Advances in Immunology Volume 4

ADVANCES IN

Immunology VOLUME 4

CONTRIBUTORS TO THIS VOLUME SYDNEYCOHEN JOSEPH

D. FELDMAN

ROBERTA. GOOD H. G. KUNKEL MACLYNMCCARTY STEPHENI. MORSE J. MUNOZ BEN W. PAPERMASTER RODNEYR. PORTER

RAMSEIER HANSRUEDY EMANUEL SUTER E. M. TAN

ADVANCES I N

Im munology E D I T E D BY

F. J. DIXON, JR.

J. H. HUMPHREY

Divirion of Exporimental Pathology Scrippr Clinic and Reroarch Foundation La lolla, California

Division of Immunology National Indituto for Modical Research Mill Hill London, England

VOLUME

4

1964

ACADEMIC PRESS

New York and London

COPYRIGHT @ 1984, BY ACADEMICPRESSINC. ALL RIGHTS RESERVED. NO PART O F THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

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United Kingdom Edition publkhed by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W.1

LIBRARY OF CONGRESS CATALOG CARDNUMBEA: 61-17057

First Printing, 1964

Second Printing, 1988

PRINTED I N THE UNITED STATES OF AMERICA

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

SYDNEYCOHEN,Department of Immunology, St. May’s Hospital Medical School, London, England (287) JOSEPHD. FELDMAN, Division of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California (175) ROBERT A. GOOD,Department of Pediatrics, University of Minnesota, Minmapolls, Minnesota ( 1)

H. G. KUNKEL, The Rockefeller Institute, New York, New York (351) MACLYNMCCARTY,The Rockefeller Institute, New York, New York (249) STEPHENI. MORSE,The Rockefeller Institute, New York, New York (249) J. MUNOZ,National Institutes of Health, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana ( 397 1

BEN W. PAPERMASTER, Department of Bacteriology, University of California, Berkeley, California ( 1 )

RODNEY R. PORTER, Department of Immunology, St. Mary’s Hospital Medical School, L&, England (287)

HANSRUEDY RAMSEIER, Department of Microbiology, College of Medicine, University of Florida, Gainesuille, F1orid.u (117) EMANUELSUTER,Department of Microbiology, College of Medicine, University of Florida, Gainesuille, Florida (117) E. M. TAN, The Rockefeller Institute, New York, New York (351)

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The fourth volume of Advances in Immunology, like its predecessors, contains reviews which range over the whole field of immunology, from a study of the biological origins of specific immunologic responses, through various aspects of their protective and pathogenic effects, to the present knowledge of the chemical structure of antibodies. We think that by including contributions of so wide a range, we not only recognize the very broad approach of immunologists to their subject but also acknowledge its fundamental unity, For example, the problems of the complexity of the immunoglobulins, on which chemical and genetic analyses are now beginning to shed light, are intimately bound with those of their evolutionary and cellular origins and their biological functions. This is one of the main reasons why immunology fascinates those who study it. The first chapter by Robert A. Good and Ben W. Papermaster provides a comprehensive review and comparison of the ontogeny and phylogeny of adaptive immunity. It deals with the acquisition of passive and active immunity by the developing fetus in relation to the phylogenetic development of active immunologic responses and relates immunologic capacity to the fmction of the thymus and of the bursa of Fabricus-a study which is ripe for review and in which the group at Minnesota has played a notable part. The second chapter by E. Suter and H. Ramseier concerns the killing of parasites by phagocytic cells and discusses the evidence for the existence and importance of mononuclear phagocytes which have an enhanced capacity to destroy intracellular parasites, apart from recognizable assistance by specific antibodies. The possible connection between such cellular immunity and delayed-type hypersensitivity is a matter of great practical interest. Current knowledge of the chemistry of cell wall antigens of gram-positive bacteria is reviewed in the fourth chapter by M. McCarty and S. I. Morse. In contrast to the soluble capsular antigens, especially those of pneumococci, the true cell wall antigens have only relatively recently been available in undegraded form suitable for chemical and immunochemical analysis; and even now it is mainly the carbohydrate and teichoic acid rather than the protein components which have been studied. Since the cell walls appear likely to provide the best source of immunizing antigens, a review of their structure should be timely as well as interesting. The fact that bacteria and their products can affect immune responses other than by acting as a source of antigenic materials has been recognized for many years. In the last chapter J. J. Munoz has Vii

viii

PREFACE

drawn together much relevant information concerning the adjuvant effects of mycobacteria, the action of endotoxins of gram-negative bacteria on antibody responses, and the peculiar activity of Bordutella pertussis in enhancing anaphylactic sensitivity in rodents. Although none of these actions has a satisfactory explanation, the reader will find a full account of the attempts to provide one. The third chapter by J. D. Feldman, Ultrastructure of Immunologic Processes, shows how electron microscopic studies have verified visually some immunologic processes whose nature was already surmised and have deepened our understanding of others, notably of the pathologic consequences of antigen-antibody interaction in the renal glomerulus. H. G. Kunkel and E. M. Tan, in their review on Autoantibodies and Disease, provide both an up-to-date account of the autoantibodies which have been discovered in autoimmune diseases and a comparison of the reactivity against the Gm groups on human y-globulin of rheumatoid factors on the one hand with the “serum normal agglutinators” on the other. The latter are thought to often arise as a result of isoimmunization of the fetus by maternal y-globulin. This chapter includes a discussion of the possible beneficial as well as pathologic effects of certain autoantibodies. Finally, S . Cohen and R. R. Porter have contributed a clear and full review of the Structure and Biological Activity of Immunoglobulins which discusses not only the evidence supporting the current views of the structure and linkages of the four recognized chains of immunoglobulins, the positions of their various genetic markers, and the nature of the antibody-combining sites, but also considers the mounting indirect evidence for the existence of two separate chains within the A or heavy chain. We are deeply grateful to the authors for the effort which they have put forth into making their chapters both informative and stimulating and to the publishers and printers for maintaining their high standards of presentation; we are confident that this volume will prove to be at least as useful as its predecessors.

September, 1964

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

CONTENTS LIST OF CONTRIBUTORS ..............................................

v

PREFACE ...........................................................

vii

Ontogeny and Phylogeny of Adaptive Immunity

ROBERTA. GOODAND BEN W . PAPERMASTER I. I1. I11. IV.

General Introduction ......................................... Ontogeny of Immunity ........................................ Phylogeny of Adaptive Immunity ............................... Concluding Statement ........................................ References ..................................................

1 4 80 94 98

Cellular Reactions in Infection EMANUEL SUTER AND HANSRUEDY RAMSEIER

I. I1. 111. IV . V. VI .

Introduction ................................................ Chemotaxis ................................................. Phagocytosis ................................................ The Normal Phagocyte in the Postengulfment Period . . . . . . . . . . . . . . The Immune Phagocyte ...................................... Conclusion ................................................. References .................................................

117 119 122 129 144 165 167

Ultrastructure of Immunologic Processes

JOSEPHD . FELDMAN

I. I1. I11. IV. V.

Prologue ................................................... Antibody ................................................... Antigen .................................................... Ultrastructure of Immunologic Reactions ........................ Epilogue ................................................... References .................................................

ix

175 176 189 196 235 237

c0”Ts

X

Cell Wall Antigens of Gram-Positive Bacteria

.

MACLYN MCCARTY AND STEPHENI MORSE I. I1 I11 IV V VI .

. . . .

Introduction ................................................ Isolation and Composition of Cell Walls of Gram-Positive Bacteria ... Cell Wall Protein Antigens .................................... Cell Wall Polysaccharide Antigens .............................. Cell Wall Teichoic Acids ...................................... Intracellular. Capsular. and Diffusible Antigens Related to the Cell Wall of Gram-Positive Bacteria ................................ References .................................................

249 250 252 255 269 277 282

Structure and Biological Activity of Immunoglobulins

SYDNEY COHENAND RODNEY R . PORTER I. I1 111. IV V

. . .

Introduction ................................................ Physical Studies ............................................. Chemical Properties .......................................... Biological Properties of Immunoglobulins ........................ Comments .................................................. References .................................................

287 289 291 319 341 342

Autoantibodies and Disease

H. G. KUNKEL AND E . M . TAN I . Introduction ................................................ I1. Widely Prevalent Autoantibodies ............................... I11. Specific Diseases Associated with Autoantibodies .................. References .................................................

351 352 368 389

Effect of Bacteria and Bacterial Products on Antibody Response

.

J MUNOZ

. .

I I1. I11 IV V VI VII

. .

. .

Introduction ................................................ Acid-Fast Bacteria ........................................... Effect of Endotoxins from Gram-Negative Bacteria ............... Effect of Bordetella pertussk ................................... Miscellaneous Bacteria and Bacterial Products ................... Possible Mechanisms of Bacterial Adjuvants ...................... Summary .................................................. References ..................................................

397 400 411 420 428 429 431 431

AUTHORINDEX ......................................................

441

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

473

Ontogeny and Phylogeny of Adaptive Immunity' ROBERT A. GOOD2 AND BEN W. PAPERMASTERS Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, and Deportment of Bacteriology, University of California, Berkeley, California

I. General Introduction ........................................ 11. Ontogeny of Immunity ...................................... A. Relationship of Fetus to Mother .......................... B. The Immunoglobulins ................................... C. Passive Transfer of Immunoglobulins to Developing Embryo ... D. Transfer of Antibodies to Fetus ........................... E. Ontogeny of Immunoglobulin Synthesis .................... F. Ontogeny of Antibody Production ........................ G. Development of the Lymphoid Tissue ...................... H. Ontogenetic Development of Transplantation Immunity . . . . . . . I. Ontogenetic Development of the Delayed Allergic Response . . . J. Immunologic Development As Studied in Cell Transfer Systems K. Immunologic Tolerance in Relation to Development of Full Immunologic Capacity ................................... L. Role of the Thymus and Bursa of Fabricius in Ontogenesis of Adaptive Immunity .................................... 111. Phylogeny of Adaptive Immunity .............................. A. Immune Responses in Invertebrates ....................... B. Immune Responses in Vertebrates ......................... IV. Concluding Statement ....................................... References ................................................

1 4 4

5 6 11 13 18 26 34 37 41 46

49 60 62 71

94 96

1. General Introduction

Although we recognize as hazardous an attempt at a critical review of the broad field we have chosen and appreciate the special treachery of writing a review of the subject during the period of its most rapid change, our own pressing concern with developmental immunobiology permits us to address this task. It seems to us that understanding of the origin of immunity in both ontogenetic and phylogenetic terms could well be a basis of operational answers to some of the most basic questions of immunology, as well as approaches to manipulation and control of immunologic processes. Already observations are at hand which 1 Original studies included were aided by grants from the U.S. Public Health Service, The National Foundation, the American Heart Association, Minnesota Heart Association, and the Minnesota Division of the American Cancer Society. 2 American Legion Memorial Heart Research Professor. 8 This work was done while Dr. Papemaster was a graduate student trainee under grant GM-24-04, Department of Microbiology, University of Minnesota.

1

2

ROBERT A. GOOD AND BEN W. PAPERMASTER

sketch out a sequence of events in the ontogeny of immunologic responsiveness and which suggest that true adaptive immunity is a characteristic only of vertebrates, These findings suggest further that the thymus plays a key role in both the ontogenesis and phylogenesis of the specific adaptive process which we recognize as immunity. In spite of the popularity of theorizing about the nature of the immunologic process ( Mudd, 1932; Pauling, 1940; Haurowitz, 1952; Jerne, 1955; Talmage, 1957,1959; Burnet, 1959; Lederberg, 1959; Karush, 1962), neither instructive nor selective theories have yet led to experiments that establish the nature of antigenic stimulation. Further the concept that specific immunologic negativity is based on a single mechanism (Mariani et al., 1959; Martinez et al., 1959; Medawar, 1961; Michie and Howard, 1962), rather than taking many forms, demands even more that these phenomena be fully explained in operational terms. Both of these central problems in immunobiology, the nature of specific positive immunologic adaptation and the nature of specific negative immunologic adaptation, seem approachable in a most direct manner through an analysis of the development of immunologic reactivity. Considerations of ontogeny and phylogeny raise basic questions concerning the process of cell adaptation and the essential role of the immune response in the body economy. Certainly, to consider these problems effectively in this review, it is necessary that we define our conception of the immune response. The group of defenses historically considered to be in the category of acquired, specific, immune responses are those that were distinguished from innate protective mechanisms at the end of the 19th century by such workers as Pasteur (1881), Metchnikoff ( 1905), Behring and Kitasato (1890), Nuttall (1888), and Ehrlich (1892). The classical acquired, immune response is antitoxin formation ( Behring and Kitasato, 1890). During subsequent investigations allergic reactions were defined (von Pirquet, 1906) and grouped into immediate and delayed types (reviewed by Chase, 1958; Lawrence, 1956). Both forms of hypersensitivity are clearly representative of acquired immune responses. Finally, homotransplantation immunity has been shown to be a manifestation of acquired immunity (Medawar, 1943, 1944, 1945; reviewed by Lawrence, 1959). In the mammal, all these responses (antibody formation, immediate allergy, delayed allergy, and homograft immunity) are well developed. All encompass specific recognition of antigenic structural patterns; some-and perhaps all-are based on active secretion of specific globulin molecules; and each is activated with the development of a pattern of spec8c memory or anamnesis. The cellular basis for acquired immunity is the lymphoid family of

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

3

cells, as first postulated by Pfeiffer and Marx (1898) and L. Deutsch (1899). From the studies of Nossal and Makela (1962) and Makela and Nossal (1962), reviewed by Nossal (1962), and those of Urso and Makinodan (196l), and Vazquez (1961, 1964), it is clear that adaptive immunity is essentially a proliferative process, also involving cellular differentiation. Pathologic (reviewed by Good, 1957a), cytologic ( Kolouch, 1938; Bjgrneboe and Gormsen, 1943; Fagraeus, 1948), histochemical (Ehrich et al., 1949; T. N. and S. Harris, 1949), immunohistochemical (Coons et al., 1955; Leduc et al., 1955), and tissue culture studies (Nossal, 1958, 1959a,b, 1960) inextricably link the proliferative process of immunity to the lymphoid system of cells. The consideration of ontogeny in mammals, as well as consideration of the phylogeny of immunity, of necessity becomes, at least in part, a study of the ontogeny and phylogeny of the development, distribution, morphology, and adaptive potentialities of the lymphoid cells. The lymphoid system of cells is a system that develops and differentiates relatively late in the ontogenetic and phylogenetic sequences. Thus, it will be shown in this review that adaptive immunity, which we are here considering, although perhaps having roots in more primitive processes, is primarily a function achieving full expression late in phylogeny and ontogeny. Recently, Burnet (1959) has referred to this system of cells as “immunologically competent cells,” and still more recently, Dameshek ( 1964) used the term “immunocyte” to describe the elements of this line. Whatever terminology ultimately holds sway, it is important to recognize that this system of cells represents an advanced stage of evolutionary adaptation for specific reactivity. The most prominent functional characteristic of these cells is their highly developed capacity to react to antigens, to proliferate in the presence of antigenic stimulation, and, as a line of cells, to remember prior experiences with antigens. In this review, we shall be concerned primarily with the origins of immunity as a biologic phenomenon embodying primary and secondary responses, with antibody synthesis and release, reactions of immediate and delayed allergy, and homograft immunity. We shall not concern ourselves with a whole host of other complex mechanisms of defense such as natural antimicrobial factors, enzymes in body fluids, polyelectrolytes capable of electrostatic interaction, complement, and phagocytosis. Some of these defense mechanisms have been reviewed in recent years (Suter, 1956; Skarnes and Watson, 1957), and the comparative aspects of some of these have been considered (Huff, 1940; Bisset, 1947; Cushing and Campbell, 1957; Sirotinin, 1960). By using this relatively narrow definition of immunity, which we

4

ROBERT A. GOOD AND BEN W. PAPERMASTER

prefer to call adaptive immunity, we may, of course, be avoiding a central issue, namely, the basic origin of this process or these processes in other bodily activities such as those involving protein synthesis, protein-protein interactions, transport mechanisms, and functional complementarity of macromolecules. This question concerning the basis of adaptive immunity in pre-existing physiologic processes is, we believe, a separate question, which at this moment has been too little studied and is far too speculative for review. We shall attempt to consider the ontogenesis of immunity in terms of the following relationships and processes : interrelationship of fetus and mother, particularly serum immunoglobulin and antibody transfer; the beginning production of immunoglobulins during fetal and neonatal life; the origins of antibody-producing capacity and ability to initiate and express delayed allergic responses; the development of capacity for homograft rejection and the decrease of susceptibility to production of tolerance in the fetal and neonatal period; and the key role of the thymus and other central lymphoid tissue in the ontogeny of the lymphoid system and adaptive immunity. Further, we shall summarize present knowledge of the phylogeny of immunity and attempt to show that, here too, the development of the lymphoid system, particularly the thymus, plays a key role in development of the potential for adaptive immune response. II. Ontogeny of Immunity

A. RELATIONSHIP OF FETUS TO MOTHER The developing fetus in mammals is essentially in the position of a well-tolerated homograft. Abundant evidence is at hand (reviewed by Greene, 1955; Nace, 1955, 1957; Woerdeman, 1955; Goldstein and Baxter, 195s) that the embryo and fetus develop characteristic individual specificity at an early stage of development and, thus, should be rejected by the mother were normal transplantation immunity to operate. Such a reaction does not regularly occur. Although the well-known lack of immunologic reactivity during early fetal life and the effective separation of the fetus from the cells of the maternal circulation preclude reactivity in the direction of fetus toward maternal antigens, the mechanisms by which rejection of the fetus by the mother is avoided are far from clear. The fetal membranes appear in some species to be lacking certain transplantation antigens ( HaikovP, 1981); however, studies by Dancis et al. ( 1962) suggest that both transplantation immunity and immunologic tolerance can be induced by injection of placental cells. It seems possible from available data that the placental membranes in this situation

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

5

function as does the membrane of the hamster’s cheek pouch in the studies of Billingham et al. (1960). Their experiments have shown that skin homografts to which the hamster was completely reactive at a skin site were well tolerated when the cheek pouch membrane was interposed between the graft and connective tissue bed. If this is, indeed, the basis for toleration of the fetal transplant by the maternal host, it would be essential that the fetal membranes themselves lack effective transplantation antigenicity; then the source of the antigenicity demonstrated in the studies of Dancis et al. (1962) would have to be blood cells or cells on the fetal side of the membrane. Whatever its mechanism, it remains that the fetus is a homotransplant usually well tolerated without evidence of rejection. There can be no question that in spite of the effective separation of maternal and infant serums, cells with antigenic characteristics foreign to the mother occasionally gain access to the maternal circulation, giving rise to an immune response, transmission of the antibody to the fetus, and a destructive attack on fetal cells. Red cells (Pickles, 1949), platelets ( Shulman et al., 1962; Vandenbroucke and Verstraete, 1955; Schoen et al., 1956), and white cells (Stefanini et al., 1959; Hitzig, 1959), are known to accept the brunt of such immunologic attack in man. That maternal cells may gain access to the fetal circulation has also been demonstrated (Lee and Vazquez, 1962), and it has been postulated that such transfers account for the more favorable treatment of maternal skin grafts by offspring (Peer, 1958), and might, under unusual circumstances, provide a basis for cellular chimerism and graft-versus-host reactions as a basis for human disease. Studies by Lengerovd (1957), Nathan et al. ( 1960), and Najarian and Dixon (1963) have shown that manipulations designed to alter the permeability of the placenta result in a high incidence of tolerance to reciprocal skin transplants of mother and offspring. B. THE IMMUNOGLOBULINS Electrophoretic analysis, coupled with immunologic studies, established that, of the proteins of the serum, those with the lowest charge density at neutral or slightly alkaline pH are those that contain the bulk of the circulating antibodies ( Tiselius and Kabat, 1939). These antibodycontaining globulins, the y2- and the yl- or (3Z-globulins7can be further divided into a low molecular weight component(s), 7S, and a high molecular weight component, 19S, on ultracentrifugal analysis (H. F. Deutsch et al., 1946). With the introduction of immunoelectrophoretic techniques by Grabar and Williams (1953), it became clear that four immunochemically distinct fractions comprise the yz and p2 components.

6

ROBERT A. GOOD AND BEN W. PAPERMASTER

These include the classical yz-globulins, which are a single, immunochemically identifiable family of protein molecules with electrophoretic mobility extending from the slowest migrating serum protein constituents as far as the a range on electrophoresis (Grabar, 1956); the (32M ( Y l M ) component, which has been shown to be the same as the 19s or yl-macroglobulin fraction (Burtin et al., 1957); and the P2A ( ~ I A )fraction which is said to have a sedimentation constant of 7s and to be characterized by a high carbohydrate concentration (Heremans et al., 1959). The (3zM- and yz-globulins are known to contain antibodies, and some evidence is at hand that the B2* component also contains antibodies (Fireman d al., 1963). The 7s or y2-globulin possesses the bulk of the circulating antibodies, among them virus-neutralizing antibodies, precipitating antibodies, complement-fixing antibodies, antidiphtheria and antitetanus antibodies, incomplete hemolysins, hemagglutinins, and such agglutinating antibodies as antiflagellar antibodies (Enders, 1944; H. F. Deutsch et al., 1946; reviewed by Fahey, 1962). The fhM( y I M ) or 19s globulin contains such antibodies as Wassermann reagins (B. D. Davis et al., 1945), heterophile antibodies (Kunkel, 19€@), rheumatoid factor (Franklin et al., 1957), cold agglutinins ( Gordon, 1953), isohemagglutinins ( Pedersen, 1945), and other agglutinins such as antityphoid 0 ( H. F. Deutsch et al., 1946). The pZlL fraction is less well understood, but evidence is beginning to accumulate that this is the protein containing the heat-labile skin-sensitizing allergens (Rockey and Kunkel, 1962; Fireman et al., 1963; Heremans and Vaerman, 1962). A fourth constituent which appears in the immunoglobulin area on immunoelectrophoresis, but is less clearly classifiable as an immunoglobulin, is the yx of Heremans (1960), subsequently defined as C-reactive protein. The behavior of this protein in specific protein-protein interactions may justify its classification as an immunoglobulin; however, since this protein, unlike the other immunoglobulins, behaves as an acute phase reactant, it will not be considered in any detail in this review. Because of clear differences in the developmental biology of these separate protein fractions, it has been necessary, in considering the development of immunologic potential, to bear in mind the three separate components of this family of immunoglobulins.

C. PASSIVE TRANSFER OF IMMUNOGLOBULINS TO DEVELOPING EMBRYO Although proteins, produced for the most part by the liver (L. L. Miller and Bale, 1954), appear in the serum at an early stage, no species thus far studied produces demonstrable amounts of immunoglobulins

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

7

during normal fetal life. Instead, an elaborate complex has evolved by which antibody-containing proteins are transmitted to the fetus and to the newly hatched or newly born animal. In the chicken, the antibodycontaining (3-globulins are transmitted from the hen to the ova 4 3 days before ovulation via the follicular epithelium (Patterson et al., 1962) and then to the blood of the embryo late in incubation via the yolk sac ( Weller and Schechtman, 1957, 1962). In the porcine and bovine species, probably because of the highly developed placental membrane structure (Mason et al., 1930), no immunoglobulins whatever are transmitted to the fetus during gestation, and, since none are produced by the fetus, these animals are entirely lacking in antibodies and y-globulin at birth. However, antibody-containing lactoglobulins are delivered in high concentration into the colostrum and thence gain access to the circulation of the neonate which remains highly permeable to protein for several days after birth (Mason et al., 1930; Hansen and Phillips, 1949; McCarthy and McDougall, 1953). The permeability of the gut for protein in newborn calves is not entirely selective (Bangham et d.,1958b), and proteins other than the immune lactoglobulins are also absorbed during this period. Studies by Pierce (1959, 1961) have shown that the low molecular weight proteins, absorbed as whole molecules by the newborn calf, are eliminated by a proteinuria which parallels the period of whole protein absorption by the gut. A similar proteinuria was observed earlier in calves by T. Smith and Little (1924) and in lambs by McCarthy and McDougall ( 1953). Apparently the proteinuria is a necessary corollary of the nonselective uptake of so large a protein load over so short a period. In 1953, Moog had observed that alkaline phosphatase appears in the intestinal mucosa of suckling mice at about 14 days of age under normal conditions, but occurs 2 4 days earlier if the animals are given cortisone or ACTH. Halliday (1956, 1957, 1959), using rats and mice, found that corticosteroids, given orally or parenterally, induced premature appearance of alkaline phosphatase in these species, and that this was correlated with premature cessation of transmission of immunoglobulins and antibodies across the gut wall. Thus, in calves, lambs, mice, and rats, antibodies are transferred from maternal serum to offspring via the colostrum. In ruminants the intestinal absorption of the newborn is relatively nonselective; in rats and mice this absorption is more selective (reviewed by Brambell, 1961, 1962) and may be under endocrinologic control. It is apparent that both in the chicken and in these mammals, the means of providing passive immunologic protection to the young during the period of transition from immunologic inactivity to full reac-

8

ROBERT A. GOOD AND BEN W. PAPERMASTER

tive capacity involves transmission of antibody-containing globulin across epithelial barriers. Studies by S. L. Clark (1959) and Feldman (196l), indicate that vigorous pinocytosis is involved in the process. Brambell (1958) has shown that transmission of immunity before birth in the rabbit, guinea pig, and rat takes place by way of the fetal yolk sac and not by way of the placenta. This membrane corresponds to the yolk sac of the chicken, long known to be the avenue of transmission of the immunoglobulins in birds (Levaditi, 1906). In rats and mice, passive transmission of antibody occurs also by means of the colostrum and milk. In these animals, y-globulin may be absorbed for a prolonged period, indeed for the entire period of lactation or as long as 19 days post partum in the rat and 16 days in the mouse (Brambell, 1961). Thus, passive immune protection tides these animals over until there has been rather complete development of the ability to respond to antigenic stimulation with vigorous production of competent antibodies. In primates, albumin, y-globulin, and a variety of a- and p-globulins appear in the serum relatively early in the gestational period, and all except the y-globulins are produced by the developing fetus (Dancis et al., 1957). The @2A- and PzM-globulinsare absent from the human circulation until the postnatal period (Hitzig, 1957; Scheidegger and Martin du Pan, 1957); neither is produced by the normal fetus. Following birth, both proteins begin to accumulate, first the @2M and later the S 2 A (Karte, 1959; Roth, 1962). The y2-globulin appears in the human fetal serum during the third gestational month; it derives from the maternal circulation and, like the other immunoglobulins, is apparently not produced by the normal fetus. Although the question of placental production of serum proteins has been raised, the most comprehensive investigations of placental protein synthesis indicate that this is not a significant source of fetal serum proteins (Dancis et al., 1957; Bardawil et al., 1958). Until Brambell's publications (1958) it had been assumed, since the fetus does not produce y-globulin and since both human beings and primates usually complete gestation with a y-globulin level slightly higher than that of the mother, that these proteins gained access to the fetus across the placental barrier. Brambell, reasoning from his studies with rabbits and rodents suggested that the route of transfer of maternal yglobulin to the offspring had not been established, and proposed that, in primates, as in several other species he had studied, the transfer of y-globulin might be by way of the yolk sac into the amnion, and hence across the gut into the circulation of the developing fetus. Studies by Slater (1954), and Bridges et al. (1959) of offspring of hypergammaglobulinemic and hypogammaglobulinemic mothers provided the strongest

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

9

possible argument in favor of the origin of the y-globulins in the maternal circulation, In a most comprehensive study of this problem, Bangham (1960, 1961) and Bangham et al. (1958a) provided clear evidence that placental transfer is indeed the route of passive immunity in primates. y-Globulin is found in the serum of monkey fetuses at 16-20 weeks and reaches a concentration equivalent to that of the maternal serum at about 35 weeks. By contrast, the y-globulin concentration of the amniotic fluid is low, regularly considerably below that of the fetal serum. Labeled maternal proteins turn up in the fetal blood, whereas labeled proteins introduced into amniotic fluid do not appear in fetal serum. The transfer of proteins from mother to fetus appears to be largely unidirectional: if the fetus is injected with labeled y-globulin, little label gets back to the mother and very little goes into the amniotic fluid. However, this work suggests that some albumin and a little y-globulin can be selectively returned by way of the placenta to the maternal circulation. Some maternal albumin is transmitted to the fetus, although not as easily as yglobulin; almost none of the other major serum protein fractions cross the placenta in the monkey (Bangham, 1961). In man, the immunoglobulin transferred during gestation is almost entirely the y2 component, and very little or no /32M- or /3zA-@ObUlingains access to the fetus. The lack of the PzM component in the serum of the neonate is not surprising; since the molecular weight of this protein is nearly 1,000,000, the size of these molecules alone could be invoked as an explanation of failure of transfer. Failure of transfer of the Pz~-globulin is not so easily explained: work to date suggests that the sedimentation constant is in the range of 75 and, thus, the lack of transfer in significant amounts to the fetus cannot readily be explained on the basis of molecular size, since the yz-globulins, which pass with ease, are molecules of similar size. One difference between the PzA- and yz-globulins, in addition to differences in mean charge density of the molecules, is the high carbohydrate content of Pzll (Heremans et d.,1959). However, thus far, there have been few studies of the physical and chemical properties of highly purified preparations of &A, and a number of other factors, not yet investigated, could be responsible for failure of transfer across the placental barrier. It is of interest that the placenta provides a significant barrier to transfer of C-reactive protein (CRP), another yglobulin component ( Rozansky and Berkovici, 1956; Nesbitt et al., 1960; Philipson and TveterHs, 1957; Good, 1952). During acute disease and in many apparently normal pregnancies, the maternal serum may have reIatively high concentrations of CRP, but the cord blood of the infant

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ROBERT A. GOOD A N D BEN W. PAPERMASTER

can be completely lacking in this component. For example, Rozansky and Bercovici (1956) studied the sera of 119 pregnant women and 71 women in labor. Of this group 32% of the pregnant women and 66% of the women in labor showed positive tests. Only 1 of 23 infants showed CRP in the cord blood sample. In a similar but less extensive study by Good (1952), an analysis was made of women with either malignant disease or acute infection at the time of delivery; although each of 12 patients had strong positive serologic reactions for CRP, in only 1 of the 12 infants was CRP present in the cord blood, and in that instance the positive reaction was equivocal. Mention is made of this relationship since CRP appears among the immunoglobulin fractions on immunoelectrophoretic analysis of the serum and since this protein has a reactivity TABLE I RELATIONSHIPOF PLACENTAL STRUCTUREAND MODEOF PASSIVETRANSFER OF IMMUNOGLOBULINS TO THE FETLJS~

Species Pig Ruminants Carnivores Rodents Ape, man (I

Tissue layers between maternal and fetal circulation at term 0

5 3 2 2

Placental or amniotic transmission

k

+ (placental) (yolk sac) +++

Importance of colostrum

+++ +++ + +-++-

Adapted from Vahlquist (1958).

with pneumococcal polysaccharides very reminiscent of immunologic reactions. Although human milk contains all of the immunoglobulins, yz, P ~ A , and PZM, in appreciable concentration (L. A. Hanson, 1959), there is no evidence that the intestinal route plays a significant role in immunoglobulin transfer to the newborn infant (Gugler and von Muralt, 1959; Schneegans et al., 1962). In summary, in all species of birds and mammals studied thus far, elaborate mechanisms are provided by nature to insure transfer of immunologically active proteins from mother to offspring. Table I summarizes available data concerning the characteristics of the placenta and the major routes of transfer of immune globulin to the fetus. There appears to be an excellent correlation of placental structure and the mechanism for passive transfer of immune globulin to the newborn. The primates, including man, apparently differ from all other species in that transfer of the y-globulins occurs across the placenta and does not involve the gastrointestinal mucosa or the epithelial layers of either the

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

11

mammary gland or yolk sac. These mechanisms provide protection for the offspring during the period of immunologic inadequacy. In man, the immunoglobulin transferred is entirely or almost entirely the yz component; the Pu- and PzM-globulins are neither produced by the fetus nor provided by passive transfer in demonstrable amounts.

D. TRANSFER OF ANTIBODIESTO FETUS In man, where the studies have been most extensive, passive transfer of antibodies to the fetus probably begins at about the same time as the appearance of y-globulin in the fetal circulation. Differences in relationship of mother and baby exist with respect to different antibodies (reviewed extensively by Vahlquist, 1958; Hitzig, 1959; Freda, 1962; von Muralt, 1982), apparently as a consequence of the different types of protein molecules, some having molecular weights as high as 1,000,000 and others as low as 150,000 (Franklin and Kunkel, 1958). Such antibodies as antitoxins against diphtheria, tetanus, and erythrogenic toxin, appear in the fetal circulation at term in essentially the same concentration as that of the maternal circulation. This relationship almost certainly holds true for all the antibodies belonging to the yz-globulins or 7s class of proteins. Included among these may be listed incomplete Rh antibodies ( conglutinins) ( Wiener and Berlin, 1947), certain antipertussis agglutinins (P. Cohen and Scadron, 1943, 1946), and virus neutralizing antibodies such as antimeasles ( Herrman, 1923), antipoliomyelitis ( Aycock and Kramer, 1930), antivaccinia ( Kempe and Benenson, 1953), anti-herpes-simplex ( S. G. Anderson and Hamilton, 1949), and antiJapanese B encephalitis (Hale and Lee, 1954) antibodies. Almost all the complement-fixing antibodies, save the reagins against syphilis, are well transmitted to the offspring (Vahlquist, 1958; Hitzig, 1959; von Muralt, 1962; Freda, 1962). The latter include antibodies to mumps, virus influenza, and toxoplasmosis. Among the antistreptococcal antibodies, antistreptolysin 0 is known to be well transmitted to the fetus (Vahlquist et al., 1950). Thus far, although almost certainly of the yz class (Wannamaker, 1958), the other antistreptococcal antibodies, such as antistreptokinase, antihyaluronidase, antideoxyribonuclease B, and antidiphosphopyridinenucleotidase, might be expected to be well transmitted to the fetus, although no definite studies have been done to our knowledge. Among the antibodies not well transmitted to the fetus, most are known to belong to the yl ( 19s) globulin or PPJd fraction of the serum proteins (Franklin and Kunkel, 1958). These include anti-A and anti-B isohemagglutinins (Wiener and Silverman, 1940), complete or saline Rh agglutinins ( Broman, 1947; Wiener and Berlin, 1947), Wassermann re-

12

ROBERT A. GOOD A N D BEN W. PAPERMASTER

agins ( Dunham, 1932), certain hemolysins against sheep cells (Richou and Ramon, 1945), and certain antibacterial agglutinins such as antityphoid “0”( Timmerman, 1931), and colon bacillus antigens “ 0and “ H( Adamson et al., 1951) . The skin-sensitizing allergens are also very poorly transmitted to the fetus (W. B. Sherman et al., 1940); the mother may possess very high titers of such antibodies, but the neonate will have no sensitivity demonstrable either by direct test or by passive transfer. This observation seems consonant with the finding of Sehon (1959, 1960) that these antibodies, too, belong to the 19s class of y,-globulins. However, more recent studies by Heimlich et al. (1960), in Campbell’s laboratory, suggest that the skin-sensitizing antibodies do not have as high a sedimentation coefficient as the studies of Sehon seem to indicate. Further, chromatography on diethylaminoethyl (DEAE ) cellulose columns ( Augustin, 1960) seem to place these antibodies in a fraction quite distinct from that in which the 19s component is located. Preliminary observations in several laboratories (Fireman et al., 1963; Rockey and Kunkel, 1962; Heremans and Vaerman, 1962) suggest that the skin-sensitizing antibodies may, indeed, be contained in the Pzn or ylA fraction which, although probably having the ultracentrifuge characteristics similar to y2-globulin, does not traverse the placental barrier in significant amounts. The question of access of maternal antibodies to the fetus is of importance in a consideration of the ontogeny of immunity from two points of view. First, the antibodies traversing the placental barrier provide passive immunologic protection of the newborn. There is, however, another side to the coin. It has been observed repeatedly that artificially initiated, active immunologic responses in young infants are adversely influenced by the presence of the specific antibody. Vahlquist et al. ( 19-48), using diphtheria toxoid, showed clearly that passively acquired diphtheria antitoxin interfered with the active production of antitoxin. Osborn et a2. (1952a) confirmed this observation, but concluded that the interference could be overcome by using larger doses of toxoid. Perkins et al. (1959) have shown that such interference occurs in poliomyelitis immunization as well. In experimental animals, Uhr and Baumann (1961) have observed and studied a similar phenomenon. Using soluble, serum protein antigens and specific antibodies, they showed that passively transferred specific antibody not only inhibits the initiation of an immune response, but that passively administered antibody may even stop production of antibody once the active immune response has been initiated. Although the mechanism of the inhibitory effect of antibody may simply be inactivation of

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

13

antigen through immunologic combination, the studies of Uhr and Baumann suggest that a more intricate feedback mechanism may be operating.

E. ONTOGENY OF IMMUNOGLOBULIN SYNTHESIS So far as can be ascertained with present methodology, virtually no synthesis of any of the immunoglobulins occurs in the developing fetus. However, as a consequence of passive transfer of y-globulin from mother to fetus, Longsworth et al. (1945) showed that human infants are born with slightly more y-globulin on the average than their mothers. This observation has been extensively confirmed (Moore et al., 1949; Orlandini et al., 1955; Oberman et al., 19%; Mellander et aZ., 1959), but there is some disagreement as to whether the y-globulin levels are significantly higher in the baby’s serum than in that of the mother (Zak and Good, 1959; von Muralt and Gugler, 1959). In cattle, sheep, and pigs, where y-globulin is not transferred across the placental barrier, the neonate is essentially agammaglobulinemic; however, it seems likely that the lack of y-globulin in these animals is not absolute. For example, Franek et al. (1961) have claimed that a Iow molecular weight y-globulin is already present in low concentration in the newborn pig. The minute amounts of y-globulin were demonstrated by an immunochemical method, and were attributed to the piglet’s own protein productive mechanisms. This y-globulin was considered a precursor of the y-globulin and antibody to be formed later in response to antigenic stimulation. Studies by Hoerlein (1957) and by Segre and Kaeberle (1962a,b), and Segre et al. (1963) have presented these relationships of y-globulin and antibody in the piglet in another dimension. Hoerlein showed that pigs 3-8 weeks of age formed antibody when allowed to suckle colostrum from their dams, suckling that was associated with passive transfer of y-globulin and antibody from the mother in appreciable concentration. Piglets not provided with this source of y-globulin and antibody by passive transfer were most feeble in antibody synthesis. Segre and Kaeberle (1962a) confirmed Hoerlein’s observations and showed that suckling of colostrum, administration of diluted hyperimmune serum of swine and horse origin, or administration of normal serum from older colostrumdeprived pigs would overcome the immunologic deficiency. In addition, they (1962b) confirmed the presence of small amounts of y-globulin in newborn pigs, and found that this small amount declined to even lower levels in colostrum-deprived animals. The immunologic inadequacy of these animals was greatest with reference to antigens not normally en-

14

.

