccharide aggregates but do not give details of heat stability or reaction with zymosan. The conglutinin preparation as prepared above is usually contaminated with lipid, some lipoprotein, and occasionally with a little of a macroglobulin component as recognized by immunoelectrophoresis. Zymosan is very strongly clumped by conglutinin and most of this contaminating material is coagglutinated, the degree of contamination varying with the efficiency of washing. If at this stage the preparation is centrifuged at 20,000 g for an hour to remove lipid and then given a further cycle of absorption and elution from zymosan, a preparation that gives a single line on immunoelectrophoresis is obtained (Lachmann and Richards, 1964). The same zymosan can be used repeatedly. Reduction of zymosan with mercaptoethanol followed by alkyIation enhances (often considerably) its reactivity with conglutinin ( P. J. Lachmann, unpublished observations, 1966). Defining one dose of conglutinin as the minimum required to give a positive in the standard test system, the activity to protein ( A / P ) ratio of conglutinin preparations are given in Table I. The repurified preparation behaves physicochemically as a single protein. When used to immunize a rabbit it produces, after repeated c o m e s of injection, three contaminating lines of precipitation when the antiserum is diffused against electrophoresed bovine serum ( Lachmann and Richards, 1964). These trace contaminants can be further reduced by making use of the higher assymetry of conglutinin which causes it to be totally excluded from Sephadex G-200 but to sediment in a sucrose density gradient with the 7 S material (Section 111,B,2). By applying


485

these procedures to zymosan-purified conglutinin, preparations of very high purity can be obtained ( A / P 100,00&125,000). A further procedure ( P. J. Lachmann, unpublished observations, 19W) that has recently been found very useful in the purification of conglutinin depends on its resistance to mercaptoethanol. If bovine TABLE I APPROXIMATE ACTIVITYTO PROTEIN RATIOSOF CONGLUTININ PREPARATIONS A/Pa

Conglutinin

Bovine serum (with good titer of conglutinin) Bovine euglobulin Conglutinin preparation cycled once on zymosan Conglutinin preparation cycled twice on symosan a

70 700 30,000 100,000

Values in doses per milligram of protein.

euglobulin-or any source of conglutinin-is treated with 0.1 M mercaptoethanol and then fractionated on Sephadex G-200 equilibrated in a buffer containing 0.01 M EDTA and 0,025M mercaptoethanol, conglutinin will be found in the exclusion peak whereas all the immunoglobulins (and complement components) are eluted later in the fractionation. When the exclusion peak is precipitated as a euglobulin, the precipitate washed, and then redissolved at physiological pH and ionic strength, it is found that whereas the great bulk of the conglutinin goes freely into solution, most of the contaminating material-including the lipid material which is generally the most persistent contaminant-has been rendered quite insoluble. The conglutinin solution is then allowed to reoxidize by dialysis against saline through which air is bubbled. Bovine euglobulin fractionated in this way yiclded a conglutinin preparation with an A/P ratio of up to 50,000. Being free of immunoglobulins and complement components, this preparation is suitable for most purposes. Conglutinin preparations cycled once on zymosan can be substantially further purified by merely treating with mercaptoethanol and reprecipitating as the euglobulin. However this combination of a cycle of preparation on zymosan-which allows large volumes of serum to be handled-followed by Sephadex G-200 filtration in mercaptoethanol and subsequent reoxidation is perhaps the readiest method for obtaining conglutinin of very high purity ( A / P 100,000). Taking an A/P ratio of around 100,000 as the value for pure conglutinin, it can be calculated that the conglutinin concentration of a bovine serum with a high conglutinin titer (say 1OOO) is around 5 mg./ 100 ml. (Lachmann and Richards, 1964).

>

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P. J. LACHMANK

Taking the molecular weight of conglutinin as 750,OOO (Section III,B,2), one “dose” of conglutinin in the standard test system can be pmoles. calculated to correspond to 1x I am indebted to Dr. D. G. Ingram for bringing to my notice the work of Fesce (1961a,b) who independently had used the technique of absorption of bovine serum (although unheated) onto zymosan followed by elution by removing calcium ions to purify conglutinin. Few details of his product are given but electrophoretically it contained “only gamma globulin.” A different method of purification was used by Sage et al. (1963). These workers prepared an antigen-antibody-C’-conglutinin complex using dextran as antigen and fresh bovine serum as source of all the other components. They extracted the precipitate with EDTA and then fractionated the eluate by two cycles of diethylaminoethyl (DEAE)cellulose chromatography. Their final product was purified 1000-fold compared with bovine serum and showed three lines of precipitation in an Ouchterlony plate with an antiserum prepared against the crude EDTA eluate.

B. PROPERTIES OF BOVINE CONGLUTININ

1. Solubility Conglutinin is a euglobulin and is precipitated by 33% saturation ammonium sulfate (Coombs, 1947). It is soluble at physiological ionic strength and pH to a concentration of (at least) 5 mg. per milliliter. It is more readily soluble in the presence of EDTA (Sage et al., 1963). 2. Sedimentation and Diffusion Sage et aZ. (1963) quote a value of s:O0,,,. of 6.6 S and a molecular weight of 650,000 obtained by the method of Trautman and Crampton { 1959). Lachmann and Richards (1964) found a value of s!o,w of 7.8 S with marked self-sharpening of the ultracentrifugal peak and extreme dependence of the sedimentation coefficient on concentration: szovw= S L ,-~ 5.5 C,where C is the protein concentration in grams per 100 ml. Measuring the diffusion coefficient by the L-plate diffusion method of Allison and Humphrey (1960) gave a value of D = 0.9 x lo-’ cm2 per second. From the amino acid composition a value of 0.72 was calculated for the partial specific volume. These parameters give a value for the molecular weight of about 750,000; and for the frictional ratio ( f / f o ) of around 4.


487

A frictional ratio of 4 denotes a degree of molecular asymmetry that is comparable with that of myosin but quite exceptional for a serum protein. Using Perrin’s equations, an axial ratio may be calculated from the frictional ratio which is found to be in the region of 100. This calculation is based on the assumption that molecular asymmetry alone is responsible for the high frictional ratio and that there is no hydration of the molecule. The calculation, therefore, gives a value that is certainly too high but does provide an upper limit. The self-sharpening of the peak in the ultracentrifuge and the steep dependence of sedimentation coefficient on concentration are both features found with highly asymmetrical molecules. In sucrose density gradient zone ultracentrifugation molecules band according to their sedimentation coefficient, and conglutinating activity is to be found in the “7s” region of bovine serum. If concentrated conglutinin solutions (say 5 mg. per milliliter) are used the sedimentation coefficient is about 5 S and the conglutinin band is found even higher in the tube. On the other hand, molecules are excluded from Sephadex gels probably as a function of their diffusion coefficient, and conglutinin with a diffusion coefficient of 0.9 is completely excluded from Sephadex G-200. Thus the sequential use of these two procedures on a source of conglutinin (Lachmann and Coombs, 1965) allows the conglutinin to be separated from relatively symmetrical molecules of all sizes within the range of the fractionation. In the case of serum this includes not only all the immunoglobulins but also most of the other proteins. 3. Electron. Microscopy

Electron microscopy provides an alternative to hydrodynamics in the study of molecular size and shape. A conglutinin preparation was, therefore, examined in the electron microscope using 20% sodium tungstoborate as negative stain and “holey films” (Lachmann and Cruickshank, 1966). A difficulty was encountered in that the material tended to aggregate during handling. However a number of structures believed to be single molecules could be distinguished especially if the grids were prepared at low-protein concentration. These structures were elongated rods about 450 A. long. Their width was difficult to measure accurately but was between 30-60 A. Similar structures were not seen when trypsin-treated conglutinin was used. These measurements correspond to a molecular volume (assuming the molecule to be cylindrical) of 3 to 13 x 105 A.3 and an axial ratio of 8 to 15.The molecular volume calculated from a molccular weight of 750,000 and a partial specific volume of 0.72 (i.e., specific gravity 1.4) comes to about 9 x lo5 A.3, showing that the observed

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P. J . LACHMANN

structures are of the right order of size. The calculated axial ratio, as anticipated, is considerably more than that observed in the electron microscope and hence it may be concluded that conglutinin is markedly hydrated in solution. There was no evidence of beading, as seen in fibrinogen (Haggis, 1964). 4. Antigenic and Electrophoretic Properties

For the study of the antigenic nature of conglutinin, an antiserum raised in rabbits against zymosan conglutinated with heat-inactivated bovine serum and then well washed has proved particularly valuable (Lachmann, 1962). This antiserum contains powerful precipitins not only to conglutinin but to the whole range of bovine immunoglobulins as described by Pierce and Feinstein (1965) and to several unidentified bovine serum proteins. A highly purified conglutinin preparation gives only a single line of precipitation with this antiserum as it does with an antiserum to alum-precipitated bovine globulins and with an antiserum to the conglutinin preparation itself. This line shows complete nonidentity with any other line of precipitation given by these antisera with whale bovine serum. Precipitating the antiserum at optimal proportions with purified conglutinin yielded a supernatant containing neither detectable conglutinin activity nor the ability to form the conglutinin precipitation line when difhsed against electrophoresed bovine euglobulin; thus demonstrating the identity of the conglutinin activity with the antigen producing the precipitin line. In all immunodihsion experiments with conglutinin the agar should be thoroughly dialyzed against dilute EDTA to prevent the interaction between agar and conglutinin which occurs in the presence of calcium ions ( Lachmann, 1962; Section II,C,2). The position of the conglutinin line in immunoelectrophoresis on agar is in the region of the origin and lies within the IgM line. On Pevikon block electrophoresis, conglutinin activity is found just to the anode side of the main Forssman antibody activity. For these reasons conglutinin was considered to be a P-protein (Lachmann, 1962). Sage et al. (1963) measured the electrophoretic mobility of conglutinin by free electrocm.?/volt-seconds at pH phoresis and obtained a value of -1.86 = 8.6 and considered conglutinin, therefore, to be a y-globulin. The difference may be only semantic. However, the electrophoretic mobility of highly assymetric molecules shows a dependence on concentration ( Abramson et al., 194%),and measurements made at appreciable protein concentrations may give anomalous results. This may explain the diffcrences between the free eIectrophoresis measurement done at high-con-


489

glutinin concentration and the agar or Pevikon block electrophoresis done at a negligible conglutinin concentration.

5. Optical Rotation Sage et al. (1963) state that “optical rotary dispersion data showed that conglutinin had a very low &-helixcontent.” 6. Chemical Composition of Conglutinin

The amino acid composition of conglutinin is given by Lachmann and Coombs (1965). The only noteworthy feature is the relatively high content of glycine (and to a lesser extent, proline), These amino acids are, of course, those found in very high quantity in many fibrous proteins, and one may picture that this amino acid composition to some extent reflects the molecular asymmetry. The carbohydrate content of conglutinin is less than 1%.

7 . Chemical Stability of Conglutinin These studies were carried out on purified conglutinin, and the effect of the treatments listed below tested on its reactivity with alexinated cells, on its appearance as an antigen in immunoelectrophoresis, and (only where stated) on its sedimentation and diffusion behavior (Lachmann and Coombs, 1965). Judged by these criteria conglutinin resisted the following treatments: a. Heating at 56°C. for 30 minutes. b. Treatment with 0.1 A.l ammonia. This treatment destroys C’4 and C’3. c. Reduction of disulfide bonds with 0.1 M mercaptoethanol followed either by alkylation or by allowing the product to reoxidize. This procedure destroys the activity of IgM and much IgA antibody. d. Reduction and alkylation followed by treatment with 1 M acetic acid. This treatment splits IgG into its chains. e. Treatment with sodium dodccyl sulfate. The solution of conglutinin in the detergent could be shown to bc active simply by dilution. f. Treatment with pepsin, at pH 3.0, using either 1%or even 10% (w/w) of enzyme. This degree of pepsin resistance is remarkable. When the pepsin-treated conglutinin was reduced and alkylated (as in paragraph c above) activity was still retained. This treatment again degrades IgG. g. Treatment with neuraminidase.

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h. Treatment with a “cellulase” preparation. This prep:iration is virtually ground up fungal mycelium and contains a number of depolymerizing enzymes. The reason for using this enzyme was that it attacks the conglutinogen. On the other hand, several treatments were found that did destroy the conglutinin activity: a. Treatment with sodium dodecyl sulfate and mercaptoethanol. The sedimentation coefficient ( sz0.w ) of the product (measured at the protein concentration 1.8 mg. per milliliter) was 3.5. On immunoelectrophoresis the material did not migrate from the antigen well, and in the L-plate only a rather indistinct precipitation line was found whose position gave a value of the difision coefficient of about 3.1. These values for sedimentation and diffusion coefficients give a molecular weight of around 100,OOO. These results suggest that conglutinin is made up of several-somewhere in the region of eight-polypeptide chains held together by a combination of disulfide and weak intermolecular bands, both of which have to be broken before the molecule splits apart. b. Conglutinin is extremely sensitive to trypsin, 0.5% (w/w) of enzyme for 15 minutes at 37°C destroying all conglutinin activity. The fragments appear to be electrophoretically somewhat heterogeneous in that they gave two spurring lines on immunoelectrophoresis. In the Lplate, a single line of precipitation is obtained giving a diffusion coefficient of 3.1. In the ultracentrifuge the trypsin-treated conglutinin sediments as a single peak with slo, of 3.5 (at a protein concentration of 1.8 mg. per milliliter). These values give a molecular weight of around lo0,OOO and a frictional ratio of around 2. The trypsin fragments thus seem to be of about the same size as the fragments produced by reduction and denaturation. There is, however, so far no evidence that they are otherwise the same. The trypsin fragments are still wholly excluded from Sephadex G-200, confirming that they are still markedly asymmetric. c. Papain also rapidly destroys conglutinin. Although large molecular weight fragments are found initially these fairly rapidly break down further. d. Chymotrypsin, on the other hand, is inefficient at breaking down conglutinin and does so only incompletely. None of the fragments produced by any of the above methods were active in inhibiting conglutination by native conglutinin even when the fragments are present in 1000-fold excess. Alexinated cells treated with


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trypsin fragments were not agglutinated by an anticonglutinin antiserum (P. J. Lachmann, unpublished observations, 1966). It would, therefore, appear that such fragments do not have the capacity to combine with conglutinogen. The most obvious feature of the properties of conglutinin so far discussed is their marked contrast to those of the immunoglobulins. For this reason alone one can be confident that conglutinin is not an antibody. C. THEREACTIVITYOF CONGLUTININ

1. The Reaction of Conglutinin with Zymosan Coombs (1947) first observed that bovine serum clumped against cell wall polysaccharide preparations ( zymosan ) and suggested this was due to conglutination. The observation was confirmed by Leon (1957) who showed that this reaction with zymosan was also calcium dependent. Both these workers interpreted their findings as due to the formation of a zymosan-antizymosan antibody-complement complex which then interacts with conglutinin. Although such complexes are certainly formed they do not wholly account for the conglutination of zymosan, which was observed by Pernis et al. (1959a) to occur independently of the presence of complement. The use made of this reaction in the purification of conglutinin has been discussed in a previous section. The reaction with zymosan in the absence of other serum factors was found not to be given by rabbit immunoconglutinins ( Lachmann, 1962; Section IV,E ) . Immunoconglutinins were found to react with zymosan but only in the presence of complement; and then the reactions were not calcium-dependent. The reaction of these I-K’s with zymosan, therefore, does seem to be wholly consequential to the formation of zymosan-antibody-complement complexes. Surprising though it may appear at first sight there is good evidence that the reactants-both conglutinin and conglutinogen-in the conglutination of alexinated cells and of zymosan are, in fact, the same. Thus conglutinin preparations (including the highly purified protein ) always showed the same relative activities on alexinated cells and on zymosan whether prepared by the zymosan method or by entirely physicochemical techniques. Absorption with either alexinated cells or zymosan removed all conglutinating activity to both indicators. Finally it has been possible to perform “mixed conglutination” between alexinated cells and zymosan (Lachmann et al., 1965). In this system specific mixed aggregates of alexinated cells and zymosan are produced by con-

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glutinin in the presence of calcium ions-a h d i n g that shows convincingly that the one substance does react with both types of particle. That the same reactive site on the conglutinin is involved in both cases is strongly suggested not only by the fact that both reactions are mediated by calcium ions but also by the observations that the “soluble” K-gen from yeast inhibited the conglutination of alexinated cells under conditions where the conglutinin was not precipitated. Also, N-acetyl-Dglucosamine which inhibits the conglutination of alexinated cells ( Leon and Yokohari, 1964; Section VI,F) has been shown in our laboratory to have the same effect on the reaction of conglutinin with zymosan.