ROBERT A. GOOD AND BEN W. PAPERMASTER

countered by pigs. Segre and Kaeberle concluded that initiation of antibody synthesis in the pig is facilitated by the availability of small amounts of y-globulin and specific antibody provided by a minimal transplacental transfer, and that the immunologic inadequacy of colostrumdeprived pigs at 3 to 8 weeks of age reflects the loss of the minute amounts of antibody transferred across the rigid placental barrier in this species. Studies by Kim and Watson (1963), on the other hand, indicate that the neonatal piglet is completely devoid of immunoglobulins and can be induced to antibody synthesis with suitable antigens even when given no colostrum or exogenous source of y-globulin. As indicated earlier, in man, the serum y-globulin level of the newborn is at least as high as that of the mother. Beginning almost immediately after birth, the y2-globulin concentration begins to fall, and for a variable period, usually for several weeks, the y-globulin levels in the serum drop sharply (Bridges et al., 1959; Zak and Good, 1959). Beginning from 3 to 10 weeks after birth in the normal baby, the y-globulin curve levels off, a plateau or trough occurs, and then the curve rises. Adult y-globulin levels are generally reached some time between the end of the first year and the age of four. This y-globulin curve has been attributed to several relationships during development. First, there is the failure of synthesis of y2-globulin by the fetus, combined with passive transfer of y-globulin across the placental barrier. Then, there is the continuing deficiency of synthesis of y-globulin in the neonatal period, combined with the growth of the yglobulin space. Production of sufficient y-globulin to compensate for the decay rate and the growing y-globulin space begins at varying times in individual infants, but usually some time between 3 and 12 weeks. After that time the y-globulin concentration rises until total production and total degradation reach an equilibrium at a circulating y-globulin level between 0.6 and 1.5 gm./100 ml. of serum, the level of healthy immunologically mature persons. During the period of relative deficiency of yglobulin production in human newborns, especially prematurely born babies, the y-globulin can decline to levels quite low by adult standards: y-globulin concentrations of less than 100 mg./100 ml. have frequently been observed in apparently normal infants. Occasionally, assumption of normal y-globulin production by an infant is delayed, and a state of transient agammaglobulinemia of infancy may be observed (N. H. Martin, 1954; Good and Zak, 1956). Although the basis for such a delay has not been ascertained in any instance, a few patients with this entity have been studied in our clinic and elsewhere. The delay in production

ONTOGENY AM) PHYLOGENY OF ADAPTIVE IMMVNITY

15

of normal amounts of y-globulin may extend up to 6 or 8, or even 10 months of age, and at subsequent study a year or two later, the child may be perfectly normal by accepted immunologic and immunochemical criteria, During this period of transient agammaglobulinemia, it is not possible, without recourse to specific antigenic stimulation, to separate such patients from those having true persistent immunologic deficiency disease (Good et al., 1962a). An interesting view of the development of y-globulin production in man has been provided by patients with agammaglobulinemia. During the course of extensive investigations of patients with this disease (Bridges et al., 1959; Zak and Good, 1959; Good et al., 1962a) we have had an opportunity to study two children born of a mother with extreme hypogammaglobulinemia, apparently of the acquired type. In addition, we have now studied 11 children born of 5 mothers who had previously given birth to boys with agammaglobulinemia, presumably of the sexlinked recessive type. The two children born of an agammaglobulinemic mother were agammaglobulinemic at birth, having levels of y-globulin of less than 10 mg./100 ml., in the same range as the maternal y-globulin (Bridges et al., 1959; Zak and Good, 1959). This observation is compatible with the conclusion that the fetus and neonate form little or no yglobulin. Further, in both children, the y-globulin concentration remained at this extremely low level until the 35th-42nd day of life in the first child, and the 21st-28th day of life in the second. Rapidly thereafter, both infants developed full immunologic competence, and levels of yglobulin within the normal range were achieved by the time these children were 6 months old. On the other side of the coin were the children born of 5 mothers who had previously given birth to agammaglobulinemic male offspring. Of these 11 children, 4 were girls who had normal serum proteins and normal immunologic responses; 4 were boys who developed normal immunologic responsiveness; and 3 were boys who had persistent and presumably permanent agammaglobulinemia. All 11 had levels of y-globulin at birth essentially equal to or slightly higher than those of the mother. During the several weeks after birth all showed a decline in y-globulin concentration. From the studies performed, it was not possible to ascertain which of the male children would be immunologically normal until several months after birth. Then, the children destined to develop full immunologic capacity showed a decrease in the rate of y-globulin decline, beginning formation of isohemagglutinins, and ultimately increasing y-globulin concentrations. Those destined to be agammaglobulinemic showed continued, essentially logarithmic decline in y-globulin levels to 20 mg./100 ml. or less. These boys had failure of

16

ROBERT A. GOOD AND BEN W. PAPERMASTER

plasma cell development and defective lymphoid tissue, with lack of secondary follicle formation, whereas the other group had normal development of the lymphoreticular tissues. We concluded that in these several groups of patients we had observed the developmental biology of y-globulin in bold relief. Although there are no data in these studies to argue conclusively against the conclusion of Trevorrow ( 1959) that appreciable amounts of y-globulin are being produced in the neonatal period in man, we find little support for this contention in our observations. Further, attributing the decline in y-globulin level in the newborn entirely to growth of the infant seems to be fallacious, since growth is even more rapid during the late fetal period when y-globulin is accumulating by passive transfer from the mother, and since no sudden change in the growth rate occurs during the first year of life which can, by itself, account for the cessation of the decline in y-globulin levels. Finally, the rapid rise in y-globulin concentration between the second and sixth months in both of the children of an agammaglobulinemic mother must reflect either a change in m e of production or rate of destruction of y-globulin. We thus interpret these and all other data to be compatible with the hypothesis that yglobulin is not produced in significant amounts by the fetus or newborn. Initial investigations with the immunoelectrophoretic technique, carried out primarily by the Swiss and French investigators, support these concepts. y2-Globulin is the only one of the immunoglobulins present in the circulation of the human neonate in significant amounts (Hitzig, 1957; Scheidegger and Martin du Pan, 1957). With later variations of the immunoelectrophoretic technique, very minute amounts of P ~ Mand p 2 A have been demonstrated in a significant percentage of cord bloods and sera from infants during the first week of life (von Muralt and Gugler, 1959; von Muralt, 1962; Vivell et al., 1960). In healthy babies, the p2M level begins to rise during the second and third months of life, and the p2.4 usually slightly later (Hitzig, 1957; Scheidegger and Martin du Pan, 1957). The Panl rises quickly to adult levels, whereas the rise in 0 2 6 parallels that of the y-globulin. Koch et al. (1959) showed that, upon falling ill, a newborn infant is ready to produce the y-macroglobulin by three weeks of age, a finding that agrees well with the early appearance of the flzM band, as observed by West et al. (1962) and others (Dietel and Lohmann, 1960; Hitzig, 1961). Further, it appears that P2M can reach full adult concentrations in the face of stimulation at a time when y-globulin production is grossly defective and the component has still not appeared in the serum (West et al., 1962). These observations are particularly significant in the

OF~TOCENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

17

light of the recent observations (R. T. Smith, 1960; Uhr et al., 1962b; Fink et al., 1962), to be discussed in detail below, that the initial immunologic response in both premature and full-term infants involves antibody primarily or entirely of 19s globulins. Immunoelectrophoretic studies of Vivell and Sick (1960), are of real interest. They, in concert with others using the immunoelectrophoretic method, have observed an adult y-globulin band in serum of the newborn, with a lack of P2M and PzA. They noticed, however, that a doubling of the y2-globulin band often occurred at about the time that the infant’s own y-globulin production was, from indirect evidence, presumed to begin. The adult immunoelectrophoretic pattern was again regularly present by 2 years of age. These authors concluded that the y-globulin newly formed by the infant was slightly different in its immunochemical properties from that received across the placenta. Among the most recent studies of the developmental biology of the immunoglobulins are those of Roth (1962) in which quantitative immunochemical techniques have been used. In general, this author’s findings parallel those of West et al. (1962). With a very sensitive immunochemical method, Roth showed that minute amounts of both p 2 A and f 3 2 ~are present in the cord blood serum. Whereas the concentration of p2= averages 160 mg./100 ml. in adults, with a range of 60 to 480 mg./100 ml. in healthy persons, cord blood contains from 3 to 24 mg./100 ml. of this protein. Rising concentrations of P2M may be noted in the first 10 days of life, especially in the presence of perinatal disease. From these studies it appears that adult concentrations of (32M are reached during the third year in normal children. The mean serum level of p2* globulin in normal adults is 200 mg./100 ml., with a range from 75 to 600 mg./100 ml. In cord blood the range of p2* is from 0.1 to 1.0 mg./100 ml., with a mean of 0.3 mg. The P2A concentration does not begin to increase until after the twentieth day, and then shows a gradual increase until adult concentrations are reached sometime after 3 years of age. In summary, it seems clear that little or no immunoglobulin synthesis occurs in the fetus, but that protection is afforded by passive transfer of y2-globulin across the placenta. The production of y2- and PzA-glObulins gets off to a slow start in the neonatal period; however, (32M production may be initiated in the immediate neonatal period, especially in the presence of disease, and appreciable amounts of this protein may accumulate within the first few weeks of life. This may well be an early adaptation to exposure to antigenic stimulation in the extrauterine environment. Far too few comprehensive studies of the developmental biology of

18

ROBERT A. GOOD AND BEN W. PAPERMASTER

immunoglobulins have been carried out in animals other than man; consequently, these have not been emphasized in this review. However, the data available indicate that in a number of species there is neonatal sluggishness in establishing production of y-globulins. Both chickens and mammals raised in a germ-free environment show declines in y-globulin concentrations to low levels (Thorbecke et aZ., 1957; Wostmann, 1959). In the most completely genn-free environments obtained, y-globulin concentrations approaching those of agammaglobulinemic patients have been recorded (Gustafsson and Laurell, 1959). Exposure of such animals to antigenic stimulation reveals, here also, a sluggishness in establishing immunologic competence, similar to that ordinarily observed in the neonatal animal.

F.

ONTOGENY OF ANTIBODY PRODUCTION

1. Development of Capacity to Produce Antibody in Response to Specific Antigenic Stimulation Specific antibody production does not ordinarily take place during embryonic development in birds and mammals; and the central issue is whether the embryo is incapable or defective with respect to immunologic response or whether antigenic stimulation is simply not available. Studies attempting to resolve this question go back many years, and it is only very recently that a reasonably clear picture of the developmental biology of antibody production has emerged. Much remains to be done : there have been studies of too few species, with too few antigens, and a lack of recognition of variability from organism to organism, species to species, and extraordinary variability from antigen to antigen, Interpretation is also difficult because much of the work has not been quantitative. As early as 1907, Rywosch reported failure of the chick embryo and young chick to respond to antigens of Escherichia coli. This observation has been confirmed and extended by several investigators using a variety of antigens (Grasset, 1929a; Weinberg and Guelin, 1936; Burnet, 1941; Beveridge and Burnet, 1946; Fox and Laemmert, 1947; Wolfe and Dilks, 1948; Burnet et al., 1950; Wolfe et al., 1957). In these studies, Weinberg and Guelin (1938) showed that neither chick embryos nor newly hatched chicks would respond to antigens of CZostridium sporogenes. Burnet (1941) found that influenza virus inoculated on the twelfth day of incubation did not stimulate antibody production in the developing chick; and later, Beveridge and Burnet ( 1946), in studies designed primarily to produce immunologic tolerance, found no immune response to bacteriophage in chick embryos. Although Fox and Laemmert (1947) confirmed

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

19

these observations in essence, using a yellow fever virus vaccine, they found that the virus persisted in a few embryos until hatching and that the chickens subsequently developed antibody. Wolfe and Dilks (1948) stimulated chicks with serum protein antigens, and found that the deficiency of the newly hatched animal was extreme and that antibodyproducing ability developed gradually up to 4 weeks of age. Between 4 and 5 weeks of age a rather sudden maturation took place and, on the average, antibody titers of birds given antigen at 5 weeks were much greater than that of birds stimulated at 4 weeks. Those injected with antigen at 6-12 weeks produced approximately the same levels of antibody as the 5-week-old birds, and the authors concluded that immunologic maturity in the chicken is reached at about 5 weeks after hatching (Wolfe and Dilks, 1948). Subsequent studies by Wolfe et al. (1957), using quantitative methods, c o n h e d these findings, and extended the observations to a correlation between the size of the spleen in the young chicks and their antibody-producing capabilities. These studies showed, too, that maturation of immunologic capacity continued throughout the life of the chicken; for example, when bovine serum albumin (BSA) dosage was calculated on a weight basis, the l-year-old chicken produced 400% more antibody than the chick stimulated at 6 weeks of age. Burnet et al. (1950) found that stimulation with washed human cells led neither to an immunologic response nor to tolerance in the chick embryo. The studies were extended to mammals, first to the rabbit and later to the guinea pig. Moll (1908), who studied only two 3-week-old rabbits injected with cholera vaccine and horse serum, found that they were grossly deficient in immunologic response compared to adult animals. He concluded that the newborn animal is defective in response to both protein and bacterial antigens. Subsequent studies by Friedberger and Simmel (1913), Coca et a2. (1921), Nattan-Larrier et al. (1927), Grasset (1929b), Polk et al. (1938), Valtis and Saenz (1928), and others seemed to establish that embryos and newly born or newly hatched animals are defective in their response to some antigens. Freund, in 1930, showed clearly the relative refractoriness of the newborn rabbit to antigenic stimulation: in rabbits less than 20 days of age, the response to typhoid bacilli, sheep red cells, horse serum, and ovalbumin was strikingly less intense than in adult rabbits immunized the same way. He showed further that true Arthus reactions could not be produced in young rabbits immunized with horse serum or egg white. Baumgartner, in 1934, reviewed the relation of age to immunologic reactivity, and concluded that the ability to be immunized was qualitatively and quantitatively inferior in the neonatal period.

20

ROBERT A. GOOD AND BEN W. PAPERMASTER

In recent years, Sterzl and Trnka ( 1957), Dixon and Weigle (1957), Deichmiller and Dixon (1957), and Bridges et al. (1959) have made similar comprehensive and quantitative studies of the development of responsiveness to protein antigens in the rabbit. In all of these studies the neonatal rabbit was far less responsive than the adult, indeed often unresponsive. The antibody response appeared along with capacity to produce plasma cells during the fourth week after birth. In later experiments, Deichmiller and Dixon (1960) found that large doses of bacterial and erythrocyte antigens in newborns resulted in y-globulin synthesis at an earlier age; Sterzl and Trnka (1957) and Riha (1961) made a similar observation studying antibody responses to bacterial antigens. Bridges et al. (1959) also forced maturation of immunologic response to BSA in the rabbit by administering it with Freunds adjuvant. With this intense stimulus, antibody to BSA was produced as early as 16 days of age, but not earlier. The earliest evidence of the morphologic response of antibody synthesis, pyroninophilia and plasma cell development, occurred 12 days after birth (Bridges et al., 1959). The general conclusion of these several groups of investigators was that immunologic unresponsiveness continues for some days beyond birth, and that adult capacity is not achieved until at least 4 weeks of age in the rabbit. Sterzl and Hrubesova ( 1956) presented observations suggesting that ribonucleic acid (RNA) obtained from spleen cells of adult rabbits experiencing an immunologic response might induce immunologic responsiveness of neonates. Several investigators have attempted to reproduce these results, without success (Stavitsky, 1958; Janovid et al., 1959; Sorkin, 1963), but Hrubesova et d. (1959) were able to obtain very low agglutination titers in very young rabbits by this type of transfer, and concluded that RNA might enhance antibody production in a nonspecific way during the neonatal period. Sterzl et al. (1960) and Franek et al. (1961) studied newborn pigs and found that these animals were also immunologically inadequate. As antigens they used Brmcella, simple protein antigens, and the very E. coli organisms that were providing the first antigenic stimulation by the gastrointestinal route. Unpublished observations from our laboratory (Zak, 19f30) confirmed the immunologic inadequacy of the piglet during the first several weeks of life, revealed by early failure to respond to soluble protein antigens. The studies of Franek et al. (1961) suggested synthesis of a small amount of a y-globulin-like protein even during the period of gross immunologic inadequacy. As noted earlier, the work of Segre and Kaeberle (1962a) appears to indicate that passively transferred specific antibody, either received in the colostrum or by placental transfer,

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

21

or provided artificially, serves to initiate immunologic responsiveness in the pig. Kim and Watson (1963) have shown, however, that with adequate antigenic stimulation and very sensitive antibody detection techniques, antibody synthesis is demonstrable in newborn piglets, even in the absence of colostrum, as early as 3 4 days. Bishop and Gump (1961) studied immunologic responsiveness in neonatal guinea pigs, with interesting results. They found that newborn guinea pigs given bovine y-globulin and adjuvant immediately after birth produce circulating antibody as detected by passive cutaneous anaphylaxis (PCA) but not by agar diffusion. By the fifteenth day all the injected animals gave a positive PCA reaction, but the amount was much less than in adult animals. The authors concluded that humoral antibody synthesis is present in this species, but that the rate of production is reduced. R. T. Smith (1960), using Salmonella flagellar antigens, showed that neonatal rabbits can respond with synthesis of a heavy antibody, presumably 19s; however, its precise characteristics were not determined. S . Harris et al. (1961) have also demonstrated significant antibody response to bacterial antigens in the newborn rabbit, and Condie et al. (1960) have shown that injections of hemocyanin in neonatal mice and rabbits will result in appearance of circulating antibody as early as 1 week after birth. Another recent investigation (Silverstein et ul., 1963) involved antigenic stimulation in utero. Fetal lambs were stimulated with diphtheria toxoid, Bacillus Calmette-Gudrin ( BCG ) , bacteriophage +X 174, ferritin, ovalbumin, and Salmonella, as early as the sixty-sixth day of gestation. Significant amounts of antibody to the phage, ferritin, and ovalbumin were produced, but the other antigens were ineffective under these conditions. The antibody was apparently 19s. Fennestad and Borg-Petersen ( 1962) studied antibody-producing capacity of bovine fetuses. Eight cows were subjected to laparotomy and injected intraplacentally with Leptospiru saxkoebing. Six of the fetuses had produced agglutinins against this antigen when tested 3 M 2 days later. Leptospiru was isolated from the kidneys of two of the fetuses. Plasma cells were found in the kidneys of five of the older fetuses, and transitional cells were present in the spleen and kidney of the youngest fetus, inoculated at 132 days of gestation and examined 32 days later. These authors concluded that bovine fetuses can produce Leptospiru agglutinins at less than 164 days of gestational age, and that, even at this early age, antibody synthesis is accompanied by formation of plasma

22

ROBERT A. GOOD AND BEN W. PAPERMASTER

cells. The titers of antibody of these fetuses were appreciable, but comparisons were not made with the adult response to the same antigen. Perhaps the most dramatic of the studies of early antibody synthesis was made in marsupials. This is the investigation reported by Kalmutz (1962). This investigator showed that the opossum embryo can respond to bacteriophage stimulation with antibody production at an extremely early age. These animals are born 12-13 days after copulation when no organ systems have been defined, although the vital ones are functional. Lymphopoiesis begins in the thymus 5 days after birth. A similar development occurs in the lymph nodes 10 days after birth and in the spleen 15 days later (Kalmutz, 1962). Using bacteriophage as the antigen, Kalmutz found antibody synthesis as early as 11 days after birth, at a time when only the thymus and the very earliest lymph nodes are present. By 22 days of age, antibody production of unquestionable significance was at hand. Studies have not yet been made on the nature of this antibody, i.e., whether it is of high molecular weight or of the classical 7s variety, probably, at least in part, because of the difliculty in obtaining samples. Additional studies on the opossum have recently been reported by La Via et al. (1983). The antigen used was a flagellar preparation of Salmonella typhi, and it was administered at various times from the sixth to the sixteenth day after birth. Antibody was detectable in sera from animals stimulated on the eighth day or later. The authors emphasized that this antibody was produced at a time when normal opossums show no lymphoid development in the spleen and have neither plasma cells nor secondary lymphoid nodules. In summary, these studies of antibody synthesis in embryonic and neonatal life in chickens and mammals indicate that all embryos and neonatal animals studied critically are immunologically deficient, and that maturation of the immunologic mechanisms to adequacy by adult standards occurs some time during the first weeks after birth. Some degree of maturation in the chicken and the mouse continues for most of the first year. However, recent studies, using certain bacterial antigens, bacteriophage, and hemocyanin, show that immunologic capacity is present in some species in the neonatal period and during embryonic life. Striking daerences in the development of immunologic responsiveness are revealed by the use of different antigens. Certain antigens, presumably the more potent ones such as hemocyanin and bacteriophage, induce antibody production in animals that appear to be grossly deficient in responsiveness when stimulated with other antigens, e.g., serum protein antigens.

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23

2. The Development of Antibody-Producing Capacity in Man The observations on the development of immunologic capacity in man are strikingly parallel to those described for experimental animals. As early as 1920 (Happ, 1920) it was found that isoagglutinins against heterologous blood groups are absent from the circulation during the first months of life and regularly appear during the second half of the first year or during the second year. Frankenstein (1920) found that infants respond poorly to typhoid antigens: in his series, only 3 of 30 young infants produced significant titers of agglutinins after injection of typhoid vaccine. By contrast, adults regularly responded to the same vaccine. Ribadeau-Dumas et al. (1925) found newborns to be refractory to antidiphtheria immunizations, although some did show slight response. These early observations were confirmed by several investigators, and, together with the findings in experimental animals, were cited by Baumgartner (1934) as evidence for the immunologic inadequacy of the very young mammal, including the human infant. On this basis, it was popular among pediatricians to delay immunization procedures until the second half of the first year or the second year of life. This delay in immunizations, even until 3 months after birth, was unfortunate since, for certain of the diseases against which routine immunization was performed, the earliest possible protection was most contributory. Sako (1947) was perhaps the first to express the view that infants have fully developed capacity to form antibodies against pertussis and other antigens shortly after birth. Sako et al. (1945) had studied routine immunizations initiated soon after birth and showed that such early immunizations provided effective antibody levels. Bradford et al. ( 1949) also pointed out that, from a practical point of view, it is possible to obtain good protective levels of antibody in infants from 1 to 3 months of age against alum-precipitated diphtheria and tetanus toxoids and pertussis vaccine. Vahlquist et al. ( 1948) and di Sant’Agnese (1949) reinvestigated the immunologic responsiveness of human infants, and were forced to the conclusion that, although immunologic responses can be obtained shortly after birth, both premature and full-term infants are slow to form antibody and are somewhat defective in their total immunologic capacity. The subsequent studies of Vahlquist and Nordbring (1952), in Sweden, and Osborn et al. (1952b) in the United States, established clearly the feebleness and slowness of the immunologic response to diphtheria toxoid in both premature and full-term infants. Both investigations also showed that antibody synthesis can be initiated in extremely premature babies and early in the neonatal period. In the studies of

24

ROBERT A. GOOD AND BEN W. PAPERMASTER

Osborn et al. (1952a), a rather striking inhibitory effect of passively transferred maternal antibody was demonstrated. Paradoxically, these studies established that immunologic maturation is clearly not a consequence of chronologic maturation alone, since extremely small premature infants seemed to gain full immunologic competence at about the same time after birth as full-term infants. It seemed reasonable to conclude that an environmental factor, possibly antigenic stimulation itself, is responsible for the final stage of maturation of immunologic capacity. Scattered observations, such as those of A. S. Sabin and Feldman (1949) and Shinefield and Townsend (1953), suggested that host immunologic response might be elicited at a very early age by virus or other infections. Koprowski et al. (1956) gave polio virus to infants under 1month of age who showed a surprisingly adequate and prompt ability to form neutralizing antibodies to these live agents. While the response was smaller than that of older children, it seemed sufficient to contain the infection and provide immune protection. Interestingly, passively transferred antibody did not seem to interfere with the antibody response of these newborn infants to polio virus. Stulberg et al. (1956) studied babies involved in an epidemic of E . coli 0127:B8 infection. About one-third of the infants developed antibodies, demonstrable by hemagglutination but not by agglutination techniques; two-thirds showed no antibody at all. By contrast, volunteer adults infected with the same agents developed both rising hemagglutination titers and bacterial agglutinins. Eichenwald and Kotsevalov (1960) reported observations on a number of full-term infants naturally infected with adenovirus types 1, 2, and 3, and ECHO virus 9, 18, and 20, during the first few weeks of life. Serologic studies revealed that these babies responded immunologically, and that their response was independent of age and birth weight, but that it varied with the type of virus involved. Passively transferred antibody specific for the virus agent did not seem to interfere with active formation of antibodies. These authors recognized that successful immune response to virus infections may not be the general rule for premature and newborn infants, and cited the aberrant behavior of newborns to herpes simplex, cytomegalic inclusion virus, and certain Coxsackie viruses as examples. They also raised the important question of whether deficiencies other than those involving the immune response might explain the peculiar vulnerability of the neonate to these virus agents. Bridges et al. (1959) and Zak and Good (1959) studied two infants born of an agammaglobulinemic mother whose immunologic response was extremely defective in the neonatal period. Indeed, these babies did

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25

not show response to the antigens studied until they were more than 1 month old in one instance and 6 weeks old in the other. Smith and his co-workers (R. T. Smith, 1960) reported their observations that premature and full-term human infants, as well as newborn rabbits, can, indeed, form substantial amounts of antibody against certain antigens within a short time. The antigens used in most of their studies were flagellar antigens of a variety of Salmonella. The antibody response was striking in that it occurred at a time when immunologic response to other antigens, such as somatic antigens of the same organisms, was defective. Further study of this antibody showed it to be a high molecular weight antibody, presumably of the 19s class. This heat-stable 19s antibody was said to be broken down to smaller units with 2-mercaptoethanol, with retention of at least some of the immunologic reactivity (R. T. Smith, 1960). In Smith's studies the 19s type of antibody seemed to be the only one present for a period of about 2 months, followed by appearance of 7s antibody against these antigens. The variance of Smith's data and those of Bridges et al. (1959) and Zak and Good (1959) is probably a reflection of the different antigens used. Fink et al. (1961, 1962) have done similar studies, using flagellar antigens of Salmonella tzjphi. In their investigation, however, the antibody produced seemed to lose all immunologic activity, in usual fashion, upon exposure to 2-mercaptoethanol, The results Uhr et al. (1962b) have obtained, using bacteriophage $X 174 in premature infants, are similar to those of R. T. Smith (1960) and Fink et al. (1961, 1962) with bacterial flagellar antigens. The babies produced antibody to the phage as promptly and in as large amounts as did older children; however, all of the early antibody produced in response to this potent antigen was of the 19s class. These authors pointed out the clash between these observations and the past studies demonstrating the incompetence of premature and full-term human infants toward other antigens. The conclusion seems inescapable: using the right antigens, immunologic capacity of a primitive type can be induced in the human newborn. Kallings and Alvin (1962) have studied inactivation of Tz coliphage by infant sera. They found that neutralizing antibodies against coliphage are absent from cord blood, but appear in some infants during the first 14 days of life and in most by the age of 30 days. The presence of an inhibitor in cord blood was suggested by the finding that cord blood serum inactivated the effect of adult serum on the virus. Thus, the low rate of virus inactivation by cord blood and the apparent rise in phage-

26

ROBERT A. GOOD AND BEN W. PAPEXMASTER

neutralizing activity during the first few weeks after birth might reflect loss of an inhibitor rather than an immunologic response. Eichenwald ( 1963) has recently studied several virus infections of the fetus which also give evidence of 19s antibody production. In this instance, the antibody response occurred even before birth of the infant. In summary, the studies of immunologic response in human infants appear to establish the existence of an immunologic deficiency which is gradually replaced by adult-type immunologic reactivity during the first few weeks of extrauterine life. The maturation of immunologic potential appears to be more a function of separation from the protected environment of the uterus than a function of chronologic maturation, since this development in prematures parallels that in full-term infants. The earliest antibody response is a partial one, evoked by certain virus infections and antigenic stimulation with bacteriophage or flagellar antigens of Salmonella, and involving 19s or BZM-globulins.The sequence agrees well with the observations of the early appearance of P z M - g l O b U h in infants, especially in association with infections (von Muralt and Gugler, 1960; Roth, 1962). A number of studies, in different species and with different antigens, have suggested that earliest response of the young animal involves 19s globulins (R. T. Smith, 1960; Uhr et al., 1962a,b). There are also preliminary indications, as will be discussed in the phylogeny section, that the antibody response of some lower vertebrates involves high molecular weight globulins. G. DEVELOPMENT OF THE LYMPHOID TISSUE Because of the clear association of lymphoid cells and tissues with all forms of immune response, consideration of the ontogeny of the immunologic processes must focus as well on the development of the lymphoid tissue. Although extensive investigations on the development of the lymphoid tissues were made more than 50 years ago (Saxer, 1896; Kling, 1904; F. R. Sabin, 1905, 1909; Hammar, 1905, 1911; Bell, 1906; Maximow, 1909, 1912), some of the most important contributions have been made only recently ( Ackerman and Knouff, 1959; Ackerman, 1962; Auerbach, 1980, 1961a,b; Ball and Auerbach, 1980). The concepts of the ontogeny of the lymphoid tissues are changing as intensive investigations are pursued in many laboratories, and conclusions must be considered somewhat tentative at present. In mammals, the key organs and tissues containing the lymphoid system and the bulk of the lymphocytic elements are the thymus, lymph nodes, spleen, and organized lymphoid tissue of the gut tract, including the tonsils, adenoids, Peyer’s patches, appendix, and, in such mammals as the

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27

rabbit, the intestinal tonsil or sacculus rotunda. Phylogenetically, lymph nodes were, until very recently (Kent et al., 1964) considered to make their first appearance, in small numbers, in birds. However, most birds have a lymphoid organ not found in other forms, the bursa of Fabricius, originally defined as a lymphoid organ by Forbes (1877) and restudied by Jolly (1915) and others (Boyden, 1922; Calhoun, 1933; Ackerman, 1962; Ackerman and Knouff, 1959, 1964). The bursa is a blind, plicated, saclike outpouching extending posteriorly and superiorly from the cloaca. The parenchyma of this organ consists almost entirely of lymphoid follicles, and recent studies of Glick et a2. ( 1956), Glick (1958, 1964), Chang et al. (1957), and A. P. Mueller et al. ( 1960, 1962, 1964) have established the intimate association of the bursa of Fabricius with the development of immunologic processes in chickens. Consequently, its ontogenetic development deserves detailed consideration in this section of the review. In all animals in which direct comparative studies have been done, the thymus is the first organ to develop as true lymphoid tissue. In man, for example, Hammar (1905, 1921) showed clearly that the thymus has a well-developed lymphocyte population by the third gestational month, prior to the appearance of recognizable lymphoid structure in the other organs that ultimately house lymphoid tissue. Knoll ( 1929), studying the development of hematopoiesis in man, described the following sequence. Lymphopoiesis is well established in the thymus by the 35-mm. stage. The spleen, as late as the 90-mm. stage, shows only erythropoiesis and myelopoiesis, but no lymphopoiesis. The ultimate establishment of lymphopoiesis in the spleen follows by at least 2 months the development of granulopoiesis in that organ. Knoll found further that a low level of circulating lymphocytes is present some time before there is discernible evidence of lymphopoiesis in the spleen. He concluded that the thymus as a source of lymphocytes would explain the small percentage of circulating cells having the staining and morphologic characteristics of lymphocytes, in the absence of evidence of lymphopoiesis in the organ which later assumes the primary lymphopoietic function. Rumpianesi and Finotti (1957), studying the embryologic development of the spleen in man, again pointed out that the spleen is reticular until 2 months of gestation, granulopoietic and myelopoietic until 4 months, and does not become lymphopoietic until 5 months of gestation, fully 2 months following development of lymphopoiesis in the thymus. Studies of the development of lymph nodes in man are also pertinent. It is clear, from the extensive studies of Wischnewezkaja (1932), that this is a late development for the most part, proceeding rapidly during the first year of postnatal life and continuing until puberty. Similar ob-

28

ROBERT A. GOOD AND BEN W. PAPERMASTER

servations were made by Campana (1938). Gulland (1894), Saxer (1896), Kling (1904), F. R. Sabin (1905, 1909), A. H. Clark (1912), Latta ( 1921) , Ando ( 1930), Gilmour ( 1941) , and Hellman ( 1921,1943) , as well as Wischnewezkaja (1932), made extensive studies of the ontogenesis of the lymph nodes in man and other mammals. From these investigations, it is clear that the lymph nodes develop at the sites of confluence of the lymph vessels and lymph sacs, with substantial mesenchymal contributions. In each species, primary, secondary, and tertiary nodes can be distinguished, and the primary nodes are the first to develop and become lymphopoietic. There is some dispute about the beginnings of lymphopoiesis. The bulk of the evidence is consistent with the concept that lymphopoiesis begins in the primary lymph nodes, and that this development occurs a good deal later than the establishment of lymphopoiesis in the thymus. For example, the data of Hammar (1905, 1921) and Gilmour (1941) establish the beginning of lymphopoiesis in the thymus prior to the 35-mm. stage in the human embryo. Lymphopoiesis in the primary lymph nodes is described as beginning in the 48-mm. stage by Gilmour (1941), the 50-mm. stage by F. R. Sabin (1905), and at the fourth month by Hellman (1943). In Gilmour’s paper, however, there is the claim that the very earliest lymphopoiesis in the body is in the connective tissue of the lymph plexuses in the 26-mm. embryo. Whether the primary site of lymphopoiesis is at these sites or in the thymus remains to be determined by careful future study. Lymphoid tissue develops in the gut at a considerably later period in most species. In recent years, ontogenetic development of the lymphoid tissues has been reinvestigated, and several important contributions have emerged. Particularly contributory have been the investigations of Auerbach (l960,1961a7b),Ball and Auerbach (1980), Ackennan (1962), and Ackerman and Knouff (1959, 1964). The mouse, like other mammals, shows the first lymphoid development in the thymus. In the 12-day mouse embryo, there is no organized lymphoid tissue; the thymus at this stage consists of an epithelial anlage surrounded by a condensation of mesenchymal tissue (Ball and Auerbach, 1960). The thymus develops lymphopoietic activity by the fourteenth day of gestation, and at 16 days that organ is primarily lymphoid. The lymph nodes and spleen of the mouse do not develop organized lymphoid structure until after birth. Ball and Auerbach (1960) developed a technique for culturing the mouse thymus anlage at a glass clot interface. When the twelfth-day epithelial thymus was cultured in this manner, it spread and showed lobulation, but it remained epithelial, and no lymphoid cells developed.