2. The Distribution of Conglutinogen (Other Than in Complement) In this context the presence of conglutinogen is defined by a reaction with conglutinin in the absence of other serum factors and mediated by calcium ions. a. In Yeast (Saccharonzyces cereuisiae). Conglutinogen is found in the yeast cell walls as shown by its presence in zymosan, in freshly prepared yeast wall fractions, and on the cell wall of living yeast cellswhich are readily conglutinated. The cell wall, however, is not the only fraction of yeast to have conglutinogen. The supernatant, after centrifuging disrupted yeast at 10,OOOg for 1 hour, has marked conglutinogen activity and will actually precipitate with conglutinin. This precipitate partially redissolves in EDTA and allows the conglutinogen (after removal of conglutinin) to be partially purified (Lachmann and Coombs, 1965). This preparation of conglutinogen is far more active on a weight basis than is zymosan. It is not, however, truly soluble but can be sedimented at lo0,OOO g for half an hour, and thus is included in the so-called “microsomal fraction.” Some of its properties are described below. b. In Other Microorganisms. No comprehensive survey of microbial species has been reported but conglutinogen is not found in examples of bacterial genera such as Escherichia, S a l m l l a , Bacillus, or Staphylococcus ( Henson, 1967). On the other hand, J. C. Jaton (personal communication, 1966) has found conglutinogen in Histophasma capsulatum. This is of particular interest in that Wu and Marcus (1964) have shown that the “cytopepsis” of these organisms is increased by a fraction of bovine serum that is likely to contain conglutinin and which retains its activity on heating. Fesce ( 1 9 6 1 ~ )reported that his conglutinin preparation reacted in the absence of complement with “various strains of Saccharomgees and of Schizomycetes”-the latter including Pseudom o m , Salmowlla, Escherichia, Staphylococcas, Streptococcus, Bacillus, and Corytdxzcterium. These experimental data are at variance with


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much other work and are difficult to account for. The assessment of conglutination of the bacteria was based only on the agglutination produced and no evidence that conglutinin could be fixed by the bacteria or eluted from them is given. Any contamination of the conglutinin preparations with immunoglobulins might produce such agglutination and the possibility of contamination cannot be excluded on the basis of published data on the preparation used. Fesce considered that his work showed that conglutinin was not an “antibody to fixed complement but rather ‘polyvalent antibodies’ to yeast and in lesser degree to many other microbial genera.” For reasons already given this view seems unlikely to be correct. c. In Agar. Conglutinin has been found to react with agar (Lachmann, 1962). The reactivity was of a degree comparable with that of yeast cell walls. However, to achieve this reactivity the source of conglutinin had to be mixed with molten agar. If heated bovine serum is mixed with fragments of agar gel, conglutinin did not enter the gel and its concentration relative to other proteins was consequently increased. This “exclusion” from agar gel was attributed to reaction of conglutinin with conglutinogen at the surface of the gel particles. Filtration through columns of broken up agar gel provides a method of purifying conglutinin by making use of this exclusion effect. Because of this reactivity, immunodiffusion studies of conglutinin in agar gel need to be done in the presence of EDTA. Agar is difficult to deplete of calcium ions and it has been found necessary to dialyze the gel against large volumes of dilute EDTA for some days before use. This treatment, however, tends to make the agar “sloppy.”

3. The Reaction of CongZutinin with Fixed Complement The nature of the “conglutinogen” in fixed complement is discussed in detail in Section VI. For the present it will suffice to say that conglutinin clumps cells that have complement fixed upon them (Bordet and Gay, 1906). To demonstrate this phenomenon on erythrocytes a complement such as equine, whose conglutinating titer greatly exceeds its hemolytic titer, is generally used, but conglutinin has been shown to react with all mammalian complements so far investigated in this connection (Section VI,D ) . It should be emphasized that the reactant involved is in the fixed complement itself. It has been shown using a fluorescein-conjugated anticonglutinin serum on a model system comprising nuclear antigens, lupus erythematosus (LE ) serum, and guinea pig C’ (Lachmann, 1962), as well as by red cell agglutination studies with anticonglutinin, that in the absence of complement fixation no con-

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glutinin is bound. Thus the suggestions that complement is rcquired only for the manifestation of conglutinin activity are invalidated.

4. The Role of Calcium Ions in Conglutination That the conglutination of erythrocytes by fresh bovine serum required calcium ions and would break up in EDTA was first observed by Leon ( 1957). Subsequently (Lachmann, 1962) it was shown that conglutinin was eluted from the alexinated cell by EDTA and that both the eluted conglutinin and the alexinated cell from which it had been eluted would participate in conglutination again if supplied with further calcium. On this basis it was suggested that calcium ions form a ligand between K and K-gen. However, the possibility that a calcium ion forms an integral part in maintaining the configuration of the K-gen or even of the K without actually being a ligand cannot be discounted. Nevertheless the differences here are subtle. If calcium ions are a part of the combining site of the K-gen or the K, it would be accurate to describe this as a ligand. Only if the calcium ions were bound other than at the reactive site, but required to maintain the reactivity of that site, i.e., were acting in an “allosteric” way, would the term “ligand” be inappropriate. The observations on factors in chickens that appear to resemble antibodies to K-gen and are ( t o a considerable extent) EDTA reversible (Price and Lachmann, 1966; Section IV,E) may suggest the possibility that calcium is concerned in the structure of the K-gen. Further information on the structure of K-gen is needed to clarify the situation. D. THE DISTRIBUTION OF CONGLUTININ AMONG DIFFERENT SPECIES

A most unusual feature of conglutinin is that it is found in certain mammalian species but not in others. From the species so far tested, it would appear that conglutinin is found only in the Bovidae and not even perhaps in all of these. Ruminants other than Bovidae remain to be tested. Conglutinating activity has in the past been observed in the sera of most animals studied (Coombs et al., 1961) but it is only recently that it has been possible to distinguish conglutinin activity from I-K activity. For screening sera, the easiest methods for distinguishing the activities are to reverse the conglutination with EDTA-which leaves only (though possibly not entirely all) conglutination due to I-K-and to test mercaptoethanol treated sera which detects (virtually) only conglutinin, if the usual “resuspension” technique for measuring conglutination is used. However, to confirm that a given reactivity is truly conglutinin requires


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that a number of the distinctive properties of conglutinin that distinguish it from antibody should be demonstrated. Some degree of isolation, as by absorption and elution from zymosan, is probably necessary. R. W. F. LePage and R. R. A. Coombs (unpublished observations, 1964) found conglutinin not only in cattle but also in a number of African ruminants-the African buffalo, water buck, Uganda kob, Kenya kob, dik-dik, topi, and Jackson’s hartebeest-the last-named having a particularly high titer1 The conglutinating activity in a number of sheep sera was, however, all due to I-K. Large numbers of mouse, rat, rabbit, guinea pig, equine, and human sera tested in this laboratory have never shown any conglutinin activity. Neither has conglutinin been found in smaller numbers of cat, dog, or pig sera. Bienenstock and Bloch (1966a) using criteria similar to those described above confirmed in a detailed study that the conglutinating activity in human serum was entirely I-K. This occurrence of a serum protein in one group of mammdsruminants-whereas it is completely absent in quite closely related groups, e.g., horses, is most remarkable. This may, in fact, indicate that the protein is made only in one order of mammals, but some other explanations can be offered. Thus it is possible that a homologous protein does occur in other species but that it is, for one reason or another, inactive in producing conglutination. It could be envisaged in this connection that tryptic activity in serum would inactivate conglutinin. For this reason human and rabbit blood was taken directly into trypsin inhibitor, but without exposing any conglutinin activity. An attempt to find a protein antigenically related to bovine conglutinin either in the Sephadex G-200 exclusion peak of human serum or in that fraction of it sedimenting in the upper part of a sucrose density gradient (i.e., the asymmetrical fraction) was also unsuccessful ( P. J. Lachmann, unpublished observations, 1966). These experiments do not exclude that in uho breakdown of conglutinin is the reason that it is not found but provide no further evidence for this view. A second possibility to be considered is that conglutinin in other species might circulate in a complex with conglutinogens. This possibility can be tested by treating serum so as to destroy any conglutinogen. Sodium periodate has been used for this purpose. It was found that periodate treatment did slightly increase the conglutinin titer of heated bovine serum, suggesting that some conglutinogen was circulating but no conglutinin activity was unmasked in human or rabbit serum ( P. J. Lachmann, unpublished observations, 1966).

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A third possibility is that conglutinin is really a tissue protein which in ruminants finds its way into the serum. However, an extensive survey of bovine tissue with fluorescein-labeled anticonglutinin ( Lachmann and Coombs, 1965) failed to localize any conglutinin in bovine tissue. It was not even possible to localize the site of formation by this technique, suggesting that conglutinin is not stored in cells in any quantity. The original contention-nameIy that conglutinin is a protein made only in ruminants-may, therefore, well be the correct one. Variations in conglutinin levels in cattle disease are discussed in a later section.

E. ANALOGYWITH OTHERSERUMFACTORS

1. C-Reactive Protein The analogy of conglutinin with C-reactive protein on the basis of their nonimmunoglobulin nature, requirement for calcium ions, and reactivity with specific polysaccharides has already been mentioned. The two factors, however, are quite distinct. Conglutinin does not react with C-polysaccharide nor does C-reactive protein clump alexinated cells. Nor is conglutinin an acute phase protein. Nevertheless, the two proteins may be considered as belonging to the same cIass of immunological phenomena.

2. Properdin Properdin has now been isolated (Pensky et al., 1964; Lepow 1965) and found to be a nonimmunoglobulin serum protein with a molecular weight of 230,OOO and a sedimentation constant of 5.2. These figures give a frictional ratio of above 2, assuming V to be in the range 0.72-0.76, so that properdin is an asymmetric molecule. Although properdin, like conglutinin, reacts with zymosan, its interaction requires the presence of complement and of magnesium ions. Furthermore properdin is heat-labile-and it will not clump alexinated cells. Points of differences between the two thus rather overshadow the points of similarity. IV.

The Congfutinogen of Yeast

A. CONGLUTINOGEN IN YEAST CELLWALLS Lachmann and Northcote (quoted by Lachmann and Coombs, 1965) tested various purified components of yeast cell walls for K-gen activity. They found that all three purified yeast wall polysaccharides-glucan, mannan, and glycogen-were inactive in fixing at least two doses of conglutinin per milligram. Of the three cell-wall mucopolysaccharides


497

described by Korn and Northcote (1960), Fraction A was found to be the only one with an activity comparable to that of intact cell walls. This is a soluble fraction containing mannose, glucosamine (including N acetylglucosamine), and about 12% protein. The greater part of the activity of cell walls is, however, not recovered in any of these fractions and on an activity/weight basis the cell-wall mucopolysaccharide fraction ( A ) was only as active as whole yeast cell walls; less than half as active as zymosan; and less than 1%as active as the “microsomal” yeast K-gen. Leon et al. (1966) have reported that a mannose-peptide showing K-gen activity in microgram amounts can be eluted from zymosan with hot neutral buffers. K-gen activity can also be eluted from zymosan with crude fungal “cellulase.” The K-gen may be detected in the eluate after heating at 100°C.to destroy the enzyme (P. J. Lachmann, 1966, unpublished observations). The name cellulase is applied to this enzyme preparation by the suppliers. There is however some evidence that the enzymatic activity observed on zymosan is not due to cellulase (i.e. p-1:4 glucan hydrolase) activity. A more accurate designation for the effect on zymosan will require further purification and study of the enzyme concerned.

B. CONGLUTINOGEN IN THE “MICROSOMAL FRACTION” OF YEAST This K-gen has been studied by Lachmann et a2. (1966). The supernatant from centrifuging disrupted yeast cells at l0,OOO g for 1 hour contains K-gen. If this supernatant is centrifuged at 100,OOOg for 30 minutes the K-gen is sedimented. On this basis the K-gen is regarded as being in the microsomal fraction. The microsomal K-gen can be considerably purified by precipitating the original yeast supernatantreduced in mercaptoethanol to increase its activity-with conglutinin. The K-gen is harvested from the precipitate, after solution in EDTA and alkylation, by 100,OOOg centrifugation. Trypsin treatment ensures that all conglutinin has been removed. One milliliter of the final product, containing about 1 mg. organic solid, inhibits 40,000doses (about 400 pg.) of conglutinin. This reactivity is about 200 times greater than that of the most active zymosan. Chromatography of the hydrolyzed material showed mannose and a trace of glucose as the only sugars. The protein content was rather more than 10%. C. CHEMICALSTABIL~~Y OF YEAST CONCLUTINOGEN Yeast conglutinogen-whether

in zymosan or microsomal fraction-

is resistant to treatment with trypsin (which is made use of in the prep-

aration), hydrolysis with dilute acid at 37°C. or heating at 100°C. The

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activity is potentiated by reduction with mercaptoethanol followed by alkylation. Conglutinogen is destroyed by oxidation with sodium metaperiodate. D.

ASTICOhXPLEMEhTARY ACTIVITY OF YEAST

CONGLUTINOGEN

The microsomal yeast K-gen shows only minor anticomplementary activity. Such anti-C’ activity as is found could not be demonstrated on EAC’142. There is thus no suggestion of a selective inhibition of C‘3 components, nor does the conglutinogen seem to be identical with those antigenic determinants that make zymosan capable of forming an R3. The reaction of K and K-gen was found not to fix guinea pig complement to a measurable extent.

E. ANTIGENICITYOF YEAST CONGLUTINOGEN Antibodies to K-gen should demonstrate I-K activity on alexinated cells and this should be abolished by absorption with zymosan (in the absence of other serum factors). A rabbit immunized with yeast K-gen failed to produce any such antibodies or any evidence of reaction with the K-gen (Lachmann and Coombs, 1965). Nor are any of the rabbit or human I-K’s that have been studied absorbable by zymosan. Evidence for the antigenicity of yeast K-gens in these species is thus lacking, and it is possible that this is owing to tolerance toward this reactant. The situation may, however, be different in chickens. Here immunization with yeast antigen or zymosan does produce a low titer of conglutinating activity against alexinated cells. This activity is not only removable with zymosan but is largely EDTA reversible, although neither as rapidly nor as completely as the EDTA reversibility of bovine conglutinin. Some reactivity of the same kind has been observed at low titer in occasional unstimulated chickens (Price and Lachmann, 1966). These reactants are resistant to trypsin, susceptible to mercaptoethanol, and can be raised by immunization. They would, therefore, appear to be I-K’s with the specificity of antibodies to K-gen. Their EDTA reversibility is very unusual for I-K-the great majority even of chicken I-K is EDTA stable-and may suggest that calcium ions are part of the K-gen. V.

lmmunoconglutinins

Immunoconglutinins may be defined as a group of antibodies showing specificity toward antigenic determinants exposed in fixed complement components but not available for reaction when complement is free in solution (Coombs, 1947). This definition is really ‘bperational” in that


499

the activity is the essential factor. Evidence that I-Ks have the other properties associated with antibody is given below. A. THE STIMULATION OF IMMUNOCONGLUTININS

Immunoconglutinins may be stimulated in a number of different ways.

I . Hetermtimulation of I-K Immunization of rabbits with bacteria sensitized with antibody and alexinated with guinea pig complement was the technique of raising I-K’s originally used by Streng ( 1930). Subsequently Coombs ( 1947) used kaolin particles exposed to fresh horse serum to provide a source of “fixed complement.” The essence of this technique is to immunize with heterologous complement in the “fixed” configuration. Besides antibodies that react with the free serum proteins of the species supplying the complement, immunoconglutinins are also formed, often at high titer. The use of Freund’s adjuvant with these antigens gave rise to prolonged I-K formation. Although these heterostimulated I-Ks are of interest with regard to the antigenic relationships between the fixed complements of different species, they represent, as far as is known, an entirely artificial phenomenon and so do not share the biological significance which may be ascribed to the autostimulated I-K’s. The appearance of I-K has also been observed in antisera to foreign serum protein in solution (Waartiowaara, 1932). Thus, a rabbit antiserum to human plc-globulin ((2’3) was found to have a particularly high titer of I-K when tested on fixed equine complement (Lachmann and Coombs, 1965). Similarly strong I-K activity was not found in antisera to IgG or to &-globulin (C’4). This I-K activity of the anti-C’3 serum, however, was abolished when the antiserum was absorbed with a source of human C’3 in solution. This type of I-K is, therefore, distinct from the original type of heterostimulated I-K and seems to depend on the existence of antigenic determinants which are accessible in human C’3 in solution but revealed in equine C’3 only after fixation.

2. Autostirnulation Waartiowaara (1932) was the first to observe that conglutinating activity could be found in rabbits injected with bacteria per se, and this observation lead him to doubt Streng’s view that I-K’s were formed in response to immunization with fixed complement. The phenomenon was extensively investigated by Blomfield (1952a) and Coombs and Coombs (1953) who came to the conclusion that these autostimulated I-Ks rep-

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resented autoantibodies to the animal’s own fixed complement-a view which all subsequent evidence has tended to confirm (vick infra). The I-K activity not infrequently found in some normal members of many animal species and that found in association with disease is considered to be of this “autostimulated” kind and it is now believed that the appearance of these I-Ks is an index of in zjirjo complement fixation. It is, therefore, these autostimulated I-K’s which have attracted interest in recent years and whose formation and properties have been studied in detail. Wherever in this review the term I-K is used, it may be understood to refer to autostimulated I-Ks unless heterostimulation is specifically mentioned.

3. Experimental Autostimulation of I-K The optimal conditions for I-K production were studied by Blomfield (1952a) (see also Coombs and Coombs, 1953). She found that repeated intravenous injections of killed bacteria-Salmonella pullmum and Proteus 0x19-was the most effective way of raising I-K sera in rabbits. Intraperitoneal or subcutaneous injection was much less effective. A subsequent course of injections gave a bigger response than was found with the first course. Gram-negative bacteria were in general much better stimulators than gram-positive ones. Two courses of immunization with egg albumen, a soluble antigen, other than serum protein gave rise to negligible I-K production. The I-K production after intravenous bacterial immunization was rapid, maximal titers being achieved after 4 to 6 days and activity having fallen sharply to a low level by 12 days. Antibodies to the immunizing bacteria, on the other hand, although detectable before the onset of I-K production, rose to a maximum in about 10 days and thereafter stayed at a high level. Further studies were carried out by Ingram (1958) who used mice and injected them intraperitoneally with a large variety of substances. He failed to provoke I-K production with a number of sugars and amino acids, cholesterol, sulfadiazine, cellulose, dextran, or inulin. A number of products given to supply “surfaces” for in vivo complement fixation-glass, lamp black, and bentonite-were also without effect. On the other hand, relatively strong I-K production was induced by hog gastric mucin, casein hydrolyzate, peptone, and a lesser degree by zymosan, agar, gelatin, kaolin, and lactalbumin hydrolyzate. None of the levels achieved by these means were as high as those (of heterostimulated I-K) given by immunization with serum proteins. Ingram concluded that his results were compatible with the view that in virjo com-


501

plement binding was taking place on his effective “antigens.” As further evidence for this view he was able to show a fall in both complement and preexisting I-K titer when an animal was reimmunized with bacteria; a new rise in I-K following rapidly thereafter. Treatment of animals with high doses of cortisone or with 6-mercaptopurine were shown to inhibit the I-K response to intravenous injection of bacteria. Colchicine was also slightly inhibitory ( Ingram 1962a, 1955). Henson (1967) has reinvestigated the factors involved in autostimulation of I-K in rabbits, partly with the aim of devising reliable methods of raising high titer I-K’s. He has shown that the presence of high titers of complement-fixing antibodies to the injected bacteria are an important prerequisite for strong I-K formation and that if intramuscular immunization with killed bacteria in Freund’s adjuvant precedes the intravenous course, very high titers can be achieved, and that the difference in stimulating potency between gram-negative and grampositive organisms is then no longer found. He also investigated the effect of treatments that affect lysosomal membranes, on the grounds that, if in uiuo complement fixation generates the antigen, this process might well be enhanced or prolonged by allergic inflammation. Treatment with hydrocortisone, which stabilizes lysosomal membranes ( Weissmann and Thomas, 1964) was found-in agreement with Ingram (1962a)-to reduce the I-K response to intravenous bacteria though the reduction was modest. Treatment with toxic doses of vitamin A [which causes lysosomal breakdown (Dingle, 196l)], on the other hand, gave no consistent effect on the I-K response. These studies are all in agreement with the hypothesis that I-K’s are autoantibodies to in viuo fixed complement. The kinetics of the response resemble those of IgM antibody production. The question whether “secondary responses” of I-K production occur is difficult to answer with confidence. After repeated courses of intravenous injections the peak levels of I-K achieved still fall very rapidly, although the “basal level” does rise somewhat. These responses appear to represent bursts of IgM antibody formation, and this is confirmed by showing all the activity to be mercaptoethanol-sensitive throughout. However, the efficiency of detection for IgG I-K is probably very low (see Section V,B) and a secondary I-K response associated with IgG formation could be difficult to detect if it were not very intense.

4. I-K Formation in Diferent Species The capacity to form I-Ks of the autostimulated variety-either without obvious stimulating cause in normal animals or following spon-

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taneous or experimental infection, or following experimental autostimulation-appears to be a general property of the animal species investigated. The distribution of I-Ks in apparently normal rats, mice, guinea pigs, and pigs and their response to immunization with bacteria is given by Coombs ( 1954). Rats and mice showed a good response to experimental autostimulation, and I-K formation in mice was subsequently studied in great detail by Ingram ( 1958). The response of guinea pigs to autostimulation was poor, but definite autostimulated I-Ks in guinea pigs have since been shown (Ingram et al., 1959; Lachmann and Coombs, 1965; Henson, 1967). Normal pigs had a rather high I-K level so that the significance of the rises obtained on autostimulation were difficult to assess. On the other hand, Wiggin (1955) found negligible I-K in normal horses and Coombs (1954) failed to produce I-K in donkeys by immunization with Salmonella pullorurn. The I-Ks in all these experiments were tested on horse complement and the possibility that the equine I-K’s (if formed) did not react well on the equine indicator must be considered. Using the more sensitive sedimentation pattern technique, howe\7er, autoreactive I-Ks in apparently normal horse sera have been encountered. Immunoconglutinins are also found in dogs (Ingram, 1963), in chickens (Price and Lachmann, 1966), and in sheep (R. W. F. LePage and R. R. A. Coombs, unpublished observations, 1964). LePage and Matson (1965) were also able to show I-K fmmation in cows after infection (see Section VIII,B,l). Coombs (1947) had previously shown that cows form heterostimulated I-K. Immunoconglutinins in rabbits were discussed above; their occurrence in man is discussed in Section VIII,B,3.

B. THE NATUREOF IMMUNOCONGLUTININS

The evidence from the methods of stimulating I-K‘s indicate that these factors are antibodies. It can also be shown that they have the properties of immunoglobulins and that the great majority of autostimulated (but not of heterostimulated) I-K activity is IgM antibody. Thus rabbit and human I-Ks, which are the most studied, are mereaptoethanol-sensitive, excluded from Sephadex G-200, sediment in the 19 S region of zone ultracentrifugation, and show the antigenicity of IgM. They react with alexinated cells in the absence of calcium ions (Lachmann, 1962; Lachmann and Coombs, 1965; Bienenstock and Bloch, 1966a ) . Heterostimulated rabbit I-K’s, on the other hand, are generally IgG antibodies ( Lachmann and Coombs, 1965).


503

The association of (autostimulated) I-K with IgM is not, however, absolute. Kunkel and Coombs ( 1960) found that on zone-ultracentrifugation of a high-titer rabbit I-K serum, some activity was found in the 7 S region. High-titer rabbit I-K's fractionated on Sephadex G-200 (Lachmann, unpublished observations, 1966) have also been found to have minor activity peaks in the second (IgG-containing) peak. The possibility, therefore, arises that the association of most I-K activity with IgM is a consequence of the fact that only agglutination tests for this activity have so far been devised. Since IgM is, molecule for molecule, several hundred times as efficient an agglutinator as is IgG (Greenbury et al., 1963),the system is greatly weighted toward detecting this type of antibody. This weighting is increased by the usual method of reading conglutination-by centrifugation and resuspension-which is designed to suppress weak agglutination. It may, therefore, well be the case that IgG I-K's are formed-at least in amounts comparable to the IgM I-K's-but that we fail to detect them. It seems unlikely, however, that large amounts of IgG I-K would be missed since heterostimulated I-K does react in the usual test system and is IgG. Certainly, methods aimed at producing high-titer IgG antibody, such as immunization intramuscularly with adjuvant, are bad methods for producing autostimulated (but good for heterostimulated ) I-K, whereas methods for getting high IgM titers-particulate antigens ( Torrigiani and Roitt, 1965) intravenously-give the best autostimulated I-K titers. The situation in this respect is similar to that found with rheumatoid factors and natural isohemagglutinins where the IgM component predominates over the IgG. The reason why certain systems behave in this way is not clear.

C. THEISOLATION OF IMMUNOCONGLUTININS There is unfortunately no easy way of isolating IgM antibodies occurring as a minor part of the total IgM. A Sephadex G-200 exclusion peak of the I-K serum represents quite a marked concentration of the activity. Centrifugation of this material in a sucrose density gradient allows the I-K to be recovered from the rapidly sedimenting fraction and separates it from conglutinin. It is, however, possible to achieve a fair degree of purifkation of I-K's by making use of the relatively easy dissociability of macroglobulin antibodies at low pH ( Kunkel, 1960). An antigen-antibody precipitate, prepared at optimal proportions, is alexinated and conglutinated with I-K. Elution of the washed, conglutinated complexes at p H 4.5 dissociates

,504

P. J . LACHMANN

the bulk of the I-K without redissolving the precipitate. This procedure achieves a 50- to 100-fold purification (P. J. Lachmann, unpublished observations, 1966). The elute may be passed through Sephadex G-200 or centrifuged to sediment the 19 S material.

D. ANALOGYWITH RHEUMATOID FACTORS Just as conglutinin appears to belong to the same class of immunological phenomenon as C-reactive protein, so do I-K’s appear to belong to the same class as the rheumatoid factors. Both I-K‘s and rheumatoid factors are groups of predominantly IgM antibodies; both appear to be directed, against the products of an immunological reaction-the fixation of antibody in the case of rheumatoid factors and the fixation of complement in the case of I-Ks. There are rheumatoid factors to multiple determinants in (fixed) IgG and I-K’s to multiple determinants in fixed complement. However the alloreactivity that rheumatoid factors are known to exhibit (see Kunkel and Tan, 1964) is as yet only suspected for I-K’s. There may also be significant differences in the kinetics of formation and in the biological significance of the two sets of factors. VI.

The Reaction of Conglutinin and lmmunoconglutinin with Fixed Complement

A. THE REACTXON SEQUEXCE IN COMPLEMENT FIXATION In recent years the knowledge of the nature of complement and the reaction sequence leading to hemolysis has increased rapidly. Figure 1 shows in schematic form a reasonably contemporary (October, 1966) account of the pathway leading to hemolysis, with the effects of decay of the intermediates and the effects of chelating divalent ions. References to most of the steps portrayed are given by Polley and Miiller-Eberhard (1967). Although the scheme has been worked out largely on guinea pig and human complement, it is generally believed that basically the sequence of events in complement fixation is the same for all species. In a discussion of complement fixation in relation to manifestations of C’ activity such as conglutination, it is perhaps unwise to focus attention exclusively on the hemolytic pathway which is a rapid “short-term” went. In ciuo complement fixation sites-whether or not they successfully achieve the E* state, have a “long-term” existence which may be anticipated to be more important from the point of view of their “antigenicity.” Such long-term intermediates are unlikely to have C‘2 on them since this rapidly decay off the cells. They are also unlikely to ha\ie

505


active C’1 on them since there is not only a C’1 inhibitor (Lepow and Leon, 1963) which reacts with fixed C’l, but the fixation of C’3 itself appears to lead to the inhibition of the activity of fixed C’1 (Lachmann, 1966a). Whether in these situations C’1 as an antigenic entity remains on the cell for a prolonged period is not known. On the other hand, they will have C’4 and C’3 on them. Both these intermediates are firmly bound to the cell apparently by hydrophobic bonds ( Muller-Eberhard et al., 196613) and they can be shown antigenically at old sites of complement fixation both on red cells and in tissue sections (Harboe et al., ~‘2ad

\\\

4

-..._ .,

/-------‘

EAC‘4

\

\

\

\

\ \

\

\

Caz+

\

\

C’lqrs

EA

\

EDTA

-EAC‘la42a-n/\n--cEAC‘42a Me+

EAC‘la -EAC‘la4

‘

,EACr(la)423-----*EAC’(1a)43,

C‘8

E* ---+

+ C‘9

(half-life about 7 minutes)

EDTA WvW-

e

EAC’(la)423 567

Hemolytic pathway

- - -Decay * at 37°C.

Take part in Conglutination, Immune Adherence, Phagocytosis.

Treatment with EDTA briefly in the cold

C’567

I

Chemotactic factor

FIG. 1. The reaction sequence in complement fixation.

1963; Lachmann et al., 1962; Hatfield and Muller-Eberhard, 1965). Of the two components, C’3 is bound in very much the larger amount (Muller-Eberhard et al., 1966b) and what is believed to be bound C’3 can be visualized in the electron microscope for some distance around complement-fixation sites ( Feinstein et al., 1967). Of the components acting after C’3, C’5, C’6, and C’7 are believed not to be bound to the complement-fixation site (Miiller-Eberhard et al., 1966a), and so far little is known of C’8 and C‘9 in this respect. On present knowledge it would, therefore, seem likely that C’4 and particularly C’3 would be the components bound on the long-term comple-

506

P. J. LACHMANN

ment fixation site and, therefore, of particular interest in regard to phenomena such as conglutination.

PATTERN TECHNIQUES FOR B. “RFSUSPENSION”AND SEDIMENTATION MEASURINGCONGLUTINATION The conglutination phenomenon has so far always been detected by its “clumping” activity on alexinated matter, i.e., as an agglutination test. This is generally done (following Coombs, 1947) by centrifuging the reaction mixture and resuspending briskly. The absence of unagglutinated cells in the resuspended mixture is then taken as the criterion of conglutination. This “resuspension” technique measures only strong agglutination and has, therefore, the great merit that weaker agglutinating systems do not produce false positives. In a conglutination reaction such weaker agglutinating systems may result from the agglutinating action of the antibody used in making EA; from the presence of antibodies to sheep red cells, of rheumatoid factors, or even of I-Ks in the complement source; or from antibodies to sheep cells or of rheumatoid factors in the source of conglutinin. Except for antisheep cell antibodies in the I-K sera, which are easily removed by absorption, it is rare for any of these factors to give false positives in the resuspension technique. Except where the contrary is stated it may be assumed that all the work on conglutination described has been carried out by such a resuspension technique. There are, however, also disadvantages to using a method designed to ignore weak agglutination. There is a loss of sensitivity in the detection of K and I-K’s. For example, for the detection of conglutinin the usual test system (vide infru) requires about 900 molecules of conglutinin per red cell pair (Lachmann, 1966b). Potentially more significant, however, than the loss of sensitivity for “antibody” is the restriction that a resuspension method imposes on the “antigens” which “antibodies”-even in ample quantity-can detect, for if the “antigen” is present only sparsely on the cell, sufficiently strong agglutination cannot be produced. As an example, Boursnell et al. (1953) estimated the number of rhesus ( D ) sites of human red cells to be around SO00 and the number of Paul-Bunnell sites to be above 100,000. Anti-D antibodies do not give agglutination by the resuspension technique; Paul-Bunnell antibodies do so. In relation to conglutination, therefore, the resuspension technique may involve a restriction of the complement antigens to which K or I-Ks can he detected. To investigate this point a sedimentation pattern technique for conglutination has been developed (Lachmann, 1966b). An


507

IgM fraction of antibody to purified Forssman hapten is used to make EA, so as to minimize agglutination by the antibody and reactivity with rheumatoid factors. Reagents from guinea pig complement are used to alexinate. Conglutination is assessed by settling pattern in an agglutination plate. About 90 molecules conglutinin per red cell pair can be detected in this system. Its use in the investigation of the specificity of K and I-K toward complement components is described in Section

v1,c.