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29

However, when the epithelial anlage was grown in the anterior chamber of the eye of a mouse of the same strain, the epithelium became lymphoid within several days. This lymphoid development was extremely sensitive to the homotransplantation reaction, and even in the anterior eye chamber of the allogeneic mouse, differentiation of the epithelial anlage to lymphoid tissue did not occur. Auerbach then made use of an incisive technique developed by Grobstein (1956) for study of embryogenesis of other organs such as the kidney. He placed the epithelial component of the 12-day mouse thymus, separated from the mesenchymal component by tryptic digestion, in tissue culture, using a hanging drop technique; and showed that this epithelial component would develop into lymphoid tissue when brought into contact with mesenchyme. His most critical experiment was to place the epithelial anlage and mesenchymal tissue in direct apposition on the two sides of a millipore membrane in tissue culture. The membrane was 20 mp in thickness, and had pores small enough to prevent ingress or egress of ceIIs. As controls, in the same tissue culture preparation, he used an epithelial anlage isolated from mesenchymal tissue. Surprisingly, perhaps, but in keeping with the earlier contributions of Beard (1894), Bell (1906), and others, the lymphoid cells developed in the epithelial component in the tissue culture rather than in the mesenchymal component. In an extensive series of studies, Auerbach ( 1960, 19f34) then established that mesenchyme from several different sources, and even from different species, would serve to induce development of lymphoid tissue from the epithelium of the thymus anlage. In all these studies, mesenchyme was most effective as an inducer of lymphoid development if it had been taken from an area in contact with epithelial anlagen. Auerbach (1961b) postulated, as had Ruth (1960), that the thymus is a primary lymphoid organ that disseminates lymphoid cells to mesenchymal tissues, such as the spleen and lymph nodes, where their immunologic function might be exercised. The development of the remainder of the lymphoid tissue in the mouse is clearly secondary, from a temporal viewpoint, to the development of the thymus. Recent investigations, to be summarized in a later section, give further support to the thesis that the full development, both morphologically and functionally, of the secondary lymphoid tissues is dependent on thymic function. In a parallel series of studies, Ackerman and Knouff (1959, 1964) and Ackerman (1962) have presented substantial evidence that the bursa of Fabricius of chickens also develops primarily by direct transformation of an epithelial anlage in the presence of a mesenchymal contact. Early investigations by Forbes ( 1877), Stieda ( 1880), Wenckebach ( 1896),

30

ROBERT A. GOOD AND BEN W. PAPERMASTER

and Retterer and Lelidvre (1913) had led them to the conclusion that the lymphoid tissue of the bursa of Fabricius was of epithelial origin. However, Jolly (1915) and others (Boyden, 1922; Calhoun, 1933; and Kirkpatrick, 1944) concluded that mesenchymal components invaded the epithelial anlage and contributed the lymphoid development. Hammar ( 1905), Maximow ( 1909), and others ( Badertscher, 1915; Kingsbury, 1915) reached similar conclusions regarding the thymus. The best current evidence argues for direct development of these lymphoid organs from their epithelial anlagen under the inducing, and perhaps organizing, influence of the mesenchyme. In the chicken embryo, the thymus again is the first lymphoid organ to develop. The epithelial component is evident before the ninth day of incubation, and the thymus is a fully developed lymphoid organ by the twelfth day of incubation. Between the twelfth and fourteenth day, budding of the epithelial folds of the bursa of Fabricius is observed, and on the fourteenth, fifteenth, and sixteenth days the lymphoid structure begins to develop by direct transformation of epithelial cells to lymphoid cells. By the eighteenth day of incubation, the bursa has a well-organized lymphoid structure. Thus, 3 days before hatching, only the thymus and the bursa of Fabricius have identifiable lymphoid tissue (Papermaster and Good, 1962). The spleen is still entirely erythropoietic and myelopoietic; the intestine contains no follicular lymphoid tissue; and the skin is lacking in lymphoid nodules. In this species, prevention, by hormonal means, of the lymphoid development of the thymus or bursa, or both, or their surgical removal at hatching or shortly thereafter, inhibits the development of the growing chicken’s immunologic capabilities ( Click et ul., 1956; A. P. Mueller et d.,1960, 1982, 1964; Papermaster et al., 1962a,b; Warner and Szenberg, 1962, 1964; Aspin84 and Meyer, 1964; Szenberg and Warner, 1W2; Warner et ul., 1962). Studying the maturation of the lymphoid tissue of the chicken thymus and the bursa, using the Coulter Counter technique employed by Ball (1963) and Auerbach ( 1964) to estimate the number and size of the free lymphoid cells in the mouse thymus, Peterson and Good (1964) found a very different maturation of the lymphoid cells in the bursa and thymus of the chicken. By the time of hatching, the thymic cells had matured to a population with the largest number of lymphoid cells having a modal volume of 70 p3. By contrast, the bursa1 population, even as late as two or three months after hatching, contained few such small cells but had a preponderance of cells of 106 p3 in volume. These clear differences in the differentiation of thymic lymphoid cells and cells of the bursa of Fabricius might account for the differences in development of potential for im-

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31

munologic function claimed to exist in the two populations by Warner and Szenberg (1964) and Szenberg and Warner (1962). Recent studies in our laboratories by Kelly (1963) and by Archer et at. (1963a,b, 1964) have been concerned with the sequential development of the lymphoid tissues in a variety of species. The studies of Archer et at. (1963a,b, 1964) demonstrate that, in the rabbit, the thymus is the first organ to become lymphoid, and the spleen, peripheral lymph nodes, and gut become lymphoid secondarily. The apparent “peripheralization” of the lymphoid structure to spleen and lymph nodes is variable about the time of birth in the rabbit, having occurred in some animals as early as the twenty-eighth day of gestation (total period of gestation about 32 days) and occurring in others as late as 1 or 2 days after birth. Neonatal thymectomy in the rabbit interferes with the lymphoid development of the spleen, lymph nodes, and some of the lymphoid tissue of the gut; however, the intestinal tonsil and the appendix apparently have a lymphoid development independent to a degree from thymus. This development occurs in close association with epithelial outpouchings-a development which shows some striking histologic parallels to bursa development in the chicken (Archer et al., 1963b). These authors have suggested that the appendix and intestinal tonsil (sacculus rotunda) may, indeed, in the rabbit be the homolog of the bursa of Fabricius. Indeed, direct evidence for participation of the appendix in development of both morphologic characteristics and functional capacity of the peripheral lymphoid tissue has recently been presented (Sutherland et al., 1964). In these studies it was found that, although neonatal thymectomy reduced the circulating lymphocyte count, depleted organized lymphatic structures in spleen and lymph nodes, and interfered with development of immunologic capacity in the rabbit, these animals seemed to recover to near normal structure and function between nine and 16 weeks after birth. Removal of the appendix also interfered with development of lymphoid structure and immunologic capacity. However, when both the thymus and appendix were removed in the neonatal period, immunologic capacity was depressed more regularly and more completely then when either organ was removed alone, depletion of lymphocyte count and organized lymphoid structure was more profound, and the deficiency thus induced persisted far longer. Paradoxically, however, it was observed (Good et al., 1964b) that rabbits subjected to neonatal thymectomy and appendectomy frequently developed Coombs positive anemia, amyloid lesions in liver, spleen, and kidney, and abundant plasma cells in their otherwise severely depleted lymphoid tissue, These observations suggest that the thymus and appendix (central lymphoid organs) possess mecha-

32

ROBERT A. GOOD AND BEN W. PAPERMASTER

nisms for controlling the development of the peripheral lymphoid tissues, not only of great importance in its full functional development, but possibly also important in managing the processes of self-recognition. In the mouse, hamster, and, to a lesser extent, in the rat, neonatal thymectomy has a profound effect on development of immunologic capacity (J. F. A. P. Miller, 1961; Good et al., 196213; Arnason et al., 1962a; Jankovib et al., 1962; Waksman et al., 1962; J. D. Sherman et al., 1963; Roosa et al., 1963; Defendi et al., 1964). In each of these species, Archer et al. (1963a, 1964) found the thymus to be the primary lymphoid organ at birth. In the dog (Kelly, 1963) the developmental sequence is similar: the thymus is lymphoid first, followed in turn by central lymph nodes, spleen, gut, and peripheral nodes; however, the relationship of this development to birth is different. The central lymph nodes (cervical nodes) of the dog develop lymphoid structure between the fortieth and forty-eighth day of gestation and the spleen usually by the fifty-fourth day; all the lymphoid organs have quite well developed structure at birth. In this species, where peripheralization of the lymphoid tissue is highly developed at the time of birth, neonatal thymectomy has little effect on the ultimate development of immunologic capacity. Table I1 presents a summary of the state of development of the lymphoid tissue at birth or hatching in several species, in relation to the effectiveness of neonatal thymectomy in inhibiting their ultimate development of immunologic capacity. Although the most detailed studies of lymphoid development in the opossum have not yet been published (Block, 1964), the data available thus far support the conclusion of the foregoing studies regarding the earliest lymphoid development in the thymus. Kalmutz (1962) demonstrated an antibody response to phage administered 11 days after birth, when only the thymus and some of the lymph nodes show lymphoid development. In the experiments of La Via et al. (1963) a similar correlation of the beginnings of immunologic reactivity and earliest lymphoid development in the thymus and some lymph nodes was observed. Another aspect of the development of the lymphoid tissues which warrants discussion in the context of development of immunologic capacity was emphasized in the studies of Bridges et al. (1959). In most species, whatever the state of peripheralization of the lymphoid tissues at birth, the lymphoid development proceeds in the postnatal period. In man, this maturation is rapid during the first several months of life, slows down to some extent, but certainly continues until puberty (Wischnewezkaja, 1932). Many investigators have shown ( Gundobin, 1906; Barnes, 1909; Nagoya, 1913; Hellman, 1921; Foerster, 1923; Wetzel, 1926;

ONTOGENY AND PHYLOGENY OF ADAPTIVE IMMUNITY

33

W. Ehrich, 1929;Denz, 1947;Gyllensten, 1950; Bridges et al., 1959) that germinal centers, both primary and secondary follicles, are largely a postnatal development in many mammalian species. Plasma cells, the cells responsible for antibody synthesis, are generally not present at birth (D. J. Davis, 1912; Sturgeon, 1951; Glaser et al., 1950; Bridges et al., 1959),except perhaps in some guinea pigs (Gyllensten, 1950),but develop as a consequence of antigenic stimulation after birth. Bridges et al. (1959)showed that, in man as well as in the rabbit, plasma cells develop in the gut, bone marrow, and other tissues in the postnatal period, and that their formation can be accelerated by antigenic stimulaTABLE I1 RELATION OF DEVELOPMENTAL STATUSOF LYMPHOID TISSUE AT BIRTH TO EFFECTIVENESS OF NEONATAL THYMECTOMY IN LIMITING IMMUNOLOGIC DEVELOPMENT

Animal Rabbit Mouse Rat Hamster Chicken5

Dog Cat

Thymus

Spleen

Gut -

Node -

++++ + o r -- - ++++ ++++ + o r -- -- -++++ ++++ ++++ ++++ ++++ ++ ++ ++++ ++++ ++ ++

of immunologic capacity J,

But variable

J, J, J,

J,

Homograft

J/ Antibody

No cffect Not known

5 The first row represents the chicken thymus, and the second the bursa of Fabricius (From Archer et al., 196313).

tion. Nonetheless, a considerable lethargy, with respect to both plasma cell formation and antibody response to soluble protein antigens and certain bacterial antigens, was observed in both the human newborn and the neonatal rabbit. There now seems to be no question that sufficiently intense antigenic stimulation will induce both plasma cell proliferation and antibody production even prior to birth in mammals. Grbgoire (1945) reported that injection of antigens would induce late embryonic and neonatal guinea pigs to develop basophilic lymphocytes as well as germinal centers. In man, in intrauterine infections with several different organisms, plasma cells are often found, and Thorbecke and Benacerraf (1962)have produced plasma cells as early as a few days after birth in the appendix of rabbits stimulated with antigen in utero. Porcile (1904)found plasma cells in the liver of two syphilitic newborns, and Pund (1953) reported plasma cell infiltration in the placenta in congenital lues, as well as plasma cells in the liver of the newborn. Paige

34

ROBERT A, GOOD AND BEN W. PAPERMASTER

et al. (1942) studied three cases of toxoplasmic encephalitis and observed plasma cells in the lesions in the patients who died in the early neonatal period. In a recent investigation, Silverstein and Lukes (1962) assessed development of the lymphoid tissues and plasmacytosis in 19 fetuses, 16 that had congenital syphilis and 3 congenital toxoplasmosis. Gestational age was estimated at 29 weeks to full-term. All 19 had mature plasma cells, and most had precocious lymphoid tissue development as well. Thus, it seems clear from these studies that most mammals have at least feeble immunologic capacity in late embryonic and early neonatal life, and that antibody synthesis is attributable, at least to some extent, to the capacity for plasma cell development even before birth. There seems to be little question, however, that the characteristic morphologic responses to antigenic stimulation, namely, lymphoid follicle development and plasma cell production, are largely postnatal events. The late embryo and the neonate show considerable inertia in all three capacities, an inertia which is lost during the first few postnatal weeks in man and over a much longer period in some species, including the mouse (Makinodan and Peterson, 1962).

H. ONTOGENETIC DEVELOPMENT OF TRANSPLANTATION IMMUNITY Numerous reviews and symposia in recent years have been concerned, at least in part, with the ontogenetic development of transplantation immunity (Hagek et al., 1961, 1962; Ebert and Delanney, 1960; Brent, 1958; Chase, 1959; Billingham, 1958; Billingham et aZ., 1956; Billingham and Brent, 1959). Although Billingham (1958) has, with some vigor, made the point that maturation of transplantation immunity does not correspond temporally with birth in many species, the development of full capacity to reject homotransplants generally proceeds in the posthatching or postnatal period. On the whole, its development correlates strikingly with the maturation of antibody-producing capabilities. In his early studies of the development of transplantation immunity in the chick embryo, hlurphy (1913, 1914, 1916) showed that foreign grafts of mammalian and avian tissues, as well as a variety of tumors, are not rejected prior to the eighteenth embryonic day. These observations were confirmed and extended by numerous investigators (Stevenson, 1917a,b, 1918; Waddington and Schmidt, 1933; Sandstrom, 1932, 1936, 1940), and many rather fantastic experiments have been performed in transplanting homologous and heterologous tissues to avian embryos.

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Willier and Rawles, in 1940, discovered that neural crest cells transplanted to embryos of the same species may survive beyond hatching and even into adult life. This appears to involve a kind of tolerance, although the critical test of capacity to accept transplants during adult life was not carried out. On the other hand, Eastlick (1941) found that limbs grafted from embryonic buds of one species to embryos of another species were tolerated up to the sixth week after hatching. Ebert and Delanney (1960) have reviewed this problem, and pointed out that homografts and heterografts are accepted by embryos and are maintained for most, if not all, of the embryonic period. Many of these homotransplants and heterotransplants performed by embryologists produce chimeric conditions in which donor and host components can be distinguished by differences in cell size, chromosome number, or such somatic characteristics as pigment. Early studies by Danforth (1929) and Danforth and Foster (1929) indicate that skin grafts show prolonged survival in newly hatched chickens. This finding agrees well with the later work of Cannon et al. (1958) who not only observed prolonged survival of skin homografts in newly hatched chickens, but found that a relatively small proportion of newly hatched fowl apparently accepted skin from adult or other newly hatched chickens on a permanent basis. The ontogenesis of transplantation immunity in ducks, turkeys, and chickens has been a focus of studies by Hraba and HaSek (1956), HaSek ( 1957,1961) and HaSkovh ( 1957). Prolonged survival of skin homografts regularly occurs in newly hatched ducks, but occurs less often in newly hatched turkeys and chickens. In an excellent study, Schinckel and Ferguson (1953) carried out skin autografts and homografts in fetal lambs between the gestational ages of 80 and 117 days. Using histologic criteria and the second-set homograft response after birth, it was well established that homografts are actively rejected by the fetus in this species. Even grafts taken from the mother were rejected by the fetus, indicating that the rejection was a consequence of immunologic response of the fetus and not the ewe. Studies of in utero transplantation in rabbits, carried out by Egdahl ( 1957), suggested the presence of vigorous homograft immunity even before birth in this species, a finding given further support by the later observation of K. A. Porter (1960) that it is difficult to induce tolerance in rabbits by injection of spleen cells during the last week or so of gestation. By contrast, cells administered prior to the twenty-second gestational day regularly produce a tolerant state in rabbits. In an interesting recent study by IvAnyi and IvAnyi (1961) skin grafts

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ROBERT A. GOOD AND BEN W. PAPERMASTER

exchanged between newborn rabbit siblings had a longer period of survival than those exchanged between unrelated rabbit newborns; some of the grafts between siblings even took permanently. All grafts between unrelated animals were rejected within 20 days, with an average survival of 10.7 days, while 55% of the grafts between siblings survived for more than 20 days, 44% for more than 30 days, 17% for more than 70 days, and 5% for 210 days or more. IvPnyi and IvPnyi concluded that the homograft response of the rabbit is weak enough in the neonatal period to be affected significantly by the diminished antigenic disparity between siblings as compared to unrelated animals. T. N. and S. Harris (1960) also found homograft immunity to be present in newborn rabbits. However, whereas the homograft response to lymphoid cells could be demonstrated as circulating antibody in adult animals, only “cellular immunity” was present in the newborns. Rawles (1955), and subsequently Medawar and Woodruff (1958) showed that newborn rats certainly have a deficient homograft response, and the latter investigations have even demonstrated production of tolerance by skin grafts placed after birth in some rats. A well-developed capacity for homograft rejection is present in newborn piglets, at a time when antibody production, at least to certain antigens, is not demonstrable (Sterzl et al., 1960). Eakin and Harris (1945), and subsequently Hildemann and Haas (1M2), studied development of homograft immunity in bullfrog larvae. They showed that the capacity to reject homotransplants develops at about the time metamorphosis is beginning, and Hildemann found that the time of appearance of homograft immunity coincided with the appearance of small lymphocytes in the animal’s circulation. In a study of homograft immunity in human, premature and fullterm infants, Fowler et al. (1960) noted a vigorous capacity to reject skin homografts. In summary, studies of developmental biology of the homograft reaction reveal gross deficiency in early developmental stages and maturation at or close to the time of delivery or hatching in most species. The period of inadequacy tends to parallel that of the other forms of immunologic reactivity. To some degree, perhaps based on quantitative factors, the capacity to reject homografts appears to develop earlier than most immunologic phenomena. There is considerable variation from species to species. In such animals as sheep, transplantation immunity is well developed before birth. In the mouse, rat, chicken, and duck, full development of the homograft response does not take place until some time following birth or hatching. Even in the rabbit, where the ability

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to reject homotransplants is clearly present prior to birth, further development of this capacity takes place after birth, as evidenced in studies in which weaker antigenic stimulation was provided by tissue from siblings.

I. ONTOGENETIC DEVELOPMENT OF THE DELAYED ALLERGIC RESPONSE In early experimental studies, Petroff and Stewart (1926)recognized that young guinea pigs cannot be sensitized to tuberculin by vaccination with killed tuberculin bacilli as readily as can older and larger guinea pigs. This phenomenon was investigated incisively by Freund (1927, 1929). In these experiments Freund demonstrated that young, experimentally infected guinea pigs react poorly or not at all to intradermal injection of aqueous extracts of tubercle bacilli, even though tuberculous lesions were present in the regional lymph nodes and spleens. Valtis (1928)confirmed Freund’s observation, using old tuberculin to test for sensitivity. In the later studies, Freund (1929)found that tuberculous guinea pigs less than 1 month of age had a negative or at most a minimally positive reaction to intracutaneous injection of tuberculin; however, they were about as sensitive to the toxic action of old tuberculin injected intraperitoneally as were adult tuberculous guinea pigs. He concluded that the mechanism which mediates systemic hypersensitivity is fully developed in guinea pigs at less than 1 month of age, when the dermal reaction is very slight or negative. Freund found further that hypersensitivity to the intracutaneous injection of tuberculin is not transmitted from tuberculous female to offspring. The deficiency in the newborn tuberculous guinea pig, Freund concluded, is not in the mechanism of developing sensitivity, shown to be intact upon intraperitoneal injection of tuberculin, but in the anatomic or physiologic characteristics of the skin of the young animal. Several other reports in the late 1920’sserved to underscore the difference in responsiveness of the skin of human infants and young experimental animals. For example, Cooke (1927) found that the skin of newborn babies was not sensitive to the injection of a considerable amount of “scarlet fever toxin,” and showed that this lack of reactivity was not a reflection of antitoxin in the blood of the infants studied. Adelsberger (1927)studied the effect of a number of irritants on infant skin, and found blister formation to be much more easily induced in babies from 8 to 12 months of age than in those under the age of 3 months. In tests involving both human infants and rabbits, Friedberger and Heim (1929)observed less sensitivity to mustard oil and eel serum in infants and newborn rabbits than in control adults or grown rabbits. These authors also concluded from their studies that a negative Schick

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reaction in young infants does not necessarily reflect the action of antitoxin, but may merely be a reflection of the unresponsiveness of the infant's skin to irritation. Calmette and Gubrin (1929) observed that young calves infected with small doses of virulent tubercle bacilli and protected from re-infection did not develop reactions until 4 to 6 months later. Weiss (1958), on the other hand, found that guinea pigs injected with killed tuberculin organisms as long as 45 days before birth frequently showed dermal hypersensitivity when tested 8 weeks after birth. Uhr (1960) and Uhr et al. (1960) have studied development of delayed hypersensitivity in guinea pig embryos and in human neonates, both premature and fullterm. In the guinea pig studies (Uhr, 1960), the embryos were injected in utero from 1to 2 weeks before birth with diphtheria toxoid, ovalbumin, or BSA incorporated in Freunds adjuvant. When skin was tested on the day of birth, 31 out of 83 animals showed the tuberculin-type of hypersensitivity. Of particular interest was a size differential: the guinea pigs weighing more than 90 gm. at birth generally were sensitive at birth, whereas those weighing less than 90 gm. were not. Uhr concluded that delayed allergy can be induced in utero, and that larger fetuses are more susceptible to development of delayed allergy than are the smaller ones. Rees and Garbutt ( 1961) described the development of hypersensitivity in mice by prenatal introduction of tubercle bacilli, but their experiments were not critical with respect to the time of development of delayed allergy during the neonatal period. The results of Sterzl's (1959b) studies in piglets parallel those of Freund in the guinea pig: the animals, although not expressing the skin reaction of delayed hypersensitivity, could in reality develop delayed allergy as revealed by capacity to express systemic sensitivity and transfer the reaction. In 1958, Waksman and Matoltsy, while studying passive transfer of delayed allergy to tuberculin in guinea pigs, found that newborn animals did not serve well as recipients of local passive reactions. Their studies in adults had suggested that passive delayed reactions depend upon proliferation and/or differentiation of the transferred cells at the test site. Consequently, they reasoned that the failure of development of visible reactions in the newborn could reflect lack of differentiation of the transferred cells toward mature histiocytes or macrophages because of the inadequacy of the neonatal cellular environment. Warwick et d. (196Oa) studied the passive transfer of delayed hypersensitivity in the newborn rabbit. By using a streptococcal infection to develop delayed allergy in the adult donors, they observed regular

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failure of passive transfer to recipients during the first and second weeks of life, an irregular response in recipients between 2 and 3 weeks of age, and regular success at 4 weeks and older. The striking parallel with the development of antibody-producing capacity with respect to certain antigens was noted. On this basis, these investigators suggested that perhaps passive transfer of delayed allergy is not entirely passive and that newborns, as well as patients with Hodgkin’s disease, both of which are refractory to the development of delayed allergy and also resist its passive transfer (Kelly et al., 1958; Warwick et al., 1961), may lack the capacity for active participation in the “passive transfer” reaction. Salvin et al. (1962) studied the passive transfer of delayed hypersensitivity in newborn guinea pigs after sensitization of the donor with purified soluble antigens. They found that the neonatal animals did not demonstrate passively transferred delayed allergy by skin testing during the first 2 weeks of life, but developed skin reactions comparable to adults thereafter. On the other hand, tests for systemic delayed allergy in the newborn recipients of cells from sensitized donors demonstrated toxicity and pyrogenicity, interpreted as evidence of passive transfer. Newborn guinea pigs in Salvin’s studies produced antibody and showed Arthus reactivity. However, passive Arthus reactions were not produced as readily in the newborns as in adults. By contrast, contact allergy and allergic encephalitis were produced in newborn guinea pigs. This differentiates the guinea pig from the neonatal rabbit which resists production of allergic encephalomyelitis ( Waksman and Morrison, 1951). Finally, Salvin et al. (1962) showed that newborn guinea pigs which had been actively sensitized to soluble antigen had cells capable of passive transfer of delayed allergy, although they did not themselves show delayed skin hypersensitivity, Studies of the ontogeny of the delayed allergic response in man have been consistent with those reported in experimental animals. The early observations of Bernard and Debre (1929) indicated that the skin sensitivity of tuberculous infants develops slowly and that more than 3 months may elapse before skin hypersensitivity is demonstrable. These observations were confirmed by several investigators ( Amick et al., 1950; Grady and Zuelzer, 1955; S. F. Davis et al., 1960; Kendig, 1954; Kendig and Angell, 1950; Kendig and Rodgers, 1958) studying tuberculosis acquired congenitally and neonatally. Kendig ( 1954), Kendig and Angell ( 1950), and Kendig and Rodgers (1958) have described several infants born with tuberculosis in whom positive Mantoux reactions developed between the nineteenth and thirty-first days after birth. Tudvad et al. (1956) studied a large group of babies immunized in the neonatal period with

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BCG and found that virtually all the children had developed delayed allergy when tested at 2 months of age. Straus (1931) reported successful sensitization of human newborns to poison ivy. In these experiments, the infants were sensitized by topical application of poison ivy paste in the scapular region or were fed a poison ivy extract, After an interval of 2 to 4 weeks, the infants were tested and 73% showed well-developed allergic reactions. A recent study of delayed hypersensitivity in human newborns is that of Uhr et al. (1960),in which contact sensitivity was induced with 2,4dinitrofluorobenzene, These authors found that older infants were readily sensitized by percutaneous application of a vesicant dose of this chemical, but that premature and full-term newborns were sensitized only occasionally. These findings indicate that in the human newborn, delayedtype allergy, like antibody synthesis, can be induced only with difficulty and irregularly, and that the premature babies are no more deficient than the normal newborns. The data of Uhr et al. suggest further that the maturation of the capacity for delayed allergic response is completed during the first year of life. Although it is accepted that mothers do not regularly passively transfer tuberculin sensitivity to their babies, Schlange (1954) reported that tuberculin allergy could be passively transferred to human newborns by exchange transfusions from patients with positive tuberculin reactions. Warwick et d.(1960b), skeptical about the small size of the lesions reported by Schlange, restudied the question of transferability of tuberculin sensitivity to human newborns. Although exchange transfusion alone, peripheral leucocytes alone, exchange transfusion plus peripheral white blood cells, and peripheral white blood cells plus bone marrow cells from donors of known sensitivity were used, none of the neonatal recipients showed delayed reactivity. In separate and simultaneous experiments, passive transfers to immunologically mature recipients with peripheral white blood cells from sensitive donors were successful in most instances. These authors concluded that the human newborn, like the neonatal experimental animal, is defective with respect to the capacity to accept transfers of delayed hypersensitivity. In summary, it is apparent that certain difficulties, particularly focused about reactivity of the skin, make interpretation of experiments on the ontogeny of the delayed allergic response difficult. However, it seems likely that capacity for achievement and expression of delayed reactivity is present in late embryonic life in most species and that its development continues after birth. In all species studied the parallel with develop-

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ment of antibody-producing capacity is close. Present knowledge is deficient, however, and based on woefully inadequate studies from the standpoint of the number of species and kinds of antigenic stimuli investigated, as well as design of critical experiments. It seems probable, considering the late development of this form of immunologic response suggested by current data, that the delayed allergic response would be subject to manipulation and control in late fetal life or the neonatal period. J. IMMUNOLOGIC DEVELOPMENT As STUDIED IN CELL TRANSFER

SYSTEMS 1. Studies of the Development of Immunologic Capacity Employing Transfer of Cells from Adults to Embyos and Newborn Animals Since immunologic competence develops progressively in late gestational life and in the neonatal period, as outlined in the foregoing sections, it seemed likely that important contributions to understanding of the immunologic deficiency of young animals might be made by study of the immunologic function of adult cells in the neonatal environment, on the one hand, and the function of neonatal cells in an adult environment, on the other. With these techniques, many investigators have contributed to understanding of immune processes; the extensive recent literature was recently ably reviewed by Cochrane and Dixon (1962) and need not concern us here. The studies attempting to utilize these transfer techniques to analyze the ontogenetic development of immunologic competence have, unfortunately, been contradictory. The earliest experiments addressing this problem, using rabbits, were presented by Dixon and Weigle (1957). Indeed, their findings appeared to provide a rational explanation for the deficiency of antibody production in neonatal rabbits. They found that sensitized lymphoid cells from adult donors failed to produce antibody in newborn recipients. The transferred cells appeared to survive, but did not undergo maturation toward plasma cells in the milieu provided by the neonatal rabbit. By contrast, after similar transfer of stimulated adult lymphoid cells to irradiated adult recipients, maturation to plasma cells occurred, and antibody was produced and released (Dixon and Weigle, 1957). These authors concluded that neonatal rabbits do not provide an adequate environment for synthesis of antibody by adult cells. These early studies suggested further that, once under way, synthesis of antibody by transferred adult cells could proceed quite well in the newborn rabbit. Early experiments based on transfer

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of stimulated neonatal rabbit cells to adult rabbit recipients indicated that, in many instances, the neonatal cells functioned quite well in the adult environment ( Dixon and Weigle, 1959). Although studies by Nossal (1959a), using neonatal rats and Hall Institute mice, an outbred stock, seemed to support the observations of Dixon and Weigle ( 1957), neonatal C3H mice, a highly inbred line, appeared to support antibody production by syngeneic adult cells quite well. In the latter situation, the role of homograft immunity was eliminated. Observations by Sparck (1959) were consonant with the findings of Dkon’s group. Here, too, rabbits were employed, and its was found that cells stimulated 4-96 hours prior to transfer could produce antibody in the neonatal rabbit. Cells stimulated a shorter or longer time prior to transfer functioned poorly in the neonatal environment. Sparck concluded that the prime difEculty in the newborn was maintaining the adult cells in the neonatal environment long enough to get antibody synthesis started. Even in the earliest studies of this problem, Sterzl (1954) concluded that neonatal rabbits will support antibody synthesis by adult cells. He even presented evidence that nucleic acids derived from adult lymphoid cells might enable neonatal rabbits to respond more vigorously to antigenic stimulation than untreated animals (Sterzl and Hrubesova, 1956). Then Trnka ( 1958), studying antibody synthesis by isolated hen spleen cells injected into young chicks, concluded that extremely young animals, although feeble in their own immune response, will support antibody synthesis by adult mesenchymal cells. Sterzl ( 1958a,b) reconsidered the problem in the light of the data presented by Dixon and Weigle (1957), and suggested that the deficiency of the environment of the neonatal rabbit was probably a reflection of rejection of the transplanted cells by the neonate before they could make an adequate immune response. As evidence, he showed that adult lymphoid cells transferred to neonatal recipients could respond to delayed injections of antigen for only 3 to 4 days after transfer (Sterzl, 1958b). Sterzl (1958a,b, 1959a) and Sterzl and Rychlikova (1958) maintained that neonatal rabbits, whatever the immunologic deficiency characteristic of their age, had the capacity to exercise a homograft reaction. These findings were in keeping with the observation of Egdahl (1957) that skin transplants placed on rabbits several days before birth may already be in the process of rejection at birth. This problem was then approached by Sibal and Olson (1958) who

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studied the capacity of adult chicken spleen cells inoculated onto the chorioallantoic membrane (CAM) of the chick embryo. These investigators found that injection of cells on the CAM with BSA, or injection of cells on the CAM following exposure to BSA, resulted in no antibody formation. By contrast, spleen cells taken from adult animals and stimulated with BSA 36-48 hours before injection on the CAM produced antibody. Spleen cells taken 7 days after stimulation with antigen again produced no antibody when injected on the CAM. Here again, there seemed to be evidence that ongoing synthesis of antibody might be supported by the embryonic environment but that initiation of antibody synthesis in this environment is difficult. Trnka and Riha (1959), in similar studies, showed that adult chicken spleen cells stimulated in vitro with either bacterial or protein antigens produced excellent antibody responses when injected onto the CAM of the developing chick embryo. The antibody response in the 18-day chick embryo was much greater than that produced by adult cells similarly stimulated and administered to 2-day-old chicks. These investigators concluded that a primary antibody response by adult cells can be made to proceed in embryos, and vigorously supported the concept of Sterzl (1958a,b) that the only deficiency of the neonatal milieu is the homograft reaction of which many neonatal animals are capable. Returning to studies in neonatal rabbits, Holub and Riha (1960) transferred adult rabbit spleen cells to newborns, but placed the cells in millipore chambers to protect them from the homograft reaction of the neonatal recipient. In these experiments they obtained primary responses of the adult cells to both bacterial and soluble protein antigens; however, the evidence for the primary response was not good, the antibody appearing very late and only in chamber fluids. In further studies in the chicken, Sterzl and Trnka (1959a) carried out experiments in 2-day-old chicks comparable to those carried out earlier in rabbits by Sterzl (1958b). They found that adult chicken spleen cells remain responsive in the newly hatched chick for a very short time, even shorter than that of adult rabbit cells in the newborn rabbit. Dixon and Weigle (1959) reasoned that if the homograft reaction were to account for the inadequacy of the neonatal rabbit in supporting production of antibody by transferred adult cells, the neonatal rabbit must be more vigorous in exercising homograft rejection than is the adult. The studies of Najarian and Dixon (1962)seem to indicate that this may, indeed, be the case. In their comparison of rejection time for skin homografts in neonates and adults, they showed the neonates rejected the skin

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in 8-7 days, and the adults in 8-9 days. By the fourth day there was good microscopic evidence of rejection in the neonate. Barnett and Grey (1962)found that irradiation of neonatal rabbits interfered with homograft rejection and fostered antibody synthesis by transferred adult cells in the neonatal environment. Grey (1962) reported, however, that antibody formed by adult cells in the neonatal environment was qualitatively different from that formed by adult cells transferred to other adults. Mark and Dixon (1963) compared antibody synthesis in syngeneic and allogeneic neonatal mice. Using soluble protein antigens as the stimulus, they showed clearly that syngeneic newborn mice will foster immunologic function of transferred adult spleen cells although not to as complete a degree as adults, whereas allogeneic neonatal mice will not do so. Studies by T. N. Harris et al. (1962)also indicated that the neonatal rabbit supports antibody synthesis by transferred adult cells but to a lesser degree than adult recipients. They concluded that either the homograft reaction of the neonate is better than that of the adult, or the transferred cells were able to get an antibody response under way faster in the adult than in the newborn. Harris et a2. also found that, when transfers were made to the neonate, a greater antigenic stimulus to the adult cells was required to invoke an immune response. In further studies, S. Harris et al. (1962) compared the homograft reaction to injected leucocytes in newborn and adult rabbits. They showed that administration of leucocytes in the neonatal period produces immunization resulting in rejection of donor leucocytes and inhibition of formation of antibody by transferred cells, It was of significance, however, that circulating antibody did not seem to be involved in this response of the neonate, in contrast to the role of antibody in adult animals similarly stimulated. Thus, serum from rabbits given spleen cells in adult life readily transferred immunity to spleen cells, but such transfer was not possible with serum from animals injected with leucocytes in the neonatal period. Cellular transfer was effective, however. We conclude, then, from this group of studies that the neonatal tissues will support antibody production by adult cells, probably somewhat less efficiently than the adult environment, but that the capacity for homograft rejection, often quite well developed in newly hatched or newly born animals, is of sufficient vigor to interfere with immunologic function of allogeneic adult lymphoid cells. Consequently, studies employing transfers of functioning lymphoid cells to neonates should take this reaction into account.

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2. Anulysis of Ontogenesis of Immunity by Studies of Transfer of Neonatal Cells to Adult Recipients In the early work of Dixon and Weigle (1959), as cited above, lymphoid cells from neonatal donors, stimulated with antigen in uitro and injected into adults, produced a respectable immune response in many instances. By using neonatal cells stimulated with bacterial antigens before transfer to adult rabbits, S. Harris et aZ. (1962) also observed an antibody response, but a much less vigorous one than that formed by adult cells under the same circumstances. Indeed, in one study (S. Harris et al., 1961) it was reported that the stimulated neonatal rabbit cells were not capable of significant antibody synthesis upon injection into an adult environment until the donor rabbits had reached 1 month of age. Makinodan and Peterson (1962) studied antibody formation by transferred spleen cells in mice as a function of age of the donor. Using an in uiuo culture model presented by 12-week-old syngeneic mice treated with 800 r total body irradiation, they compared antibody formation of spleen cells of donor mice between the ages of l week and 29 months. In these studies they were able to demonstrate a gross deficiency of lymphoid cells from mice sacrificed at 1 week of age. Indeed, at this age the amount of antibody produced was approximately 1% of the levels reached at immunologic maturity. They found that the immunologic capacity of these cells increased rapidly between 1 week and 1 month of age and that it continued to increase until the donors were 8 months old. Following this peak, immunologic capacity decreased to the end of the observation period, 29 months. Although the most rapid development of immunologic capacity occurred during the first month of life, it is of interest that, in this test system, the spleen cells of %monthold mice were only capable of one-third the response of cells of 8-monthold donors. In a recent review of the earIier observations from his laboratory, coupled with a reassessment of the problem, Sterzl (1963) has presented observations, now confirmed in many quarters (Mitchison, 1957; Papermaster et al., 1959, 1962a; Nossal, 1959a; T. N. Harris et al., 1959, 1962), that neonatal or newly hatched animals can support antibody synthesis well enough to permit study of the problem. With this model, he found that, in rabbits, the peripheral lymphoid cells are deficient immunologically during the first few weeks of life, as revealed by transfer studies employing newborn rabbits as recipients. By the time donors are 30 days of age, the lymphoid cells of rabbits are capable of vigorous immunologic

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response even when placed, following stimulation, in the neonatal environment. We would conclude from these observations that much more extensive study, using appropriate cell transfer systems, especially those employing irradiated inbred animals, might clarify the controversy which exists and, more importantly, supply critical information on the development of the immunologic capacity of the lymphoid cells through life. The limited data now available demonstrate that, in one strain of mice at least, the immunologic capabilities of the peripheral lymphoid tissues and the lymphoid cells may increase for a very long period of time-far beyond the neonatal period. Recent studies in mice by Dalmasso et al. ( 1963,1964),employing the Simonsen graft-versus-host reaction ( a homotransplant reaction) as an assay system (Cock and Simonsen, 1958), seem to support these observations of Makinodan and Peterson (1962). In this system, too, development of immunologic capacity of the peripheral lymphoid tissue was shown to proceed far beyond the immediate neonatal period. The development of the peripheral lymphoid tissues was shown by the study of Dalmasso et al. (1963) to be dependent upon the thymus. Removal of the thymus in the neonatal period or at 6, 14, 25, or 35 days of age appeared to arrest the development of the lymphoid tissue at the stage reached when thymectomy was carried out. The rabbit, too, appears to show at least some degree of development of the immunologic capacity of its peripheral lymphoid cells during the early weeks of life. Whether the development of the immunologic capabilities of peripheral lymphoid cells in early life is primarily a result of increased cellularity of the peripheral lymphoid tissues or of maturation of the capacity of the individual cells themselves needs to be studied critically.

K. IMMUNOLOGIC TOLERANCE IN RELATIONTO DEVELOPMENT OF FULL IMMUNOLOGIC CAPACITY An additional way to view the ontogeny of immunity is to consider it in terms of susceptibility to production of specific immunologic negativity. This phenomenon, commonly referred to as immunologic tolerance, had its origin in the observations of Owen (1945) and Owen et al. (1946) that fraternal twin cattle often have two blood types-their own and that of their fraternal twin. From this most provocative experiment of nature, Owen reasoned that fraternal twin cattle, frequently synchorial (Lillie, 1917) had exchanged their unique blood cell precursors during fetal life, and that each had accepted these precursors as functioning homovital grafts which produced red blood cells for a prolonged period

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in postnatal life. D. Anderson et at. (1951) and Billingham et al. (1952) showed further that these chimeric fraternal twins will accept skin grafts from one another, although they are fully capable of rejecting grafts from both parents and from siblings of separate birth. Reasoning from the observations of Owen (1945) and Owen et al. (1946), and from the finding of Traub (1935a,b, 1936a,b,c, 1938, 1939) that intrauterine infection with lymphocytic choriomeningitis virus renders mice incapable of later immunologic response to the virus, Bumet and Fenner (1949) postulated that antigen administered during embryonic life might thereafter be considered as “self” and thus incapable of giving rise to a positive immune response in such a conditioned host. The initial experimental attempts to test this hypothesis in the chicken embryo yielded negative results (Bumet et al., 1950). In 1953, Billingham et al. showed, in their classical experiment, that immunologic tolerance of homografts in inbred mice can be produced by injection of cells from the prospective donor strain into the prospective recipients during late fetal life. During recent years, the phenomenon of immunologic tolerance has been studied most extensively and has been the subject of a number of most provocative reviews (Billingham and Brent, 1959; Billingham et al., 1956; Brent, 1958; Chase, 1959; Lawrence, 1959; Woodruff, 1960; Mitchison, 1961; Haiek et al., 1961). Indeed, an entire volume devoted to expression of the present state of knowledge of immunologic tolerance has recently been published (HaXek et al., 1962). Tolerance, or as we prefer to consider the phenomenon “specific immunologic negativity,” can be produced not only with living cells of prospective donors of tissue homografts, but by simple defined protein antigens (Cinader and Dubert, 1955, 1956; Dixon and Maurer, 1955; Hanan and Oyama, 1954; Wolfe et al., 1957; R. T. Smith and Bridges, 1958; Tempelis et al., 1958; Terres and Hughes, 1959; Dresser, 1961); bacteria and bacterial products (Felton and Ottinger, 1942; Felton, 1949; Buxton, 1954; Felton et al., 1955a,b; Friedman and Gaby, 1960; Gowland and Oakley, 1960; Weiss and Main, 1962); viruses (R. J. C. Harris and Simons, 1958; Hotchin and Weigand, 1961); and parasites (Kerr and Robertson, 1954). This phenomenon has been produced in many different species and certainly occurs in man as well (Woodruff, 1957; Woodruff and Lennox, 1959; Good, 1957b, 1960; Fowler et al., 1980; Albert et al., 1959; Lejeune, 1962). Although immunologic tolerance at first seemed to be interpretable in terms of Burnet’s postulate, further study has indicated that susceptibility to tolerance induction varies greatly from species to species (Woodruff

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and Simpson, 1955) and from strain to strain (Billingham and Brent, 1957; Martinez et al., 1958), and that it is also, in part, a function of the nature and quantity of the antigen (reviewed by R. T. Smith, 1961 and Halek d al., 1961). Initially, a “tolerance responsive period” was proposed (Billingham et al., 1956; Billingham and Brent, 1959). However, it became clear, particularly from work with defined antigenic systems (R. T. Smith and Bridges, 1958), that production of tolerance is, at least to some degree, dependent on dosage of antigen administered over a rather extended period around the time of birth. Further studies by Dixon and Maurer ( 1955), by Johnson et al. ( 1955), by our own group with a variety of systems (Condie et al., 1957; Aust et al., 1957, 1960; Mariani et al.,1959, 1960a,b; Martinez et al., 1959, 1960a,b, 1963a,b; Shapiro et al.,1961; LaPlante et al.,1962; Forsen and Condie, 1963), and by Simonsen ( 1960) indicated that specific immunologic negativity, tolerance if you will, can be produced not only in the newborn but through adult life. This concept and its experimental support, deriving both from work in our own laboratory and that of others, using techniques of persistent antigen overloading (Condie et al., 1957; Mariani et al., 1959, 1960a,b; Shapiro et al.,1961; Brent and Gowland, 1962; Wigzell, 1962; Martinez et al., 1963a,b; HaBek, 1963), parabiosis (B. A. Rubin, 1959; Mariani et al., 1959, 1960a,b; Martinez, 1960a,b; Naki6 and SilobrEi6, 1958; Naki6 et al., 1960; Skowron-Cendrzak and Konieczna-Marczynska, 1959; Edgerton and Morrell, 1961; Hardin and Werder, 1961; E. Jensen and Simonsen, 1962), or administration of antigen by unusual routes (Battisto and Miller, 1962) or in special form (Dresser, 1962), now establish beyond reasonable doubt that a state entirely comparable to classical immunologic tolerance can be produced during adult life in several species. Indeed, the long-lasting tolerance produced by neonatal allogeneic cell injection in mice may be a function of tolerance of host antigens induced in the injected adult cells (Martinez et al., 1962d). The “tolerance responsive period has proved to be a variable one, depending upon the tolerance-inducing stimulus, an observation quite compatible with findings indicating that the vigor of the immunologic response develops gradually in late gestation and in early neonatal period in certain mammalian species and that its evocation in the early period depends in large measure on the nature of the stimulus. Viewing the phenomenon of immunologic tolerance in terms of the development of the lymphoreticular system and immunologic responsiveness, the most reasonable thesis seems to be that specific immunologic negativity is not a phenomenon peculiar to embryologic development or

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neonatal life, as was originally postulated, but a phenomenon that can be produced at any time during life, and is inversely related to the state of development of the lymphoid tissue (see Section 11, G of this review, and Archer et al., 1964) and directly related to the dose of antigen administered (R. T. Smith and Bridges, 1958; Mariani et d., 1959). It is also, in a manner as yet unknown, a function of the quality of the antigenic material employed (Dresser, 1962) and possibly even a function of the route of administration of the antigen or hapten (Battisto and Miller, 1962). A corollary is that the relative ease of induction of specific immunologic negativity during embryonic life and the neonatal period is a reflection of the relatively poor development of the lymphoreticular mass at this stage of life and the consequent inadequacy of the positive adaptation to antigenic stimulation, i.e., antibody production and graft rejection. Thus, it is attractive to consider immunologic tolerance as an example of a general phenomenon, specific immunologic negativity, which must ultimately be based on the same mechanisms as those responsible for Felton’s immunologic paralysis (Felton and Ottinger, 1942; Felton, 1949; Felton et al., 1955a,b), now known to be a central failure of immune response (Sercarz and Coons, 1959; Gitlin et al., 1958), and the immunologic failure (Chase, 1946) produced by feeding haptenic materials to guinea pigs. Predictable from this view as well would be the observations which establish that manipulations such as X-irradiation (Main and Prehn, 1955; Trentin, 1956) and treatment with 6-mercaptopurine (Sclbwartz and Dameshek, 1959; LaPlante et al., 1962; Forsen and Condie, 1963) facilitate production of specific negativity during adult life by bringing about a regression toward the fetal or neonatal state of relative inadequacy of the lymphoreticular system of cells and consequent inadequacy of positive immune response. In summary, then, it is relatively easy during gestation and early postnatal life to overload the lymphoreticular system with antigen and produce tolerance. With further development of the peripheral lymphoid tissue and the growing capacity for positive immunologic adaptation by t h i s system of cells, it becomes more difficult-but not impossible-to produce a specific negative adjustment to antigenic exposure.

L. ROLEOF

THE THYMUS AND BURSAOF FABRICIUS IN ONTOGENESIS ADAPTIVEIMMUNITY Until a few years ago two of the most enigmatic organs in experimental biology were the bursa of Fabricius of birds and the mammalian thymus. Studies of the bursa had suggested that it participated in some endocrinologic relationships ( reviewed by Glick, 1964); similar sugges-

OF

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tions had been made for the thymus on the basis of experimental findings (Gyllensten, 1953; Ehrich and Seifter, 1948) and clinical links to thyroid disease and conditions involving adrenal-pituitary dysfunction (reviewed by Fisher, 1964). The mouse thymus was known to be the site of development of many leukemias, and in 1944 it had been shown that removal of the thymus at the appropriate time sharply reduced the incidence of such leukemias ( McEndy et al., 1944). An increasing number of studies had linked the mammalian thymus to lymphocytopoiesis: Metcalf ( 1956) had prepared a lymphocytosis-stimulating factor from thymus which was an effective stimulus to lymphocyte production in baby mice, and Schooley and Kelly ( 1964), Bierring ( 1960), Metcalf (1960), Yoffey (1957)) and others had demonstrated that thymectomy of adult animals depressed lymphocyte levels in thoracic duct lymph and circulating blood, and reduced the weight of peripheral lymphoid organs. Fichtelius (1953, 1958), in a series of experiments with PS2 labeling, had suggested that thymocytes were involved in immunologic processes. The thesis of an immunologic function of the thymus was not new; this had been advanced repeatedly by such investigators as Hammar (1938) and T. N. Harris et al. (1948), but their extirpation experiments in young or adult animals showed no significant effect on antibody production. Two developments in the early 1950’s set the stage for a renewed experimental attack on the problem of the thymus. The first of these was the finding, in 1953, that a patient with acquired agammaglobulinemia had developed a thymoma at about the time of onset of his clinical disease (Good, 1954; Good and Varco, 1955; MacLean et al., 1956, 1957). This was considered by Good (1954) to be a highly provocative linking of two very rare conditions, and it prompted further pursuit of a link between the thymus and immunologic function. In experiments involving thymectomy of young rabbits and subsequent stimulation with BSA, no effect could be demonstrated ( MacLean et al., 1958, 1957). The second development occurred in 1954, when the bursa of Fabricius was linked to antibody production in the chicken by the serendipitous discovery of Glick and associates (Glick et al., 1956; Glick, 1958; Chang et al., 1957, 1958). Their studies established that when the bursa was excised during the first 2 weeks after hatching, the birds were deficient antibody producers in adult life. At the University of Wisconsin, a second group of investigators was engaged in endocrinologic studies which ultimately led to the first experiments on neonatal thymectomy in mammals. Meyer et al. (1959) had been studying the effects of administration of various hormones during egg incubation on the development of the bursa of Fabricius. They had found that with appropriate dosage

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of 19-nortestosterone before the lymphoid development of the bursa began, they could arrest this development. A. P. Mueller et d. (1960) studied the antibody responses of chickens treated with 19-nortestosterone on the fifth day of incubation, and found that their antibody response was even more severely affected than that of surgically bursectomized birds. These findings were confirmed by Papermaster et al. (1962a,b), who also showed that hormonal bursectomy of the host greatly enhanced the graft-versus-host activity of injected adult homologous cells, and permitted allogeneic adult spleen cells to function more effectively in antibody synthesis in hormonally bursectomized recipients than in controls. The resemblance of the bursa to the thymus has been noted repeatedly; the bursa has been called the cloaca1 thymus. The studies of Glick and of the Wisconsin group showed clearly that antibody production is affected only if the bursa is removed early in life, and A. P. Mueller et al. (1960) suggested that neonatal thymectomy might reveal a parallel in the mammal. Three entirely independent groups of experiments showed that neonatal thymectomy has a significant effect on immunologic reactivity: ( 1 )the studies by Fichtelius et al. (1961) in young guinea pigs; ( 2 ) the experiments of Archer, Martinez, Good, Papermaster, and associates in rabbits (Archer and Pierce, 1961; Archer et al., 1962; Good et al., 1962b) and mice (Good, 1961; Martinez et al., 1962a,b; Dalmasso et al., 1962b; Good et al., 196213); and ( 3 ) the studies by J. F. A. P. Miller (1961, 1962a,b, 1963, 1964). In rabbits, the effect of neonatal thymectomy on antibody production is variable both from animal to animal and antigen to antigen (Archer et al., 1962); in guinea pigs, relatively mature animals at birth, the depression of antibody response is slight but significant (Fichtelius et al., 1961); but in the mouse transplantation immunity is sufficiently affected by neonatal thymectomy to permit skin transplants across the H-2 histocompatibility barrier and even across species barriers in some instances, and antibody production to certain antigens is almost entirely eliminated (Martinez et al., 1962a,b; Papermaster et al., 1962d; J. F. A. P. Miller, 1962a,b). Parrott and East (1964) have shown clearly that the effect on antibody production is a quantitative one, and that with such potent antigens as hemocyanin and pneumococcal polysaccharides, many of the neonatally thymectomized mice produced significant amounts of antibody. As a matter of fact with these antigens antibody synthesis was almost as good as in control mice. M. Hess et al. (1963) used tetanus toxoid in recent studies; the effects of neonatal thymectomy were noted in the primary response, but were much more evident in the secondary response.

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Neonatally thymectomized mice are particularly vulnerable to homologous disease when injected with parent strain lymphoid cells (Parrott, 1962; Martinez et al., 1962~).Their lymphoid tissues show minimal lymphoid development and their circulating lymphocyte levels are greatly reduced, although most strains have adequate levels of y-globulin and appreciable, if not entirely normal, numbers of plasma cells. The cells of spleen and lymph nodes of neonatally thymectomized mice are very low in immunologic activity, evident when cell suspensions of these tissues are injected into F1 hybrid recipients; splenomegaly and other manifestations of graft-versus-host reactivity are not evident unless the dosage of cells is multiplied several times (Dalmasso et al., 1962a, 1963, 1964; J. F. A. P. Miller, 1964). The experimental evidence to date is consistent with the thesis that the mouse has one primary central lymphoid organ, the thymus, and that this is a key source of cells or humoral substances, or both, necessary to normal maturation of the peripheral lymphoid tissues and to normal development of immunologic capabilities. As indicated in the section on the ontogeny of the lymphoid tissues, the newborn mouse has no detectable organized lymphoid cells in spleen or lymph nodes; however, the fact that the neonatally thymectomized mouse retains a small measure of immunologic reactivity suggests that the thymus' influence on immunologic development may already have been exercised to some extent before birth. As will be indicated shortly, studies on the effect of thymectomy in young but not newborn mice indicates that, in this species, birth is a point on a continuum of thymus-dependent immunologic development, a continuum which extends back to the embryonic period and extends well into young adult life. Thus, in the mouse, the effect of thymectomy varies with both the time of thymectomy and the nature of the challenge to the animal's immunologic capabilities. This is well illustrated in the skin homografting and immunologic runting models. In early studies, Martinez et al. (1962a) demonstrated not only the profound effects of neonatal thymectomy on transplantation immunity but also the effects of thymectomy as late as 30 days of age on the response to skin grafts when donor and host differed at certain non-H-2 histocompatibility loci. This was true when Ce skin was grafted on C,H recipients, for example. When tumor tissue was used as the transplant, DBA/2 mice thymectomized as late as 40 days of age accepted a Balb/C multiple myeloma tumor that was rejected by sham-operated animals of the same age (Martinez et al., 1964). Thymectomy at 40 days of age also affected the susceptibility of certain F1 hybrids to runt disease following injection of parental strain spleen cells (Martinez et d.,1962c; Good et al., 1962b).

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A series of studies by Dalmasso et d.(1983,1964) demonstrates graphically that the thymic contribution to the development of immunologic reactivity in the mouse continues beyond 35 days of age, and that the degree of immunologic capability is correlated with age at thymectomy. They demonstrate, too, that animals thymectomized during this early period suffer a kind of immunologic arrest: their immunologic status remains roughly at the stage of development it had reached by the time of thymectomy, and they neither “catch up” to animals thymectomized at later ages or normal animals, nor show significant regression. The model used was the spleen assay of graft-versus-host reaction in Fl hybrid recipients of spleen cells from parent strain animals that had been thymectomized at 1, 6, 14, 25, or 35 days of age, and sacrificed 2 or 6 months later. For comparison, animals of the same hybrid strain received parental strain cells from animals sacrificed at 14 and 35 days of age, and at 2-3 months. Some recipients of cells from donors thymectomized at 14 days of age showed slight splenomegaly. Those given cells from the 25-day group had a higher incidence of graft-versus-host response and had larger spleens. Recipients of cells from parent strain donors thymectomized at 35 days all showed splenomegaly, again with an appreciable increase in the average spleen weight over the %day group; the activity of these cells was still much less than that of cells from normal 2-3-month-old donors. When the graft-versus-host activity of cells from normal 14- and 35-day-old donors is compared with that of cells from 2%- to 3-month-old donors thymectomized at 14 and 35 days, the parallel is striking. Speculation on the reason for immunologic failure following neonatal thymectomy has centered on the thymus as a source of cells or humoral factors essential to normal lymphoid development and immunologic maturation. Although until recently the evidence for egress of lymphoid cells from the thymus had been indirect, a recent study by Nossal and Gorrie (1964) provides direct evidence that such emigration does occur. They labeled thymic cells directly in situ with tritiated thymidine without labeling other lymphoid organs, and showed that small numbers of labeled cells gradually appeared in the mesenteric lymph nodes. However, the quantity of cells distributed from the thymus is very small in adult guinea pigs, and only 5 to 10 times as great in the neonatal guinea pig (Nossal, 1964). In light of the extensive proliferation that characterizes the thymus, it is clear that many cells must both arise in the thymus and be destroyed there. The recent work of J. E. Harris et al. (1964) seems to establish that bone marrow lymphocytes enter the thymus and may ultimately leave the organ. Sainte-Marie and Leblond (1964) have presented substantial evidence that thymus cells gain the peripheral

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blood. Other evidence of dispersion of thymic cells to peripheral lymphoid tissues is found in the studies of J. F. A. P. Miller (1962a) and of Dalmasso et at. (1963, 1984). Miller thymectomized newborn (Ak x T6)F1mice, and 2 days later grafted them subcutaneously with homologous thymus tissue; he showed, using the T6 marker, that cells of the thymus donor strain appeared in the spleen, although they were in all instances in the minority. Dalmasso et al. (1963, 1964) have reported similar findings, using the graft-versus-host capabilities of the peripheral lymphoid cells of the thymus-grafted animals in appropriate F1hosts as the criterion of the relative proportions of donor and host cells in the chimera. Both donor and host type cells were found in the spleen, but the host cells predominated. Reconstitution of immunologic capacity of neonatally thymectomized mice has been approached by J. F. A. P. Miller (1962a, 1983, 1984), by Parrott and East (1964), and by Dalmasso et al. (1963), with some variability in results. J. F. A. P. Miller (1982a) has reconstituted immunologic capacity of neonatally thymectomized mice by subcutaneous grafting of homologous thymus tissue from newborn donors during the first 3 weeks of life. He also restored 10-week-old neonatally thymectomized mice bearing skin grafts with isologous lymphoid cells from 8week-old donors presensitized against the strain of the graft donor. Restoration is defined in terms of a second-set homograft response to skin from the same strain as the original graft donor; when cells from nonsensitized donors were used the results were equivocal (J. F. A. P. Miller et al., 1962). In a later report J. F. A. P. Miller (1963) indicated that he had been able to restore neonatally thymectomized mice to the point of an adequate homograft response with lymphoid cells. Parrott and East (1964) used prevention of wasting disease as the criterion of restoration, and did this in neonatally thymectomized mice grafted in the kidney capsule at 12-15 days of age with whole thymus glands. They also prevented wasting disease with isologous spleen cells administered intravenously 1-2 days after thymectomy. Subcutaneous thymus grafts were not effective in preventing wasting disease in their animals. In the studies of Dalmasso et al. (1963) several regimens were used. All efforts to restore immunologic activity with cell-free preparations failed. Effects of cell administration were evaluated by three criteria: long-term survival (most neonatally thymectomized mice are short-lived), rejection of skin homografts, and immunologic activity of peripheral lymphoid tissues as assessed by graft-versus-host reactions in appropriate hybrid recipients. All the regimens, whether spleen or thymus cells were administered, increased the life span of the animals. Only adult spleen cells restored the homograft response: this was effective whether

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the cells were administered intraperitoneally at 2 days of age or intravenously at 40 days of age. With respect to restoring immunologic competence of spleen cells in the graft-versus-host model, intravenous injection of 100 million adult spleen cells in the neonatally thymectomized animal at 40 days of age was most effective. Intraperitoneal injection at 2 days of age of 10 million adult spleen cells had a definite effect, although it was less consistent than that observed in the older recipients. The inference drawn from these studies, showing that immunologic function can be restored in neonatally thymectomized mice by isologous spleen cells-not thymus cells-from immunologically mature donors is that the immunologic defect of neonatally thymectomized mice involves primarily a lack of cells. The thymic contribution is not thus explained, however, since the studies with neonatally thymectomized mice bearing thymus homografts have indicated that host cells predominate in the peripheral lymphoid tissues and that cells of the thymus-donor strain constitute a significant minority of such cells. The results cited above, involving a large measure of immunologic restoration with peripheral lymphoid cells in the complete absence of a thymus, suggest that the effect of the homologous thymus on proliferation of host cells is mediated by cells from the thymus graft. Recently, Yunis et al. (1964) have succeeded in reconstituting neonatally thymectomized mice completely from the standpoints of growth potential, immunologic capacity, and longevity, by injection of a sufficiently large number of adult syngeneic spleen or adult syngeneic thymus cells, again pointing to the essential defect in the lymphoid cells of the neonatally thymectomized mice. Further, Hilgard et al. (1964) have shown that injection of a sufficiently large number of spleen or adult thymus cells will even reverse the wasting process, whereas transplantation of whole thymuses from either newborn or older donors will not. Recently, however, Levey et al. (1963) and Osoba and Miller (1963, 1964) presented evidence arguing strongly for a humoral or preferably hormonal effect of the thymus on the development of the lymphoid tissues. These investigators placed immature syngeneic mouse thymuses in cell-tight millipore chambers implanted intraperitoneally in neonatally thymectomized mice, and showed that such treatment would permit the development of the lymphoid structure and its immunologic potential. These observations argue strongly for a hormonal effect produced by the thymus, in confirmation of the prior experiments of J. F. A. P. Miller ( 1962a) and Dalmasso et d. (1963). These findings do not, however, exclude the possibility that the thymus may function, especially early in embryonic life, as a source of lymphoid cells seeding the other, more peripheral lymphoid tissues. In two other species, the chicken and the rabbit, the thymus is the

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first organ to become lymphoid (as it is in all species that have been studied carefully from this point of view) ; however, both apparently have a second central lymphoid organ which contributes significantly to immunologic development (Archer et al., 1963b; Warner and Szenberg, 1982). These relationships have been discussed in the section on the ontogeny of lymphoid tissues. In both of these species, there is evidence of recovery from the immunologic depression induced by bursectomy in the chicken (A. P. Mueller et d.,1964) and thymectomy in the rabbit (Archer et al., 196313). Mueller et al. (1964) have emphasized that the latest primary antigenic stimulation in their bursectomized chickens was given at 22 weeks. Secondary stimulation at 34 weeks in animals not responding at 22 weeks resulted in significant antibody production. They have also documented late acquisition of rabbit hemagglutinins in neonatally bursectomized chickens, and have postulated a degree of recovery from the effects of bursectomy. It has not been possible, although extensive efforts in this direction have been made by Aspinall and Meyer (1964) and by Warner and Szenberg (1964) to interfere with development of the chick thymus during the embryonic period without affecting the bursa. However, data from both of these laboratories indicate that neonatal thymectomy of the chicken produces a defect in homograft immunity without affecting antibody production significantly (Aspinall and Meyer, 1964; Szenberg and Warner, 1962; Warner and Szenberg, 1962, 1984; Warner et al., 1962). In the rabbit, the thymus is sometimes but not always the only lymphoid organ at birth, and Archer et al. (1984) originally postulated that the variable effect of neonatal thymectomy on antibody production reflected the degree of “peripheralization” of lymphoid tissues at the time the thymus was removed. As indicated in the section on the ontogeny of the lymphoid tissues, more recent studies suggest that the rabbit has a second central lymphoid organ, the appendix, whose development is, to a degree at least, independent of the thymus. The postulate of Archer et al. (1963b) is that removal of both the thymus and the appendix of the rabbit in the neonatal period would result in more profound depression of immunologic reactivity than that observed as a result of thymectomy alone. There are two animals that present unique opportunities for study of the role of the thymus in immunologic development. One is the opossum, born after only 1213 days of gestation and completely lacking in lymphoid development at birth. Kalmutz (1962) has shown that here again the thymus is the first organ to become lymphoid, and that until this

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development occurs there is nothing approaching immunologic reactivity. He showed, too, that some degree of immune response is possible before the peripheral lymphoid tissues develop. Recent studies by Block (1964) show clearly that the thymus is the first lymphoid organ in the opossum, followed in turn by development of central lymph nodes and then spleen. He correlates development of immunologic potential with development of the lymphoid tissue, beginning with the thymus, in this animal. The hamster also presents an unusual model: hamsters thymectomized in the neonatal period have, in the studies of J. D. Sherman and Dameshek (1963, 1964), been y-globulin and plasma cell deficient. Defendi et al. (1964) noted unusual susceptibility to tumor development in thymectomized hamsters receiving polyoma viruses, and preliminary reports by J. D. Sherman et al. (1963) and Roosa et al. (1963) indicate that neonatally thymectomized hamsters are very deficient immunologically. Neonatally thymectomized rats have been studied extensively by Waksman et al. (1962), Arnason et a2. (1962a), and Jankovib et al. (1962), and the status of their transplantation immunity has been investigated by Defendi et d. (1964). Arnason et al. (1962,b) have emphasized the suppression of delayed allergic responses in these animals, but have demonstrated significant deficiencies of antibody production, Arthus reactivity, and homograft response. In general, the effects were less profound than those observed in mice. The rat is similar to the mouse in that the thymus is usually the only lymphoid organ at birth (Archer et al., 1963a, 1964).Whether the results documented by Waksman et a2. and by Defendi et al. reflect incomplete thymectomy, or a degree of “peripheralization” at birth not evident histologically, or the presence of thymusindependent lymphoid tissue which develops after birth, is not known. The dog has been studied by Kelly ( 1963) : in this species the thymus is lymphoid early, and the spleen and lymph nodes are also lymphoid well before birth. Neonatal thymectomy has little effect on the antibody response of the dog. In man, the best data also indicate that the thymus is the first lymphoid organ to develop (Hammar, 1905; Knoll, 1929), followed in turn by the primary central lymph nodes, spleen, peripheral lymph nodes, and gut, as in the dog. However, the human fetus shows peripheralization of the lymphoid tissue long before birth. In summary, of the animals studied so far, the mouse typifies the dependence of immunologic development-structural and functionalon the presence of the thymus during the first weeks of life. The development is a continuum extending back before birth and continuing to 40 days of age and beyond, and the effect of thymectomy at various times

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during this period depends on the immunologic response tested and the intensity of the stimulus, In the chicken, the bursa of Fabricius and the thymus are both related to the maturation of immunologic capabilities, the bursa affecting primarily antibody production and perhaps some forms of transplantation immunity, and the thymus affecting primarily the homograft response. In the rabbit, removal of the thymus in the newborn period affects antibody production to a variable degree and has no effect on skin homograft rejection. In both the chicken and the rabbit there seems to be a degree of immunologic recovery, most clearly documented in the rabbit; the mechanism of this recovery has not been delineated, but in the rabbit it has been proposed that the thymus-independent lymphoid tissue of the appendix exercises a function similar to that of the thymus in restoring lymphoid tissues and lymphocyte levels, and presumably immunologic function. Less is known about the degree of independence or interrelationship of the chicken thymus and bursa of Fabricius, but it may well be that the thymus ultimately compensates in part for the loss of the bursa, a development which occurs relatively late in the chicken. There are several clinical observations which serve to underline the importance of a functional thymus to immunologic development in man as well. A small group of patients has been reported who are immunologically deficient: not only hypogammaglobulinemic but lymphopenic as well. Unlike patients with classical agammaglobulinemia and hypogammaglobulinemia, whose thymus is relatively normal (Good et al., 1964a), a number of these patients have had vestigial thymus tissue weighing less than a gram in most instances, and almost completely lacking in lymphoid cells (Good et al., 1964a; Tobler and Cottier, 1958; Hitzig et al., 1958). It is of interest that, in several of these patients, the thymus was not only very small and very deficient in lymphoid development, but showed a failure of descensus. Thus, a true failure of development of the epithelial anlagen of the thymus can be postulated to lie behind the failure of development of the peripheral lymphoid tissues in these children. These patients have been unusually susceptible not only to bacterial infections, the major threat to the classical agammaglobulinemic patients, but also to viral and fungal diseases. One of these children, reported by Rosen et al. (1962) was grafted with thymus tissue, but in the 40 days before the child's death there was no restoration of lymphocyte levels or immunologic function. He had a deficient homograft response. There is another possible link of thymic deficiency to immunologic inadequacy in the human being. Ataxia-telangiectasia is a disease of chil-

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dren, characterized principally by neurologic abnormalities, but accompanied by unusual susceptibility to infection of the sinuses and lungs in most of the patients (Boder and Sedgwick, 1958). It has recently been discovered that about one-third of these patients have definable deficiencies of the immunoglobulins (Peterson et al., 1963). One patient (Peterson et al., 1963) has a very deficient antibody response, a failure of delayed allergy, and an abnormal homograft response. Interestingly, in the single post mortem mentioning the thymus at all, this organ was reported to be lacking (Boder and Sedgwick, 1958). In two additional cases of ataxia telangiectasia (Lemli, 1963; Bowden, 1963) the thymus could not be found on post-mortem examination. The initial observation that acquired agammaglobulinemia and thymic abnormality were associated has gained support over the last few years: there are now 8 cases to our knowledge (Good, 1954; C. M. Martin et al., 1956; Ramos, 1956; Lambie et al., 1957; Gafni et al., 1960; Wollheim and Waldenstrom, 1962; Rotstein and Good, 1961 ). Although the classical agammaglobulinemia of Bruton appears at this writing to be a peripheral immunologic failure, it is suggested that at least some hypogammaglobulinemia-lymphopeniapatients ( so-called Swiss agammaglobulinemia ) , and possibly ataxia-telangiectasia patients who are immunologically deficient, may have primary failure of thymus development. The finding of Page et al. (1963) that children with congenital sexlinked recessive agammaglobulinemia have an unusually high incidence of acute leukemia and lymphoma may even link this form of agammaglobulinemia to thymic malfunction, even though the thymus of most of the children with congenital sex-linked agammaglobulinemia who have died has been normal in the gross and microscopically. Recent experimental data and clinical observations have been underlining the association of thymic abnormality with a group of diseases involving immunologic dysfunction. Burnet and Holmes ( 1962, 1964) have studied a mouse strain which regularly develops a Coombs positive hemolytic anemia and abnormal thymic lesions; and Strauss et al. ( 1960), Beutner et al. (1962), White and Marshall (1962), E. V. Hess et al. (1964), and others have emphasized the association of thymic lesions, myasthenia gravis, and antimuscle and antinuclear antibodies. It has been known for some years that the most characteristic lesion of the thymus of myasthenia gravis patients is the formation of germinal centers, with plasma cells-a development rare in the human thymus under normal conditions (Castleman and Norris, 1949). Wolf et al. (1963) studied a patient with a thymoma and classical systemic lupus erythematosus (S.L.E.). Interestingly, this man was a member of a perfectly extra-

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ordinary kinship in which a number of hematologic dyscrasias and a variety of diffuse mesenchymal disorders were observed, as well as a high incidence of immunologic disorders : one patient with acquired agammaglobulinemia, three with low y-globulin levels and lack of one or both of the ~2-immunoglobulins,three with hypergammaglobulinemia, and five with significant but not diagnostic levels of antinuclear antibody. Funkhouser (1961) studied a patient with a thymoma and L.E. cells, but lacking in other evidence of lupus erythematosus. Several instances of the simultaneous occurrence of lupus erythematosus and myasthenia gravis have been summarized by E. V. Hess et al. (1964). This seems to be more than a chance incidence. Thymic tumor has also been associated with myositis and myocarditis, in the apparent absence of myasthenia gravis; aregenerative anemias and pancytopenias; endocrinologic disorders; and nephrosis (reviewed by Fisher, 1964; and Good d ul., 1964a). Although, as indicated earlier, there is evidence that histologic abnormality of the thymus is associated with immunologic dysfunction in adult life, it is clear from the rapidly accumulating experimental data on a variety of experimental animals that the major immunologic role of the thymus is exercised, depending on the species, in the late embryonic period and the early postnatal or posthatching period. Clinical observations have just begun to fit into the picture, and it seems evident, from the point of view of the role of the thymus in human disease, that we have merely glimpsed the tip of the iceberg. At this writing the thymus has been linked to a number of human diseases, although only the crudest indices of abnormality have been applied: very small size, failure of normal descent, very large size, and extremely disturbed histology; as yet, only hints of what is cause and what effect have been available. The nature of many of these relationships will undoubtedly be established as understanding of the thymus increases and as improved methods of assessing thymic function are developed. 111. Phylogeny of Adaptive Immunity

A comprehensive survey of the phylogeny of the immune response would include its origins in the invertebrate Metuzoa and its evolution to the higher vertebrates. However, it will be evident in the sections to follow that comparative immunology is still in its early stages, despite the fact that the earliest work of Metchnikoff (1884) in this field dates back to the 1880's. The effort has often been diffuse and complicated by lack of critical quantitative methodology and appropriate controls for specificity of response.

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In this review, we shall limit ourselves to adaptive immune responsiveness for the most part, defining this type of response in terms of ability to respond to antigenic material by production of specific combining substances, and to show an anamnestic response to these same antigens on subsequent exposure. Our emphasis will be on studies involving:

1. Chemical definition of the combining system. 2. Definition of the cellular basis for adaptive immunity. 3. Observations, in various species, of the capacity to react with delayed sensitivity and homograft rejection, and the relationship, if any, to the capacity for antibody synthesis. So far as is known, all antibody activity in mammals is localized in the serum globulins. Their properties have recently been discussed by Fahey ( 1962). A comparative study of y-globulin evolution might provide further insight into the structure and formation of these proteins. It is also possible that substances produced by primitive animals in response to antigenic stimulation warrant designation as antibodies although they do not fulfill the physicochemical criteria of y-globulins. In all species, evidence for an anamnestic response is of interest because of the association of plasmacytic proliferation with the secondary response (Kolouch, 1938;Kolouch et al., 1947;Fagraeus, 1948;Leduc et al., 1955;Urso and Makinodan, 1961; Nossal and Makela, 1962;Baney et al., 1962).The lymphoid cell family is the primary cellular basis for adaptive immune response in vertebrates and will be discussed in detail in a later section, emphasizing particularly the most primitive animals in which plasma cells or lymphocytes can be identified. However, the possibility that another cell system may mimic adaptive immune responses in an invertebrate species cannot be excluded at this time. The y-globulins are distinct among the mammalian serum proteins in their affinity with antigens, and the lymphoid cell family is a particularly intriguing example of differentiation. Our understanding of both the y-globulins and lymphoid cell differentiation is still very incomplete, however, and we believe that undue restriction of inquiry in the lower animals would neglect biologic adaptations of great interest. The lower forms also offer unusual opportunities for structural studies or analyses of cellular events which would be difficult or impossible in higher homothermic vertebrates. Indeed, it may be possible to dissect the complex cellular events leading to antibody synthesis phylogenetically, and to find arrested levels of maturation in various species both at the cellular and molecular levels. Finally, there may be organisms in which there is dissociation of

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adaptive immunologic responsiveness : that is, animals in which the homograft response, delayed sensitivity, or antibody-producing capacity is present in the absence of one or both of the other types of responsiveness. Such species would be of extreme interest. In the following sections, the principal focus will be adaptive immunologic responsiveness in invertebrates and poikilothermic vertebrates. Studies on innate immunity and phagocytosis will be included only where they seem to be related to the emergence of adaptive responses. A. IMMUNERESPONSESIN INVERTEBRATES Most biologists agree on the phylogenetic relationships of the various classes within the subphylum Vertebrata, but more often disagree on the phylogeny of the invertebrates. Much of this controversy is based on the lack of paleontologic evidence of the soft-bodied invertebrates. Systemic classification of the invertebrates has been discussed by Hadzi (1953) and Marcus (1958), and the general aspects of such species classification, evolutionary history, and ecology have been reviewed by E. D. Hanson (1961).A complete manual of invertebrate zoology and classification is that of Hyman (1940-1959).There is general agreement on the evolution of the Metazoa from protozoan ancestors, and on the classification of coelenterates and flatworms as simpler and more primitive than arthropods and protochordates. There have been several reviews of immunity in invertebrates (Cantacuzhe, 1923; Huff, 1940; Baer, 1944; Bisset, 1947), but none has focused specifically on induced responses. In the following sections, evidence will be presented indicating that, to the time of writing, adaptive immunologic responsiveness has not been demonstrated in invertebrates. Exceptions may, of course, be found in the future; however, if such responses are observed, they will doubtless be exceptional rather than typical. 1. Reactions to Infection with Microorganisms Reactions of higher vertebrates to infectious organisms involve many physiologic responses in addition to antibody formation. The following studies are reviewed to indicate that invertebrates are subject to infection, and to suggest, on the basis of the accumulated evidence, that their defenses are nonspecific. The response tends to vary with species, and is often relatively unpredictable even within species. Metalnikov (1920) studied the bee moth larva, Galleria mellonella, and found it to be resistant to Mycobactem'um tuberculosis, Corynebacterium diphtheriae, Pasteurella pestis, and streptococci, but susceptible

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to a variety of Gram-negative organisms, particularly Proteus and Pseud o m m , not ordinarily pathogenic in many mammals. Tarr (1937) reported that the honey bee larva was highly susceptible to Bacillus larvae infection. Infection of the crustacean sand flea, Orchestia, with a luminous bacterium was reported by Inman (1927). Other types of infection in invertebrates were discussed by Bisset ( 1947) and Huff ( 1940). Detailed studies of infections with microorganisms were carried out by Cameron (1932, 1934) in earthworms and caterpillars. Earthworms TABLE I11 PATHOGENICITY OF VARIOUS BACTERIAFOR Galleria mellonell@ Nonpathogenic Mycobacterium tuberculosis (human, bovine, avian) Bacterium dysenteriae Vibrio cholerae Bacterium typhosum Clostridium tetani Clostridium sporogenes Pneumococcus Bacterium paratyphosum B (Schottmuller) Pasteurella pestis Gonococcus Hemophilus influenzae Mycobacterium smegmatis Staphylococcus pyogenes aureus Coynebacterium

Pathogenic Streptococcus pyogenes Streptococcus faecalis Cmynebacterium diphtherhe Proteus vulgaris Bacillus mycoides Pseudomonas pyocyanez Bacterium coli communis

Variable Bacillus subtilis Staphylococcus pyogenes albus Clostridium oedematiens Vibrion septique Clostridium welchii

hofmannii 0

From Cameron ( 1934)

.

tolerated large doses of bacteria pathogenic for human beings: some were resistant to diphtheria toxin in doses as high as 300 times the lethal guinea pig dose, although the toxin induced vacuolation and destruction of the coelomic corpuscles, and necrosis of the body wall. Some degeneration of the coelomic corpuscles was also noted after injection of Shigellu dysenteriue, but it was milder than that resulting from diphtheria toxin. No pathogenic effects were noted upon administration of Staphylococcus aureus, Proteus, Vibrio cholera, hemolytic and nonhemolytic streptococci, pneumococci, and paratyphoid A. In further studies, Cameron (1934) described the effects of human parasitic and saprophytic bacteria on Galleria mellonellu, as well as the

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ROBERT A. GOOD AND BEN W. PAPERMASTER

response to organisms common in this species and its environment. Wide variations in susceptibility were noted, and in some instances virulent and nonvirulent variants of the same bacterial strain were isolated. A summary of Cameron’s bacteriologic studies on lepidopterous larvae is given in Table 111. He concluded that saprophytic organisms were generally more virulent in this species than human pathogens, but that variation in the individual properties of the bacterium determined its effectiveness in overcoming the host’s defenses. 2. Responses to Immunization with Killed or Attenwzted Bacteriu and Other Nonliving Antigens a. Humoral Immunity. Cantacuzene (1922) isolated a Vibrio from the marine worm, Sipunculus nudus, and prepared killed suspensions for vaccination. Both pathogenicity and deaths were reduced in worms inoculated with the vaccine before administration of a living culture. Paillot (1920) found that Agrotk segetum, a caterpillar, demonstrated immunity to a virulent culture of a Bacillus species 24 hours after vaccination with 3-week-old cultures of the same organism. This early immunity has been reported repeatedly in studies of invertebrates, and some investigators have presented evidence that this rapidly developing immunity is both phagocytic and humoral (Paillot, 1920; Metalnikov and Gaschen, 1921; Toumanoff, 1927). However, Zernoff ( 1928) claimed that it was possible to transfer this early immunity by inoculation of homologous cell-free hemolymph. The nature of the humoral mediators of immunity in invertebrates was only meagerly investigated in the early work. Cantacuzene demonstrated agglutinins and lysins in ascidians (1919), snails (1916), and several types of crabs (1912). A compilation of many of the other studies on humoral immunity in invertebrates was presented by Huff ( 1940). Many of these experiments did not test specificity adequately, and others had no controls for nonspecific reactions. In some instances, the nature of the protective capacity, i.e., that it was, in fact, humoral, was not ascertained. Two early reports (Chorine, 1927; Zernoff, 1931) are of particular interest in that immunity to test organisms was produced by unrelated bacteria, horse serum, and beef broth. Bernheimer et al. (1952) re-examined humoral immunity in caterpillars, using Escherichia coli B, Tz coliphage, streptolysin 0, and human erythrocytes as antigens. Hemolymph was collected by cutting tubercles of the thoracic segments, and repeated bleedings were possible. In no instance was there definite evidence for antibody formation following single or repeated injections of the antigens. When agglutination re-