C. THE SPECIFICITY OF CONGLUTININ AND IMMUNOCONGLUTININS TOWARD THE COMPONENTS OF FIXED COMPLEMENT The earlier studies on this topic have been reviewed by Coombs et ul. (1961). In a conglutinating system using sheep red cells, horse complement, and heated bovine serum as source of antibody and conglutinin Coombs et al. (1950)found evidence that C’l, C’4, and C’2 all had to react before a conglutinable intermediate was reached. No definite conclusion could be reached about the role of (classical) C’3 because the R3 reagent made by zymin absorption was found to be deficient in other complement components. Rice (1953) found in the same system that if both the horse complement and the heated bovine serum, but not if only one or the other, was absorbed with the minimum amount of zymosan to absorb (classical) C’3 conglutination was abolished. She concluded that C’3 was also required for conglutination. However, zymosan was subsequently shown not to inactivate (classical) C’3 in heat-inactivated serum (Pillemer et al., 1953), although it does absorb conglutinin, and in retrospect these results are d a c u l t to interpret. Blomfield (1952a,b) suggested that there might be two Ct4 factors in that both are ammonia sensitive, one going on to react with (classical) C’3 to produce lysis, the other, to react with conglutinin. The problem has been reinvestigated in recent years in the light of the newer knowledge of the complement system, including the finding that Cj3 as well as C’4 is ammonia-sensitive (Taylor and Leon, 1959; MiillerEberhard, 1951). It has been found (Lachmann, 1962) that EAC’142 fails to react (by resuspension) with K or with any I-K sera. However when Ct3 is reacted with this intermediate, reactivity is acquired. A more detailed study of alexinated intermediates made with guinea pig complement (Lachmann and Liske, 1966a; vide infru) confirmed that reactivity with K (whether measured by resuspension or by sedimentation pattern) is acquired only when C’3 is fixed and is then affected neither by the loss

508

P. J. LACHMANN

from the intermediate of C‘1 or C’2 nor by the subsequent interactions of C’5 and C‘6. The reactivity of conglutinin, therefore, appears to be exclusively directed against fixed C’3. With I-K’s the results obtained have been found to depend on the method used for measuring conglutination. Using the resuspension method, I-K’s, like conglutinin, have bcen found to react only with fixed C’3 (Lachmann, 1962; Lachmann and Coombs, 1965), but using sedimentation patterns ( Lachmann, 1966b) I-Ks reacting with fixed c‘4 as well as with fixed C’3 have been found very commonly. It would appear that the number of reacting C’4 sites is not large enough to produce conglutination as read by resuspension. The possible Occurrence of I-K to other fixed complement components is still under investigation. Mollison ( 1965), using an indicator comprising human red cells, human antibody, and human complement and measuring agglutination on a tile, found that the reactivity of his indicator decayed faster toward rabbit I-K than toward anti-C’3 and that the agglutination pattern produced by the I-K tended to give a “mixed field” appearance. He suggested that I-K activity toward C’2 could account for these results.

Conglutinogen Activating Factor ( KAF ) More recent studies (Lachmann and Muller-Eberhard, 1967) of the reaction of conglutinin with an alexinated intermediate built up with purified human complement components have shown the C’3 step of complement fixation to be more complex than was apparent from hemolytic studies. Thus an intermediate EA01423 which was made with purified components and reactive hemolytically, in immune adherence, with I-Ks, and with anti-C’3 antisera was found not to react with conglutinin. Reactivity with conglutinin was acquired when this EAC’1423 reacted with a further factor in human serum. This factor-to which the name K-gen activating factor or “KAF” has tentatively been givenappears to be distinct from previously described complement components and to play no essential role in the hemolytic sequence. The reaction of KAF with bound C’3 does not require C’1 or C‘2 to be present on the alexinated complex; it is not affected by the previous reaction of the a m plex with C’q, C’6, and C’7; it does not require the presence of calcium or magnesium ions, but does not proceed in the cold. At present it is thought somewhat more likely that KAF causes some alteration in bound C’3 leading to the appearance of conglutinogen, rather than that KAF is itself fixed on the alexinated complex. Following KAF treatment, EAC’1423 retains at least the greater part of its activity in hemolysis and immune adherence as well as with I-K’s and anti-C’3 antisera. However the susceptibility of the bound C‘3 to elution by enzymes was found to be greatly increased (Section VI,G,l).

CONGLUTININ AND IMMUNOCONGLUTINIKS

509

The biological role of KAF in complement action-especially in species like man which have no conglutinin-seems unlikely to be primarily concerned with the activation of K-gen as a reactant. Possibly K-gen has significant properties other than its reaction with conglutinin; possibly also the increased susceptibility of bound C’3 to enzymatic attack has some biological importance. AND PROPERTIES OF CONGLUTINABLE D. THEPREPARATION INTERMEDIATES FROM DIFFERENT SPECIE^

1. Using Equine Complement

The conventional test system for conglutination is EA alexinated with a sublytic dose of equine complement at 37°C. Sheep red cells have generally been used as source of E. Blomfield (1952b) studied the interrelationship between the species of antibody used to prepare EA and the efficiency of complement fixation achieved with different species of complement. Although several complements were found to be adequately fixed on only certain species of antibody, horse complement was found to be well fixed on all the species of antibody tested. The naturally occurring anti-Forssman antibody in bovine serum is used widely to make EA (Coombs, 1947) and has the advantage that is almost nonagglutinating at the dilutions used for complement fixation. However, rabbit or equine antisheep cell or anti-Forssman sera may be used equally well. The hemolytic titer of horse complement on EA is generally not more than 5; the titer for conglutination about 100. At 37°C. full reactivity of the alexinated complex is achieved within 10 minutes and at 15°C. within 20 minutes; at 5°C. reactivity is acquired in 3 hours; and at 0°C. hardly at all (Ingram, 1962b). The stability of the equine alexinated intermediate was reported by Ingram (1962b) to be such that the requirement for conglutinin was doubled after 9 minutes incubation at 37°C. or 17 minutes at 25°C. Lachmann and Liske (1966b) measured the halflife of this complex in terms of the decay time needed to double the initial requirement for complement and obtained value of 3 hours at 37°C. They found that the requirement for conglutinin of a complex prepared with excess complement had not doubled even after 3 hours at 37°C. The reasons for the discrepancies between these results is not clear. The designation of the equine intermediate in terms of complement components is not certainly known but is thought probably to be EAC’( 1)43 (Lachmann and Liske, 1966b).

310

P. J. LACHMANN

2. lrsing Rabbit Complement When alexination with rabbit C' is carried out for 10 minutes at 37"C., conglutination beyond the hemolytic end point is not generally seen, except from time to time with stored sera. However, cutting down the temperature and/or time of alexination eliminates the hemolysis, and a conglutinable intermediate is formed to about the same complement titer that gives hemolysis at 37"C., 5 minutes alexination at U)"C., 10 minutes at 15"C., or 30 minutes at 10°C. can be used for this purpose, the last giving in general the strongest conglutination with the least hemolysis (Lachmann and Liske, 1966b). At temperatures below 4"C., conglutinable intermediates are not formed. This "piling up" of a conglutinable intermediate during alexination is not found with guinea pig or human complement. The half-life of the conglutinable complex was found to be about 2.5 hours at 37°C. The designation of the rabbit intermediate is thought, on the basis of its stability for lysis by C' reagents as we11 as for conglutination, probably to be EAC'( 1)423.

3. With Human Complement Except occasionally with aged sera, conglutination beyond the hemolytic titer cannot be achieved by alexination with human complement by adjusting the time or temperature of alexination within the usual range of these variables. With a proportion of human sera, conglutinable intermediates can be made with zymosan-absorbed serum ( R 3 ) , which with these sera produce intermediates apparently of the form EAC'( 1)423567 (Lachmann and Liske, 1966b). The use of low concentrations of zinc ions with a critical amount of complement gives rise to an intermediate resembling EAC'1423 ( Muller-Eberhard et al., 1964) and such intermediates are conglutinable (Lachmann and Coombs, 1965). This effect of zinc ions in abolishing lysis further than conglutination is not seen with rabbit or guinea pig complement. The half-life of the intermediate made in the presence of zinc ions was about 90 minutes at 37°C. 4. With Guinea Pig Complement

With whole guinea pig serum, no conglutination beyond the lytic end point has been observed. Conglutinable intermediates have, therefore, been prepared using complement reagents (Lachmann and Liske, 1966a) (uide supra). The most convenient intermediate to prepare and use is EAC'(1)423 or its decayed form EAC'43. This has been made by treating EAC'142 (made with a guinea pig R3 reagent which differs in


511

this respect from the human reagent) with guinea pig serum heated at pH 8.5 at 62%. (C’H62)-a reagent containing only C‘3 of the classical C‘3 components (Klein and Wellensiek, 1965). It has also proved possible to prepare reagents analogous to guinea pig C’H62 from human and rabbit serum, which on reaction with guinea pig EAC’142 provide conglutinable intermediates without providing lysis. E. THEHETEROGENEITY OF THE REACTANTSIN FIXED C’3

1. K-gen The various conglutinable intermediates described in the last section all detect roughly the same titer of conglutination with a given preparation. Absorption of conglutinin can similarly be achieved with antigen-antibody complexes alexinated with different complements ( Lachmann and Coombs, 1965). The K-gen in mammalian C’3, therefore, seems to be homogeneous insofar as its reactivity with K is concerned. This may be because the K-gen is identical in all the species tested or it may be that K is not as “discriminating” in its reactivity as an antibody would be.

2. I-K-gens In contrast to the situation with K-gen, there is ample evidence that I-K-gens are heterogeneous. First, this heterogeneity involves different complement components, C’3 and C‘4, as already discussed. Second, study of other aspects of this heterogeneity has so far been done almost wholly by the “resuspension” technique and so applies only to the anti-C‘3 I-K’s. These show a marked degree of species specificity. Rabbit (autostimulated) I-K’s generally-but not invariably-show higher titers when tested on a rabbit indicator than on an equine indicator. Absorption with antigen-antibody complexes alexinated with horse complement readily removes I-K activity to the equine alexinated cells usually with only marginal effects on the reactivity toward rabbit alexinated cells. Absorption with antigen-antibody complexes alexinated with rabbit complement removes K and I-K’s rather poorly-less reactants appear to be fixed than is the case with equine complement-but the titer toward the equine indicator falls with that toward the rabbit indicator (Lachmann and Liske, 1966b). This supports the view of Coombs and Coombs (1953) that these I-Ks are autostimulated antibodies in that their reaction with equine complement behaves like a

512

P. J . LACHMANN

cross-reaction. IIc.tcrostimulated rabbit I-K’s have shown only trivial (if any) reaction with rabbit indicators, in contrast to their high titers with the equine indicator, and thus represent antibodies to different I-K-gen determinants in (2’3. When tested on the human and guinea pig indicators, rabbit I-Ks generally react to good titers (comparable to those given with equine indicator) on human but generally less well on guinea pig. There is further evidence that there is intraspecies heterogeneity of I-K-gens in rabbits. This stems from the observation that antigenantibody complexes alexinated with different rabbit complements show marked differences in their absorbing capacity toward different I-K‘s (Lachmann and Coombs, 1965); and testing on different rabbit indicators has shown distinct patterns of reactivity with different I-K’s (Menson, 1967). It has not, however, so far proved possible to establish a genetic basis for these variations, and it is not yet clear if these intraspecies differences reflect allotypy of rabbit C’3 or if they are a consequence of quantitative factors influencing the fixation of C’3. The I-K’s detecting the intraspecies differences appear, in any case, to represent only a small part of the I-K present in a given serum. In this respect, as in many others, I-Ks may show a resemblance to rheumatoid factors. True autoreactivity of rabbit I-K may be shown in vitro by alexinating EA with its own complement, when strong conglutination is found to a dilution corresponding to the complement titer. Absorption of rabbit I-K on its own complement in ~ i t by r ~adding antigen-antibody complexes to the fresh I-K serum works badly (Ingram, 1958; Lachmann and Coombs, 1965) probably owing to the generally poor activity of Ag/Ab/C’(rabbit) as an I-K absorbent; but when this experiment is done in uivo by challenging an I-K producing rabbit with bacteria, there is a sharp fall in I-K titer demonstrating the autoreactivity (Coombs et al., 1961). Human I-K’s appear to show a general similarity to those in rabbits. Reactivity with human complement has been shown on the human indicator cells ( Lachmann, unpublished observations, 1966) but mostthough not all-human I-K sera have shown higher titers on guinea pig indicator than on human indicator. True autoreactivity is readily shown on those I-K sera from whom a conglutinable indicator can be made. This autoreactivity is, in fact, a nuisance as a number of otherwise suitable human R3 reagents have to be rejected because they are agglutinated by their own I-K‘s. Ingram (1958) and Bicnenstock and Bloch (1966a) have shown that absorption of a fresh human I-K serum with


513

Ag/Ab complexes reduces its I-K activity on an equine indicator, again showing autoreactivity.

F. INHIBITION OF CONGLUTINATION BY SUGARS Leon and Yokohari (1964) tested a variety of sugars for inhibition of conglutination of alexinated cells and found N-acetyl-D-glucosamine and closely related acetamido sugars to be the most active inhibitors. LFucose was also inhibitory. D-Glucosamine and D-fucose and a wide variety of other mono- and disaccharides and their derivatives were inactive. Leon and Yokohari found that 4 to 8 x le4M N-acetyl-D-glucosamine would inhibit conglutination in their system. Bienenstock and Bloch (1966a) confirmed that 0.1 M concentration of this sugar would completely inhibit conglutination by a bovine serum with high conglutinin titer and showed that this concentration had no effect on conglutination by human I-K. In accord with these results, Lachmann et al. (1966) found that to inhibit conglutination of one dose (1.5 x loi) alexinated cells by three doses (0.03 pg.) conglutinin needed about 350 ,pg. N-acetyl-D-glucosaminne-corresponding in the test mixture to 4 )( l W M N-acetyl-D-glucosamineand about M conglutinin. The inhibition produced was virtually independent of the concentration of calcium ions present. N-acetyl-D-glucosamine also inhibited the conglutination of zymosan by K but failed to inhibit the conglutination of alexinated cells by rabbit I-Ks or even by the chicken I-K’s that behave like antibodies to K-gen. Chitin, an insoluble polymer of N-acetyl-Dglucosamine, gave no significant reaction with K. Leon and Yokohari (1964) pointed out that the specific sugar moiety-an acetamido sugar such as N-acetyl-D-ghcosamine and possibly L-fucose-might represent a portion of the conglutinin molecule, a portion of the complement component (i.e., the K-gen), or even a portion of the antibody molecule altered by complement fixation. Since N-acetylD-glucosamine inhibits the conglutination of zymosan, the last possibility can be discounted. The second possibility, i.e., that the sugar might be a “hapten” to the K-gen and can thus compete with the K-gen for a site on K, is attractive. That on a weight basis the sugar is less than 0.1% as efficient an inhibitor as the microsomal yeast K-gen is not incompatible with a “hapten-antigen” relationship. On the other hand, although C’3 does contain both hexosamine and fucose ( Muller-Eberhard et al., 1960), the microsomal yeast K-gen does not appear to contain any of these sugars. Furthermore, the observation (Lachmann et al., 1966) that the inhibitory effects of microsomal yeast K-gen and N-acetyl-D-glucosamine

514

P. J. LACHMANN

c.o~igIutinationinterfere \i.ith rather than augment each other would be more compatible with the sugar’s reacting with the K-gen rather than the K, i.e., suggesting that it is not a hapten. The sugar might, therefore, conceivably represent a portion of the conglutinin. Conglutinin, however, contains Iess than 1% carbohydrate and though the amount of sugar needed to form an active site could be undetectable at this level, the idea does not seem very likely. With present knowledge, the nature of this inhibition of conglutination cannot, therefore, be certainly decided. More recently Leon et al. (1966) have reported that trimannose acts as efficiently in inhibiting conglutination as does N-acetyl-D-glucosamine. Since mannose is known to be the main sugar component of yeast K-gen ( Sections IV,A and B ) this trimannose oligosaccharide appears very likely to represent a hapten to the K-gen.