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actions occurred, as with undiluted hemolymph and suspensions of E . coli, they were observed in both control and experimental groups. When the hemolymph was diluted there was no specific agglutination. Ability to agglutinate human erythrocytes was stimulated by ovalbumin as well as the red cell antigen. This agglutinin was labile and was lost after storage in the cold. Further studies by Bernheimer (1952) established that caterpillar hemagglutinins for mammalian erythrocytes were heat labile, and did not produce hemolysis in the presence of guinea pig complement. Hemolymph from Sarnia cecropia was tested for ABO specificity, but failed to differentiate these blood groups. Comparable results were obtained (Triplett et al., 1958) when the sipunculid worm, Dendrostomum zostericolum, was immunized with red cells and protein antigens. Further studies on heat-stable bactericidins in lepidopterous larvae have been reported by Briggs ( 1958). Studies of a different nature have been conducted by Phillips (1960) and Phillips and Yardley (1980) with the sea anemone, among the most primitive invertebrates of the Coelenterata. After single and multiple injections of BSA, the animals were ground and extracts tested for their ability to interfere with the combination of BSA and antibody to BSA prepared in rabbits. The extracts did interfere with this reaction, and the degree of interference increased with time after immunization. The activity of the extracts was specific to some extent, since precipitation of human serum albumin and rabbit antibody to human serum albumin was unaffected. Further studies by Phillips (1962) have indicated that the extracts from BSA-treated anemones do interfere with other antigenantibody systems, however. Experimental evidence suggests that certain invertebrates produce humoral factors following immunization which react with antigens. Chemical studies of the type described by Isliker (1957), R. R. Porter (1960), and Kabat ( 1961)would be useful in assessing the resemblance, if any, of these combining substances to the antibody y-globulins of higher animals. Neither complementarity nor inducibility per se is an adequate criterion for y-globulin-antibody formation. Mora and Young ( 1962) showed that such polyelectrolyte macromolecules as heparin, protarnine, DNA, RNA, and polyglucose sodium sulfate can interfere with phage neutralization by rabbit antibody. Indeed, electrostatic attractions, hydrogen bonding, and the stereochemical complementarity and close fitting characteristic of antigen-antibody bonding may occur when chemical groups of two macromolecules are in appropriate apposition (Talmage and Cann, 1961) , However, the careful chemical characterization of any induced, specific substance produced in response to antigenic

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ROBERT A. GOOD AND BEN W. PAPERMASTER

stimulation in an invertebrate would be of great interest even without resemblance to y-globulins. b. “Cellular Immunity,” Phagocytosis, and the In$.ummatoy Reaction. The concept of cellular immunity and the role of phagocytosis in protection of the organism was originated and developed most thoroughly by Metchnikoff. In his classical report of 1884, he described the result of infection of the fresh water crustacean, Daphnia, with the blastomycete, Monospora bicuspidata. The course of infection and resistance was followed in the small transparent animals under the microscope. When introduced into the animal, the spores penetrated the intestinal wall and came into contact with phagocytic leucocytes which engulfed and digested them. On the other hand, little or no phagocytosis was observed when spores of the fungus Saprolegnia germinated outside the animal and infected it by inward penetration of the mycelia (Metchnikoff, 1892). Further research on phagocytosis is chronicled in Metchnikoffs lectures on the comparative pathology of inflammation (1892) and in his book on immunity (1905). Experiments were done in most of the phyla and in many vertebrates including the higher fishes. The inflammatory process in invertebrates consists of phagocytosis and digestion of the foreign material. When indigestible material is involved, the process is one of encapsulation, the formation of one or more layers of leucocytes around the material, which may remain within the organism or be extruded through nephridia, external pores, or other channels. In almost all species there are certain organisms that do not stimulate phagocytosis or continue to multiply actively within the phagocytes, achieving either destruction of the host or a symbiotic relationship within host cells. Many subsequent investigations of phagocytosis in invertebrates were conducted, a number of them by MetchnikoEs students. None of these studies was as complete as Cameron’s (1932) experiments with the earthworm. He studied phagocytosis and the reactions of the cells lining the coelomic cavity to India ink, carmine particles, milk and fat globules, colloidal iron, dust, and coal, as well as homologous and heterologous cells, and endogenous and foreign bacteria. All the inert materials, except fat or milk, were phagocytized within a few minutes, and the phagocytic process seemed to be autonomous in each segment. Mammalian blood cells and spermatozoa were all taken up within 48 hours. The reaction to bacteria varied with the organism, as noted in the last section. One of the most interesting findings was that homologous annelid spermatozoa remained within the coelomic cavity longer ( 4 or 5 days) than mam-

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malian spermatozoa, suggesting a degree of heterologous tissue recognition. Further studies by Cameron involved the reaction to injury with a hot needle or strong acid. This reaction consisted of: an increase in the number of coelomic corpuscles within the first hour, proliferation of parietal coelomic epithelium near the site of injury, formation of undifferentiated tissue at the site with the appearance of numerous acidophilic cells, removal of dead tissue by desquamation and phagocytosis, ingrowth of epithelial cells from the superficial epithelium of the healthy margin, and gradual formation of muscle and fibrous tissue with ingrowth of new blood vessels. Complete repair was effected within 10 days. The process is not unlike the repair effected after removal of segments in annelids. In subsequent studies with caterpillars, Cameron (1934) showed that the cells of the hemolymph were most active in phagocytosis, but that pericardial cells and cells of the fat body were also phagocytic. Cells which Cameron identified as lymphocytes seemed to show a relative increase during the course of a reaction and to decline when the foreign material had been removed. Tubercle bacilli remained viable within cells of the blood and the pericardial cavity throughout metamorphosis, and were virulent for guinea pigs after isolation from adult moths. Response to other organisms was variable, as shown in Table 111. Recent studies of inflammation in invertebrates are those of Schlumberger (1952) who used methylcholanthrene and talc in the body cavity of the cockroach, and of Bang (1961) who investigated reactions to injury in the oyster. Little attention was given to the immunologic aspects of inflammation and phagocytosis in the early literature, although Metalnikov (1927) mentioned that immunized caterpillars showed an increased phagocytic rate for bacteria. Triplett et al. (1958) found no increase in either the phagocytic rate or the number of phagocytes during immunization in the marine worm, Dendrostomum zosterico2um. Phillips’ recent results (1982) suggest that, in the anemone, the rate of specific intracellular digestion of BSA conjugated with C14-labeled p-aminobenzoic acid, and the uptake of Tz and Ts coliphages, are both enhanced by prior injections of the antigen. These observations suggest an induced cellular uptake of antigenic material and bear on the question of the nature of cellular sensitization to protein materials. The studies of the specificity of this reaction have not been completed, but these experiments are a beginning in the study of cell reactions to well-defined proteins in invertebrates.

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ROBERT A. GOOD AND BEN W. PAPERMASTER

Clearly, phagocytosis and intracellular digestion are the major determinants of immunity in the invertebrates. The inflammatory reaction of earthworms resembles that of the vertebrates, except that the characteristic sequence of granulocytes followed by lymphocytic and monocytic infiltration does not occur. Instead, the coelomic corpuscles proliferate and differentiate at the site of injury to form fibrous connective tissue support for the ingrowing muscle fibers and epithelial cells. So far, no reports of increased proliferation of any cell type as a specific response to antigenic stimulation have been confirmed. Of particular relevance is the absence of evidence of recall, as indicated by increased proliferation of phagocytes or free cells of the blood, hemolymph, or coelom in immunized animals. However, exhaustive efforts to demonstrate cellular sensitization to antigens have not been made. Attempts to elicit specific phagocytic proliferation, and even production of humoral substances, should be continued with relatively well-defined soluble or particulate antigens, the responses to which are detectable by precise means and, in the case of circulating antibodies, in small quantities. An example of such an antigen is bacterial viruses, such as the coliphages, which have the added advantage of being readily detectable in small quantities themselves as a consequence of their biologic activity. Only after such antigens have been administered over a period of months, and no immune response observed, can it be concluded that a given species lacks immunologic reactivity. All such studies should include rigid controls for specificity, as well as attempts at chemical definition of the substances apparently reacting with the antigen, 3. Invertebrate Blood Cells The blood cells of invertebrates are not easily classified along the lines established for the higher vertebrates. Kollmann (1908) made a rather complete study of invertebrate blood cells, and regarded the leucocyte with scanty cytoplasm as the most primitive cell type. In the earthworm, Cameron ( 1932) identified acidophilic and basophilic cells, and granular chloragogen cells whose granules had great affinity with many anionic and cationic dyes. Only the basophilic and acidophilic cells were active in phagocytosis. Cameron (1934) described the cells of the hemolymph of caterpillars as lymphocytes, granular leucocytes, and spherule cells. The lymphocyte-like cells were the most active during phagocytosis; they appeared to proliferate during the course of an infection, but not in a manner specific to any one organism. Further infonnation on invertebrate blood cells is included in Cameron’s (1932, 1934) papers, and in reviews by Huff (1940) and Jordan (1938).

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We believe that important distinctions between vertebrate and invertebrate immunity have been blurred by application of terms used for higher vertebrate leucocytes to blood cells of invertebrates. Little is known about the developmental sequence of any of the cell types in invertebrates, and, aside from phagocytosis, virtually nothing is known about function of the different cell types and their contents, e.g., the granules. Although the small basophilic cells with scanty cytoplasm in invertebrates have been called ‘lymphocytes,” there is no evidence that the analogy with the cells of higher vertebrates is valid. Indeed, it seems to us that one of the important distinctions between immunity in vertebrates and invertebrates rests on the presence or absence of definitive lymphoid tissues and lymphoid cells. Cuenot ( 1896) described lymphogenous organs along blood sinuses in invertebrates, but did not establish their role in lymphopoiesis. Moreover, lymphocytes and other cells of the lymphoid series have been clearly linked with specific immunologic reactions in the higher vertebrates, whereas circulating monocytes, and macrophages and reticular cells of the lymphoreticular organs have been associated with phagocytosis. As noted earlier, Cameron (1934) attributed a major role in phagocytosis to lymphocytes in the caterpillar. In the section dealing with immunity in vertebrates, evidence for the association of the evolution of lymphoid tissue with the development of adaptive immunity will be presented. For the present, in the absence of critical functional or chemical markers, conclusions about phylogenetic homologies among the blood cells are purely speculative. 4. Homotransplantation Immunity Homotransplantation is easily achieved in most invertebrates. Loeb ( 1945) reviewed the evidence for successful homotransplantation in coelenterates, planarians, earthworms, insects, and echinoderms. Additional data on transplantation in invertebrates were presented by Cushing and Campbell (1957)and discussed in a review by Favour (1958). In annelids, heterologous grafts do not succeed as well as homologous ones, and rejection time seems to be related to genetic distance: the greater the disparity, the more rapid the rejection. Plagge (1939)transplanted caterpillar organ rudiments between members of different genera and even different families. The transplants survived and developed normally in the foreign host, and success or failure seemed to depend more on graft size than on degrees of donor-host relationship. Ovaries transplanted from Drosophila melunogaster to Drosophilu funebris did

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ROBERT A. GOOD AND BEN W. PAPERMASTER

not function, a failure attributed to species specificity of a hormone necessary for maturation of the ova (Vogt, 1940). Recent experiments in marine invertebrates have been conducted by Cushing ( 1957, 1962). In the mollusk, Pecten irrudiuns, homografts were retained for 14 days; however, the series was too small to be conclusive. Triplett et al. (1958) studied skin and tentacle transplants in the marine sipunculid worm, Dendrostomum. Technical difficulties prevented adequate assessment of skin graft reactions, since neither autografts nor homografts healed. However, tentacle transplants in the coelomic cavity permitted studies of graft survival up to 70 days. The reaction to autografted or homografted tentacle was encapsulation by coelomic cells. Second set grafts were studied in worms preimmunized with injections of chopped tentacle. Encapsulation was less evident in the preimmunized group than in the controls. Thus, these experiments suggest that there is not only an absence of homograft recognition in this organism, but also a lack of ability to replace cells which had encapsulated the small bits of tentacle injected. Preliminary experiments on transplantation immunity in the octopus by Cushing (lSe2) suggest that there is no difference between the response to homografts and autografts up to 40 days after grafting. In summary, present evidence indicates that invertebrates fail to distinguish between autologous and homologous tissue, and, in some instances, between these and heterologous tissue. When grafts were not retained, the failure was attributable to technical failure or nonimmunologic mechanisms.

5. Anaphylaxis and Hypersensitiuity Several claims of active anaphylaxis in invertebrates were included in the early literature. Krafka (1929) described anaphylaxis in crawfish made sensitive to human serum. Studies by Ramsdell (1927a,b) were quoted by Huff (1940) as evidence for hypersensitivity in invertebrates. In these experiments the smooth muscle reaction in the earthworm and the swimming motion in the paramecium were lost following passive sensitization with ovalbumin and rabbit antibody. However, none of these results has been substantiated when adequate controls for specificity and reaction in noninjected animals were included. We are aware of no reports dealing with delayed sensitivity in invertebrates. 6. Proteins in Body Fluids Studies on the protein chemistry of invertebrate blood, hemolymph, and coelomic fluid have been reviewed by Engle and Woods (1960),

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and comparative electrophoretic patterns, by paper and moving boundary electrophoresis, have been compiled by H. F. Deutsch and Koenig ( 1956). Proteins with the electrophoretic mobility of y-globulins have not been detected in most studies of invertebrate body fluids; however, Condie (1962) has isolated a protein from the keyhole limpet, a mollusk, which has electrophoretic and ultracentrifugal properties of y-globulin. The functional properties of this material are not known.

7. The Nature of Immunologic Activity in Inuertebrates Invertebrates are clearly capable of protecting themselves from pathogenic and saprophytic microorganisms; yet this protective capacity does not appear to be closely related to the specific mechanisms that characterize adaptive immunity in mammals. Reactions against parenterally introduced foreign substances commonly involve phagocytosis and encapsulation by the ameboid cells of the blood or coelomic fluid. The evidence relating any of the free cells of the invertebrates to the lymphoid cells of the higher vertebrates is meager and difficult to interpret in view of the lack of specific functional markers for lymphoid cells. There is no evidence of organized lymphoid tissue in invertebrates. The ability to distinguish antigenic surface patterns appears to be limited in most invertebrate species. When observations suggesting such capabilities have been presented, evidence for their immunologic nature has been lacking. The studies reviewed here do not exclude the possibility that a prototype of the adaptive immunologic response exists in some invertebrates. The time factor has not been considered carefully enough in previous studies, including the interval between presentation of the antigen and subsequent test, and the duration of exposure to the antigen. Reactions should be tested weeks or even months after repeated antigenic stimulation before concluding that there is no response.

B. IMMUNE RESPONSES IN VERTEBRATES Interpretations of immune responses in vertebrates are less difficult. Vertebrates are all members of a single subphylum, whereas the invertebrates represent many phyla. The phylogenetic relationships among the various vertebrate classes are also more definite, since they have been based on studies of fossils and correlated closely with geologic time. In addition, homologies of organ structure and anatomic position in living vertebrate representatives are more obvious, with less deviation from a basic body form; and similarities in the circulatory system and blood

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ROBERT A. GOOD AND BEN W. PAPERMASTER

permit use in lower vertebrates of many of the techniques developed for study of mammals. Immune responses in vertebrates have been reviewed by Bisset ( 1947), Cushing and Campbell ( 1957), Favour ( 1958), Sirotinin ( 1960), and Hildemann (1962). Recent advances in the study of immune responses in the lowest vertebrates (Papermaster et al., 1962c, 1963) have suggested that adaptive immunity evolved in the lower marine vertebrates and that this development was correlated with the earliest appearance of a thymus and definitive lymphoid tissue. 1, Reactions to Infection with Microorganisms

Studies of the reaction of poikilothermic vertebrates to various pathogens and saprophytes were a natural extension of the work on invertebrates by Metchnikoff and his associates, Their experiments indicated that survivors of a first exposure to a pathogen were less susceptible to the same organism upon re-exposure ( Metchnikoff, 1905). Studies on the effect of temperature on pathogenicity of infections such as anthrax have yielded variable results (Gibier, 1882; Sabrazhs and Colombot, 1894; Mesnil, 1895). Gibier (1882) found that high temperatures resulted in loss of “immunity” to anthrax in frogs, but Sabrazhs and Colombot (1894) and Mesnil (1895) found susceptibility to anthrax in cold-blooded vertebrates to be unaffected by temperature changes. The Sabrazhs and Colombot findings were, however, an exception to the general rule ( Bisset, 1947) that poikilothermic animals are completely “immune” to anthrax. Bisset (1947) suggested that some of the contradictions reflected the focus on the host’s defenses alone, without consideration of the effects of temperature on the invading organism. In addition, the inconsistencies may be attributable to changes in other physiologic processes which in turn affect host-parasite relationships. More typical is the response of trout to a Gram-negative rod, Bacterium salmonicida, which causes furunculosis ( Bisset, 1948). Young fish are more susceptible to this disease than older ones, and outbreaks are more common in warm, slow-moving water. Gee and Smith (1941) showed that protection from this organism could be conferred by immunization with killed organisms.

2. Response to Immunization with Killed Organisms and Other Antigens and the Effect of Temperature The studies of Metchnikoff and his associates had clearly demonstrated that resistance to infectious microorganisms in poikilothermic vertebrates is temperature dependent; however, in many of their studies

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the effects of temperature were not controlled. The variable results in studies of fish, frogs, and alligators prompted a study by Widal and Sicard (1897) on frogs and other animals placed at their disposal by Metchnikoff. In frogs, they found that a variety of species produced agglutinins above 21" but not at 12"C, but they emphasized that large doses of antigen were needed to elicit agglutinin formation in these animals. Similar results in frogs were reported by Friedberger and Seelig (1908), Wollman (1938), and F. W. Allen and McDaniel (1937), and in fish by Nybelin (1934) and Pliszka ( 1939). In all these studies it was apparent that poikilothermic animals formed antibody more rapidly at warmer temperatures, and that they produced it at a slower rate and in smaller amounts than mammals and birds. That antibody production is not completely inhibited in fish at colder temperatures was reported by W. Smith (1940) who showed that agglutinins to Bacterium salmonicida were formed at 1O"C, provided the antigen was given in sufficient quantity and sufficient time allowed following immunization. A study by Cushing (1942) on the rate of antibody production by fish to sea urchin spermatozoa at 15" and 20°C also showed a delay in antibody response in the cold. The animals were bled at 3-4 day intervals up to 25 days after the injection of the sperm suspension. The fish kept in the warm tank showed a rise in antibody titers by the eleventh day, while those kept at 15°C did not show a rise in titer until the fifteenth day. Bisset (1948) found that frogs immunized at 8°C produced no antibody, but that antibody rapidly appeared in the serum when the animals were warmed to 20°C. Conversely, frogs immunized at 20°C and then kept at 8°C had much lower titers than control animals which remained at 20°C. In further experiments, Bisset (1949) demonstrated that the antibody titers of fish and frogs at low temperatures could be raised significantly by injections of corticosteroids. No further investigations of this phenomenon have been reported, and the mechanism of adrenal steroid action on immunity in lower vertebrates remains ill-defined. This topic will be considered further in the section on vertebrate lymphoid tissue. Use of a sensitive detection technique, bacteriophage neutralization, enabled us (Papermaster et al., 1962~)to detect small amounts of neutralizing antibody to Tz coliphage in the bullhead, Ameiurus melas, held at 10°C for 14 days after a single injection of 1O1O phage particles (Fig. 1). The temperature at which antibody formation may be inhibited apparently reflects the temperature of the animal's normal habitat. With the California hagfish, an animal adapted to cold marine waters, the temperature could not be raised above 25°C because of aberrant myo-

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ROBERT A. GOOD AND BEN W. PAPERMASTER

cardial contraction and other physiologic dysfunction. An animal adapted to high temperatures, the desert iguana, Dipsosuurus dorsalis, was studied by Evans and Cowles ( 1959).Agglutinin formation to Salmonellu typhosa H antigen was indicated by titers of 1:lO or less at 25°C and by titers of 1:80 to 1:640 at 35°C.Thus, the animal's normal environment must be considered in studies of the effect of temperature, and in many instances, comparisons of response among groups of animals may not be possible. The inhibitory effects of low temperatures on antibody formation in

I

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**Bullhead 10' 14 days

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FIG.1. Relative neutralization rates of T, phage by serum from immunized hagfish, bullheads, and mice. The number of days between stimulation with 1010 phage particles and bleeding are indicated for each curve. In the hagfish the results were the same at 4 and 7 days, regardless of the temperature. (From Papermaster et al., 1982c, with permission of Nature.)

many poikilothermic animals deserves further investigation. Indeed, such problems as the mitotic rate of antibody-forming cells, effects of steroids on cell lysis or secretion, inhibition of antibody production by 6-mercaptopurine and other metabolic antagonists, immunologic paralysis, and separation of the sensitization and secretory phases of antibody production, might be profitably investigated at lower temperatures in poikilothermic animals. 3. Evolution of Antibody-Forming Capacity in Vertebrates

Although much controversy remains concerning the systematics of vertebrate evolution, the evidence available appears to indicate pro-

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gressive development of complexity in body form which should be considered together with the development of immunologic capacity. The important characteristics of the chordate phylum are: gill clefts in the adult or young larva and embryo; dorsal, hollow nerve chord; notochord; segmentation; a coelomic cavity; and a tail (Young, 1950; E. D. Hanson; 1961; Romer, 1962). Study of existing species has suggested that the vertebrates arose from the tunicates whose body form is bilaterally symmetrical and has a dorsal nerve chord, notochord, gill slits (Berrill, 1955); however, the origin of the vertebrates is still contentious, since the fossil records of the protochordates are incomplete. A further step toward vertebrate evolution is represented by Amphioxus (the lancelet) ; this animal lacks jaws, is a filter feeder, and has segmentally arranged axial musculature ( Young, 1950). The vertebrates presumably evolved from similar forms, and the most primitive lacked jaws but had a notochord and other chordate characteristics. These primitive, jawless fish were the ostracoderms. Romer (1962) believes that they developed in fresh water; however, Robertson (1957) concluded that the earliest vertebrate forms arose in salt water on the basis of paleontologic evidence and recent studies of the plasma osmolarity in cyclostomes. Much anatomic evidence indicates that the cyclostomes are the living representatives of the most primitive vertebrates. Among the most significant characteristics are the lack of jaws and complete vertebrae, the cartilaginous skeleton, the persistent notochord, spherical gill pouches, and two semicircular canals in the internal ear (Hubbs, 1961). The two major groups of cyclostomes are the hagfishes (Eptatretidae) and the lampreys (Petromyzonidae) . Lampreys are found in both fresh and salt water, but the marine forms spawn in fresh water. The hagfishes, on the other hand, are entirely marine, and are considered to be more primitive than the lampreys on the basis of anatomic features (Hubbs, 1961), physiologic studies of the heart ( D. Jensen, 1961), and biochemical studies of the plasma ionic concentration (Robertson, 1954) and hemoglobin structure and function ( Manwell, 1958; Ingram, 1962). Indeed, hagfish hemoglobin resembles that of the invertebrate sea cucumber. These vertebrate ancestors probably developed in the late Ordovician period about 480 million years ago. The higher vertebrate fishes evolved from cyclostome-like precursors in the late Silurian and early Devonian seas. From progenitors probably resembling the placoderms, now extinct, two branches of fish evolution proceeded, the bony fishes and the cartilaginous fishes ( Colbert, 1955; Romer, 1962). Modern representatives of the early bony fishes are the sturgeon, garpike, paddlefish, and the dogfish or bowfin. The cartilaginous fishes include the sharks, skates, and

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ROBERT A. GOOD AND BEN W. PAPERMASTER

rays. Amphibia evolved from a specialized group of bony fish, the crossopterygians, of which the African and Australian lungfish are existing species. Primitive reptiles developed from the amphibians toward the close of the Paleozoic era, with birds and mammals evolving in the Mesozoic, and finally man in the Quaternary period about 1 million years ago (Colbert, 1955; Romer, 1962). Figure 2 summarizes Class Osteichthyes BONY FISHES

Boss

Class Chondrichthyes CARTILAGINOUS FISHES

Sturgeon

Bowfin

Pike Perch Bullhead

TO PRIMITIVE AMPHIBIANS

,c ,' Coeioconths

Chimeras

Class Agna tha

?

OSTRACOOERMS JAWLESS VERTEBRATES

OF SILURIAN 8 DEVONIAN PERIODS

RAT FISHES

PLACODERMS PALEOZOIC JAWED VERTEBRATES

II I

FIG. 2. A simplified evolutionary development of the fishes, showing the position of the bowfin, lamprey, haghhes, rays, and other fishes included in the studies documented. (From Finstad et al., 1964.)

the taxonomic relationships of the several groups of fishes, and indicates those we consider to be key forms for studies of the evolution of immune reactions. Many of the relationships among genera, species, and phyla are being examined with the use of iminunogenetic and immunochemical techniques. Immunogenetic blood grouping in fowl was described by Irwin (1947,1949) and in fishes by Cushing and Sprague (1953) and Fujino and Cushing ( 1959). Immunochemical protein analysis and studies of serum protein relationships in primates by Goodman (1961,1962) and

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in reptiles by E. Cohen (1955) have been useful in establishing phylogenetic relationships. A comprehensive discussion of comparative serology is beyond the scope of this paper, but some of the studies have been reviewed by Cushing and Campbell (1957), in two recent symposia (C. Cohen, 1962; Cushing and Stormont, 1962), and in the papers cited above. The coupling of immunologic data with biochemical and physiologic findings may well help to resolve some of the problems of taxonomy and evolution of organ systems in ontogeny and phylogeny. Further studies in this expanding field of research may elucidate the role of antigenic polymorphisms in evolution and differentiation. Previous work on the evolution of antibody formation has focused either on the invertebrates or vertebrates at the level of the teleost fishes or above it. We are aware of no previous studies on the cyclostomes and the lower ganoid fishes. Engle and Woods (1957) and Woods and Engle (1957), and more recently Clem and Sigel (1963) have studied the electrophoretic patterns of elasmobranch sera. With the exception of a study by CantacuzBne (1919) on ascidians, the protochordates have also been neglected. Thus, the transitional protochordates, cyclostomes, and lower fishes appear to provide the most promising area of research on the evolutionary origins of adaptive immune responses. a. Protochorhtes. Although studies on the natural bacteriolysins of ascidians have been reported by CantacuzBne (1919), no experimental attempts at active immunization of the protochordates were found. The ascidians and Amphioxus are of particular interest in this group, since they represent the most likely forebears of the primitive vertebrates. b. Cyclostomes. The cyclostomes are the most primitive of the true vertebrates and, as such, represent a unique group for studies on the phylogenetic origin of the antibody response and other forms of immunologic reactivity. Our studies started with the hagfish, Eptatretus stoutii (Good and Papermaster, 1961; Papermaster et al., 1962~).These animals are marine thoughout their life cycle and spawn at the ocean bottom. The embryo develops directly into a form resembling the adult without a larval stage (Young, 1950). The hagfish preys upon dead or dying fish, entering their mouths and eating the flesh from the inside. In our laboratory, hagfish were maintained in buckets of sea water at 10°C for most experiments, without feeding, for up to 7 months in good condition. All attempts to induce antibody formation in the hagfish with diverse antigens have been unsuccessful. The antigens used for immunization included killed Brucella abortus, typhoid-paratyphoid A and B vaccine, BSA, hernocyanin, and the bacterial viruses Tz (Escherichia coli phage)

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ROBERT A. GOOD A N D BEN W. PAPERMASTER

and MSP-8 ( Streptomyces griseus actinophage ) . Numerous injections were given over periods up to 3 months, and many of the antigens were administered with complete Freunds adjuvant in an effort to elicit an antibody response; but no agglutinating, complement-fixing, or neutralizing antibodies, or antigen-binding globulins were found. As a control on conditions of temperature and starvation, the freshwater teleost bullhead, Ameiurus mehs, was immunized with 1Olo T2 phage at 10°C and kept without feeding for the same periods of time as the hagfish. Under these conditions, neutralizing antibody to T2 phage could be demonstrated in the serum by the fourteenth day after immunization in the bullhead but not in the hagfish. No circulating phage particles could be detected in the 1Cday sample of the bullhead, while as many as 4OOO particles/ml. were found in the serum from hagfish given the same amount of phage. At U)"C, a temperature barely tolerated by the hagfish, a 7-day study was completed. Both hagfish and bullheads received an injection of 1Olo particles of phage, and their serum was tested for the presence of phage or neutralizing antibody. In the bullhead, no phage particles were detectable by the fourth day after injection, and neutralizing antibody was present in small amounts (Fig. 1).The hagfish had neither cleared the phage nor formed antibody by the seventh day, at which time the experiment was terminated because the hagfish appeared moribund. In these studies, incubation of phage and serum was continued for periods up to 72 hours at 0" and 37°C in order to detect neutralizing activity, but in no case was the rate of neutralization greater in immunized hagfish serum than in the diluent control or in normal undiluted hagfish serum. In all studies with bullhead or other fish sera, a heterologous phage was included in the reaction tubes as a control on the specificity of antibody inactivation. A next step in vertebrate evolution is represented by the lamprey, Petromyzon. As indicated earlier, the marine forms are anadromous, but in the Great Lakes and other areas, these animals have adapted completely to fresh-water existence. An opportunity was available in the spring of 1962 to study the immune response in spawning lampreys collected by the U.S. Fish and Wildlife Service. In the early experiments, no significant difference in the primary response of nonimmunized control lampreys and those intensively stimulated with Tz phage was observed. More recent studies by Finstad d d.(1964) and Good and Finstad ( 1964) have established that the lamprey possesses an epithelial thymus with occasional lymphoid cells, has a family of lymphocytes, including

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large, medium, and small, in the peripheral blood, and has small foci of lymphocytes in the primitive spleen and primitive bone marrow. Immunologic responses to bovine albumin and bovine y-globulin are lacking. Formation of neutralizing antibody against bacteriophage is at best equivocal and hemagglutinating antibody production against hemocyanin is feeble or lacking entirely. Agglutinating antibodies are, however, produced against Brucella antigens, and these animals accept autografts and reject homografts of skin. Evidence for a delayed immune response was also obtained. Thus, at this writing, we have evidence that the lamprey possesses a very primitive lymphoid tissue and has a low level of immunologic competence. C. Elasmobranchs. The chondrichthyes and osteichthyes represent independent lines of development from placoderm-like forebears which became extinct at the end -of the Devokan period (Fig. 2 ) (Romer, 1962). In an effort to include the principal groups of lower fishes in our study, antibody formation was followed in the guitarfish, Rhinobatos productus, and other elasmobranchs. In the guitarfish group, a welldeveloped response was demonstrated in a 7-day bleeding following secondary antigenic stimulation with lo1' phage particles. The primary response was poorly developed, and the formation of neutralizing antibody and clearance of phage from the serum were considerably slower than in the secondary response (Table IV). Sharks were capable of producing high antibody titers to influenza PR-8 virus in the studies of Sigel and Clem (1963).In our own studies (Papermaster et al., 1964) the relatively primitive shark, Heterodontus francisci, produced antibody against both hemocyanin and bacteriophage, although not with great vigor. d . Chondrosteans and Holosteans. The lower bony fishes were of particular interest since they include, we believe, several modern representatives of evolutionary stages ( actinopterygians or ray-finned fishes ) from which the teleosts developed; to our knowledge immune responsiveness had not been studied in any of these forms. The representative of this group included in our series was the holostean fish, Amia calua, the bowfin or dogfish of the northern United States. The antibody response was similar to that found in the elasmobranchs, in that secondary and tertiary stimulation with T2 phage resulted in increasing amounts of neutralizing antibody. The evolutionary development of immunologic competence, as revealed using hemocyanin and bacteriophage T2 as antigens, is set down in Table IV. Further studies are being pursued in

TABLE IV PINLOGENETIC DEVELOPMENT OF IMMUNITYIN THE LOWERFISHES

Fish studied Bullhead (Ameium melas) Bass (Microptow dnwides) Bowfin (Amia calva) Shark (Heterodontus francisci) Guitar6sh (Rhinobatos productus) Lamprey (Petromyzon mudnus) Hagdish (Eptatretus stoutii)

Antibody to hemocyanin PrecipiHemagglutating tinating Not studied Not studied

++

-

-

++ +

Neutralizing antibody to phage

+ Not studied +

+

+ +

2

2

-

4

Spleen develop ment

+ ++ + + + 2

Thymus develop ment

+ ++ + +

+(Larval) - (Adult -

8 tr

2 3 z

3 cd

3*

E $i

I

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the bowfin, sturgeon, garpike, paddlefish, and other representatives of the primitive bony fishes. e. Teleosts and Higher Vertebrates. The many references cited in earlier sections of this review attest to the ability of all vertebrates above the level of the lower fishes to make antibody following appropriate antigenic stimulation. Other aspects of immune response in these animals will be covered in subsequent sections. f. Summary. The studies on the cyclostomes and the cartilaginous and lower bony fishes have indicated that the capacity to form circulating antibody and demonstrate immunologic memory is present in the elasmobranchs and lower bony fishes, may be developed to a slight degree in the lamprey, and is not present in the hagfish. Thus, the capacity for this particular adaptation appears to have evolved in the fishes of the Devonian period. Correlation of this response with the evolution of the lymphoid tissue will be discussed later. Knowledge of the developmental sequence involved in this large step of evolution, namely the establishment of all the different forms of immunologic reactivity, may be filled in by further study of the lamprey and the primitive fishes, including representatives of the actinopterygians or crossopterygians. A less pleasing alternative may be final burial of these evolutionary steps with the fossil forebears of the recent fishes, placoderm-like forms. 4. Churacterization of Antibodies and y-Globulins The y-globulins in mammals are now known to include a heterogeneous group of serum proteins. Details of nomenclature and electrophoretic behavior have recently been reviewed by Fahey (1962). Designation of a protein as a y-globulin merely on the basis of its migration toward the cathode at pH 8-9 in an electrophoretic pattern is at best tenuous, without further physicochemical characterization. This would hold particularly fcr the lower vertebrates, where none of the correlations of chemical properties with antibody activity exist as they do in mammals and avians. Nevertheless, electrophoretic analysis under similar conditions can provide an initial guideline and can indicate the species in which further physicochemical studies are warranted. Engle and Woods ( 1960) have reviewed electrophoretic analyses of invertebrate and vertebrate serum proteins. In original studies by these authors (Engle and Woods, 1957; Woods and Engle, 1957; Engle et al., 1958), none of the invertebrates gave evidence of proteins migrating with the characteristics of y-globulin in human sera, whereas eight species of elasmobranchs all had bands which behaved electrophoretically like

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ROBERT A. GOOD AND BEN W. PAPERMASTER

y-globulin. Preliminary studies on immunoelectrophoretic characterization of serum proteins in cyclostomes have recently been completed ( Papermaster et al., 1 9 6 2)~. Immunoelectrophoretic studies of hagfish sera indicated that no bands in the regions associated with y-globulin migration in human serum could be detected (Fig. 3). In the lamprey, a single band migrating with the mobility characteristic of p- or fast y-globulin in mammals was found in occasional sera; further chemical studies of this material are being made. In the holostean fish, Amia calua, bands with the immunoelectrophoretic properties of y-globulins were clearly demonstrable. These results on the serum proteins of the hagfish support the data on their lack of antibody-forming ability. The occurrence of y-globulins in amphibians has been reported by Evans and Horton (1961) and by Elek et al. (1962), and in reptiles by Baril et al. ( 1961). Other electrophoretic studies of higher vertebrate sera are summarized by Engle and Woods (1960). Recently, Uhr et al. (1962a) have studied the antibody produced by teleost fish, frogs, and chickens to the coliphage +X 174. In all three species, the antibody activity was found in electrophoretic fractions corresponding to human y-globulin. A shift from a component which sediments rapidly in the ultracentrifuge (19s) and is mercaptoethanol sensitive, to a slowly sedimenting ( 7s ) mercaptoethanol-resistant fraction occurred in the course of immunization in the chicken. In both the goldfish and the frog, however, phage neutralizing activity from both early and late sera was lost following treatment with B-mercaptoethanol. Studies on the sera of the elasmobranch guitarfish (Rhinobatos productus) used in our experiments, shown to have Tz phage neutralizing activity and antibody to hemocyanin following immunization with these antigens, have been performed by Grey (1963a). Grey’s study indicated that the globulin antibody was 2-mercaptoethanol sensitive and sedimented rapidly as 19s. The antibody to hemocyanin could be specifically localized in the cyptoplasm of lymphoid cells from spleen imprints by the fluorescent antibody method. As with the ontogenetic development of y-globulin in mammals, in which a shift from 19 to 7s y-globulin occurs during the neonatal period (see section on ontogeny) , the most primitive form of y-globulin of proved antibody activity in phylogenetic development may also be the 19s type, as seen in the guitarfish. Continued study of the y-globulins in elasmobranchs and other primitive fishes may provide worth-while information on the composition and

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FIG. 3. Immunoelectrophoresis of serum from hagfish (top), lamprey, Amla calua, and immunologically normal human being. The increasing complexity of the y-globulins is evident in the higher forms. (From Papemaster et al., 1984.)

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ROBERT A. GOOD AND BEN W. PAPERMASTER

evolution, not only of the y-globulin components, 19 and 7S, but of y-globulin subunits. The clarification of the sedimentation properties of lower fish y-globulins may indicate that the ontogenetic pattern of antibody formation recapitulates a phylogenetic sequence at the molecular level. Support of this possibility could well derive from studies of the subunit developments in both ontogeny and phylogeny. The specificity of lower vertebrate antibody and its ability to distinguish small structural differences in hapten groups compared to mammalian antibodies should also be studied in more detail. Our phage neutralization studies indicated that bowfin and guitarfish antisera would not neutralize antigenically unrelated phage. Further studies to clarify the limits of resolving power with lower vertebrate antibody would be of interest, since clues for the evolutionary development of the pattern for active site assembly may result. Subunit analysis might also clarify the nature of peptide chain assembly, in that primitive vertebrate yglobulins might differ altogether in the types of peptide chains they exhibit, or give evidence of a simpler chain structure that is retained along with further chain addition, as in hemoglobin evolution (Manwell, 1958; Ingram, 1962). 5. Antigenicity in Lower Vertebrates Bacterial antigens uniformly stimulate agglutinating antibody production in elasmobranch and teleost fishes, and in amphibians and reptiles. Bacterial and other viruses also appear to induce production of neutralizing antibody. Precipitating antibodies are rarely observed: to the present time the only precipitating antibody we have observed in lower vertebrates has been antibody to hemocyanin in the holostean bowfin. Numerous attempts have been made by us and by Grey (1963b) to stimulate fish with BSA, with and without emulsification in Freund's adjuvant, but no precipitating or hemagglutinating antibodies or antigencombining globulins have been demonstrated. BSA continues to circulate for more than 30 days when administered to elasmobranchs, teleosts, and frogs. Similar results with BSA in turtles were obtained by Thorbecke (1961). These findings may mean that BSA is not very antigenic in the lower vertebrates, or that the animals are easily paralyzed with this protein. They may also reflect the relative insensitivity of methods for detecting small amounts of antibody to serum proteins when compared to the bacteriophage neutralization technique; however, it seems most likely to us that they reflect the inadequacy of serum proteins as antigens, perhaps related to differences in particulate size of these proteins compared to viruses and bacteria. Anaphylactic reactions to whole serum

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in teleost i&h were reported by Dreyer and King (1948), as will be described in the next section. Small doses of bacteriophage and bacteria appear to be less effective in stimulating antibody production in fish and frogs than in mammals and birds. We were able to provoke what we interpret as secondary responses to TS phage in elasmobranchs and teleosts with doses of 10'l to 10l2 phage particles, whereas Uhr et al. (1962a) were not able to elicit a secondary response in goldfish with 1O1O (pX174 phage particles. With each species a very wide dose range must be used to evaluate antibodyproducing capacity. 6. Immediate and Delayed Sensitivity

Early studies on anaphylaxis in the lower vertebrates were reported by Friedberger and Mita ( 1911) . Goodner, in 1926, reported unsuccessful attempts to sensitize frogs to sheep serum, and attributed failure to differences in smooth muscle innervation in the frog. The Schultz-Dale phenomenon could not be demonstrated in frog smooth muscle. Some reaction could, however, be demonstrated in the heart after intracardiac injection of small amounts of sheep serum. Downs (1928) produced active anaphylaxis in turtles by injection of mammalian serum proteins. Anaphylactic reactions of the affected heart included a decrease in the rate and force of auricular and ventricular beats. More critical studies were reported by Dreyer and King, in 1948. One immunizing injection of horse serum was given intraperitoneally to a variety of common teleost fish (perch, sunfish, rock bass, and goldfish) held at 18°C. Anaphylactic sensitization was indicated by fanning of the dorsal fin, increased excursions of the gill clefts, and sinking to the bottom of the container with the dorsal fin contracted against the body within 10 minutes of the second injection of protein. The reaction was specific for horse serum, and could be diminished by epinephrine. In the frog, no evidence of anaphylactic sensitization could be demonstrated using similar immunization procedures. We are aware of no previous literature dealing with delayed sensitivity in the lower vertebrates. In our own laboratories, several preliminary investigations of delayed reactions have been undertaken. In the hagfish, no evidence of delayed reactions to BCG or old tuberculin could be demonstrated, despite repeated attempts at sensitization ( Papermaster et al., 1962~).In Amia calva, the dogfish, a well-defined indurated area could be detected in the axial musculature, the skin of the sides, and on the upper lip, 3 days after challenge with Ascaris antigen. The challenging dose was administered 30 days after initial sensitization with Ascaris

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ROBERT A. GOOD AND BEN W. PAPERMASTER

antigen in complete Freund’s adjuvant. Reactions of this type were not observed in nonsensitized controls. Induction of delayed sensitivity in chickens was recently reported by Warner and Szenberg ( 1964). Comparative studies on immediate and delayed sensitivity reactions are meager, and interpretation is difficult. Anaphylactic reactions are variable in mammals, and even more so in the lower vertebrates. Such factors as temperature, length of time for immunization, amount of antibody produced, and the nature and extent of the inflammatory reaction need to be evaluated in the interpretation of hypersensitivity reactions in lower vertebrates. The present studies represent only a beginning in this important area. 7. Transplantation Zmmunity Although the literature on homotransplantation in mammals and avians is extensive, comparatively few detailed studies have been performed with poikilothermic vertebrates. The early investigations were conducted to elucidate patterns of inductive tissue interaction and were not concerned with tissue incompatibilities. A complete compilation of the early work is included in the monograph by h e b (1945).More recent reviews of comparative transplantation are available in several of Hildemann’s papers (Hildemann, 1957,1962;Hildemann and Haas, 1959, 1962) and an article by Favour ( 1958). Quantitative studies on scale homograft reactions were reported by Hildemann in 1957 and 1958 on the goldfish, Carassius uuratus. The highly significant observation contributed by this study was the more rapid breakdown of second-set scale homografts in sensitized recipients, as measured by the median survival time and the inflammatory reaction. Thus, rejection of scale homografts in this teleost species was shown to be unequivocally immunologic in nature. Although rejection was slower in fish kept at lower temperatures, significant differences in rejection time of first- and second-set grafts were still evident at 10°C. Genetic studies of histocpmpatibility differences in teleost fish were described by Kallman and Gordon (1958).Fins were grafted in inbred strains of platyfish (Xiphophorus maculatus) at temperatures varying between 21 and 27°C. Isografts and parent to Fl hybrid grafts were completely accepted. The patterns of rejection of grafts from F1 hybrid to parent and from parent to Fz were similar to those established for mammals. Thus, where genetic diiferences existed, fin grafts were rejected, the interval between grafting and rejection varying from 3 days to 3 months, depending on the genetic relationship between donor and host.

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The ability to reject homografts correlates well with the development of other immune mechanisms. Moreover, the homograft reaction can be inhibited by metabolic antagonists in teleost fish (Goss, 1961) as in mammals (Meeker et al., 1959). Many of the earlier contradictory reports on homograft reactions in amphibian larvae can now be interpreted in the light of the data of Hildemann and Haas (1962)on the ontogeny of the homograft reaction in bullfrogs. In their studies larvae were tolerant of homografts to about 40 days posthatching; this was followed by a transitional period of several days, and finally establishment of well-developed rejection mechanisms. The median survival time in the species studied, Ram cutesbeianu, at a temperature of 25"C., was 12-14 days for h t - s e t homografts and 5 days for second-set grafts. There was approximately a threefold prolongation of rejection time when the temperature was maintained at 15°C. In a study by Erickson (1962)skin graft rejection did not develop as rapidly in the salamander, Triturus uiridescens, as in the fish and anurans studied by Hildemann and Haas. Survival times averaged 23 days for primary skin grafts and from 9 to 17 days for second-set grafts. The variation in graft rejection times among various vertebrate species is influenced by temperature and degree of immunologic maturation at the time of grafting. However, as indicated above, intraspecies antigenic polymorphism may account for the largest differences. Compared to rabbits and random-bred mice, adult Syrian hamsters reject homologous grafts slowly over periods of months ( Billingham and Hildemann, 1958). Likewise, graft rejection in Triturus is slower than in the anuran species studied. Studies with isolated bullfrog populations, nearly all of whose skin grafts were rejected, indicated that every tadpole had at least one isoantigen different from those occurring in any other tadpole (Hildemann and Haas, 1981). Such studies are amenable to population genetic analysis (Owen, 1959); such a study was recently performed on mice by Owen (1962). The subject of sexual dimorphism and its relationship to histocompatibility reactions of amphibia has been discussed by Pizzarello and Wolsky ( 1960). The evolution of the homograft reaction may or may not follow the same pattern as the antibody response: the evidence is not yet complete. The studies on invertebrates, discussed in an earlier section, indicate that recognition of allogeneic tissue as antigenic is not characteristic of this group. We attempted to study the homograft response in the hagfish. Adequate evaluation of skin transplants was prevented by failure of

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ROBERT A. GOOD AND BEN W. PAPERMASTER

both autografts and homografts to heal properly (Papermaster et al., 1982c), as in the studies of Triplett et d. (1958) in Dendrostornum. Implantation of presumable allogeneic liver failed to provoke an inflammatory reaction; however, these studies are not complete, and a final evaluation of the homograft reaction in the hagfish is dependent on further experimentation. Similar studies on lampreys, elasmobranchs, and holostean fishes should be completed.

8. Evolution of the Lymphoid Tissue Present evidence suggests that the evolution of lymphoid tissue as well as the specific immune reaction takes place within the vertebrates. Although a precise definition of the lymphocyte in functional terms is not yet available, the phylogenetic origin of lymphoid cells can now be sketched on the basis of recent experimental studies. a. Hematogenous Organs and Evolution of the Spleen. As in the developing mammalian embryo, erythrogenic cells are the first hematogenous cells to be encountered in the lowest vertebrates, as described by Jordan (1938) and Jordan and Speidel (1930). In hagfish peripheral blood, large nucleated erythroid cells are the most nbmerous cell type. Occasional cells with granules, and with bilobed or multilobed nuclei, are presumed to be protogranulocytes. Another distinctive cell type is the spindle cell, thought to be a thrombocyte. A smaller round cell, with a thin rim of basophilic cytoplasm, reminiscent of the lymphocytes of higher vertebrates, is seen in the hagfish. Jordan (1938) described this cell as a lymphoid hemoblast, and suggested that it is a primitive precursor of the other blood cells. In the absence of studies with isotope labels and other markers, conclusions about the interrelationships of the blood cells in this species must remain tentative. However, the failure of immunologic response, the apparent lack of lymphocytopoietic centers, and the absence of other cells resembling the lymphoid cell line of higher vertebrates suggest that the small round cell of the hagfish peripheral blood may be a precursor of the erythrocyte or thrombocyte cell lines rather than a lymphocyte as we conceive of this cell in mammalian blood. It is clear, from extensive studies of the hagfish involving vigorous efforts at immunization with a variety of antigens, that it lacks cells morphologically identifiable as being of the plasma cell system. Further evidence of the hagfish's lack of a lymphoid cell system comparable to that of higher forms is derived from examination of the hemocytopoietic tissue in the submucosa of the gut tract and liver. The gut tract accumulation of cells represents the most primitive state of splenic

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development. These cells, budding from the vascular endothelium in the gut and liver, are the main source of the primitive erythroblasts of the peripheral blood; however, there is little difference in the stages of cellular development observed in the peripheral blood and in these hematogenous centers scattered diffusely throughout the length of the gut tract. Most significant from the immunologic standpoint is the absence of focal accumulations of lymphocytes anywhere in the hagfish-a conclusion we reached after thorough examination of sample serial sections through these animals. In the lamprey, there is an advance of splenic organization into a central hemocytopoietic organ. The epithelium forms a fold inward and surrounds the hemocytopoietic tissue more or less as a pedicle in the anterior gut. This is true of the larval lamprey and recently transformed young adult; but in the spawning lamprey, the gut tract spleen has been replaced with fibrotic tissue, and the hematogenous tissue is found in the cartilaginous vertebral arch. This represents the most primitive beginning of intramedullary hematopoiesis. No clear lymphoid follicles or aggregates have been seen in these hematopoietic centers; however, the lamprey's peripheral blood contains cells which appear to represent a lymphoid developmental sequence. In the elasmobranch and holostean fishes, and in all higher vertebrates, the spleen, in addition to being erythropoietic and granulocytopoietic, also contains focal accumulations of large, medium, and small lymphocytes, and, after antigenic stimulation, basophilic cells resembling the plasma cell series. There is red and white pulp in the spleen of elasmobranchs and bony fishes, but the follicular accumulations of lymphoid cells are not as well organized as in chickens and mammals. Thus, the mere appearance of splenic tissue in evolution does not correlate with the evolution of adaptive immunity, but the appearance of organized lymphoid tissues in the fishes above the cyclostome level correlates very well with the presence of immunologic reactivity (Papermaster et ul., 1963). This relationship becomes even more obvious after a consideration of the phylogenetic development of the thymus and its role in lymphocytopoiesis. b. Evolution of the Thymus. In contrast to the paucity of studies on the immune response, hemocytopoietic tissue, and blood cells of lower vertebrates, is the abundant literature on the comparative anatomy, embryology, and histology of the thymus up to the 1930's, stimulated by the intense interest in the thymus as an endocrine organ around 1900. Studies on the phylogeny of the thymus have been reviewed by Maurer

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ROBERT A. GOOD AND BEN W. PAPERMASTER

(1906), Salkind ( 1915), and Hammar (1936). Miiller ( 1871) found no evidence of definitive thymus tissue in the protochordates and hagfish. His observations were confirmed by Cole (1906) and Stockard (1906) in their studies of the hagfish thyroid. In the lamprey, there is no clear indication of development of a lymphoid thymus, but a primitive epithelial thymus seems to exist (Salkind, 1915). Our own studies on the hagfish and on larval and adult lampreys have confirmed the findings of these early investigators. An epithelial thymus is evident in the early embryonic thornback ray, but in newborn rays the thymus is lymphoid and remains SO, as was demonstrated by Beard in 1894 and 1900. Studies on the thymus of teleost fish were reported by Maurer (1886) and Hammar (1908). All species of teleost fish studied possess a clearly definable lymphoid thymus ( Salkind, 1915). A relatively complete study of the development of the thymus in the dogfish, Amia culua, was reported by Hill (1935). Investigations on the development of the thymus in amphibia before, during, and after metamorphosis were first reported by Maurer (1887) and extended by others (Rogers, 1918; Baldwin, 1918; B. M. Allen, 1920; Hoskins, 1921; Choi, 1931a,b). Hammar (1936) reviewed anatomic studies of the thymus of reptiles, avians, and mammals. Earlier reviews were those of Salkind (1915) on fish, reptiles, and avians, of Jolly (1923) on avians, and of Maximow (1909) on mammals. In those fishes in which'the thymus is unequivocally present as a lymphoid organ, elasmobranchs and all higher fishes, it is usually associated with every gill pouch, where it develops in a position dorsal to the gill arch epithelium. Examination of our own material has confirmed the histologic observations of Beard ( 1894). A well-developed cortex and medulla were noted in the thymus above the gill arches in the day-old guitarfish (Rhinobatos productus), a common California ray referred to earlier. A similar situation exists in Amia culuu and the higher bony fishes. In amphibians, during the larval period, the thymus is intimately associated with the gill arch, but during metamorphosis, these form more compact bodies (Maurer, 1886) and are more like the structures encountered in higher vertebrates, avians, and mammals. In reptiles and avians, the thymus is a lobulated mass of tissue running the length of the neck, bilaterally, whereas in mammals the thymus is a pair of glands in the anterior, ventral part of the thorax. The thymus may, however, be cervical in mammals, as in the guinea pig, and cervical and thoracic bodies may be present. In other instances, as in man, the thymus may be primarily thoracic, but ectopic thymus tissue may reflect the complex embryologic origin in the gill pouches of the embryo.

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9. The Vertebrate Immunologic System and the Cellular Basis of Adaptive Immunity

a. Vertebrate Zmmunologic System. The foregoing discussion has emphasized the difference between the protective mechanisms of invertebrates and vertebrates. That the invertebrates have ample means of protection from saprophytic and pathogenic microorganisms is evident, particularly from the studies of Cameron (1932, 1934). These protective devices consist primarily of phagocytosis and encapsulation; however, humoral bactericidal substances are also a protective adjunct, and have been most extensively investigated in Lepidoptera (Bernheimer et al., 1952; Briggs, 1958).Surviving infection in the invertebrates depends on the outcome of the host-parasite relationship just as it does in mammals; and, from studies reported thus far, the invertebrate host seems to be no less successful than the mammal in the outcome of infection. The evolution of the vertebrate immunologic system, if it is considered in a broader biologic perspective, presents a more complicated picture. Even a superficial examination of the reactive elements in an advanced vertebrate’s immunologic armamentarium discloses that all or nearly all the invertebrate mechanisms are retained in some form. Histiocytes and macrophages in tissues, and polymorphonuclear leucocytes of the peripheral blood actively phagocytize foreign particles. The distribution and function of these cells was quite completely described by Aschoff (1924) who termed the functioning elements the reticuloendothelial system. Most authors have also included immunologically competent cells as part of the reticulo-endothelial system in mammals, and justifiably so. As Thorbecke and Benacerraf (1982) and Biozzi et al. (1960) have shown, the phagocytic uptake of bacteria is enhanced in the immune host. Also, in the in uitro system of antibody production under study by Fishman (1959, 1961), phagocytic elements seem to play a key role in initiation of antibody synthesis. The normally occurring bactericidal substances in mammalian serum have also been studied carefully; a complete discussion of these is found in the review by Skarnes and Watson (1957). Thus, with the evolutionary legacy from the invertebrates consisting of both humoral and cellular components, we may usefully inquire into the reasons for the development of immunologically reactive cells in the more advanced vertebrates. b. Immunocytes (Lymphoid Cells). In earlier portions of this review, the lymphoid cells of the vertebrates above the level of cyclostomes have been deaIt with as a group, with little consideration of

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specific cell types. As has been noted, present knowledge of these cells is inadequate: nomenclature, based on morphology, is controversial, and functional markers for lymphocytes are lacking. Most morphologists agree that lymphocytes can be classified as small, medium, or large, on the basis of cell diameter, and nuclear and cytoplasmic morphologic features. Another cell of the lymphoid series, of obvious importance as an immunocyte, is the plasma cell, the antibody-secreting cell. The relationship of lymphocytes and plasma cells has been discussed in previous reviews by Sundberg (1955,1960) and Good ( 1957a). Recently, Gowans (1962) has completed experiments on the transformation of the small lymphocytes to the immunocyte system in immune reactions in rats. By using lymphocytes labeled with Hs-adenosine, Hs-thymidine, and HSleucine, and chromosome marker differences between rat and mouse cells, he has shown that the small lymphocytes can transform, directly or by division, into cells with the morphologic appearance of young plasma cells. Production of a thoracic duct fistula, with subsequent depletion of the circulating small lymphocytes, removes the ability of the deprived animal to give a primary response to antigenic stimulation ( McGregor and Gowans, 1963). In bullfrog larvae, Hildemann and Haas (1962) were able to correlate the ontogenetic development of homograft immunity with the appearance of small lymphocytes in the peripheral blood. In chickens, both Terasaki (1959) and Szenberg and Warner (1961) have found large lymphocytes to be the reactive cells in the graft-versus-host reaction. Species differences, notwithstanding conflicting studies on the nature of reactive immunocytes, will likely be resolved when the entire cycle of lymphocytopoiesis and lymphocyte maturation is understood more completely. The inclusion of the thymus in this process has been discussed in the section on ontogeny. At present, the following concepts appear to be consistent with experimental data. Antigens in the form of cells, particulate macromolecules, or smaller soluble polysaccharides are taken up by macrophages in the reticulo-endothelial organs (Campbell and Garvey, 1960) and perhaps by lymphoid cells directly. At some stage of their development, lymphoid cells-presumably small lymphocytes in most species-become sensitized to antigens and mature into antibody-producing cells or participate directly in cytotoxic reactions. The advance in vertebrate immunologic evolution appears to lie in the ability of certain cells, presumably lymphoid cells, to discern antigenic structural differences. The studies quoted above have indicated that this ability most likely became manifest early in the evolution of

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the primitive fishes. The association of the thymus with this process was also emphasized. Recently, Triplett ( 1962) has demonstrated that the immunologic recognition process can be analyzed in the ontogeny of amphibian vertebrates. In his experiments, removal of the adenohypophysis at the early larval stage, propagating it in allogeneic embryos and re-implanting it at a later stage of maturation in the host of origin led to rejection of the organ. We would suggest that the development of immunologic recognition in this species parallels the development of the thymus in the early larva. The change from a state of tolerance to recognition of antigenic properties may reflect the maturation of the thymus from a primarily epithelial organ to a lymphoid one. Triplett’s experiments illustrate the usefulness of these lower vertebrates with prolonged maturation periods in analysis of problems in developmental immunobiology. The use of amphibians in the ontogenetic studies of the cellular basis for immunologic recognition is now well established as a result of this work of Hildemann and Haas and Triplett. A concerted effort in these animals, with more detailed analysis at the morphologic level, would appear to be worth while in efforts to establish the reactions of various cell types to a variety of antigenic stimuli during development. c. The Vertebrate Immune Response and Tumor Immunity. A more refined immunologic recognition system is clearly evident in the evolution of higher vertebrates. We can hardly afford to restrict the scope of immunologic inquiry to the classical concept of infectious diseases and protection mechanisms, when the infectious nature of nucleic acids from viruses and tumor cells has been clearly demonstrated (Holland et al., 1959; Ito and Evans, 1961) in intact mammals and mammalian cells cultured in oitro. A recent report (Johnstone, 1962) collecting current information on tumor immunity provides evidence from a number of laboratories that tumor cells may be antigenically different from normal somatic cells. Antigenic differences between methylcholanthreneinduced sarcomas and normal host tissues have been reported by Prehn and Main ( 1957) and by Klein et al. ( 1960). Tumors induced by polyoma virus contain antigens not found in normal cells (Habel, 1962). Recent studies by H. Rubin (1961, 1962a) on the immunologic reactions to Rous sarcoma in chickens indicate that birds made tolerant to Rousassociated virus were susceptible to progressive tumor growth. In further studies, H. Rubin (1962b) was able to relate the limitation of tumor spread in d u o and the destruction of tumor cells in vitro directly to the cytotoxic activity of lymphocytes, With the evolution of definitive organ sites for specialized functions

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and the concomitant development of appropriately differentiated cells, a process for the sorting out and maintenance of cell structure might have evolved concurrently. Malfunction of differentiated cells in vertebrates is often characterized by tumorous or malignant growth. Balls (1962) has reviewed the types of cancer in amphibians. Huxley (1956) has indicated that metastatic tumors of a lethal nature, comparable to those in vertebrates, are poorly documented in invertebrates. The publication of Scharrer and Lochhead (1950) suggests that tumors may occur in invertebrates, but they are unusual and have often not been clearly separated from the inflammatory and walling-off reactions in these animals. We are aware of no reports of malignancy in cyclostomes. Thus, lymphoid cells, and an organ associated with their formation and control, the thymus, may have evolved to subserve the peculiar function of “policing somatic cell surfaces by detecting antigenic changes. Such surface changes may herald the formation of a cell line no longer susceptible to aggregational and other ordering forces associated with the differentiation and maintenance of normal organ structure. Immunologic regulation of somatic cell changes, and subsequent malignant development, as a result of mutation or virus cell transformation, has been suggested by Thomas (1959) and Burnet ( 1962). Indeed, adaptive immunity in vertebrates may have evolved as a mechanism for facilitating tumor cell recognition and disposal. The finding of Prehn and Main (1957) that tumors which overcome the host’s natural defenses and thus arise spontaneously in mice do not have recognizable antigenicity would be compatible with this hypothesis. IV. Concluding Statement

Without attempting to summarize what are, in reality, already summary statements on the wide range of topics considered under the overall heading of the ontogeny and phylogeny of immunity, we should like, very briefly, to suggest a view of the areas of most significant development in recent years. Surely one of these is that immunologic maturation starts earlier than we supposed, say 5 years ago, and probably continues longer. At least two major factors played a role here: the tendency to think of birth or hatching as more of a break in the continuum of immunologic development than it probably is, and the overgeneralization of findings of studies employing simple protein antigens in the newborn. It is clearly possible, with appropriate antigens, to evoke plasma cell formation and antibody production before birth in a number of mammalian species, including man. The immunologic inertia of late fetal life

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and the early months of postnatal life is real and certainly has important implications for the clinician, but a latent capacity to respond when the stimulus is sufficiently intense is clearly present well before birth in man. The role of the 19s ( fi2Y7 or yla) globulins in t h i s development is particularly significant. The progression of immunologic development well beyond the newborn period, in some experimental animals at least, and probably in mammalian species generally, has also been emphasized, in the findings of the growing immunologic competence of peripheral spleen cells of mice well into adult life, and in the experiments on “immunologic arrest” in mice totally thymectomized well after the neonatal period. A parallel development has been the shift away from the concept of “tolerance responsiveness” as an exclusive function of immunologic immaturity. Age is clearly a relative barrier here, and evidence of development of immunologic reactivity is also observed in development of the increasing barrier to production of specific immunologic negativity as the animals proceed toward maturity. Immunologic negativity, whether to homografts or other forms of antigenic challenge, and whether induced in young or older organisms, seems to be basically the same kind of specific central immunologic failure. The role of the thymus in mammals, and the bursa of Fabricius in chickens, in the development of immunologic competence both before and after birth has placed a new focus on the ontogeny of the lymphoid tissues and the lymphoid system of cells. Data on a wide range of species are accumulating indicating that the thymus is the first organ to show lymphoid development, whether this occurs well before birth, as in man, or shortly before birth or hatching, as in the mouse or chicken, or after birth, as in the opossum. It seems probable that there is no immunologic reactivity of the embryo before there is lymphoid development of the thymus. Both clinical and experimental data are accumulating in support of the thesis that absence of the thymus during the period of development of the peripheral lymphoid tissues results in both abnormal lymphoid development and immunologic deficiency. A consideration of the role of other primary lymphoid tissue, presumably thymusindependent, is just beginning. In phylogeny, the data suggest that the transition to adaptive immunity occurs in the lower vertebrates and that it is paralleled by the development of a lymphoid thymus, other organized lymphoid tissues, and the lymphoid system of cells. However, thus far, the studies of the lower fishes have not been sufficiently comprehensive, and much remains to

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Cellular Reactions in Infection' EMANUEL SUTER AND HANSRUEDY. RAMSEIER2 Department of Microbiology, College of Medicine, Univerrify of Florida, Gainerville, Florida

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I. Introduction . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . 11. Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . , 111. Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . A. Opsonins ............................................... B. Virulence and Opsonization ...... .. ... . ,. .. .. .. . . .. . . .. .. . IV. The Normal Phagocyte in Postengulfment Period . . . . . . . . . . . . . . . . . A. Influence of Particle Ingestion on Metabolic Activity . . . . . . . . . . B. Intracellular Kill of Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . C. Digestion of Intracellularly Killed Bacteria . . . . . . . . . . . . . . . . . . D. Facultative Intracellular Parasites . . . . . . . . . . . . . . . . . . . . . , . . . . V. The Immune Phagocyte . , . .... . .. . .. .. .. . . .. .. .. .. .. . . . . .. .. . A. Demonstrations of Cellular Immunity . . . . . . . . . . . . . . . . . . . . . B. In Vitro Acquisition of Cellular Immunity . . . . . . . . . . . . . . . . . . . C. Properties of the Immune Mononuclear Phagocyte . . . . . . . . . . . . D. Nonspecific Manifestations of Cellular Immunity . . . . . . . . . . . . . . E. Consequences of Residence in Immune Cells . , . . . . . . . . . . . . . . . F. Transfer of Cellular Immunity . . . . . . . . . . . . . . . . .. . . . . . . . . . . . G. Relationship of Cellular Immunity and Delayed-Type Hypersensitivity . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . H. The Immune Cell in Reactions to Tissue and Cellular Homografts VI. Conclusion . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . References , . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction3

As a consequence of parasitic replication or death and of altered reactivity of the host, the dynamic equilibrium between host and parasite is subject to constant changes. Although some of these changes may be mediated by factors transported in the humors, all are ultimately a consequence of responsiveness of cells. Cellular reactions may be involved primarily or secondarily in host-parasite relations, i.e., either as response to direct stimulation by the pathogen or its products or mediated by factors liberated in the primary reaction. Most of this review will 1 The authors' experimental work quoted in this review was supported by Grant AI-01302-06 of NIAID of USPHS. 2 Trainee supported by 5TI A1 128-02 of NIAID of USPHS. Present address: The Wistar Institute, University of Pennsylvania, Philadelphia, Pennsylvania. 3 The following abbreviations are used: PMN, polymorphonuclear leucocytes; MP, macrophages, monocytes, histiocytes; RES, reticuloendothelial system.

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deal with primary responses; it will be further limited mainly to reactions to foreign materials introduced into the host by infectious agents, such as viruses, rickettsiae, bacteria, protozoa, and fungi. Traditionally, cellular reactions in inflammation are looked upon as a problem of population, namely, the mobilization and accumulation of cells. More recently, reactions studied at the single cell level have become increasingly more important, such as metabolic and physical alterations resulting in a more or less efficient cell frequently referred to as a stimulated cell in contrast to a resting cell. Such studies have been conducted successfully in uitro with established human cell lines, e.g., HeLa cells (Puck, 1981)and lymph node cells derived from antigenically stimulated animals (Nossal and Makela, 1982). It seems essential that similar techniques be developed for the study of RES cells. Immunity and immune reaction mean different things to different investigators depending on past experience and present interest. A definition of these terms, or better the description of a point of view, is therefore in order. In this review the terms “immunity” or “immune reactions” will be used in their broadest meaning encompassing reactions of a higher organism to infection, homologous and heterologous tissue transplantation, and to intentional immunization with soluble antigens or other materials. This implies that immune reactions may be specific and nonspecific and can be, but are not necessarily, dependent on antibody formation. When viewed from this standpoint, phagocytes have distinct and varied functions in immune reactions, although all cells of an organism contribute toward the state of immunity at any given time. As pointed out by Taliaferro (1949), the phagocytes take a central position in these reactions because of their ubiquity by distribution or ability to be mobilized, their content in hydrolytic enzymes and inhibitors, and possibly their multipotentiality exhibited by at least some of the younger forms. Major phases of interactions between host cells and pathogens are the establishment of physical proximity and contact between parasite and host cell, phagocytosis, intracellular interactions between cell and parasite, and events secondary to interactions, such as cellular hypersensitivity and antibody formation. In all steps of this interaction, the final outcome of the response is a resultant of several components. For the sake of convenience or convention, innate or native responses are separated from acquired ones to which specificity is usually attributed. In recent years it has become increasingly evident, mainly as a consequence of experimentation with germ-free and pathogen-free animals, that responsiveness hitherto attributed to native resistance in reality is based on acquired

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reactivity through previous exposure to homologous or heterologous stimuli. It was surprising at first that such acquired responses would be of nonspecific nature in regard to the stimuli involved and thus were independent of the specific immune response. The mechanisms responsible for these changes are essentially unknown, and what can be presented today is an enumeration of facts rather than a real understanding of the phenomena. It is important to note that most agents and stimuli that are known to induce such altered responsiveness to infection also induce changes of the RES. This leads us to consider the RES as a composite of different cells with remarkable adaptability and responsiveness. For these and other reasons, studies concerned with cellular responses to infection should ideally be conducted comparatively in germfree, pathogenfree, “normal,” and artificially infected animals. The materials and techniques for such studies are available but have not yet been fully exploited. Thus, our judgment as to the line of demarcation separating innate and acquired responsiveness is based mainly on tradition. In this review, innate resistance or immunity is defined as the level of resistance in the absence of any manipulations intended at specific or nonspecific increase of such resistance. By contrast, acquired immunity encompasses qualitative and quantitative changes of resistance as a result of,previous experience with homologous or heterologous agents and antigenic materials. Since a number of reviews have recently been written on the subject of phagocytosis and its role in infection (Suter, 1956; Hirsch, 1959; Rowley, 1962), historic completeness is not attempted. It is rather intended to emphasize in this review postphagocytic events and to evaluate the evidence in regard to cellular immunity and the role of phagocytes in the immune response. Since cellular events of specific nature have been implicated in the host’s response to homograft and tumor, a short section has been added on this subject. For want of space, this latter part lacks the thoroughness attempted in the review, but it should suffice to point out some of the similarities of reactions and problems. II. Chemotaxir

A major prerequisite for homeostasis is the recognition of foreign material and of degraded or effete tissue constituents leading to their ultimate elimination. In the case of molecules or particles beyond a size allowing clearance by the glomerulus, phagocytosis represents the first step toward elimination or sequestration. Therefore, one may assume that

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recognition of the foreign by phagocytes is an essential part of their total functional capacity. If this assumption is correct, it logically follows that phagocytes must also be involved in the first phase leading to antibody formation, Although discrimination between self and foreign is a much talked about subject, factual knowledge concerning the molecular basis of this vital function of some cells is lacking. Recognition and discrimination must occur on or near the surface of susceptible cells and requires contact between the material to be recognized and the cell. Contact may be at random as between particle or molecule in the blood and circulating leucocytes or sessile elements of the RES lining such strategic areas in the vascular bed as sinusoids of liver, spleen, and lymph nodes. In tissues, however, contact with motile phagocytes is enhanced by directed migration, so-called chemotaxis. In view of the above-outlined role of phagocytes in homeostasis, chemotaxis may represent one possible discriminatory mechanism. However, it should be noted that many nonantigenic materials exhibit strong chemotactic activity ( McCutcheon, 1946). A systematic study of chemotactic potency of polysaccharides of bacterial, plant, and animal origin was undertaken by Meier and Schar (1957). Human buf€y coat explants in plasma, which form a symmetric, circular halo of migrated cells within 8 to 12 hours, were exposed from one side to the influence of the material to be tested. In case of positive chemotaxis, cellular migration would become asymmetric with more cells accumulating toward the chemotactically active agent, whereas negative chemotaxis resulted in a migratory defect opposite the agent. In general, extracts from gram-negative microorganisms were far more active than those from gram-positive ones, Since the various extracts were not prepared by the same methods, exact quantitative comparison is not possible, especially when considering the chances of contamination of tissue polysaccharides with bacteria and their products (Merler et al., 1980). The fact that substances lacking any antigenic potential or degree of foreigness can have a strong chemotactic effect, indicates that chemotaxis is not limited to a specialized function, such as recognition, but represents general expression of leucocyte activity. Chemotactic agents influence in an unknown manner cellular movement over surfaces independent of their chemical nature and antigenic relationship to the host or the cell. It seems well established that chemotaxis requires the presence of Ca and complement, or at least some component of fresh serum which can be removed by specific antigen-antibody complexes (Delaunay and Pages, 1946). Using a rather ingenious technique, Boyden (1962a) investigated the

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chemotactic effect of antigen-antibody mixtures on the migration of PMN through Millipore filters. It was found that antigen-antibody complexes at equivalence could elaborate in presence of fresh serum a soluble heat-stable factor which is chemotactic by inducing migration of cells through the filter pores. Boyden’s interpretation of these findings calls for a relatively involved chain of reactions triggered by the antigenantibody complex activating a heat-labile serum constituent to a reactive enzyme. The latter, in turn, converts a precursor to a heat-stable chemotactic agent, The actual participation of such components of this system has to be proven; nevertheless, the hypothesis that antigenantibody complexes exhibit chemotaxis offers an approach to a puzzling problem. It is conceivable that C’ is identical with the heat-labile system leading to the formation of the chemotactic material. Furthermore, the fact that chemotaxis appears dependent or enhanced by antibodies is of great significance for our understanding of the development of the lesion in the Arthus reaction (Cochrane et al., 1959) and of the various manifestations of serum sickness (Dixon, 1962). Boyden (1982b) also found that macromolecules have greater chemotactic activity the further they are removed phylogenetically from the species from which the test phagocytes are derived. Even insoluble substances, such as cellulose, are active; and the activity can be removed if the test is performed with serum which has been absorbed with cellulose, although other products retain their activity in the same serum, In the normal animal, then, recognition by chemotaxis would be mediated by “natural antibody.” The importance of chemotaxis in the inflammatory response has recently been questioned ( Spector and Willoughby, 1963). Multiple factors are implicated in this process, such as increased vascular permeability to protein and specific changes in vessels and leucocytes. It is difficult to quantitate the relative importance of each contributory factor, which is subject to great variations. Free phagocytic cells are supposed to exhibit random motility. Chemotaxis influences the randomness resulting in directed migration ( McCutcheon, 1946). It is difficult to assess the evidence for the randomness of cell motion since all tests involve the use of artificial surfaces. It is conceivable that surfaces such as glass or plastic exert a multidirectional chemotactic effect resulting in apparent randomness of motion. The mechanisms of ameboid movement are far from being elucidated, although numerous hypotheses have been offered, such as the fountain-zone contraction theory (Allen, 1961) and a theory explaining movement on the basis of electrical potential gradients within the cell maintained by active ion transport across the membrane ( Bingley and Thompson, 1962 ) .