011

G . THE CHEhlICAL STABILITY OF CONGLUTINOGEN COKGLUTINOGENS IN FIXED COMPLEMENT

AND IMMUNO-

Crude fungaI “cellulase” rapidly destroys K-gen in conventional equine alexinated cells whereas reaction with rabbit I-Ks is lost incompletely and slowIy (Lachmann and Coombs, 1965) Bienenstock and Bloch (1966a) have found human I-K to resemble rabbit I-K in this respect. EAC’1423 made with purified human complement components has been tested for susceptibility to enzymes before and after treatment with KAF (Lachmann and Muller-Eberhard, 1967). It has been found that EAC’1423 before KAF treatment resists the action of the usual dose of fungal cellulase as shown by the retention of its reactivity with KAF. After KAF treatment, K-gen is readily destroyed by the enzyme and cannot be restored by more KAF. Similarly K-gen in EAC’1423 treated with KAF is destroyed by very small amounts of trypsin, amounts that have no detectable effect on EAC’1423. The amounts of trypsin needed to destroy the reactivity of EAC’1423 with 1-Ks (or in immune adherence) are much larger, though again EAC’1423 treated with K A F is more sensitive than EAC’1423.

H. THE LIBERATION OF CONGLUTINOGEN AND IMMUNOCONGLUTINOGENS FROM A C’3 FRACTION OF HUMANSERUM Attempts have been made to mimic the effect of the complementfixation reactions to the extent of exposing K-gen and I-K-gen activity from a semipurified C‘3 ( PIc-globulin1 fraction (Lachmann and Coombs, 1985). Unsuccessful treatments included the Sevag procedure (in an


515

attempt to isolate the carbohydrate moiety) and mercaptoethanol treatment (which potentiates K-gen activity in zymosan), Treatment with proteolytic enzymes was also generally unsuccessful though some I-Kgen activity was produced by trypsin. Mild acid treatment was effective in exposing both K-gen and I-K-gen to some extent, but this treatment lead to precipitation of the C’3 so that no soluble material was available for study. However, coating red cells with PICpreparation by tanned cell or bisdiazobenzidine techniques was ineffective in exposing K-gen or I-K-gens, suggesting that aggregation per se is insufficient to explain the effect of the acid treatment. Crude fungal “cellulase” was found to produce some K-gen activity from the partially purified C’3 fraction. This, incidentally would also have contained KAF. C’3i, the material produced from purified C’3 by the action of EAC’142, shows no K-gen activity but does have some I-K gen activity for certain I-K’s. Soluble K-gen has still to be isolated from highly purified (73 with or without KAF. VII.

Conglutination as a Serological Tool

Many types of cell, if sensitized with a complement-fixing antibody, may be conglutinated in the presence of complement and K. This “direct conglutination reaction,” therefore, provides a test for antibody. Because the aggregation produced by conglutination is so strong, the reaction found special application in the detection of antibodies against poorly agglutinable, freshly isolated, bacterial suspensions. The use of this reaction in serodiagnosis was fully reviewed by Coombs et al. (1961), and there is little that can be added to what they wrote at that time. Perhaps the main importance of the reaction remains the direct conglutination of sheep cells-the model on which studies of the mechanism of conglutination and of complement fixation can be performed. Since conglutination, like hemolysis, is a consequence of complement binding, a complement-fixation test analogous to the hemolytic complement-fixation test but using a conglutinating indicator can be devised. Such a “conglutinating complement absorption test” was first shown to work by Streng (1910) and fully described by Hole and Coombs (1947a,b). The value of the reaction is that antibodies of certain mammalian species (and this includes some human antibodies) have been found empirically better able to show fixation of complements (particularly equine) producing conglutination than they are of fixing complements (such as guinea pig or human) that produce hemolysis (Coombs and Hole, 1948; Blomfield et al., 1949). There is, again, little to add to the account of the uses of the conglutinating complement absorp-

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tion test given by Coombs et al. (1961). Cisalpino and Hurtado (1957) found the technique more sensitive than capillary precipitation for demonstrating C-reactive protein in human sera by reaction with rabbit antiserum. Lang (1962) reported promising results with the technique in showing the production of antibodies in cats during infection with panleukopenia virus; cat antibodies being poor fixers of guinea pig complement. K. J. O’Reilly (personal communication, 1963) was able to confirm these results. It may be anticipated that this reaction will find wider use in serodiagnosis, particularly where antibodies from different animal species are to be compared. A mixed conglutination reaction has been developed (Lachmann et al., 1965) where zymosan particles are bound to regions of complement fixation by the reaction of both with conglutinin. This allows the reaction of particular cells with antibody and complement to be localized under the microscope and is applicable to the study of dead or fixed cells, or those obtained by the disaggregation of tissues, which cannot be studied by immune cytolysis. VIII.

The Biological Significance of Conglutination

A. MECHANISMS THATM A Y BE INVOLVED In discussing the biological significance of conglutination it is perhaps profitable first to enquire what mechanisms can be envisaged that would allow the presence of K or I-K’s to influence allergic reactivity in the host.

1 . Aggregation The obvious manifestation of conglutination is the clumping of alexinated matter. In reactions that depend primarily on the number of particles rather than their total bulk, such clumping may have significant consequences. This may be relevant in infections where the outcome may depend on the effective number of infecting organisms. Ingram (1959b; vide infra) showed that mice given conglutinin or I-K passively showed a higher resistance to infection, especially with small numbers of virulent organisms. This protection could be attributed to the effect of conglutination in reducing the effeetiw number of invading organisms. 2. Augmentation of C o m p l e m n t Fixation It is relatively simple to show that in the presence of K or I-K more complement is fixed by EA than if these factors are absent. In the usual


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test system with horse complement the increase was found to be in the region of 25% (Lachmann, 1962). It is much less easy to be sure whethcr this is due to the reaction between K and K-gen, or I-K’s and I-K-gens, initiating fresh sites of complement fixation, since it is difficult to produce alexinated cells that fix (or inactivate) no further complement on their own, However, Ingram ( 1958) performed complement-fixation tests with equine alexinated cells and sera containing various amounts of I-K and showed that the dilution of the I-K serum required to give complement fixation was proportional to its I-K titer. He also performed the further experiment of treating the alexinated cells with guinea pig complement in the presence of I-K sera. The naturally occurring bovine antisheep cell antibody used to make EA does not fix guinea pig complement wcll so that no hemolysis was found with the alexinated cells and the guinea pig complement per se. The addition of I-K sera, Ingram found to produce lysis whereas that of sera that contained no I-K did not. A bovine serum containing a high titer of K failed to produce hemolysis, in this system. Bienenstock and Bloch (1966a) have repeated this experiment with human I-K and obtained similar results. It, therefore, seems likely that the reaction of I-Ks-which as IgM antibodies might be expected to be C’-fixing-with I-K-gens does fix complement. Under these circumstances one would anticipate that C’ fixation followed by I-K reaction followed by more C‘ fixation, and so on, would proceed until either all the complement or all the I-K were used up. This, however, is found to be by no means the case, so some mechanism for stopping the reaction “cycling” must exist. The situation with regard to K and K-gen is less clear. Conglutinin could not be shown to fix guinea pig complement with yeast K-gens (uide supra) but complement of other species have yet to be investigated.

3. lmmune Adherence Immune adherence by alexinated matter appears to depend on an activity of fixed C’3 (Nishioka and Linscott, 1963; Nelson, 1965) and so its relation to conglutination is clearly of interest. Turk ( 1959) tested the effect of K (as heat inactivated bovine euglobulin) on the immune adherence produced by starch “sensitized with guinea pig y-globulin and by complexes of diphtheria toxin and guinea pig antibodies in the presence of horse or guinea pig complement. In all cases he obtained inhibition of immune adherence with quite modest amounts of conglutinin-just enough, in fact, to produce strong clumping of the alexinated starch. It would seem most likely that the clumping

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action of the conglutinin, by greatly reducing the number of alexinated sites available, was responsible for this inhibition. Sell (1965) performed immune adherence on tissue cells treated with antibody and complement. In this system, where the clumping effect is virtually inoperative, a very large (1000-fold) excess of conglutinin was required to inhibit immune adherence to any appreciable extent, suggesting that blockage of the “immune adherence site” on C‘3 by conglutinin is, in fact, difficult to achieve. It might, perhaps, be possible to select I-K’s with a specificity toward this site-or a region in close proximity to it-which would be more effective in this respect; and considerable biological significance could be read into such I-Ks if they were found in significant amounts.

4. Opsonizutwn In vitro study of the effect of K and I-K‘s on phagocytosis is in general vitiated by the agglutination produced, and it is still not known for certain if the combination of K or I-K with fixed C’3 would per se affect its activity as an opsonin (Nelson, 1962). In uiuo, Ingram (1958) has shown that mice given I-K passively clear bacteria (Salnwnella typhimurium) more rapidly from their circulation, mainly into liver and spleen, than do normal mice. The findings of Wu and Marcus (1964) that the “cytopepsis” of Histtoplamul cupsulatum is increased by a heated fraction of bovine serum likely to contain conglutinin has already been mentioned. This phenomenon would not necessarily involve complement since H . capsulutum itself contains K-gen.

5. Formation of Anuphylatoxin The formation of anaphylatoxin involves the action of (classical) C’3 (Osler et al., 1959). However, no effect on the production of anaphylatoxin was found in the presence of large quantities of purified K ( W. E. Jonas and P. J. Lachmann, unpublished observations, 1964). No survey of I-K’s in this system has been carried out. 6. Allergic Reactions in Viuo

Coombs and Gel1 (1963) classified the allergic mechanisms of tissue damage in uiuo into four types, and their nomenclature is used here. Type 1 allergic reactions-of which classical anaphylaxis is the prototype-are now believed to be mediated by noncomplement-fixing antibodies (see Benacerraf, 1965), so that no effect of conglutination would be anticipated. Cattle for all their conglutinin are certainly quite cap”ble of producing anaphylaxis ( Bray, 1937; Code and Hester, 1939).


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Type 2 allergic reactions are the reactions of allergic cytotoxicity. Hemolysis of EA in vitro provides a model for this type and the effects of K and I-K on this have been described in a previous paragraph. There is little information on in vivo Type 2 situations with regard to the effect of conglutination. It might be expected that effects would be greater where the cells involved could be “clumped” (for example, allergic thrombocytopenia or hemolytic anemia). Type 3 allergic reactions have the Arthus reaction as their prototype. Here the involvement of complement is believed to play a vital pathogenic role and here it would be of most interest to know whether significant modifications are produced by the presence of K and I-K. In preliminary experiments R. R. A. Coombs and D. G . Ingram (unpublished observations, 1959) found no obvious differences from controls in Arthus reactions produced in rabbits with high I-K titers. In so much as the reaction of I-K with fixed complement initiates more complement fixation, this process might play some part in prolonging the allergic inflammation of Type 3 reactions. However, experimental evidence for such prolongation is not so far available. Type 4 reactions, as exemplified by the tuberculin reaction, do not involve serum antibody and it is not known if complement fixation has any necessary part in these reactions. Cattle, again, give normal tuberculin reactions. B. THEVARIATIONS OF CONGLUTININ AND IMMUNOCONGLUTININ TITERS IN DISEASE

1. I n Cattle Streng (1909) and von Jettmar (1923) reported that in infectious diseases in cattle the K level falls. Cantoni and Casillo (1963) reported that conglutinin levels in tuberculin-positive cattle were higher than in comparable tuberculin-negative animals. LePage and Matson ( 1965) studied calves experimentally infected with Rickettsia phagocytophilia. They found a sharp fall in K titer on infection followed after some days by the appearance of I-K. There is a marked drop in K titer in normal cows at the time of calving. Conglutinin is to be found in bovine colostrum but is generally absent (by resuspension technique) in presuckling calf serum (R. W. F. LePage and R. R. A. Coombs, unpublished observations, 1964; Ingram and Barnum, 1965). However, Henson (1967), using the agglutination pattern method, found that low titers were usually present in presuckling calf sera. These results indicate that although conglutinin appears to be

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consumed in allergic reactions there is no evidence that its formation is stimulated by such reactions. This is consistent with its “nonantibody” nature. 2. I n Erperinierital Animals

The formation of I-Ks in rabbits and mice in response to infection and the role that these may play in the resistence to such infections has been studied extensively by Ingram (1958, 1959a,b), Iagram et a]. (1959), and Ingram and Soltys (1960), and the results are summarized by Coombs et al. ( 1961). a. The Formation of I-K in Experimental Infections. When rabbits were infected with Listerin monocytogenes through the conjunctiva to give a local infection there was a slight rise of I-K titer at about 10 days. JVhen pregnant rabbits were so infected, a systemic infection ensued, and I-K titers were followed in a rabbit that survived this infection. The Iow I-K titer initially present fell to nothing immediately after infection. Fresh I-K activity appeared on the fifth day and reached its peak between the tenth and fifteenth day, then falling back to the preinfection level. Similar behavior was found in rabbits infected by mouth with Salmonetla typhimurium. In rabbits who have relapses these are accompanied by a drop in I-K titer followed by a further subsequent rise. The initial consumption of I-K early in the infection can be shown more readily if the animals are stimulated to produce I-K with an unrelated killed organism before infection. In general the extent of the I-K response was proportional to the severity of the infection clinically. Acute and chronic bacterial infections in mice similarly gave rise to I-K production to an extent proportional to the severity of the challenge. Animals with pre-existing antibody to the infecting organism produced higher titers. Infection of rabbits with encephalitis viruses produced a modest titer of I-K. Infection of guinea pigs with Rickettsia burnetti gave rise to two peaks of I-K activity, one at 2 to 3 weeks and one at about 10 weeks, apparently corresponding in time to antibody formation to two distinct antigens of these organisms. Infections of rabbits with trypanosomes produced the highest titers of I-K found in these studies. It was shown that successful treatment of the infection was associated with a drop in I-K titer and it was suggested that this might prove a useful parameter for controlling treatment. Cats infected with trypanosomes also produced high titer I-K. There is thus good evidence that I-K is consumed in the initial stages


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of an infection in experimental animals and its production stimulated so that maximum titers are reached about 10 days later. In acute infections the level then rapidly falls over the next 23 weeks, but in chronic infections the high I-K titer may be maintained. b. Evidence That K and I-K Increases Resistance to Infection. These experiments (Ingram, 1959a,b) were performed on mice who were given subcutaneously either heated bovine euglobulin (as source of K ) or rabbit heterostimulated I-K (raised by injecting unheated horse serum on kaolin) prior to bacterial challenge. Ingram was able to show that at the time of challenge there was conglutinating activity in the sera of his mice and that this fell sharply after challenge. If infection was performed with avirulent or nonpathogenic organisms in s&cient number to cause death in the controls by “toxemia,” no protection was afforded by the K or heterostimulated I-K. However, if the infection was with virulent organisms, which cause death by establishing themselves and dividing, then protection by both factors was demonstrated. Absorption of the bovine euglobulin or rabbit serum with alexinated cells to remove conglutinating activity abolished this protection. The protection afforded by the conglutinin and by the heterostimulated I-K were of the same order of magnitude. This evidence demonstrates that the presence of conglutinating activity can afford protection against bacterial infection. The similar activity of the two reagents tested and the restriction of the effect to the virulent organisms, where the number of infective units is likely to be important, rather than to avirulent organisms, where the lethal toxemia probably is a function of the mass of the inoculum, both suggest that the “clumping” effect of the conglutination may play an important part in this protection. If this is the case, autostimulated I-K would be anticipated to have a similar effect.

3. I-K in Man Marks and Coombs (1957) examined the sera of approximately 6OOO blood donors for I-K. Rather more than 50%had no detectable conglutinating activity and rather less than 101%titers of more than 4. When monthly samples from different localities were tested it was found that the distribution of I-K titers among this healthy population occasionally showed shifts in the direction of higher titers. There was some suggestion that this might be associated with epidemics of infectious disease (Sonne dysentery and influenza). Marks and Coombs (1957) also studied in some detail the distribution of I-K in various diseases. They found elevated levels in acute, and

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particularly in chronic, bacterial infections and in acute viral infections ( mainly poliomyelitis). In the acute bacterial infections the I-K titers showed some proportionality to the duration of symptoms (within the first 2 weeks), suggesting that the kinetics of I-K stimulation here are similar to those observed in rabbits, Elevated titers were also found in patients with acute nephritis, ankylosing spondylitis, rheumatoid arthritis, acute rheumatic fever, and mitral stenosis. Another survey of I-K in blood donors has been carried out in Warsaw (Jasser and Bragiel, 1962). The titers found here were rather higher than those found by Marks and Coombs (1957)-30% of sera having titers above 4 and only another 30% having I-K in undiluted serum or none at all. The authors attributed this to a higher incidence of subclinical infection in the Polish population. However, differences in the reactivity of indicator systems and observer daerences in reading end points may affect comparisons between results at different centers enough to account for this degree of variation. Immunoconglutinin titers have been reported in a number of other human diseases, Pernis d al. (1959b) reported raised levels in patients with silicosis. Caspari and Ball (1962) confirmed the raised level in rheumatoid arthritis and reported consistently high levels in Hashimotds disease. However in multiple sclerosis, both acute and chronic, normal values were found. Bienenstock and Bloch (196613) surveyed I-K titers in a number of diseases. On the basis of a group of 50 blood donors they considered a titer of 16 as the upper limit of normal in their test system and estimated the percentage of patients with titers higher than this figure for various diagnoses. Raised levels were found in over two-thirds of patients with Hashimoto’s disease and primary gout; between onethird and two-thirds of patients with Sjogren’s syndrome, systemic LE, myasthenia gravis, rheumatoid arthritis, and ankylosing spondylitis; and in less than one-tenth of patients with myocardial infarction, osteoarthritis, or traumatic arthritis or of the blood donors. In general these findings are all compatible with the view that I-K formation reflects in vioo complement fixation. Bienenstock and Bloch‘s finding of high levels in primary gout is perhaps surprising. However Barnett et uZ. (1968) have reported finding IgG, IgM, and C’3 (PICglobulin) as well as urate crystals in macrophages from synovial fluid in gout, so that the urate crystals may be providing a focus for a complement-fixing system. Analogous, perhaps, to Ingram’s ( 1959a) finding in rabbits that challenge with bacteria leads to a sharp fall in I-K, Bienenstock and Bloch (1966b) found that in a patient with systemic LE, acute exacerbation was accompanied by a drop in I-K titer. Similarly


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a drop in titer was found in five patients within 2 days of receiving a kidney graft. All the above I-K estimations were carried out on equine alexinated cells by the “resuspension” technique. However, by the “sedimentation pattern” technique on guinea pig alexinated cells (Lachmann, 1966b) no human serum so far tested has given an entirely negative result, and it seems likely that some I-K activity-and, therefore, probably some in uiuo complement fixation-is the normal state of affairs. By this method, too, high values have been found in rheumatoid arthritis and chronic bacterial infection and particularly high values in patients with West African trypanosomiasis ( Lachmann and Watson, 1966). It seems likely, therefore, that the general pattern of reactivity in disease will prove to be similar by both techniques. There is as yet insufficient data even to speculate whether the anti-C’4 I-Ks which can be detected only by the sedimentation pattern method will show a distribution in disease different from that of the anti-C’3 I-K’s; and whether measuring I-Ks on a number of species of complement, including human, will yield greatly differing results. IX. Summary

The phenomenon of conglutination is brought about by two distinct and apparently unrelated serum factors. Conglutinin is an unusual serum protein found only in ruminants that react with its “antigen” (the conglutinogen) through the mediation of calcium ions. It is a highly asymmetric molecule and its hydrodynamic and chemical properties distinguish it sharply from the immunoglobulins. Conglutinogen is found not only in fixed complement. It occurs also in yeast-both in the cell wall and in the microsomal fraction-and in certain related organisms. Conglutinogen is predominantly polysaccharide. The conglutinogen in fixed complement is found in fixed C’3 of all species tested. Immunoconglutinins are a group of predominantly IgM autoantibodies formed apparently in response to the antigenic stimulation of the animal’s own fixed complement. They are heterogeneous in their reactivity. The bulk of I-K is anti-C’3, but anti-C’4 I-K has also been identified. Even the anti-C’3 I-Ks are still found to be markedly heterogeneous in their reaction with different species of complement and even within a single species. Immunoconglutinins are formed in all mammalian species tested and in chickens. With regard to their biological significance some tentative conclusions may be made: (1) both K and I-Ks are consumed in in uiuo

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complement-fixing actions; ( 2 ) although the formation of K is not stimulated by in v i m complement fixation, the production of I-Ks correlates with this process both in experimental and clinical situations; ( 3 ) the presence of K and I-K can afford some protection against infection by virulent organisms. Except for the third conclusion there is, however, little information as to whether the involvement of K or I-K in complement-fixing reactions in d c o is harmful or beneficial to the host. In particular it is not yet knovr7n what effect, if any, the involvement of K or I-K might have on the part that fixed complement plays in the allergic mechanisms of tissue damage-the Types 1 4 allergic reactions of Coombs and Gel1 (1963).In the context of the human autoallergic diseases, it is, therefore, not clear whether the raised I-K titers should be viewed with approbation, indifference, or dismay! With regard to conglutinin the possibility should also be considered that its real significance lies in its reactions with the K-gens in yeasts and related organisms and that its reaction with fixed complement is merely a bizarre cross-reaction. ACKYOWLEDGMENT The author is much indebted to Professor R. R. A. Coombs for his interest and help. In so far as work done by the author is concerned, a grant from the Nuffield Foundation is gratefully acknowledged.

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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. Numbers in italic show the

page on which the complete reference is listed.

A Aarons, I., 53, 79 Abrams, R., 156, 205 Abramson, H. A., 488, 524 Abramson, S., 117, 218 Abruzzo, J. L., 234, 251, 467, 477 Achter, E., 128, 206 Ackerman, G. A., 402, 455 Ada, G. L., 275, 334, 386, 420, 449 Adams, E., 260, 334 Adamson, R. H., 199, 205 Adinolfi, hl., 377, 435 Adler, F. L., 174, 212, 257, 265, 289, 295, 296, 328, 330, 331, 384, 414, 421, 435, 441 Adlersberg, D., 371, 453 Adorjin, S., 443 Aggeler, P. hi., 179, 223 Aho, K., 467, 477 Ainis, H., 256, 268, 272, 328, 361, 435 Ainsworth, E. J., 407, 435 Aisenberg, A. C., 186, 205, 402, 435 Al-Askari, D. J., 297, 301, 332 AI-Askari, S., 159, 210, 303, 304, 328, 330 Alberts, E., 375, 458 Albright, J. F., 170, 218, 256, 271, 315, 323, 328, 329, 333, 360, 419, 429, 438, 447 Alderman, I. XI., 407, 453 Alexander, H. L., 126, 150, 208 Alexandre, G. P. J., 183, 205 Alican, F., 165, 219 Alksne, J. F., 51, 79 Allen, P. Z., 399, 435, 472, 477 Allibone, E. C., 97, 205 Allison, A. C., 322, 328, 486, 524 Altman, K., 197, 205, 226

Amano, T,, 377, 435 Amati, A., 187, 205 Ambrose, C. T., 108, 113, 171, 180, 205, 256, 269, 270, 280, 328, 361, 435 Ames, B. N., 294, 328 Amiel, J. L., 137, 143, 194, 195, 205, 219 Amin, A., 370, 435 Amoroso, E. C., 348, 435 Amundsen, E., 356, 457 Anastasi, A., 198, 207 Andersen, B. R., 168, 214 Andersen, E. K., 406, 438 Anderson, H*w., 178, 209 J. M . 7 347, 435 Anderson, N. A., 228 Ando, K., 462, 477 An&&, J., 165, 223 AndrB, J. A., 161, 205 Andres, G. A,, 19, 25, 48, 49, 79 A* c.*370, 437 Andrews, E. C., Jr., 50, 85 Anigstein, L., 19, 79, 111, 228 Anker, H. S., 261, 267, 268, 272, 279, 314, 335, 360, 361, 454 Ansley, H. R., 288, 329 Antonchak, N., 193, 213 Apt, L., 374, 442 Arana, J,, 63, 79 Arana, J. A., 8, 22, 79 Arbesman, C. E., 115, 205 Arbovys, S., 124, 200, 201, 227 Archer, G. T., 50, 79 Archer, 0. K., 98, 205, 403, 435, 455 Arhelger, R., 40, 41, 79 Arhelger, R. B., 25, 41, 79 Arias-Stella, J., 85 Armentrout, S., 179, 227, 425, 458

529

-

530

AUTHOR MDEX

Armstrong, J. C., 183, 216 Amason, B. G., 119, 124, 201, 205, 227. 369, 435 Arnesen, K., 243, 250 Amold, H., 128, 205, 206 Aronsson, T., 31, 86, 513, 526 Aschaffenberg, R., 347, 435,444 Asherson, G. L., 68, 69, 79, 83 Ashford, A. 193, 206 Ashworth. C. T., 57, 58, 60, 82 Askonas, B. A., 256, 264, 284, 286, 289, 296, 298, 328, 336, 351, 414, 421, 423, 435, 444 Asofsky. R., 2.36, 258, 263, 273, 328, 332, 336, 426, 456 Asotsky, R. XI., 259, 262, 291, 332, 3’36 Aspinall, R. L., 96, 206, 403, 435 AssimacopouIos, C. A., 165, 206 Aswag, M., 165, 222 Attardi, G., 274, 328, 362, 436 Aubuchon, I>., 93, 208 Auerbach, R., 147, 207, 226, 257, 265, 288, 325, 331, 361, 442 August, S., 147, 214 Austen, K. F., 79, 206 Austin, C . \i., 334, 386, 420, 449 Averich, E., 11, 80 Avery, 0.T., 236, 251, 293, 329 Axelrod, E. A., 419, 436 Axtell, H. K., 122, 166, 219 Azar, I I A,, 402, 436

B Bach, F., 174, 203, 206, 215, 292, 324, 325, 329, 331 Back, N., 128, 206 Badalamenti, G., 31, 32, 89 Baer, H., 393, 436 Baglioni, C., 374, 436 Baguena, J., 111, 219 BQguena, R., 111, 219 Bailey, C. H., 52, 79 Bain, B., 203, 206, 323, 324, 329 Baker, B. L., 110, 206 Baker, J. A., 141, 226 Baker, R., 134, 206 Bale, W. F., 6, 10, 19, 23, 24, 69, 79, 88 Balish, E., 356, 450 Ball, E. J., 522, 524

B d o , T., 351, 439 B a h e , H. \V., 137, 206 Baney, R. N., 315, 329, 423, 436 Bangham, D. R., 342, 344, 436 Banks, H., 191, 210 Baram, P., 301, 329 Barandun, S., 374, 436 Barber, E., 252 Barber, N . B., 197, 217 Barber, N. D., 419, 446 Barbu, E., 232, 234, 244, 245, 250, 252 Bardos, T . J., 128, 206 Barker, H., 502, 520, 525 Barker, P., 196, 228 Barnes, B. A., 165, 206 Barnes, D. W.H., 403, 443 Barnes, W., 19, 79 B nett, E. V., 524 Barnum, C. P., 199, 228 Bamum, D. A., 519, 525 Baron, L. S., 377, 448 Baron, S., 140, 141, 212 Barr, M., 400, 436, 438 Banick, E. R., 372, 436 Barth, W. F., 402, 446 Bartlett, S., 348, 435, 444 Bartol, G. M., 8, 81 Bartz, Q . R., 178, 182, 211, 212 Baserga, R., 120, 206 Bass, A. C., 371, 449 Battisto, J. R., 93, 101, 156, 206, 213, 427, 436 Batty, I., 351, 358, 436, 449 Bauer, D. C., 383, 436 Bauer, R. Ch., 375, 436 Baum, J. H., 183, 220 Baumann, J. B., 354, 388, 400, 401, 421, 424, 425, 432, 457 Baumgartner, F. A., 103, 208 Baumgartner, L., 393, 436 Baxter, J. H., 5, 9, 13, 19, 22, 31, 32, 79, 82, 88 Bazeley, P. L., 361, 436 Beale, H. D., 358, 443 Bealmear, M., 402, 458 Beak, T. F., 19, 20, 21, 86 Beamer, P. R., 356, 452 Beardwood, C., 373, 436 Beattie, E. J., Jr., 165, 221

AUTHOR INDEX

Beaumariagc, M. L., 115, 217 Becker, E. L., 32, 88 Bednaiik, T., 361, 451 Beer, C. T., 185, 220 Begemann, H., 194, 206 Behring, 92, 206 Beiber, S., 207 Bein, H . J., 115, 216 Beiser, S., 233, 240, 246, 251, 252 Beldotti, L., 143, 162, 224 Bell, A. L., 51, 85 Bell, E. T., 52, 79 Bell, M., 151, 224 Bellanti, J., 371, 457 Bellanti, J. A., 383, 387, 393, 436 Ben, M., 199, 205 Benacerraf, B., 46, 47, 50, 51, 79, 85, 93, 101, 117, 131, 206, 212, 233, 243, 249, 252, 255, 266, 303, 304, 321, 329, 333, 336, 357, 365, 382, 387, 399, 413, 415, 416, 417, 434, 436, 442, 446, 447, 456, 466, 467, 475, 477, 518, 524 Bencosme, S. A., 12, 79 Bendich, A., 120, 151, 199, 207, 233, 240, 251 Benedict, A. A., 384, 440 Benirschke, K., 346, 347, 435, 436 Benjamin, D. C., 371, 436 Bennett, B., 309, 311, 312, 329, 334 Bennett, I . L., 45, 87 Bennett, J. C., 294, 329 Bennett, L. L., Jr., 148, 151, 219, 224 Bennett, W . E., 256, 271, 329, 360, 438 Bennison, B. E., 102, 128, 139, 157, 219 Benos, S. A,, 117, 131, 206 Bentley, M., 156, 205 Berenbaum, M. C., 102, 136, 140, 144, 157, 168, 186, 206, 358, 362, 436, 437 Berger, E., 351, 375, 436 Berggard, I., 372, 436 Berglund, K., 99, 112, 113, 114, 206, 207, 211, 277, 329, 363, 419, 436 Bergstrand, C. G., 371, 436 Berman, L., 361, 436 Bernecky, J., 9, 10, 13, 19, 25, 83, 89 Bemeis, K., 194, 196, 206 Bernier, G. M., 275, 329

531

BernovskL, J., 276, 329, 363, 436 Berns, A. W., 54, 79 Bernstein, M. T., 277, 290, 332 Bertelli, A., 187, 188, 189, 206, 207 Berthrong, M., 45, 87 Bertino, J. R., 138, 139, 207 Bertram, L. F., 115, 205 Berumen, L., 194, 219 Betke, K., 374, 437, 445 Bevans, M., 4, 5, 7, 11, 19, 29, 79, 88 Bezer, A. E., 119, 216 Bianco, A. R., 143, 213 Bickerick, R., 376, 450 Bieber, S., 139, 157, 164, 168, 170, 180, 211, 220, 409, 449 Bielschowsky, M. B., 119, 207 Bienenstock, J., 495, 502, 512, 514, 517, 522, 524 Bierman, H. R., 367, 437 Biggs, P. M., 340, 437 Bigley, H. J., 234, 243, 251 Bigley, N. J., 244, 249, 251 Billingham, R. E., 94, 100, 121, 124, 207, 353, 354, 390, 410, 411, 437 Binet, J. L., 276, 277, 329, 363, 437, 438 Bing, J., 96, 207 Biorcklund, A., 312, 329 Biozzi, G., 79, 117, 131, 206, 382, 437 Birch-Andersen, A., 20, 90 Biro, C. E., 482, 526 Bid, Z., 97, 215 Bishop, J. E., 356, 438 Bisiani, M., 189, 207 Bj@meboe,M., 96, 111, 207, 373, 437 Black, J., 119, 220 Black, L., 358, 437 Black, M. M., 288, 329 Black, P. H., 146, 218 Black, R. L., 147, 207, 221 Blair, J., 8, 22, 63, 79 Blair, J. D., 79 Blaaovii., D., 383, 455 Blau, M., 24, 26, 79 Blix, U., 231, 232, 251 Bloch, H. S., 198, 207 Bloch, K. J., 495, 502, 512, 514, 517, 522, 524 Block, M., 345, 368, 394, 430, 446 Block, M. H., 403, 451