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The mode of action of chemotactically active substances, either primary or formed secondarily upon interaction of antigen-antibody complexes with fresh serum, is completely unknown. It is hoped that further investigations will yield answers to foremost questions: ( I ) Is chemotaxis an essential contributor in initiating effective cellular reactions to microorganisms and other antigenic or foreign materials? (2) Is Boyden’s activating system of general significance pertaining to a large proportion of chemotactic phenomena? ( 3 ) Is chemotaxis due to the absorption of an inhibitor rather than the induction of a stimulator in a negative gradient toward which the cells move, as suggested by McCutcheon (1955)? I1I. Phagocytosis

Whatever the mechanisms assuring physical proximity of foreign materials with phagocytes, ingestion of the particle is the most important step in establishing contact between antigen or particle and active compartments of the cell. The consequences of this physical approximation are varied and concern the physiology of the host cell as well as the fate of the microorganism or its antigenic components. The reactivity of cells and of the host as a whole is frequently altered after such an encounter at the intracytoplasmic level. Investigations over the past 70 years established beyond doubt the importance of phagocytosis in immune phenomena especially infection immunity of metazoa, and methods of quantitation of the process were developed. The equally important role of phagocytes or their precursors in antibody formution and hypersensitivity reactions has been appreciated only recently. Most studies done in uitro or in uiuo are subject to serious limitations. In uitro systems employing isolated cells either from the peritoneal cavity of animals or from the circulating blood of man have some disadvantages inherent in most studies done outside the body, such as inadequacy of environmental conditions and insufficient homeostasis. However, this approach allows usually accurate quantitation of populations of parasite and host cells and their activities and permits analysis of factors influencing the interactions between the two. In duo studies have the advantage of taking into account factors of the internal environment, but they are limited in quantitation. In general, such investigations have confirmed results obtained from in uitro studies and were highly informative in regard to the significance of these reactions for the animal as a whole. It is not surprising that studies devoted to the question of the outcome

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of the interaction between parasite and phagocytic cells have sometimes resulted in contradictory findings and opinions. Rather astonishing is the fact that general conclusions could be reached in spite of the fact that widely different experimental systems were employed and that many variables influencing or altering host cell-parasite interactions were essentially neglected. Some of these variables are: nature of irritant to elicit peritoneal phagocytes; age and type of leucocytes; age of microbial culture; size of inoculum; cell-to-microbe ratio; microbial heterogeneity; presence or absence of surfaces promoting phagocytosis; duration of experiment; and, most important of all, various opsonizing factors. A. OPSONINS The functional importance of opsonins or opsonic systems in cellparasite interactions is established beyond doubt. The recognition that serum factors enhance phagocytosis helped greatly to bridge the gap between the irreconcilable theories of humoral and cellular immunity of von Behring and Metchnikoff (for references, see Suter, 1956; Hirsch, 1959). In spite of over 60 years of investigation, the exact nature and chemistry of opsonins is still to be determined, and the distinction between natural and acquired opsonins remains a matter of dispute. Evidence indicates that there are several types or systems of serum components involved in opsonization. With few exceptions, the relative importance of any of these factors has not been determined yet. One of these systems is heat labile and appears to be complement involving all four components (Ward and Enders, 1933; Maalge, 1946; Ecker and Lopez-Castro, 1947; Howard and Wardlaw, 1958; Slopek et al., 1960). According to Sterzl ( 1963), C' of piglet serum which does not contain any detectable amount of antibody, enhances uptake of Escherichia coli in the S-phase by the perfused rat liver. All four components of C' seem involved. Salmonella typhimurium is removed only slowly by the RES of the newborn piglet although carbon particles are phagocytized at the same rate as by the adult. Forty per cent of the organisms are found in the spleen, whereas carbon accumulates predominantly in the liver of these animals (Mouton et al., 1963). A second, heat-stable opsonizing factor is associated with y-globulin and is found in normal and immune sera. y-Globulin or the active component associated with it presumably coats the particle with a film of protein, as appears to be the case with salmonella and with bentonite particles (Slopek et aZ., 1980). The latter are easily phagocytized by human leucocytes when coated with human 7 S y-globulin (Potter and Stollerman, 1961), whereby C'l, C'2, and C'4 enhance opsonization (Rytel and Stollerman, 1963). Serum

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or y-globulin is not required in all systems, e.g., polystyrene, but not starch particles, are ingested in absence of serum (Sbarra and Karnovsky, 1959). Apparently the nature of the surface of the particle is important in determining the need for coating; the starch particles being hydrophilic and polystyrene hydrophobic. More recently, a third thermolabile component of human plasma was described essential for phagocytosis of group A streptococci in uitro in the presence of optimal amounts of anti-M antibody. Some plasmas were found deficient in this factor, called “coopsonin,” although they contained normal levels of C’, lysozyme, properdin, and bactericidal potency against gram-negative bacteria (Stollerman et al., 1963). The activity is associated with a protein of a molecular weight over 50,000.It is conceivable that coopsonin is similar, if not identical, with the phagocytosis-promoting factor of Tullis and Surgenor (1956). Further investigations have to reveal the chemical identity and the general significance of this factor. It is of interest in this context that fibrinogen is readily phagocytized upon polymerization. Heparin-precipitable fibrinogen, which represents an intermediate stage of polymerization of fibrinogen to fibrin, is phagocytized at a rapid rate by the RES whereas fibrinogen is not (Lee and McCluskey, 1962). It is likely that upon polymerization the surface of the complex is altered to such an extent as to render the protein susceptible to phagocytosis. These findings are in keeping with earlier findings by Knisely et al. (1948) that coating of inert particles with fibrin has a strong opsonizing effect. Without any doubt specific antibody is a most effective opsonin and, although it is mentioned here in fourth place, its importance was recognized very early. Studies on specific opsonization are hampered by the fact that “natural” antibodies or “nonspecific” opsonins can be present at all times even in germfree animals, either measured in clearance (Thorbecke and Benacerraf, 1959) or bactericidal tests ( Michael et al., 1962). Only small amounts of antibody appear to be required for opsonization in uiuo, e.g., 0.01 pg. antibody N/100 gm. body weight enabled opsonization of l O Q E . coZi-amounts difficult to demonstrate in any other test except passive cutaneous anaphylaxis (PCA) (Benacerraf et al., 1959). For extensive studies on opsonization, Biozzi et al. (1955, l W l ) improved their technique for the measurement of clearance of particles by the RES of the mouse. After determining for 10 minutes the rate of removal of the bacteria under normal conditions the opsonizing factor is injected intravenously and subsequently the new removal rate is measured. By this means each animal serves as its own control. Under these conditions the rate of removal of Salmonella in the normal mouse

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and the opsonizing effect of antiserum vary considerably, whereby the extent of the latter is independent of the former. The opsonizing power of an antiserum is a function of the number of bacteria used for the test, whereas the rate of removal in the normal animal is independent of the number of particles. In the case of SaZmoneZZu the opsonins are identical with antisomatic antibody, and antiflagellar antibodies do not influence clearance (Biozzi et a,?., 1963). Kupffer cells of the liver are more responsive to the opsonizing effects than are the RES cells of the spleen. Finally, the effect of antibody-dependent opsonization is reduced in decomplemented animals, but the loss of activity can be compensated for by increased amounts of antibody containing serum (Biozzi and Stiffel, 1962). Some information as to the mode of action of specific opsonins may be forthcoming from recent investigations on the chemical nature of the protein or portion of the protein responsible for activity. It is quite well established that antibodies of the 7 S variety are powerful opsonins. In addition, Rowley and Jenkin (1962) and Turner and Rowley (1963) demonstrated that the specific opsonizing activity for gramnegative bacteria of pig serum is found in the fhglobulin fraction. Digestion of the y-globulin with pepsin destroys the opsonizing power for red blood cells but leaves partial activity for the removal of E . coZi by the normal mouse (Spiegelberg et aZ., 1963). A 7 S associated opsonin for S. typhimurium retains approximately 50 % of its original activity after treatment with papain under reducing conditions. The remaining activity is found associated with fragment I after purification of the split products on a carboxymethyl-6-cellulose column, The activity is demonstrable in vitro and in uivo (Shands and Suter, 1963). It is difficult, if not impossible, to assign relative importance to any of the described opsonins except under well-defined conditions. Thus, heterologous serum can be opsonizing for one species of microorganisms but not for another, the latter being the case with gram-negative organisms; and heating at 56” C.may either enhance or reduce activity or have no effect at all, as shown with the perfused rat liver by Wardlaw and Howard (1959). These nonspecific opsonins are consumed upon injection into animals of large numbers of biologic or inert particles causing a sharp decrease of the animal’s capacity to remove particles injected subsequently. Such depletion of opsonins may well be the cause of reticuloendothelial blockade (Jenkin and Rowley, 1961) . “Natural antibodies” have been implicated in discriminatory uptake of particles by phagocytes. Thus, Perkins and Leonard (1963) demonstrated that mouse MP engulfed foreign erythrocytes in the presence of mouse serum. Phagocytosis of red blood cells was more extensive, the

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greater the phylogenetic distance between the donor of erythrocytes and the mouse. Consequently, the requirement for specific opsonins was greater for cells from genetically related donors. In similar investigations Vaughn (1963) showed that in vitro phagocytosis of foreign erythrocytes by rabbit PMN is dependent on heat-stable and heat-labile factors of rabbit serum. The latter seems identical with C’. The former is found in the y-globulin fraction and is absorbed specifically by the species of erythrocytes under test without altering the opsonizing power for red blood cells of another species, This is also the case for zymosan, cellulose, and polystyrene particles. This opsonin for foreign cells may be an isoantibody (Bennett et al., 1963). It was also noted that there was a difference in phagocytic capacity between PMN and MP in regard to homologous, effete erythrocytes. The former do not ingest effete red blood cells, whereas the latter do, requiring a factor present in normal serum. This factor is present in normal serum and is found adsorbed on MP (Vaughn and Boyden, 1984). The importance of phagocytosis by MP as a recognition mechanism is indicated in experiments by Nossal and Ada (1964)showing that phagocytosis of flagellin in the regional lymphnode leads to cellular changes resulting in antibody formation. Antigen uptake is accelerated in the immunized and tolerant animal indicating that tolerance does not abolish recognition but prevents postphagocytic events necessary for antibody formation. Similarly, effete red blood cells are phagocytized without subsequent antibody formation. B. VIRULENCE AND OPSONIZATION In many instances the interaction between phagocyte and pathogen is infiuenced by the degree of virulence of a particular type of microorganism for a given host species. The interference of the microbe’s virulence with effective phagocytic kill or elimination can be at the level of phagocytosis or intracellular destruction of the pathogen. Organisms which are highly susceptible to phagocytic antibacterial principles depend for their survival in a host on protection against phagocytosis. Representatives of this category of organisms are the pneumococci and the streptococci. The role of the bacterial capsule in the prevention of phagocytosis has been reviewed by Wood (1960). In general, a specific opsonin is required to overcome this inhibition. Other pathogens are not as readily destroyed after ingestion and either survive intracellularly or even retain their capacity for multiplication. Under these circumstances the relation between virulence and opsonization is less clear, and consequently contradictory findings have been reported. In this category of organisms belong the staphylococci, salmonellae, brucellae, and myco-

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bacteria. They are frequently referred to as facultative intracellular pathogens, by contrast to the obligate extracellular pathogens mentioned above. In presence of normal rabbit serum, the coagulase-positive “Smith strain of staphylococci is poorly phagocytized by rabbit polymorphonuclear leucocytes and survives, whereas the coagulase-negative strain is rapidly taken up and digested by the same cells. Treatment of the virulent strain with specific antiserum increases its rate of phagocytosis and destruction. This indicates that both variants of staphylococci are susceptible to leucocytic antimicrobial substances, although there is a quantitative difference between the two strains in that fewer coagulasenegative staphylococci survive intracellularly ( Cohn and Morse, 1959). Similarly, peritoneal MP of the rabbit were reported to inactivate coagulase-positive and coagulase-negative staphylococci in the presence of immune and normal serum, respectively, although at a slower rate than PMN (Mackaness, 1960). The resistance of the Smith strain of staphylococci to phagocytosis is attributable to an antigen located on the surface of the cocci. It appears to be a complex consisting of carbohydrate, glucosamine, and amino acids ( Morse, 1962) . These findings are somewhat at variance with results obtained by other investigators. The discrepancies, however, are more apparent than real and can be explained by differences of technique, strain used, cell type, and interpretation. Thus, Staphylococcus aureus was found to survive but not to multiply within neutrophiles and monocytes from normal rabbits and within normal human blood leucocytes, whereas Staphylococcus epidermidis was destroyed under similar conditions. Rat monocytes, on the other hand, destroyed both types of organisms equally well (Kapral and Shayegani, 1959). These &dings were in agreement with earlier reports that human leucocytes would phagocytize virulent and avirulent staphylococci equally well, but that a certain proportion of the former would survive ingestion and resume intracellular multiplication ( Rogers and Tompsett, 1952). More recently, the relatively rapid rate of phagocytosis of virulent strains in presence of “normal” human sera has been attributed to a phagocytosis-promoting antibody present in all human sera, which most probably is a specific antibody (Rogers and Melly, 1960). Accordingly, a large proportion of even virulent staphylococci are inactivated, but some survivors resume intracellular multiplication in the damaged leucocyte (Melly et al., 1960). Quite similar conclusions were drawn by Shayegani and Kapral (1962). It has to be assumed that opsonization not only influences the rate of uptake of bacteria, but also the ultimate fate of the organism in the intracellular environment by

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activation of intracellular bactericidal mechanisms, possibly by rapid ingestion of large numbers of staphylococci (Melly et al., 1960). It should be noted in this connection, that antibodies against staphylococci were found in all of 36 nonimmunized, pathogenfree rabbits (Cohen et al., 1961) , Similarly, the presence of precipitating antistaphylococcal antibodies in a large number of human sera has been described (Jensen, 1958; Rogers and Melly, 1960) , although their phagocytosis-promoting property has not been studied. Virulent strains of Salmonella, when opsonized with normal serum, are relatively poorly phagocytized by mouse peritoneal macrophages and multiply after ingestion. Pretreatment of these organisms with immune serum increases rate of phagocytosis and extent of intracellular kill; thus the virulent organism has been rendered avirulent (Jenkin and Benacerraf, 1960). In the presence of normal serum, virulent organisms multiply readily within the cells in uitro, provided the period of observation is extended beyond 4 to 6 hours after phagocytosis, and the phagocytes are eventually destroyed by the bacterial population. There is a correlation between the degree of virulence and the extent of intracellular multiplication and phagocytic destruction, i.e., virulent strains multiply to a greater extent and produce more cell damage than do attenuated or avirulent ones (Saito et al., 1957; reviewed by Ushiba et al., 1959; Mitsuhashi et al., 1961). Intracellular kill of Salmonella typhimurium is supposed to occur rapidly within peritoneal macrophages of mice and to be independent of the virulence of the strain. The difference between virulent and avirulent organisms is manifested in the rate and extent of phagocytosis and the susceptibility to opsonization rather than by intracellular behavior (Whitby and Rowley, 1959; Rowley and Whitby, 1959). A similar correlation between virulence of organisms and rate of removal by the RES was observed in in uiuo studies (Jenkin and Rowley, 1961). Some of the discrepancies of findings by different groups of investigators are explainable by the techniques used and especially by the differences in duration of experiments. Prolonged cultivation usually reveals that a sizable proportion of the phagocytized organisms survives and eventually multiplies, This latter phase of the cell-parasite interaction may well escape observation, if the experiment is terminated prematurely, Of further importance is the influence of opsonizing serum components, especially antibody, on the subsequent fate of the organisms after ingestion. Thus, virulent S . typhimurium are readily phagocytized by peritoneal hlP from rabbits, multiply intracellularly, and lead to destruction of the cell population. If specific antiserum against the 0 antigen is present during phagocytosis, destruction

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of cells is reduced and postponed, although the bacteria retain their ability to multiply (Gelzer and Suter, 1959). Later studies by Jenkin (1963) showed that the enhancement of the rate of phagocytosis alone, e.g., induced by adsorption of bacteriophage, does not alter the ability of the pathogen to multiply intracellularly, but that adsorption of opsonin, especially antibody, may alter the intracellular fate. Both investigations indicate that phagocytosis and intracellular fate are two independent events which may or may not be influenced by humoral factors. As a consequence, great care has to be taken in the interpretation of findings obtained in mixed systems, e.g., mouse MP and pig serum versus homologous serum. Furthermore, recent yet unpublished experiments show that the physiological state of the phagocyte, especially of MP, has to be considered. Thus, peritoneal MP of the mouse require serum opsonins for phagocytosis of S. typhimurium in suspension or when settled on glass immediately prior to the test, whereas the same cells will readily phagocytize organisms in the absence of serum after they have been maintained on glass for a period of 24 to 48 hours. A similar pattern of interaction is found with Brucellu. An opsonizing effect is best demonstrable when working with virulent organisms, and intracellular survival and multiplication appears directly dependent on virulence or smoothness of the strain (Holland and Pickett, 1956; Braun et al., 1958). A less pronounced role is attributable to opsonization in the interaction between mycobacteria and phagocytes. Virulent and avirulent variants of Mycobacterium tuberculosis are equally well phagocytized by PMN and by MP (Bloch, 1948; Suter, 1952), although serum opsonins may accelerate the uptake of both variants. However, Skurski et ul. (1957) reported that an attenuated strain, BCG, was much more readily phagocytized by horse leucocytes than a virulent strain, H3,Rv. Fresh serum, even when lacking specific antibody, would greatly enhance the uptake of either strain. A difference between virulent, attenuated, and avirulent strains is detectable by following the fate of mycobacteria after ingestion by MP. Virulent organisms multiply and cause cell destruction, whereas the avirulent variant does not. The attenuated group of organisms take some intermediary position by being able to multiply but causing little or no damage to the host cells (Suter, 1952; Mackaness et al., 1954).

IV.

The Normal Phagocyte in the Postengulfment Period

Phagocytosis leads to intracellular location of particles, contained within a vacuole, which is frequently referred to as phagocytic vacuole or phagosome. This vacuole is bordered by a membrane, presumably

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derived from the cytoplasmic membrane. Sequestration of the phagocytized particle has been demonstrated in many instances, e.g., staphylococci within human leucocytes (Goodman and Moore, 1956), Mycobucterium lepmemurium in spleen MP of the rat (Chapman et al., 1959), erythrocytes in mouse MP ( Essner, 1960), and Brucella suis in guinea pig MP ( Pearson et al., 1963). These observations suggest that, at first, the particle is surrounded by its own extracellular environment within the microvolume of the vacuole. The limiting membrane separates it from the cytoplasm, and the interchange between vacuole and cytoplasm is determined by the permeability properties of this membrane. The postphagocytic events have to be viewed with this spatial arrangement in mind. The establishment of such a relationship between particle and cell has a number of consequences for both: metabolic changes of the cell during phagocytosis, i.e., formation of the vacuole; effects of materials elaborated by the microorganisms or its breakdown products; action of the cell on the particle; and immunological events. A. INFLUENCE OF PARTICLE INGESTION ON METABOLIC ACTIVITY

During ingestion of particles the phagocyte experiences a number of metabolic changes affecting pathways involved in energy procurement and possibly lipid metabolism. Since the pertinent literature has been reviewed recently (Karnovsky, 1962) the present discussion can be restricted. The most striking change is an increase in oxygen consumption setting in promptly after addition of particles to the PMN or MP suspension ( Baldridge and Gerard, 1933; Stahelin et al., 1956a; Sbarra and Karnovsky, 1959; Cohn and Morse, 1960a). The stimulation is observed with PMN, MP, and alveolar MP amounting to 250, 350, and 20 %, respectively, above the resting value which is 2.5, 7.5, and 30 PI. of oxygen per hour and milligram protein. The increase is a function of the load and the size of the particles offered to the cells, with particles of less than 0 . 2 diameter ~ causing no changes. Consequently the rate of glucose utilization is enhanced, resulting in depletion of cellular glycogen and disappearance of glucose from the medium. Lactate production is increased even under aerobic conditions (Becker et al., 1958; Sbarra and Karnovsky, 1959; Cohn and Morse, 1960a). Of interest is a shift of glucose utilization toward enhanced activity of the hexose monophosphate pathway as manifested by increased conversion into respiratory C 0 2 of glucose carbon-1 over that of carbon-6 (Stahelin et al., 1957; Sbarra and Karnovsky, 1959). According to Karnovsky ( 1962) the stimulation of the hexose monophosphate pathway occurs regularly

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during phagocytosis even if the increase of glucose utilization is prevented by a metabolic inhibitor, such as cyanide (Sbarra and Karnovsky, 1959). The increase of glucose carbon-1 utilization over and above the increase of glycolysis during phagocytosis is about fourfold. A very similar metabolic shift is observed with blood leucocytes from diabetic patients or with normal cells stimulated by endotoxin (Munroe, 1963). The cells stimulated with endotoxin exhibit enhanced capacity for phagocytosis and intracellular destruction (Cohn and Morse, 1960b), whereas the phagocytic power of cells from a diabetic is unaltered. This indicates that a high rate of glucose carbon-1 utilization is indicative of, but not necessarily sufficient for, cell stimulation. These changes during phagocytosis and the effect of inhibitors on them indicate that phagocytosis is an energy-requiring process. However, the mechanisms responsible for these changes are not elucidated. Although increased permeability of the leucocyte membrane cannot be excluded, quantitative changes in the content or activation of cellular enzymes has to be considered. Since ingestion of particles involves membrane activity, the increased rate of metabolism could possibly be due to additional synthesis of lipid-rich cell membrane. Incorporation of labeled precursors into lipids was found to be from 10 to 30 % greater in phagocytizing than in resting cells, whereas incorporation of labeled leucine into cellular proteins or labeled glucose into glycogen was not increased ( Sbarra and Karnovsky, 1960). Similarly, neither acid phosphatase nor esterase activity of rabbit exudate PMN or MP was increased during particle ingestion (Dannenberg et al., 1963). In the case of PMN, the influence of these metabolic changes on the functional activities was studied by Cohn and Morse (1960a) with exciting results. Staphylococcus albus was killed rapidly in the presence of opsonic factors upon ingestion by rabbit peritoneal leucocytes. Cells which had previously ingested heat-killed staphylococci killed live organisms of the same strain ten times more efficiently than did normal cells. The pretreated cells also exhibited increased rate of phagocytosis. The bacteria used for pretreatment and subsequent testing could be either a different strain of staphylococci or a heterologous species. Thus, the ingestion of heat-killed Escherichia coli or Mycobacterium smegmutis conditioned leucocytes equally well against staphylococci. Ingestion of the heat-killed organisms was essential, e.g., heat-killed Staphylococcus aurem, which is only poorly ingested under the conditions employed, stimulated cells very little. Pretreatment of phagocytes with heat-killed Staphylococcus epidmidis did not substitute for the requirement of

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opsonins. Although no definite explanation for this stimulation of leucocytes by uptake of heat-killed organisms is available, the striking similarity to the activation of leucocytes by endotoxin was demonstrated in a further study (Cohn and Morse, 1960b). In this case pretreatment of leucocytes with 0.1 pg./ml. of endotoxin increased intracellular kill or phagocytosis of S. epidemidis one hundred times. Again, pretreatment with endotoxin did not substitute for the need of opsonization. In addition, endotoxin stimulates glycolysis as does phagocytosis. An enzyme, diphosphopyridine nucleotidase ( DPNase), produced by some group A streptococci may well interfere with the energy metabolism of PMN and damage the cell, The enzyme is separable from streptolysin 0 and is specific for DPN, i.e., it does not split triphosphopyridine nucleotide (TPN) (Carlson et al., 1957). This enzyme could possibly be responsible for the shift of metabolism during phagocytosis by removal of DPN and forcing the cell to enter the TPN-dependent carbon-1 pathway. A good correlation exists between elaboration of DPNase and leucotoxicity of the strain (Bernheimer et d.,1957). An exceedingly interesting system, originally described by Merchant and Morgan (1950), of metabolic and functional interference was studied by Ginsberg and associates. Influenza virus is adsorbed readily by inflammatory PMN of the guinea pig. Only about 20 % of adsorbed virus can be eluted, and even less than 1% of the remaining virus is detectable by infectivity (Ginsberg and Blackmon, 1956). Leucocytes that had adsorbed between 40 and 130 particles per cell showed greatly reduced anaerobic glycolysis with glucose as substrate when COz and lactate production were measured. This inhibition could amount to 90% of normal activity, required the presence of calcium, and was manifest with glucose or glucose-6-phosphate as substrates, but not with fructose-6phosphate or fructose-1,6-phosphate. This suggests that the affected enzyme is phosphohexoisomerase which catalyzes the reaction from glucose&phosphate to fructose-6-phosphate (Fisher and Ginsberg, 1956a). When the ability of leucocytes to phagocytize yeast cells, which had adsorbed virus, was studied, a marked depression of leucocytic activity was noted; and, most interestingly, this inhibition occurred only with glucose as substrate and not with fructose (Fisher and Ginsberg, 1956b). These findings provide an elegant model for the study of interference by virus with phagocytic function and presumably with host resistance to secondary bacterial invasion. Both studies by Ginsberg et al. and by Cohn and Morse are significant in regard to the physiology of leucocytes. They provide examples of stimulation or inhibition of glycolysis accompanied by increased or depressed phagocytic function.

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B. INTRACELLULAR KILL OF MICROORGANISMS

1. Physicochemical Conditions Microorganisms ingested by phagocytes are exposed to conditions of chemical and physical nature, which influence their fate within cells. Acidity in intracellular compartments has been intensively studied as one such condition. Unfortunately, accurate measurements of the pH within vacuoles are not available; all investigations had to resort to the use of indicator dyes which allow an approximation but not a measurement of the hydrogen ion concentration. Numerous observations on protozoa and phagocytes lead Metschnikoff (1902) to conclude that the content of the digestive or phagocytic vacuole was of low pH, and that disruption of the integrity of the vacuole would result in neutralization of its content by well-buffered cytoplasmic constituents. Using similar techniques as Metschnikoff, Rous (1925) arrived at very low values for the pH in granules of MP, whereas Sprick (1956) gave values between 4.7 and 5.5 for the pH of the immediate environment of tubercle bacilli ingested by peritoneal PMN and MP. The importance of low pH in uitro for survival and multiplication, or rather inhibition of bacteria in the presence of organic acids and other cellular inhibitors has been clearly demonstrated (Dubos, 1954). However, it is more difficult to assess the role of high hydrogen ion concentration in uiuo. Nevertheless, the activity of several antimicrobial factors and enzymatic reactions is favored by a low pH, in fact by about pH 5.0 (Dubos, 1954). Lysozyme, known to have a limited spectrum of activity, has been shown to lyse a wide range of bacteria under acid conditions (Hirsch, 1960a). Most important, lysis of granules of PMN upon ingestion of bacteria is facilitated by a pH 4.0 to 5.5 ( Cohn and Hirsch, 1960a). In addition, Sprick ( 1956) has suggested that the increased action of nicotinamide and pyrazinamide against intracellular mycobacteria (Mackaness, 1956) is dependent on the low pH of the vacuolar content. Finally, the metabolic shift toward increased oxidation of glucose carbon-1 can be explained by the low pH developing as a consequence of enhanced glycolysis. Acid conditions favor the lysis of granules thus releasing a DPNH oxidase regenerating DPN from DPNH. This leaves pyruvate available for the increased regeneration of TPN from TPNH, the latter stimulating the direct oxidation of glucose at carbon-1 (Evans and Karnovsky, 1961). 2. Enzymes and Antimicrobial Substances

Besides the enzymatic amatory for the maintenance of life and physiologic activity, leucocytes contain a number of enzymes and com-

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ponents which are supposed to be of special functional significance. With the exception of lysozyme (see below) there is no enzyme to which any specific antimicrobial or antitoxic function can be ascribed, although it is quite certain that some of the hydrolytic enzymes are functionally important in spite of the lack of detailed information. The knowledge concerning the function of these enzymes is scant, and the fact that some of them have been studied in greater detail, such as acid phosphatase, can be attributed to the availability of techniques rather than to their significance in leucocytic function. It has been widely accepted that the lysosomes serve as containers or storage granules for hydrolytic enzymes, such as alkaline and acid phosphatases, acid ribonuclease and deoxyribonuclease, cathepsin, nucleotidase, @-glucuronidase, and arylsulfatase (deDuve, 1959; Cohn and Hirsch, 1960a). The content in acid phosphatase of MP has been studied under a variety of conditions. Thus Grogg and Pearse (1952) demonstrated in experimental tuberculosis that the MP (epithelioid cell) of the tubercle had a high content of histochemically demonstrable acid phosphatase. This was also shown to be the case for mononuclear exudate cells of rabbits and mice vaccinated with Bacillus Calmette-GuBrin (BCG) (Suter and Hulliger, 1960; Thorbecke et al., 1961). Such animals also showed increased activity of the same enzyme in Kupffer cells upon intravenous injection with tubercle bacilli, endotoxin, or zymosan (Howard, 1959; Thorbecke et al., 1961). These changes may represent a state of general stimulation as indicated by the fact that a number of manipulations of MP, such as cultivation on glass (Weiss and Fawcett, 1953), infection in uitro with tubercle bacilli (Suter and Hulliger, 1960), or implantation of granuloma-producing substances into the subcutis ( Gedigk and Boutke, 1957) cause the appearance of acid phosphatase. The available information does not permit to judge whether the high enzymatic content is the cause of, or coincident with, the enhanced killing capacity of stimulated MP (Jenkin and Benacerraf, 1960; Cohn and Morse, 1960a). The fact that alveolar MP have less killing power for Escherichia coli and for Staphylococcus aureus than peritoneal MP, although the former contain more lysozyme and acid phosphatase than the latter, may be of significance in this respect ( Pavillard, 1963). Although there is no absolute proof that enzymes other than lysozyme found in phagocytes can be held responsible for the kill of intracellularly located bacteria, the presence of antibacterial principles, the most important being phagocytin isolated from rabbit PMN ( Hirsch, 1956a,b, 1960b), is undisputed. Phagocytin is a stable protein distinct from lysozyme and histone. It is rapidly lethal but not lytic for either gram-

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positive or gram-negative bacteria, Active material was found abundantly in rabbit polymorphonuclear leucocytes, but more sparsly in human or guinea pig leucocytes. No phagocytin has been demonstrated in MP. The bactericidal action is enhanced under acid conditions. The antibacterial action of phagocytin has been ascribed to alterations of the microorganism’s permeability ( Cohn, 1963a) . A cytoplasmic material of PMN with antibacterial activity, quite different from lysozyme or phagocytin, was observed by Fishman and Silverman (1957). Extracts of rat PMN prepared by ultrasonic vibration were found to be bactericidal for gram-positive and gram-negative bacteria with a pH optimum at about pH 7. The mitochondria proved to contain most of the heat-stable active material, which shows properties of a lipoprotein (Fishman et al., 1957). Lysozyme could well be a major antibacterial substance of PMN. Exudate leucocytes contain approximately 1 to 2 mg./ml. packed cells (Myrvik and Weiser, 1955). A similarly high amount of 2 to 5 mg./ml. packed cells has been reported in rabbit alveolar MP (Myrvik et ul., 1961), whereas peritoneal MP of the same species contain only 0.5mg./ ml. packed cells, i.e., about 5 times less than alveolar cells. According to Brumfitt and Glynn ( 1961), rat peritoneal MP contain 0.85 X pg./ cell (or 0.1 mg./ml. packed cells), whereas the value for guinea pig peritoneal MP is 0.2 x pg./cell or 0.1 mg./ml. packed cells (Ramseier and Suter, 1964). Lysozyme is limited in its antibacterial spectrum to bacteria which possess the lysozyme susceptible P-1,4-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in an accessible position. Some conditions widen the range of microorganisms attacked by lysozyme, such as chelating agents (Repaske, 1956), acid pH range ( Hirsch, 1960a), and glycine ( Ralston et al., 1961). Furthermore, Amano et ul. (1954,1955) have shown that a lysozyme-like substance isolated from guinea pig peritoneal leucocytes accelerated in vitru complement and antibody-induced lysis of smooth forms of Vibrio tytrogenus, Vibrio comma, and Type I of Hemophilus pertussis. Finally, the basic nature of lysozyme could possibly exert antibacterial activity in addition to its enzymatic action. Direct evidence for the importance of lysozyme as an intracellular bacteriolytic agent was provided by studies of Brumfitt and Glynn ( 1961) . Lysozyme resistant and sensitive strains of Microcuccus lysudeikticus were phagocytized equally well by rat peritoneal PMN and MP and human PMN in the presence of heterologous serum. However, the intracellular fate of the two strains in either type of phagocyte was different. Whereas the egg-white lysozyme-sensitive strain was rapidly

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killed and lysed, the highly resistant strain of M . lysodeikticus showed no loss of viability. A strain of the same species which was moderately resistant to lysozyme was destroyed at a rate intermediate between the two other strains. Deacetylation of the strain resistant to 4000 pg. lysozyme/ml. did not affect the viability of the bacteria but restored its susceptibility to intracellular lysozyme. Furthermore, egg-white lysozyme did not differ from lysozyme extractable from rat MP or human PMN in regard to physiochemical properties. However, egg-white lysozyme was immunologically distinct from leucocyte lysozyme. Thus, antibody prepared against egg-white lysozyme in the rabbit inhibited the in vitro lysis of the sensitive strain of Micrococcus lysodeikticus by the former, but it had no effect on the lysozyme-like substance extracted from MP or PMN. Basic nuclear proteins, such as histones and protamines, are known to have antibacterial properties. Like all other cells, phagocytes contain histones. Miller et al. (1942) described the sensitivity of a number of gram-positive and gram-negative organisms to protamine sulfate and thymus histone. In a more extensive study, Hirsch (1958) confirmed these results and demonstrated the bactericidal action of histones on susceptible bacteria, such as Escherichia coli, Sdmonella, Shigella, Pseudomas, Klebsiella, and Staphylococcus epidermidis. Proteus, Serratiu, S . aureus, and various types of streptococci were found to be less susceptible. As shown recently by Ramseier and Suter (1964), virulent tubercle bacilli (HS7Rv)are resistant to calf thymus histone or histone Fraction B of Crampton et al. (1955). Histones are present in all cells, but their intranuclear location requires passage to the phagocytic vacuole for intracellular bactericidal action. Whether this may occur under physiologic or pathologic conditions remains to be determined. In the past, many antibacterial substances of leucocytic origin have been described (Skarnes and Watson, 1957). Unfortunately, most of these are only poorly characterized, One of these, termed leukin, was studied more fully by Skarnes and Watson ( 1958). Extraction of rabbit peritoneal PMN in dilute acid yielded a soluble and heat-stable antibacterial fraction active against staphylococci, pneumococci, and streptococci but not against gram-negative organisms. This leucocytic factor has greatest activity at pH 8.2,and it has been identified as a protamine or protamine derivative. From these studies it appears that the PMN is well equipped for intracellular inactivation of ingested bacteria. Relatively little is known in this respect of the MP, which plays a crucial role in immune reactions to infection, especially in response to facultative intracellular bacteria.

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Like in PMN, parasitization of MP results in low intravacuolar pH. Whether this represents the only antimicrobial activity of MP remains to be investigated. The situation is especially puzzling since phagocytin could not be found in MP. Also, their content in lysozyme seems to be only about 10 to 20 % of that reported for PMN. Alveolar MN are a notable exception to this. However, in spite of a high content in lysozyme and acid phosphatase, lung MP of the rat kill E . coli and S . uureus less readily than do MP from the peritoneal cavity (Pavillard, 1963). 3. Degranulation of Polymorphonuclear Leucocytes Criticism raised as to the physiological significance of antimicrobial substances extracted from PMN has been satisfied fully by results of investigations by Cohn and Hirsch (196Oa). They succeeded in isolating the cytoplasmatic granules from sucrose-lysed PMN by differential centrifugation. These granules, which sedimented at 8200 g, were found to lyse in weak acids or upon addition of surface-active agents. Lysis of the granules was optimal at pH 4.0 whereby their content became nonsedimentable. It was established that these granules contained 70 to 80 % of the total cellular phagocytin in bound form, which was liberated upon lysis. In addition, the granules proved to contain a number of hydrolases, many of which are active under acid conditions, about 50 % of the total cellular lysozyme, and cathepsin. These granules then resemble morphological entities of liver cells, called ‘lysosomes,” which contain a similar collection of acid hydrolyases (deDuve, 1959). Lysosomes, when isolated in sucrose, have been reported to be enzymatically inactive. They release all internal enzymes in soluble and fully active form under a number of conditions injuring the lysosome membrane. The significance of PMN granules and their content in the intracellular events following ingestion of bacteria has been demonstrated by Hirsch and Cohn (1960) and Cohn and Hirsch ( 1960b). Following phagocytosis of various microorganisms including cell wall preparations of yeast by rabbit or human PMN, a marked reduction in the number of stainable cytoplasmic granules was observed. The process, termed degranulation, took place within 30 minutes after initiation of phagocytosis and could be correlated directly with the amount of material ingested by the cells. However, no degranulation was found to occur in PMN upon contact with endotoxin which is known to activate leucocytes (Cohn and Morse, 1960b). In addition, electron micrographic observations have confirmed degranulation following particle ingestion. Phagocytosis of large numbers of bacteria was demonstrated to be followed by a marked reduction of phagocytin, whereas nuclear antibacterial substances were not altered.