532

AUTHOR INDEX

Bloedow, C., 198, 226 Blomfield, A. hl., 499, 500, 507, 509, 515, 524, 525 Bloom, B. R., 172, 207 Blozis, G. D., 56, 57, 59, 63, 79 Blum, I., 507, 527 Blum, I. H., 377, 450 Blum, K. U., 194, 206 Blumenstock, D. A,, 141, 145, 207, 214, 21,5, 226 Blumenthal, H. T , 54, 79 Bodel, P., 183, 218 Bogdanov, B., 189, 207 Bohle, A., 8, 85 Bohonos, N., 171, 222 Boisvert, P. J., 104, 105, 222 Bollag, W.,194, 196, 206, 209, 223 Bollinger, F . W., 22. 82 Bond, V. P., 298, 330 Bonmassar, E., 188, 207 Bonting, S. L,, 11, 79 Booth, B. A,, 167, 207 Bordet, J., 480, 493, 524 Borek, F., 417, 437 Bore], Y., 156, 159, 207 Borenfreund, E., 199, 207 Borg-Petersen, C., 342, 394, 441 Borisova, N. B., 234, 252 Ro~tree,,4. L., 348, 440 Borum, K., 114, 207 Rosch, H., 97, 215 Boss, J. H., 11, 13, 19, 21, 22, 23, 79 Bossak, E. T., 371, 453 Boult, E. H., 41, 89 Bourne, G. L., 346, 436 Bourseaux, E., 128, 205, 206 Boursnell, J . C., 506, 524 Bowser, H. T., 393, 436 Boyd, 11'. C.. 377, 437 Boyden, S. V., 50, 88, 406, 437 Boyer, G., 208, 251, 278, 329 Boyns, A. R., 469, 477 Boyse, E. A., 309, 311, 312, 329, 334 Bradley, S . G., 128, 208, 348, 359, 388, 393, 395, 401, 403, 445, 449 Bragiel, I., 522, 525 Brambell, F. W . R., 347, 348, 350, 351, 371, 400, 436, 437, 443 Brandes, \\'. C., 376, 454

Brandis, It., 405, 437 Brandriss, M. W., 119, 141, 146, 147, 168, 208, 303, 214, 329 Brandt, L. W., 370, 437 Brauer, J. A., 339, 437 Braun, W.,232, 233, 233, 236, 237, 238, 239, 242, 247, 248, 251 252, 277, 288, 329, 335, 404, 405, 437, 438, 443 Braun, H., 374, 445 Bray, G. W., 518, 524 Brean, H., 45, 52, 88, 127, 223 Brecher, G., 233, 251, 361, 447 Breidenbach, G., 356, 452 Brennan, hl. J., 185, 226 Brent, L., 100, 207, 299, 329, 353, 354, 390, 437 Brent, R. L., 11, 79, 80 Breuninger-Peck, E., 336 Brkzin, C., 137, 195, 205 Bricker, N. S., 39, 86 Bridges, R. A., 179, 208, 354, 372, 390. 392, 442, 453 Briggs, C., 348, 435 Briggs, D. R., 198, 207 Briggs, F. N . , 193, 227 Briggs, G. hl., 139, 146, 214 Briot, hl., 111, 214 Bristow, E . C., 111, 196, 228 Brit, J., 206 Brittain, R. S., 122, 123, 124, 166. 224 Broberger, O., 309, 310, 312, 329. 334 Brock, N., 128, 205, 206 Brock, T . D., 178, 208 Brocklenhurst, W. E., 99, 208 Brodie, B. B., 193, 218 Brody, G. L., 136, 208, 216, 438 Brody, J. L., 356, 375, 438 Brondz, B. D., 309, 311, 312, 329 Bronsky, E. A., 112, 209 Brooke, hl. S., 139, 208, 402, 438 Brookes, P., 125, 208 Broschard, R. W., 172, 227 Brown, C. S., 168, 227 Brown, F., 191, 208 Brown, G. B., 251, 208 Brown, H., 350, 365, 453 Brown, I. N., 140, 144, 157, 168, 178, 206, 208

533

AUTHOR INDEX

Brown, L., 299, 329 Brown, R. C., 308, 310, 334 Bruch, H. E., 30, 86 Brule, G., 194, 219 Brunner, K. W., 194, 208 Brunson, J., 25, 79 Brunson, J. G., 40, 41, 79 Bruton, 0. C., 97, 208, 374, 438 Bryant, J. C., 361, 440 Burckhardt, K., 438 Bryan, C. E., 151, 224 Bryant, B. F., 216 Brzosko, W . J., 47, 50, 85 Buchanan, J. M., 182, 218 Buckler, C., 140, 141, 212 Buckler, C. E., 140, 141, 142, 212 Buckley, S. M., 149, 225 Buemann, J., 55, 88 Buffk, D., 275, 329 Bukantz, S. C., 44, 80, 89, 93, 126, 208 Bulman, H., 288, 304, 315, 316, 319, 321, 330 Bunim, J. J., 147, 207, 221 Burchenal, J. H., 120, 151, 179, 208 217 Burckhardt, K., 352, 438 Burdge, D. C., 148, 208 Burgi, E., 211 Biirgin-Wolff, A., 351, 436 Burkholder, P., 54, 83 Burkholder, P. M., 31, 80 Burkholder, P. R., 178, 211 Burnet, F. M., 96, 99, 122, 208, 227, 339, 365, 387, 389, 390, 438 Burnett, F. M., 53, 83, 208 Burnett, J. P., Jr., 185, 216 Burtin, P., 275, 329 Buscheck, F. T., 361, 436 Buskirk, H. H., 128, 208 Bussard, A., 171, 218, 275, 276, 332, 363, 437, 444 Bussard, A. E., 276, 277, 329, 363, 438 Butler, N. R., 400, 438 Butler, V. P., Jr., 233, 240, 251 Butler, W . T., 128, 149, 157, 177, 180, 181, 184, 185, 208, 425, 441 Buxton, A., 347, 386, 393, 438 Buxton, C. L., 371, 448 Byers, H. D., 165, 214

C Calabresi, P., 159, 169, 217 Calcagno, P. L., 5, 80 Calcott, M. A., 505, 510, 526 Calkins, E., 53, 80, 87 Callahan, S., 164, 211 Callaway, J. M., 122, 189, 229 Calman, R. M., 351, 448 Calne, C. F., 55, 56, 86 Calne, D. B., 134, 208 Calne, R. Y., 123, 160, 208 Cameron, G., 28, 84 Cameron, I. L., 405, 442 Cameron, J., 115, 208 Camp, W. E., 103, 208 Campbell, A., 294, 329 Campbell, B., 96, 106, 107,208, 3, 21 Campbell, D., 4, 28, 84 Campbell, D. H., 5, 6, 39, 42, 84, 289, 298, 329, 335, 400, 421, 423, 438, 464, 468, 477 Campbell, P. N., 351, 435 Cann, J. R., 427, 456 Cannon, J. A., 122, 180, 208 Cantoni, C., 519, 524 Capalbo, E. E., 256, 271, 315, 323, 329, 333, 360, 438 Cardinali, Giuliana, 185, 209 Cardinali, G., 185, 209 Carey, J., 96, 209 Carey, W . F., 377, 448 Carlinfanti, E., 426, 438 Carpenter, R. R., 168, 214, 303, 306, 329 Carr, 0. B., 25, 79 Carstairs, K., 361, 438 Carter, H. E., 178, 209 Casey, T. P., 120, 209 Casillo, S., 519, 524 Caspary, E. A., 522, 524 Caspersson, T., 173, 174, 209 Catron, D. V., 350, 365, 453 Cattan, A., 194, 219 Cavalieri, L. F., 149, 225 Cavelti, E. S., 56, 68, 80 Cavelti, P. A., 56, 68, 80 Caviles, A. P., 179, 223 Cebra, J. J., 275, 329 Celada, F., 232, 233, 251

,334

AUTHOR INDEX

Ceppellini, R., 232, 233, 251 Centeno, E., 23, 66, 68, 70, 85 Cerny, J., 353, 444 Cerrini, L., 189, 207 Chachulska, W., 405, 457 Chang, S. C., 8, 83 Chang, T. S . , 96, 213 Chapeau, l l . L., 4, 19, 25, 48, 49, 79, 88 Chapman, J. .4., 361, 441 Chapman, J. B., 151, 224 Chapman, N. D., 267, 324, 326, 329 Char, D. F . B., 373, 438 Charlier, H., 357, 451 Charlwood, P. A., 438 Chase, J. H., 110, 210 Chase, 31. W.,100, 172, 207, 209, 358, 389, 427, 428, 438 Chaube, S., 199, 220 Chen, K. H., 8, 83 Cherry, W. B., 355, 375, 438, 439 Cheville, N. F., 157, 216 Chew, if'. B., 200, 209 Chezzi, I., 450 Chiappino, G., 275, 329, 334 Chieco-Bianci, L., 361, 447 Chikamitzu, €I., 11, 19, 80, 89 Chin, D., 297, 301, 331 Chirigos, M. A., 143, 215, 216 Chmelai, >I., 377, 445 Chniielewicz, L. F., 128, 206 Chiistensen, J., 115, 209 Christensen, P. A, 400, 447 Christian, C. L., 33, 80, 234, 251, 428, 451, 467, 477 Christie, A., 393, 450 Churg, J., 4, 8, 10, 40, 80 Ciminera, J. L., 455 Cinader, B., 390, 400, 438 Cisalpino, E., 516, 525 Claman, H. N., 112, 209, 427, 456 Clark, D. S., 136, 189, ,209,213 Clark, J . G., 123, 228 Clark, R. F., 163, 217 Clark, S. L., 351, 438 Clarke, D. A., 150, 168, 209, 222 Clarkson, B. D., 125, 148, 150, 183, 217 Clausen, J., 377, 438 Clawson, B. J., 52, 79, 117, 209

Clegg, R. E., 370, 437 Clemmensen, J., 406, 438 Cloud, S., 147, 161, 227 Coburn, A. F., 104, 105, 209. Cochran, T. H., 87 Cochrane, C. G., 9, 10, 31, 3% 33, 34, 38, 43, 44, 45, 50, 51, 52, 80, 83, 90, 131, 209 Cochcrane, Ch. G., 358, 360, 438 Code, C. F., 518, 525 Coe, J. E., 416, 438 Cohen, A. S., 53, 80 Cohen, C., 399, 438, 469, 477 Cohen, E. P., 296, 299, 300, 319, 329, 423, 438 Cohen, J. O., 355, 375, 438, 439 Cohen, hi. W . , 260, 334, 398, 427, 439, 450 Cohen, S., 99, 120, 209, -233, 240, 251 Cohen, S. G., 347, 439 Cohen, S. L., 121, 220 Cohn, hf., 274, 328, 362, 436 Colberg, J. E., 275, 329 Cole, L. R., 22, 80 Cole, Q. P., 148, 222 Collins, D. N., 53, 83 Collins, J. A., 144, 145, 207, 226 Colombani, J., 203, 210 Colter, J. S., 235, 251 Comploier, F. C., 68, 88 Condie, R., 160, 219 Condie, R. M., 133, 135, 151, 153, 154, 158, 159, 161, 162, 163, 209, 212, 215, 217, 218, 219, 221, 372, 405, 406, 439, 442 Condit, P. T., 138, 209 Confalonieri, A., 189, 207 Connoly, J. M., 96, 209, 274, 332, 362, 423, 424, 446, 458 Controulis, J., 178, 209 Conway, H., 213 Cooke, R. A., 200, 228 Coombs, A. M., 466, 468, 476, 477, 480, 481, 494, 499, 500, 507, 511, 515, 516, 520, 525 Coombs, R. R. A., 377, 455, 466, 468, 476, 477, 480, 481, 483, 486, 487, 489, 491, 492, 494, 496, 498, 499,

535

AUTHOR INDEX

500, 502, 503, 504, 505, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 518, 520, 522, 524, 525 Coons, A. H., 96, 113, 128, 149, 157, 170, 171, 177, 180, 181, 184, 185, 205, 208, 209, 221, 225, 256, 259, 260, 265, 269, 274, 280, 281, 290, 295, 314, 315, 323, 328, 329, 332, 333, 334, 361, 362, 390, 418, 423, 424, 425, 426, 427, 428, 429, 435, 439, 441, 446, 448, 452, 458 Cooper, H. L., 317, 329 Cooper, M. D., 97, 98, 124, 209 Cooper, N. S., 31, 32, 50, 79, 89, 233, 243, 249, 252 Cora-Figueroa, E., 233, 238, 239, 241, 242, 252 Corcoran, A. C., 7, 19, 81 Cordova, C. C., 294, 333 Cornes, J. S., 98, 209 Cornwell, S., 117, 209 Corper, H. J., 125, 126, 215 Corvazier, P., 257, 314, 331 Corvazier, R., 352, 446 Cosulich, D. B., 172, 227 Cotchin, E., 348, 435 Cote, W. P., 403, 448 Cottier, H., 97, 226, 423, 439 Coulter, J. R., 376, 439 Coulthard, S., 363, 441 Courcon, J., 374, 442 Cowart, G. S., 375, 438 Cowen, D. M., 124, 290, 214, 331, 365, 417, 422, 442 Cox, H . R., 119, 220 Craddock, C. G., 232, 233, 252, 406, 439 Craig, J. M., 11, 79, 94, 97, 111, 209, 213, 374, 442 Cramer, L. M., 108, 189, 209 Crampton, C. F., 486, 527 Creasey, W. A., 167, 185, 207, 209 Cree, I . C., 135, 209 Creech, O., Jr., 123, 124, 145, 222, 228 Creger, W . P., 351, 439 Cremer, N. E., 257, 272, 274, 289, 329 Cress, H., 257, 272, 330 Crim, J. A., 128, 208 Crockett, K. A., 105, 222

Cromartile, W. J., 22, 80, 100, 216, 389, 426, 445 Crosby, L. K., 296, 300, 329 Crosby, W. H., 168, 211, 227 Crossland, A., 13, 19, 21, 22, 28, 83 Cruchaud, A., 181, 182, 209, 425, 439 Cruickshank, A. H., 200, 209 Cruickshank, B., 13, 19, 21, 22, 28, 80, 83 Cruickshank, J. G., 487, 526 Culling, C. F. A,, 308, 312, 335 Cummings, M. M., 117, 118, 209, 224 Cunningham, A. J., 277, 330 Cuppage, F., 57, 58, 60,61, 62, 82, 83 Cuppage, F. E., 35, 57, 86 Cutler, J. L., 405, 439 Cutts, J. H., 185, 209, 220

D Daft, F. S., 356, 450 Daguet, G., 137, 195, 205 d‘Alessandri, A., 194, 209 Dalmasso, A. P., 97, 214, 505, 526 Dalton, M. L., Jr., 165, 214 Dameshek, W., 93, 151, 155, 160, 161, 163, 168, 205, 210, 221, 224, 402, 407, 408, 428, 452 Dammin, G. J., 44, 80, 89, 94, 124, 126, 137, 150, 183, 205, 220 Dancis, J., 352, 383, 387, 393, 411, 439, 449, 457 Dandeu, J. P., 234, 244, 245, 250, 252 Danes, B. S., 361, 439 Daniel, T., 180, 181, 210 Daniel, T. M., 180, 227, 425, 458 Daniels, Ch., 377, 435 Danielson, D., 291, 331 Dannenberg, A. M., Jr., 117, 218 D’Antone, N., 405, 439 Darken, M . A., 171, 210 Darrach, M., 111, 220 Dausset, J., 203, 210 David, G., 11, 80 David, J. R., 159, 176, 181, 210, 303, 304, 328, 330 Davies, A. J. S., 340, 439 Davies, C. H., 99, 168, 212 Davis, E. V., 361, 447

536

AUTHOR INDEX

Davis, P., 198, 226 Day, E. D., 19, 24, 26, 79, 80 de Bodo, R. C., 373, 458 de Bruin, H., 11, 79 DeBruyn, P. P. H., 191, 210 de Duve, C., 110, 116, 210 Defendi, V., 439 Deicher, H . R. G., 232, 233, 234, 251 Deichmilier, M. P., 45, 90, 353, 359, 371, 439 Deitch, A. D., 233, 251 de la Chapelle, A., 317, 331 de la Haba, G. L., 171, 228 Delanney, L. E., 380, 440 Demiral, B., 362, 448 Demis, D. J., 168, 211, 227 Dempster, W . I., 56, 84, 94, 122, 210 Dent, J. fI., 117, 210 DeOliveira, L., 23, 80 Der, B. K., 96, 221 Derbes, V. J., 117, 210 Derick, C. L., 104, 210 Desai, R. G., 351, 439 DeSimone, A. R., 234, 251 de Somer, P., 356, 441 Dethier, F. M., 13, 88 Dettmers, A. E., 354, 451 Dethviler, €I. A., 393, 4.39 Deutsch, H. F., 370, 447 Deutsch, L., 358, 439 c k Vaux St. Cyr, C., 369, 435 de Vries, T., 259, 332 Dewey, V. C., 143, 217 DeWitt, C. W., 123, 124, 222 Deysson, G., 138, 226 Diamond, H. D., 153, 213, 428, 442 Diamond, L. K., 138, 211 Dickinson, T . C., 94, 166, 224 Didakow, N. C., 118, 221 Diefenbach, H., 113, 210, 228 Dienes, L., 415, 439 Dilks, 393, 458 Dillon, H., 179, 208 Dilworth, M. J., 119, 213 Dingle, J. H., 467, 478 Dingle, J. T., 501, 525 DiPaolo, J. A., 102, 210 DiSant'Agnese, P. A., 393, 439

Dische, R. hi., 11, 80 Dische, Z., 11, 80 Dishon, T., 404, 443 Dixon, F. J., 4, 5, 7, 8, 9, 10, 11, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 80, 81, 82, 84, 89, 90, 92, 93, 100, 113, 131, 209, 210, 222, 315, 329, 351, 353, 358, 359, 360, 371, 389, 395, 396, 406, 411, 419, 423, 424, 426, 428, 436, 438, 439, 449, 450, 458 Dixon, J., 137, 220 Djordjevic, B., 210 Doak, P. B., 124, 220 Doak, S. A l . A., 340, 439 M b i b , G., 351, 439 Dobriner, K., 120, 221 Dodd, Al. C., 234, 243, 244, 249, 251 Doebbler, T. K., 93, 222, 308, 310, 312, 334 Doerr, R., 377, 439 Dolowy, W. C., 356, 439 Done, A. K., 106, 210 Dong, E., Jr., 165, 218 Dong, L., 8, 33, 40, 86 Doniach, D., 63, 87 Donnell, G. N., 32, 33, 84 Donohue, D. XI., 139, 207 Domer, M. M., 392, 439 Dossetor, J. B., 123, 218 Doty, P., 238, 251, 399, 442 Dougherty, T. F., 109, 110, 111, 118, 210, 214, 227, 228 Douglas, S. R., 412, 458 Drabkin, D. L., 278, 330 Drapiewski, V. A., 11, 80 Dralkoci, M., 192, 193, 210, 216 Dray, S., 275, 329, 373, 440, 463. 477 Dreesman, G., 384, 440 Dresser, A. M., 270, 273, 276, 330 Dresser, D. W., 93, 101, 210, 339. 390, 391, 421, 428, 440, 448, 449 Drews, J., 194, 196, 206, 212 Dreyer, W. J., 294, 329 Drummond, K. N., 93, 166, 167, 205, 219

AUTHOR INDEX

Dubiski, S., 390, 438, 463, 466, 469, 477, 478 DuBois, S., 199, 213 Dubos, R. J., 355, 440 Dudziak, Z., 469, 477 Duhamel, L., 374, 451 Duke, W. W., 102, 210 Dulaney, A. D., 68, 88, 2.43, 250, 386, 440 Dulbecco, R., 382, 383, 440 Dumonde, D. C., 55, 68, 69, 79, 80, 83 Dunne, H . W., 348, 440 du Pan, M., 371, 440 D u Pan, R. M., 371, 448 Duplan, J. F., 440 Dustin, P., 183, 210 Dutton, A. H., 169, 210, 257, 263, 265, 269, 278, 279, 280, 281, 284, 314, 315, 330, 336, 360, 457 Dutton, R. W., 158, 169, 210, 257, 260, 263, 264, 265, 266, 267, 269, 270, 271, 272, 278, 281, 329, 330, 333, 334, 336, 279, 284, 288, 289, 304, 314, 315, 316, 317, 319, 320, 321, 322, 323, 324, 360, 362, 406, 423, 440, 451, 457 Duvall, L. R., 182, 280, 211 du Vigneaud, V., 198, 212, 217, 356, 440

E Eady, J. D., 279, 288, 289, 315, 316, 319, 321, 330, 406, 440 Eagle, H., 271, 330, 360, 440, 462, 477, 480, 525 Earle, D. P., 30, 81 Earle, W. R., 361, 440 East, J., 402, 444, 449 Eastoe, J. E., 11, 12, 90 Ebaugh, F. G., 361, 447 Ebert, J. D., 380, 440 Edelman, G. M., 370, 401, 440, 447 Edgington, 62, 81 Edney, M., 339, 389, 438 Edsall, G., 387, 440 Edwards, P. C., 151, 224 Egdahl, R. H., 157, 219, 257, 272, 297, 299, 330, 333, 354, 380, 410, 423, 440, 447 Ehrich, W., 51, 81

537

Ehrich, W. E., 4, 8, 10, 33, 40, 42, 43, 46, 81, 105, 184, 212 278, 290, 330, 331, 450 Ehrlich, J., 178, 211 Ehrlich, P., 92, 211 Eichenwald, H. F., 347, 394, 440 Eichhorn, G., 399, 442 Eigner, J., 238, 251 Eisen, A. Z., 147, 207, 221 Eisen, B., 168, 211 Eisen, H. N., 8, 10, 11, 19, 22, 23, 24, 25, 26, 81, 86, 87, 110, 211, 258, 269, 276, 278, 284, 285, 331, 332, 379, 401, 409, 415, 417, 418, 422, 440, 445 Eisler, D. M., 375, 440 Eitzman, D., 353, 371, 451, 457 Eitzman, D. V., 383, 387, 393, 436, 442, 453 Elberg, S., 297, 301, 331 Elberg, S. S., 412, 413, 440 Elder, C. C., 182, 212 Elgart, M. L., 168, 227 Elias, D., 497, 513, 526 Eliel, L. P., 120, 221 Elion, G. B., 101, 139, 148, 151, 157, 164, 167, 168, 169, 170, 180, 207, 209, 211, 215, 220, 407, 409, 444, 449 Elkins, W. L., 56, 81 Ellen, K. A. O., 235, 251 Ellinger, P., 191, 211 Elves, M. W., 292, 330, 360, 361, 440, 441 Ely, R. S., 106, 210 Emmelot, P., 125, 211 Enderlin, M., 6, 19, 36, 39, 42, 88 Enein, M. A., 185, 209 Engbaek, H. C., 52, 89, 111, 225 Engel, H., 458 Engelman, M., 149, 213 English, J. P., 148, 222 Epp, M., 441 Epstein, L. B., 316, 330 Epstein, R. B., 145, 211 Eraslan, S., 165, 214 Erdmann, G., 6, 81 Erlanger, B., 240, 251 Erlanger, B. F., 233, 240, 246, 251, 252

538 Eschbach, J. W., 145, 211 Estrada-Pama, S., 233, 240, 251 Estrin, I., 190, 222 Ethridge, C. B., 86 Etteldorf, J. N., 371, 449 Evans, D. G., 380, 441 Evans, E. P., 403, 443 Evans, L. D., 117, 224 Ecans, L. W., 323, 328 Evms, V. J., 361, 440 Everbeck, H., 371, 440 Eyal, Z., 192, 194, 211 Eyquem, A., 156, 176, 221 Eyssen, H., 356, 441

AUTHOR INDEX

Fennelly, J. J., 199, 228 Fenner, F., 100, 208, 293, 330, 389, 438 Fennestad, K. L., 342, 394, 441 Ferger, M. F., 212 Ferguson, K. A., 345, 410, 452 Ferguson, L. A., 259, 268, 314, 332, 360, 446 Fernandes, hl. V., 310, 312, 332 Fernando, hi. V . P., 98, 220 Ferraro, A., 119, 212 Ferrebee, J. W . , 13, 88, 141, 144, 145, 207, 914, 225, 226 Fesce, A., 486, 492, 525 Field, E. J., 107, 119, 161, 212 Figueroa, J. E., 123, 220 F Finalli, hl., 522, 527 Faber, L. P., 165, 221 Finch, S. C., 375, 438 Fngraeus, A., 96, 112, 113, 184, 206, Findley, A., 213 211, 257, 330, 358, 360, 419, 436, Finger, H., 119, 130, 196, 211, 227 -1.11 Finger, I., 381, 441 Fahey, J. L., 233, 251, 373, 441,444,452 Fink, C. W., 383, 441 F d e y , G. H., 128, 211 Finkelstein, M. S., 103, 212, 352, 354, Fanger, H., 13, 88 378, 383, 387, 388, 401, 421, 424, Farber, \I. B., 355, 358, 393, 395, 443 432, 441, 457 Farber, S., 138, 173, 174, 209, 211 Finland, hl., 109, 117, 217 Farhi, A,, 179, 211, 217 Finstad, J., 136, 189, 213, 370, 442, 450 Faris, T . D., 123, 166, 203, 225 Firschein, I . L., 174, 215, 292, 332 Farquhar, 31. G., 26, 51, 81 Fischel, E. E., 110, 111, 114, 207, 212 FCm,L. E., 3, 4, 5, 9, 39, 40, 81, 88 Fish, A. J., 166, 167, 219 F x r , R. S., 94, 100, 101, 190, 191, 192, Fish, L. A., 370, 441 210, 211, 216, 217, 223, 426, 441 Fishbein, W. N., 199, 221 Fmconnet, hl., 156, 233, 207, 252 Fisher, E. R., 9, 81 Fci\oiir, C . B., 117, 218 Fisher, J., 464, 478 Faucett, D. W'., 12, 81 Fisher, J. P., 200, 228 Fecsik, A. I., 425, 441 Fishman, M., 173, 174, 212, 257, 265, Fedoroff, S., 376, 441 289, 295, 296, 298, 299, 330, 331, Feingold, S . , 203, 210 360, 414, 421, 441 Feinstein, A., 450, 471, 477, 488 505, Fitch, F. W . , 101, 212, 334, 363, 392, 525, 527 401, 441, 449, 451 Feldman, J, D., 8, 9, 27, 39, 40, 46, 48, Fitzgerald, J . D. L., 47, 85 49, 50, 57, 63, 64, 70, 80, 81, 85, Fitzgerald, P. J., 10, 11, 23, 24, 25, 26, 89, 90, 93, 96, 210, 211 86, 87 Feldman, M., 101, 155, 211, 361, 428, Flanagan, C., 43, 44, 46, 81 447, 448 Flax, M. H., 165, 206 Felix, A., 386, 441 Fleischman, J. B., 257, 285, 331, 470, Fellmer, K. E., 194, 206 477 Felton, L. D., 100, 211, 389, 426, 441 Flick, J. A., 22, 28, 84 Fennell, R. H., Jr., 49, 81 Flinner, R. L., 123, 124, 222

539

AUTHOR INDEX

Floersheim, G. L., 108, 137, 143, 185, 194, 195, 196, 212 Florman, A. L., 351, 441 Folsch, E., 194, 196, 206, 212 Fogelman, J. A., 54, 85 Foley, G. E., 173, 174, 209 Folkes, J. P., 178, 212 Fong, J., 297, 301, 331 Foote, F. W., 25, 86 Forbes, I. J . , 292, 331 Forbes, M., 376, 399, 453 Ford, C. E., 124, 214, 290, 331, 403, 417, 442, 443 Forman, B., 200, 228 Forman, C., 43, 81, 105, 184, 212, 278, 330 Forman, C. W., 4, 10, 33, 40, 42, 46, 51, 81 Forsen, N. R., 151, 154, 212 Foschi, G. V., 402, 440 Fouts, P. J., 7, 19, 81 Fowler, R., 410, 441 Fox, C. L., Jr., 93, 220 Fox, J. P., 339, 441 Fox, M., 137, 212 FranBk, F., 348, 357, 359, 361, 370, 372, 384, 441, 450 Frangione, B., 119, 215, 373, 441 Frankel, H., 40, 89 Franklin, E. C., 32, 89, 99, 212, 259, 273, 297, 332, 373, 374, 383, 387, 441, 457 Franklin, R. M., 191, 212 Franzl, R. E., 109, 218, 405, 441 Frazier, L. F., 196, 228 Freeman, E. B., 93, 220, 222 Freeman, M . J., 260, 335 Frei, E., 324, 334 Frei, E., 111, 218 Frei, P. C., 93, 101, 212 Freireich, E. J., 142, 215 Frenger, W., 186, 212 Frenkel, E. P., 199, 212 Freter, G. G., 401, 456 Fretzdorff, A. M., 196, 227 Freund, J., 354, 386, 393, 411, 441 Frick, E., 56, 81 Friedell, G. H., 108, 224 Friedewald, W. F., 69, 83

Friedman, D. I., 96, 221 Friedman, H., 297, 299, 300, 308, 309, 312, 331, 423, 441 Friedman, H. P., 276, 289, 296, 298, 331 Friedman, R. M., 119, 140, 141, 142, 146, 207, 212 Friou, G. J., 233, 251 Frisch, A. W., 99, 168, 212 Froh, C., 497, 526 Frohardt, R. P., 182, 212 Frontino, G., 188, 206 Fudenberg, H., 188, 190, 225, 373, 441, 505, 525 Fudenberg, H. H., 373, 374, 441, 469, 477 Fujikawa, K., 377, 435 Fujimoto, T., 4, 8, 9, 23, 32, 35, 39, 81 Fujimoto, Y., 172, 227 Fullmer, A. M., 12, 81 Fulmor, W., 172, 227 Fumarola, D., 405, 439 Funi, H., 277, 319, 332 Furth, J., 68, 81 Fusari, S. A., 182, 212

G Gabrielsen, A., 98, 209 Gabrielsen, A. E., 93, 96, 97, 100, lU, 136, 189, 213, 214, 368, 402, 442 Gaines, S., 405, 445 Gaisford, W., 400, 450 Gale, E. F., 178, 212 Gale, G. R., 199, 213 Gall, C. L., 177, 228 Gallagher, R., 203, 227 Gambini, G., 491, 522, 527 Gammeltoft, A., 55, 88 Gammon, A. M., 102, 228 Gammon, G. A., 119, 213 Garattini, S., 137, 195, 205 Gardella, J. W., 13, 88 Garin, A. M., 226, 251 Garrison, F. E., Jr., 164, 223 Garvey, J. S., 258, 265, 266, 269, 270, 289, 295, 298, 329, 331, 335, 361, 421, 423, 438, 442, 443 Gault, M. H., 123, 218 Gaunt, R., 193, 213 Gay, F. P., 480, 493, 524

540

AUTHOR INDEX

Gaynor, E. C., 7, 11, 19, 88 Gcll, P. G. H., 117, 213, 303, 317, 329, 331, 335, 415, 416, 434, 436, 442, 463, 473, 474, 475, 477, 518, 524, 525 Gell, P. H., 275, 334 Geller, B. D., 177, 213 Gellhorn, A., 149, 213 Grnghof, D. S., 156, 213 Gengozian, N. 197, 215, 359, 396, 442, 447

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