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Concomitant with the engulfment of microorganisms, a decrease in the activity of phosphatases, p-glucuronidase, and cathepsin in the fraction containing the cytoplasmic granules was observed, while increased activity of these enzymes was found in the supernatant fraction. The finding that the over-all enzyme content of resting and phagocytizing cells was the same is further indication for the release of granule components into the cytoplasm of the phagocyte. Interestingly, phagocytin could not be recovered after phagocytosis, confirming earlier observations by Hirsch (1956a) that the substance is irreversibly adsorbed by bacteria. Finally, Hirsch ( 1962) showed in phase-contrast microphotographs that lysis of the cytoplasmic granules begins early in the course of ingestion of particulate matter by human, rabbit, and chicken PMN. Only granules adjacent to the invaginating cell membrane surrounding the engulfed microorganisms would rupture, presumably contributing to the formation of a vacuole. Electron micrographs show that the membrane of the granule fuses with that of the phagocytic vacuole. This finding establishes conclusively the discharge of the granule content into the vacuole ( Zucker-Franklin, 1963). Following degranulation, a clear zone appeared in place of the granule, while the adjacent surface of the microorganism showed an increase in phase density possibly caused by combination of granule content either with the surface of the microorganism or the cell membrane surrounding the ingested bacterium. Within the following few seconds, the darkening of the organism fades and the clear zone contracts toward the engulfed particle. In a similar manner acid hydrolases are found up to 60 per cent in a granular, postnuclear fraction of rabbit peritoneal MP. Phagocytosis results in a shift of enzymes from the granular fraction into the cytoplasm (Cohn and Wiener, 1963a, b). These enzymes presumably become visible as amorphous material in the phagocytic vacuoles (North and Mackaness, 1963a). In this context the detailed studies on the effect of staphylococcal leucocidin, or of its two synergistically acting components leucocidin F and S, on metabolic processes and the physiologic state of the PMN of the rabbit are of interest. Leucocidin causes a reduction of glycolysis by a factor of 4 to 5 (Woodin, 1961), while simultaneously protein is lost, presumably from the granules. Extruded materials were identified as RNase, DNase, P-glucuronidase, peroxidase, lysozyme, and phagocytin, whereas the intracellular content of acid and alkaline phosphatases remained unaltered ( Woodin, 1962). Extrusion of granular proteins from leucocytes resembled the excretory process of the pancreas and required (1) collision of the granule with the surface of the cell, (2) adherence of

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the granule at the membrane, and (3) passage of the contents to the exterior. Calcium which is essential for this process accumulates intracellularly under the influence of leucocidin (Woodin and Wieneke, 1963). Electron microscopy revealed loss of endoplasmic reticulum under the influence of leucocidin with increased vesicle formations, some of which was extruded as a whole. In the absence of calcium no such formations were seen and no protein was extruded (Woodin et al., 1963). These studies are an elegant example for the action of a bacterial exotoxin and its influence on cell-parasite interaction. The significance lies in the fact that the granular content is discharged toward the exterior of the cell rather than into phagocytic vacuoles, as shown by Cohn and Hirsch (1960a) upon phagocytosis. This diversion or excretion of inhibitory material and other disturbances of leucocytic metabolism caused by leucocidin may be responsible for the ability of S. aureus to survive after phagocytosis. Very similarly, Weissman et al. (1963) demonstrated the release of p-glucuronidase and acid phosphatase from isolated liver lysosomes of the rabbit upon addition of streptolysin S. Streptolysin 0 was less active and its activity depended on addition of cysteine. Direct observation of rabbit PMN confirmed the lytic effect of streptolysins on granules with the subsequent appearance of filamentous processes on the cell membrane, cytoplasmic liquefaction, and nuclear fusion. Streptolysin 0 was more potent and acted more quickly than streptolysin S-(Hirsch et d., 1963) . Mechanisms and means by which PMN kill ingested microorganisms have thus become apparent. The acidity which develops shortly after particle ingestion due to the increased metabolic activity of the energyspending cell may or may not by itself be antibacterial. Nevertheless, acid conditions provide an internal environment for increased activity of at least two well-known antimicrobial agents, phagocytin and lysozyme. In addition, acid hydrolytic enzymes may come into play which are important for the digestion of killed parasitic microorganisms. OF INTRACELLULARLY KILLED BACTERIA ,C. DIGESTION The major function of phagocytes in higher animals is the disposal, i.e., digestion of particles, foreign macromolecules, and effete cells. Evidence for this digestive process has been obtained by various means, such as microscopic observation, fluorescent antibody labeling, and metabolic tracer studies, to mention a few. Electron micrographs present excellent evidence for the gradual intracellular digestion within the phagosome or the phagocytic vacuole (Essner, 1960) and also indicate the difference

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in behavior between virulent and avirulent variants of the same organisms (Goodman et al., 1956). An attempt at a metabolic study of intracellular survival or death and digestion was made by Stahelin et al. ( 1956b), but was rather inconclusive because complete oxidation to COZ of C1*-labeled bacteria was used as the only criterion for degradation. Similar but more extensive studies were undertaken recently by Cohn (1963a) using rabbit PMN, human and horse blood leucocytes, as well as rabbit MP. Radioactively labeled ( Psz or C14) Escherichia coli, Bmillus subtilis, Micrococcus lysodeikticus, Staphylococcus albus and aureus, and Salmonella typhimurium were phagocytized and killed rapidly (i.e., within 20 minutes) by leucocytes in the presence of normal fresh, homologous serum. As evidenced by the loss of the isotopes, extensive degradation of bacterial lipid, nucleic acid, and protein occurred following the inactivation of the bacteria in both PMN and MP over a period of 180 minutes, Experiments employing C14-labeled bacteria revealed the degradation of the labeled proteins to peptides and amino acids. Furthermore, it could be shown that only a small percentage of Cl4-labeled bacteria was completely oxidized to COz by leucocyte enzymes. Differences found in the breakdown of the various bacterial species presumably are due to the composition of the bacterial surface and not to the type or source of leucocyte. Studies on the localization of degraded material within and without the leucocytes lead to the conclusion that the initial intracellular event is the liberation of the bacteria's pool of small molecular metabolites. Following the degradation of high molecular weight bacterial components, the breakdown products are rapidly transported to the extracellular medium and no significant accumulation of small bacterial breakdown products occurs within the leucocyte. However, re-incorporation of bacterial constituents into leucocytic lipids could be demonstrated. In an extension of these studies, Cohn (1963b) found that under conditions where phagocytosis of the organism did not require an immune serum, intracellular degradation of P32-labeledbacteria by PMN or MP was delayed by immune serum as compared to normal serum. Ammonium sulfate precipitation revealed the inhibitory activity to be in the y-globulin fraction. Treatment of bacteria with immune serum prior to phagocytosis resulted in inhibition of intracellular degradation, indicating that antibody entered the cell during phagocytosis. Using fluorescent antibody, this had been demonstrated by Gelzer and Suter (1959). Immune serum, however, was found to be without influence on the distribution of bacteria within PMN, nor were speed or extent of degranulation significantly affected. Last, neither iodoacetate, arsenite,

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nor cyanide were found to influence the rate of degradation of killed bacteria in both PMN and MP once the organisms were ingested for 30 minutes. This finding emphasizes the independence of degradation from energy-yielding processes. Cohn (196313) suggests that delay of intracellular degradation caused by antibody may be due to the formation of a complex between antibody and a component of the bacterium. This complex has to be degraded by a protease, possibly cathepsin, before further enzymatic attack is possible, thus delaying the degradation process.

D. FACULTATIVE INTRACELLULAR PARASITES Most studies concerned with mechanisms of intracellular kill and digestion of pathogens were done with PMN and obligate extracellular parasites. PMN are readily obtained in large numbers either from the circulating blood or from inflammatory exudates induced in cavities. Furthermore, the end result of the interaction can be measured as loss of viability. Facultative intracellular bacteria assume with the host cell, mostly macrophages, a more subtle interrelationship which is potentially of long duration and is characterized by a delicate balance between parasitic virulence and antimicrobial potential of the cell. The organisms of this category, which belong to the genus Salmonella, Parnobacteriaceae, Brucella, Mycobacteria, and include fungi, have one property in common, namely, the ability to resist intracellular destruction. They may be capable of intracellular multiplication. Destruction of the host cell is a possible but not a necessary consequence of parasitization. It should be noted that a number of pathogenic organisms, the staphylococci for example, cannot easily be placed in either category but show characteristics of both groups. Since, by definition, obligate extracellular pathogens are rapidly killed by phagocytosis, an essential expression of virulence is their ability to resist phagocytosis, usually attributable to a surface component. Specific opsonization by antibody is required for phagocytosis. Avirulence, then, is characterized by rapid phagocytosis in absence of opsonin. In the case of facultative intracellular bacteria, such as Brucella and Mycobacteriu, opsonization plays a secondary role, and relatively little diflerence is found between virulent and avirulent strains in this regard. The most essential attribute of virulence is not resistance to phagocytosis but ability to survive and multiply intracellularly. It is to be expected that this resistance to the intravacuolar environment is dependent on some surface component preventing access of cellular enzymes to susceptible substrates. In investigations concerned with intracellular

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survival and multiplication, mycobacteria and brucellae have been studied most extensively. Virulent and avirulent strains of Brucella abortus behave differently when cultured in chick embryo cells (Holland and Pickett, 1958). The smooth strain multiplies intracellularly, whereas the rough strain loses viability within a few days under the same conditions. It has not been shown yet that the loss of a surface antigen is responsible for the vulnerability of the rough variant in the intracellular environment. Surprisingly, the rough strain inflicts greater damage on the cell culture than does the smooth strain, presumably because of the more rapid uptake of the former resulting in multiple infection (Braun d al., 1958). A similar difference in destructive power for guinea pig MP between rough and smooth B. abortus was described by Freeman et al. (1961). In their experiments the smooth strain caused only 15 to 30 % damage or destruction as measured by release of nucleic acid from the cells, whereas the destruction by the rough strain was up to 75 %. This toxicity was not observed when heat-killed organisms were used, and was dependent on the number of organisms picked up. Since no toxic factor could so far be isolated, one can only speculate that possibly the rapid intracelMar degradation of the rough strain may yield a toxic product, or that the observed high rate of glutamate oxidation by the rough strain may cause a critical intracellular depletion of an essential cellular constituent ( Dasinger and Wilson, 1962). Similar observation of relation between ability to multiply intracellularly and virulence have been made with Pasterrrellu tu2arenSis in cultured L cells (Merriott et al., 1961). Intracellular growth may have a lasting effect on microorganisms. Thus, B. abortm grown for 72 hours in vitro in guinea pig MP, when compared with slant grown organisms, exhibits enhanced resistance to the bactericidal effect of normal and immune bovine serum and of phagocytes, and survives to a greater extent in the tissues of mice (Stinebring, 1962). Virulent, but not avirulent, tubercle bacilli exert an inhibitory action on guinea pig leucocytes as manifested by reduced migration from a buEy coat explant ( Allgower and Bloch, 1949). This inhibition shows an amazing correlation with host susceptibility; thus mammalian tubercle bacilli will not inhibit chicken leucocytes, whereas avian bacilli do (Martin et al., 1950). In a very similar fashion MP are inhibited by large numbers of intracellular tubercle bacilli accumulated either by massive phagocytosis or intracellular multiplication ( Berthrong and Hamilton, 1958). It is of interest that large numbers of extracellular, virulent tubercle bacilli do not seem to inflict any injury on cultured MP (Suter, 1952). There is suggestive evidence that a surface constituent

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may be responsible for this toxicity of virulent tubercle bacilli. The component was identified as trehalose-6,6’-dimycolate ( Bloch, 1950; reviewed by Noll, 1956). This “cord factor” proved to be chemotactic and leucotoxic. A metabolic explanation for cytotoxicity is offered by findings that extracts of in viuo grown mycobacteria and of lungs from mice infected with tubercle bacilli inhibit the electron transport system of various mycobacteria and lung homogenates of normal mice (Bekierkunst and Artman, 1959). This inhibitor was shown to be a diphosphopyridine nucleotidase, and it was suggested that it is introduced with the tubercle bacilli into the phagocyte resulting in inhibition of phagocytic respiration and possibly death of the cell (Artman and Bekierkunst, 1961). Intracellular metabolism and fate of tubercle bacilli was studied by Stahelin et d.(1956b) using C14-labeled organisms and guinea pig peritoneal PMN. Exposure of heat-killed virulent tubercle bacilli to PMN resulted in little or no degradation of bacillary material to C1402 by leucocytic enzymes over a 4-hour period, whereas significant conversion of bacterial carbon to COz by leucocytes occurred after ingestion of heatkilled Mycobacterium phlei, staphylococci, or Bacillus subtilis. Living tubercle bacilli, when phagocytized, maintained the original rate of oxygen consumption and COz production, whereas ingested M. phlei showed a decline in oxygen uptake and CO, production, and bacterial components were converted into COz. Such studies allow the conclusion that intact virulent tubercle bacilli resist the attack by PMN enzymes. Unfortunately, no experiments are reported with MP as the host cell. It can be speculated that this invulnerability of virulent mycobacteria is due to some property of their surface. Some evidence to this effect is given by observations that certain nonionic surface-active polyoxyethylene ethers (Tritons) have antituberculous activity, whereas others of the same series are protuberculous, depending on the chain length of the agent. The antituberculous members of the series possibly displace hydrophobic lipids from the bacterial surface, thus rendering it accessible to cellular enzymes (Hart and Rees, 1955). It was shown by Mackaness (1954) that these surface-active agents are taken up by macrophages which then restrain in uitro intracellular multiplication of virulent tubercle bacilli. There is ample evidence that PMN and MP contain substances with inhibitory power against tubercle bacilli. This inhibition has been attributed to lysozyme which is present in large quantities (up to 4 mg./ml. packed cells) in lung macrophages (Oshima et al., 1961; Myrvik et al., 1961, 1982). For complete inhibition in uitro of virulent tubercle bacilli approximately 1200 pg./ml. of lysozyme are required (Ramseier and

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Suter, 1964). Another inhibitor was extracted by water from peritoneal MP of normal rats. However, tests were done using only an avirulent variant of human tubercle bacilli. No inhibitor was extractable from guinea pig monocytes ( Colwell, 1958). Similarly, Akiyama and Fong (1962) were unable to find an inhibitor for tubercle bacilli in watersoluble extracts from guinea pig MP. By contrast, studies in our laboratory (Ramseier and Suter, 1964) have shown that peritoneal MP of normal guinea pigs contain a substance that possesses antimycobacterial activity. It was found that lo2 to lo8 virulent (H&) or attenuated (BCG) tubercle bacilli were completely inhibited by the equivalent of approximately 4 x 106 sonically disrupted normal cells. The heat-labile factor, found to be located in the cell nucleus, proved to be different from lysozyme. A very similarly acting material was obtained by Kotani et al. (1962)but was not further characterized. V. The Immune Phagocyte

It is a well-established fact that animals which had previously experienced contact with microorganisms exhibit an increased capacity to phagocytize and destroy homologous or heterologous pathogens. Antibodies, especially opsonins, are responsible for increased phagocytosis, whereas macrophages have been recognized to acquire increased inhibitory and destructive power also demonstrable in in uitro systems. Stimulated or altered cells have frequently been called immune phagocytes, and the phenomenon has been referred to as cellular immunity. Many investigators are of the opinion that this term is inappropriate, or that “immune” should at least be bracketed by quotation marks. In view of the definition presented in the Introduction, such a restriction is unnecessary. It is hoped that in the following, sufficient evidence can be presented to justify fully the term “immune phagocytes.” Cellular immunity is a status or condition of mononuclear phagocytes or other cells which has to be defined operationally and cannot be described yet in molecular terms. Although no definition is intended, functionally the term implies that the immune phagocyte has a greater ability to restrict intracellular growth of ingested parasites or to destroy them than has the “normal” phagocyte. Characteristically, only pathogens capable of prolonged association with their host are known to induce this state of immunity. Among these, Mycobbacteria, Brucella, Salmonella, pathogenic fungi, and Listeriu have been studied most thoroughly. These pathogens induce during their intracellular residence the state of delayed hypersensitivity in their host. To what extent cellular immunity and delayed hypersensitivity are related phenomena is difficult to judge.

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A. DEMONSTRATION OF CELLULAR IMMUNITY Careful observation of the pathogenesis of the tuberculous lesion in rabbits and ingenious experimentation led Lurie ( 1933) to the conclusion that the macrophage in the tuberculous lesion plays a major role in reducing the bacterial population in the lesion or in destroying bacteria of reinfection. MP from normal and immunized rabbits were infected in uitru with equal numbers of bovine or human tubercle bacilli, and paired samples of normal and immune cells were injected into the anterior chambers of the left and right eyes, respectively, of normal rabbits. This allowed observation of the fate of ingested bacteria in a host in the absence of immune body fluids. The number of viable bacteria recovered after 10 to 20 days from each eye was compared with the number implanted and was found to be 2 to 20 times smaller in the eye injected with immune cells than in that with normal cells (Lurie, 1942). Lurie’s original observations were confirmed in in uitru studies following the fate of tubercle bacilli within MP from normal and immunized guinea pigs or rabbits. Whereas the bacilli multiplied rapidly in the former cells, their multiplication was retarded or completely inhibited by the latter (Suter, 1953). Immune serum had no additional influence in spite of repeated washing of the cells. These findings have, in general, been confirmed subsequently, with the exception of results published by Mackaness (1954) who could find no difference in the rate of multiplication of virulent or attenuated tubercle bacilli in cultures of MP from normal or BCG-immunized rabbits. Technical differences may account for these discrepant results, especially the fact that the multiplicity of infection was 10 times greater in Mackaness’ experiments than in Suter’s, thus preventing any expression of cellular immunity ( Suter and Hulliger, 1980). Mackaness’ contention that inhibition of growth by cells from the immune donor in Lurie’s experiments can be explained by the possible transfer of hypersensitivity with cells is quite irrelevant. Although it is true that Lurie noted inflammation in the interior chambers, more inflammation (if this is an expression of hypersensitivity) was found in the chamber containing normal cells, presumably caused by the greater multiplication of the bacilli. Even if transferred hypersensitivity were a mechanism responsible for growth inhibition, the importance of Lurie’s findings would not be diminished. Abe ( 1958) obtained identical results as did Suter (1953). Berthrong and Hamilton (1958) using a plasma clot technique concluded that monocytes from the peritoneal cavity of BCG-immunized guinea pigs inhibited the development of intracellular cords and the appearance of masses of tubercle bacilli which over an observation period of 9 to

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14 days would accumulate in normal monocytes and cause their disintegration. Infection of the cells with large numbers of bacilli masked the difference found by Lurie and Suter, whereas low initial infection allowed the manifestation of pronounced inhibitory effect by immune cells. A situation not very much different from that reported for tubercle bacilli was found for Brucella. Pomales-Lebr6n and Stinebring (1957) and Braun et al. (1958)examined the interaction between virulent and avirulent brucella and MP from normal guinea pigs and guinea pigs immunized with viable Brucella abortus. By comparison with normal cells, multiplication of the test organism was inhibited in immune MP. In addition, there was evidence of intracellular disintegration of virulent and avirulent Brucella in immune, but not in normal cells. Smooth strains seemed to caused greater cytotoxicity of immune cells than rough strains. Elberg et al. (1957) reported similarly that Brucella melitensis would cause little or no degeneration of cultured immune MP, whereas over one-half of the normal MP were destroyed during the same period of time. Cellular immunity against Brucella was also studied by Holland and Pickett (1958) in a system comprising guinea pig, rat, or mouse MP and B. abortus, Brucella suis, and B. meldtensis. Animals were immunized with smooth living B. abortus or B. melitensis. It was found that, while all three strains grew abundantly within normal MP, immune MP greatly restricted the intracellular growth of smooth and nonsmooth Brucella. Although brucellae, unlike tubercle bacilli, are sensitive to antibodies, the growth of smooth BruceUa within either normal or immune cells was not influenced by the addition of specific antiserum to the culture medium. Immunity could not be induced by injecting animals with heat-killed brucella, although these animals produced agglutinating antibodies. Similarly, vaccination of animals with living but rough B. suis failed to induce cellular immunity. Salmonellosis of the mouse provides another model for the study of cellular immunity. MP obtained from mice immunized with live vaccine of SalmoneZ2a enteritidis inhibited in uitro intracellular multiplication of a virulent strain of this species. This inhibition was independent of the presence or absence of specific antibody in the culture medium. As was noted with brucellae, immunity could not be induced by immunizing mice with dead salmonellae (Ushiba et al., 1959; Saito et al., 1960). Almost identical results were published by Sat0 et al. (1981)who cultured and infected macrophages obtained from the liver and subcutaneous tissue of normal and immunized mice. Protective immunity, measured as ability of MP to restrict intracellular multiplication and cellular destruction, was induced only by a live attenuated culture.

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Listeriosis represents another infection to which immunity is not transferable by serum, and, thus, the possibility of cellular immunity was considered. Exudate macrophages of normal sheep and sheep immunized with live List& monocytogenes were studied for their interaction with listeriae in uitro. Immune cells in presence of immune serum inhibited intracellular multiplication of the pathogen and escaped destruction, whereas in all other combinations of cells and serums the organisms multiplied abundantly and destroyed the cells ( Njoku-Obi and Osebold, 1962). Recently, Mackaness ( 1962) investigated the response of macrophages from normal and immunized mice to Listeriu monocytogenes. The native susceptibility of the mouse was found to be due to the capacity of the organism to survive and multiply in host macrophages as evidenced by growth in liver, spleen, and peritoneal cavity. Mice able to survive a primary infection of 0.5 LDEo,when challenged with 9 LD5,, proved to be immune. The challenge organisms failed to grow in liver and spleen and were finally cleared. Employing macrophages from the peritoneal cavity, the response of normal and immune cells of mice to infection with listeria was tested using plaque formation in macrophage monolayers as the criterion. Practically every cell of the immune animal, i.e., after recovery from a primary infection, was capable of inactivating listeriae. Whereas the plaque-producing efficiency in monolayers of normal macrophages was 91.2 %, it was only 0.9 % in monolayers of immune cells. In addition, about 50 % of the bacteria were lysed by the cells. Absolute cellular immunity was found to appear on the fourth day after infection and to last for about 3 weeks. Thereafter, the host was unable to inactivate the challenge inoculum of listeriae. When tested in uitro, immune cells were found to retain resistance for at least 3 days, despite repeated washings. This, together with the fact that serum of manifestly resistant mice could neither be shown to confer any protective effect in normal recipients nor affect the viability of listeria, indicates strongly that resistance of the host depends upon an acquired property of the macrophages. The validity of these findings is substantiated by histological observations of lesions after primary and secondary infection. Upon reinfection, sections of liver and spleen showed that the %-hour lesions were composed almost exclusively of mononuclear cells, whereas primary infection called forth the typical pattern of an inflammatory response, consisting in a central core of neutrophiles surrounded by layers of MP. Although this primary inflammatory reaction lasted for 3 days, it was completely absent in secondary infections. Primary infection elicited by the fourth day massive infiltration of mitotically active

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macrophages, whereas the lesions produced upon reinfection after 3 days showed almost no macrophages with mitotic figures. Cellular immunity appears to be of importance in the reaction of the mammalian host to Toxoplasma gmdii, an obligate intracellular parasite. Macrophage cultures from a species susceptible to infection with this protozoan parasite sustain its intracellular replication and are destroyed by the agent. Cells from natively resistant species, e.g., rats, can survive infection although the parasite multiplies to some extent. Macrophages from animals that had survived infection with an attenuated strain supported parasitic growth to a limited extent only; immune serum greatly enhanced this inhibition (Vischer and Suter, 1954). As is the case with Listeria (Njoku-Obi and Osebold, 1962), infection immunity against Toxoplasma is mediated by macrophages and serum. It seems, therefore, that depending on the parasite under study a greater or lesser role can be attributed to circulating antibody, Listeria and Toxoplasma belonging to the former and Mycubacteria, Salmonella, and Brucella belonging to the latter. Serum or serum components, however, appear to be important for the maintenance of cellular integrity upon infection in vitro and thus contribute toward the manifestation of cellular immunity. Fong et al. (1958, 1957, 1959; reviewed by Elberg, 1960) demonstrated that MP from normal and BCG-vaccinated rabbits would undergo extensive destruction in vitru within 48 hours after infection with virulent tubercle bacilli. If the normal serum of the medium was replaced by serum from an immune rabbit, cellular degeneration of immune cells was greatly reduced. The reduction of cell destruction over a 48- to 72-hour period amounted to a factor of 1.5 to 4.8. Immune cells in presence of immune serum enjoyed almost complete protection, whereas immune cells or serum alone were not as effective. The cellular component of this protection appears to be specific, i.e., no cross resistance between Salmonella, Brucella, and Mycabacteria could be observed, whereas the serum factor was nonspecific. Any serum from a stimulated animal could be active. In addition, absorption of a serum from a BCG-immunized animal with BCG, or a serum from a Salmonella-immunized animal with Salmonella, did not remove the protective factor, and the y-globulin fraction of protective sera had only little activity. The active component of serum is heat-stable (70°C. for 30 minutes) and nondialyzable. The protective effect was demonstrable on the fifth day after immunization, at a time when both skin tests and cell inhibition tests were still negative (Suter, 1953). In oitro exposure of virulent tubercle bacilli to the immune serum from BCG-immunized rabbits for 15 hours at 37°C. failed to alter the capacity of the bacteria

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to necrotize MP and multiply intracellularly. This indicates that the nonspecific serum factor has no direct effect on the bacilli. Also, in uitro cultivation of normal and immune MP in normal or immune serum was not effective in changing the native susceptibility or resistance of these cells, thus excluding the possibility that the immune serum indirectly influenced the ability of virulent tubercle bacilli to necrotize MP by increasing the resistance of the cells. Studies on the necessity of the participation of the serum factor during parasitization and cultivation revealed that effective manifestation of resistance to virulent tubercle bacilli by immune cells required the continuous presence of immune serum. The results of these experiments are somewhat at variance with those of other investigators who studied the tubercle bacilli-monocyte interaction (Lurie, 1942; Suter, 1953). This could be attributable to the fact that Elberg's group trypsinizes the MP prior to cultivation, possibly removing a surface protein. Unfortunately, there are no data available indicating whether the tubercle bacilli multiply intracellarly or extracellularly during the period of observation. In many respects, this protective substance resembles the C-reactive protein found in serum during the acute phase of a multitude of injuries, especially infection (Tillett and Francis, 1930). This protein behaves electrophoretically like a @-globulinand does not precipitate when serum is treated with ammonium sulfate at 50 % saturation; it appears within a few days after infection or antigenic stimulation (Wood, 1953; Wood et d.,1954).Sera containing C-reactive protein, or crystallized C-reactive protein, have a stimulating effect on migration of human PMN, but are toxic in higher concentrations (Wood, 1951). The possible relationship between the protective factor and C-reactive protein will have to be investigated more extensively. a m a t i v e results could help to explain the many observations in regard to nonspecific stimulation of phagocytic activity.

B. I n Vitro ACQUISITION OF CELLULAR IMMUNITY There is general agreement that cellular immunity is induced only by immunization with live vaccine, presumably because of necessity of bacterial multiplication. Studies on experimental infections with SaL moneZh and BruceZZu are in accord that immunization with killed organisms does not prevent death from a challenge infection but at best prolongs survival time (Topley, 1929; Kobayashi and Ushiba, 1952; Mitsuhashi et al., 1958) or has no effect at all (Smith, 1956). Living vaccines, on the other hand, can be highly protective (Smith, 1958; Elberg and Faunce, 1957).

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Rather extraordinary results on successful attempts to induce cellular immunity in uitro were reported by Sato et al. (1962a). MP from normal mice were cultivated on cover slips for 18 hours and were then parasitized with either living, attenuated (in a ratio of 1 organism per 30 cells), or heat-killed (10 organisms per 30 cells) Salmonella enteritidis. After incubation for 60 minutes, extracellular bacteria were removed hy washing. The parasitized cells were kept in a medium consisting of Hanks’ balanced salt solution containing 30 % horse serum, streptomycin, and penicillin. Three days after immunization the cells were infected with virulent S . enteritidis. At increasing intervals of time after challenge infection the total number of cells was counted, cells were stained, and the total number of bacteria in infected phagocytes was determined. The results showed that cultures infected in Vctro with live vaccine lost from 30 to 35 % of the original cells during the first 24 hours after challenge infection. Thereafter, no further losses occurred. On the other hand, a steady loss in viability up to 90 % was found in untreated cells or in cells treated with heat-killed vaccine. Counting of intracellular bacteria revealed that the organisms had increased by a factor of about 2.5 in untreated cells and in cells treated with dead vaccine, whereas cells that had been pretreated with viable vaccine showed no evidence of intracellular multiplication. If confirmed, these results would indicate that within 3 days after infection in vitro changes characteristic of cellular immunity take place. This would provide a unique opportunity for an analysis of the factor or factors involved. h c o m C. PROPERTIES OF THE IMMUNE MONONUCLEAR Cellular immunity appears to be one possible pathway of expression of the immune response in mostly subacute or chronic infections in which the pathogen establishes a relationship of prolonged duration. In addition to exhibiting the manifestations of cellular immunity, i.e., the restriction of intracellular multiplication of the parasite, mononuclear phagocytes undergo some characteristic changes, especially in their enzymatic makeup. Although the enzymes involved have specificity for a substrate, the stimuli responsible for the alterations are not necessarily related to the enzyme or enzymes. Peritoneal monocytes of guinea pigs infected with tubercle bacilli exhibit increased respiratory activity as measured as 0 2 consumption and COz production. This enhanced respiratory rate is more pronounced in virulent than in attenuated infections (Stahelin et d.,1957). Similarly, acid phosphatase and P-glucuronidase are found in greater quantity in monocytes from infected animals than in those from controls (Suter and Hulliger, 1960; Saito and Suter,

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1964). If such cells are cultured in vitro and are subsequently infected with heat-killed BCG or virulent tubercle bacilli, the monocytes derived from normal rabbits show an increase of their amount of acid phosphatase per cell, although as many as 60 % of the cells can be lost. By contrast, immune phagocytes lose part of their acid phosphatase, in spite of the fact that the cells show no morphologic signs of degeneration. It is tempting to suggest that the loss of enzymes upon reinfection of cultured cells with Mycobacteria is comparable to the discharge of enzymes as described by Woodin (1962) induced in PMN by leucocidin. In the former event the reaction would be mediated by cellular hypersensitivity, but is due to direct toxic action in the latter. It is of considerable interest to note that inbred rabbits belonging to a strain with high native resistance to tuberculosis have peritoneal marcophages with increased activity in a variety of metabolic tests when compared with cells from a susceptible strain (Allison et al., 1961). The enzymes studied include acid phosphatase, lipid metabolism, and enzymes of the Embden-Meyerhof pathway and Krebs cycle. Infection of representative animals of both strains with tubercle bacilli did not enlarge the observed differences (Allison et d.,1982). Such findings on immunization and genetic factors in resistance to bacterial infection suggest some connection between enzymatic activity of the cell and repression of intracellular growth, as an expression of cellular immunity. Ribble (1961) reported that an intravenous injection of typhoid vaccine, purified endotoxin, and Newcastle disease virus into rabbits resulted in an elevation of the plasma lysozyme content. According to Kerby (1952), the increase of lysozyme in the circulation can be explained by the damage of granulocytes caused by bacterial endotoxin. It is not known whether endotoxin exerts a similar action on macrophages, although it seems likely that damage takes place with the consequent release of lysozyme. A similar increase in the concentration of lysozyme in the serum of rabbits is observed after immunization with heat-killed tubercle bacilli (Myrvik and Weiser, 1951) or in guinea pigs and rabbits after the injection of heat-killed virulent tubercle bacilli suspended in paraffin oil (Janicki and Patnode, 1961) . This increase appears early in the course of sensitization and persists as long as 4 months. Another property of the BCG-induced immune cell, the increased capacity to phagocytize particles, undoubtedly is a manifestation of nonspecific stimulation. It is a well-known fact that the phagocytic activity of the RES can be increased considerably by various stimuli other than BCG immunization, such as endotoxin (Biozzi et al., 1955), zymosan (Benacerraf and Sebestyen, 1957), and transplantable tumors

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(Biozzi et al., 1958; Old et al., 1980) which all cause proliferation of RES cells. Over-all measurements of the phagocytic activity are, however, not suitable to give information on increased phagocytic activity of the single cell. It is interesting to note in this connection that Suter (1953) found no difference in the phagocytic activity of monocyte cell cultures from normal and BCG-immunized animals. Consistent with observations indicating stimulation of multiplication of phagocytic cells by BCG vaccination are observations by Myrvik et al. (1962) who found that BCG vaccination increased the number of alveolar marophages from two- to tenfold. Similarly, after a primary infection with a vaccinating dose of Listeria monocytogenes sections of liver and spleen showed mitotically active macrophages ( Mackaness, 1962). The cytoplasmic response of the immune MP to the presence of Listeria appears to be similar to that of the normal cell, but it occurs at an accelerated pace and with greater intensity (North and Mackaness, 1963b). Recent attempts to elucidate properties of macrophages in relation to tubercle bacilli have revealed that sonically disrupted MP from the peritoneal cavity of BCG-immunized guinea pigs exerted an antimycobacterial activity which was, on the average, 4 times greater than the activity of similar cells obtained from normal animals. It was found that the active substance in concentrations inhibiting the growth of virulent and attenuated tubercle bacilli completely, did not act on Brucella abortus, Brucellu melitensis, Salmonella typhimurium, Stuphylococcus aureus, or Escherichiu coli. The activity of the inhibitor was found to be independent of serum. Intravenous or intraperitoneal injection of 50 pg. endotoxin into normal animals did not result in an increase of the antimycobacterial activity of the MP when tested 96 hours after the injections. Although stimulation of the RES was not measured, it would thus appear that the increase of antimycobacterial activity induced by BCGimmunization was specific (Ramseier and Suter, 1964). A similar conclusion can be drawn from the work of Kochan and Rose (1962) who compared the fate of intravenously injected tubercle bacilli in mice 3 days after endotoxin injection or 10 days after immunization of the animals with either living or heat-killed BCG. Their findings indicate that injections of endotoxin or heat-killed BCG did not produce immunity to the challenge infection, whereas immunization with living BCG did. It appears that many of the manifestations of immune cells can be elicited nonspecifically. However, some of these nonspecific stimuli only seem to elicit short-lived activities, whereas those induced by viable particles are generally of longer duration. Studies on the immunization power of living versus dead vaccines strongly suggest that induction of

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cellular immunity depends on the reproductive capability of the inducing agent.

D. NONSPECIFIC MANIFESTATIONS OF IMMUNITY Planned or accidental introduction of viable or dead bacteria or their products into animals alters their reactivity to subsequent homologous or heterologous infection. Since no immunological relationship between these various agents has been established, it is assumed that MP participate in this increase of nonspecific immunity, especially in reaction to facultative intracellular parasites. The in vitro demonstration of cross immunity was described by Elberg and associates (1957). MP derived from rabbits immunized with viable BCG were found to resist in vitro cellular destruction not only by virulent tubercle bacilli but also by virulent Brucella melitensis, and vice versa. In both cases the effect could only be observed in the presence of immune serum of either kind. The nonspecificity of the protective serum component already described in the tubercle bacilli-monocyte system was confirmed in these studies. The protective activity was not reduced when the agglutinating action of the anti-Brucella rabbit serum was removed by absorption. Experiments presumably designed to study cellular cross immunity were reported by Sato et al. (1962b) and by Tanaka et al. (1962). Mice were immunized either with Salmonella enteritidis or Salmonella typhimurium. Macrophages from these animals inhibited intracellular multiplication of the homologous organisms and of S . enteritidis, S . typhimurium,Salmonella choleraesuis,E . coli, but not of virulent mycobacteria. The immunizing agents share somatic antigens, and it is quite surprising that E . coli should multiply intracellularly in normal mouse MY. Therefore, these results have to be considered with caution. Guinea pigs exhibit increased resistance to Brucella if injected simultaneously with virulent tubercle bacilli (Pullinger, 1936), and primary infection with Brucella reduces the severity of superinfection with Codella burnetii (Mika et al., 1954). Similiarly, mice injected with Brucellu abortus are more resistant to virulent tubercle bacilli than control animals (Nyka, 1956). Employing Brucella suis as the primary invader and tubercle bacilli as challenge organisms, Henderson et al. ( 1956) demonstrated nonspecific resistance against secondary infections provided the primary infection induced a generalized lymphatic response. This was especially the case if primary and secondary invader followed the same route, thus exposing the secondary invader to “primed”

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lymphatic pathways. Mice vaccinated with viable BCG were shown by Dubos and Schaedler (1957) to have increased resistance to intravenously injected Staphylococcus a u r w . The protective effect against the staphylococcal infection manifested itself in a prolongation of the survival time following infection and in reduction of numbers of staphylococci that could be recovered from spleen, liver, and kidney. Similar results were obtained by immunizing mice with killed Mycobacterium fortuitum. Careful studies revealed increased phagocytic capacity of the RES of mice and rabbits which had been immunized with BCG (Biozzi et al., 1958). This change could not be explained by a rise in opsonins but was attributed to cellular factors (Biozzi et al., 1963). More recently, Sulitzeanu et al. (1962,) vaccinated mice with living or killed BCG and challenged the animals with virulent B. abortus. The animals were found to be resistant to the superinfection as evidenced by a lowered bacterial count in spleen and liver. Furthermore, sera of BCG-vaccinated mice apparently contained a factor which would confer a significant degree of protection against Brucella in normal mice. Interestingly, no correlation was found between the degree of resistance to Brucella of the donor mice and the protective activity of their sera. Passive administration of anti-Brucella serum, however, did not modify a pre-existing Brmcella infection. The possibility has to be considered that the serum of infected animals stimulates phagocytic activity nonspecifically. These findings on bacterial interference are difficult to explain. Although it cannot be denied that a common nonspecific stimulus induced increased activity of the phagocytic cell, the observed heightened resistance to superinfection could in some cases be due to the increased capacity of the serum of pretreated animals to opsonize the challenge inoculum. Jenkin and Benacerraf (1960) would certainly favor the second possibility. Thus, serum obtained from BCG-immunized and endotoxin-treated mice stimulated phagocytosis and intracellular kill of virulent Salmonella strains in nitro by mouse mononuclear cells (Jenkin and Benacerraf, 1960; Jenkin and Palmer, 1980).A similar conclusion can be reached from results of experiments by Howard et a2. (1959) who observed that BCG immunization of mice rendered the animals more resistant to infection with Salmonella enteritidis as measured by the mean survival time. It was also found that radioactively labeled suspensions of this organism were cleared from the blood twice as rapidly in BCG-treated than in normal animals. Jenkin and Rowley (1963) reported that BCG immunized mice exhibit elevated opsonic power of their sera for Salmonella typhimurium. Using pathogen-free mice such opsonins could not be demonstrated, although the BCG immunized animals re-

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moved SaZmoneZZu more rapidly from the circulation than did control animals (Suter, 1964). Last, it should not be forgotten that both mycobacteria and endotoxin can act as adjuvants in the production of humoral antibodies or delayed-type hypersensitivity. At present, these findings of nonspecific stimulation of cellular immunity are difficult to interpret. They are additional evidence supporting the notion that elements of the RES react adaptively to foreign stimulation, as expressed by alterations in enzyme content, metabolic activity, and fine structure. Unlike the PMN, the MP is equipped with the machinery for protein synthesis, and the latter reacts to stimulation with increased enzyme production. It is important to note that such changes of cellular activity appear to be universal, i.e., involve many elements of the RES distant from the site of stimulation rather than only a few cells participating in actual phagocytosis. How such stimulation is transmitted from cell to cell is a mystery, but could either be based on distribution of antigen or a product thereof, or on production of an antibodylike molecule with high affinity for RES cells. E. CONSEQUENCES OF &SIDENcE IN IhXMWNE ' CELLS As has been pointed out previously, facultative and obligate intracellular parasites after ingestion by phagocytes are not easily disposed of by normal cells. Opsonizing antibodies were found to play a minor role, if any. The immune macrophage, however, is endowed to exert definite antibacterial action on these parasites. Manifestation of this activity is probably bactericidal only in few cases. More often, the action seems to result in growth inhibition and attenuation of the parasites. Fong et al. (1959) studied the capacity of virulent tubercle bacilli to cause degeneration of MP after intramonocytic residence. The bacilli were grown intracellularly for 6 days in either normal or immune monocytes maintained in normal or immune serum. Bacilli liberated from the cells were used to infect new cultures of immune or normal MP. The findings show that intracellular passage of virulent tubercle bacilli in a normal system (normal cells and normal serum) or immune system (immune cells and immune serum) did not result in the loss of the ability of virulent bacilli to cause destruction of normal and immune MP cultivated in the presence of norm,? serum. On the other hand, bacteria passaged through an immune system showed an impaired capacity to destroy normal MP cultivated in immune serum. In continuing these studies, Fong et al. (1961) found that virulent tubercle bacilli, when passaged in a normal or immune system, exhibited decreased virulence for mice as evidenced by low mortality rates after intravenous

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inoculation. Passage in an immune system proved to be more effective than passage in a normal system. This attenuation is described as the result of intracellular residence of the bacilli. However, the reduction of bacillary virulence was not a result of heritable alterations of the bacilli, since in uitro cultivation of passaged bacilli lead to reversion of virulence. Reduction of virulence was found to be accompanied by a loss of the ability to bind neutral red as well as by changes in behavior toward inhibitory substances. These cells were sensitive to inactivation by sodium oleate and by a lysate prepared from normal rabbit MP. Again, bacilli passaged through an immune system were found to be more sensitive than normal-passaged organisms. On the other hand, immune-passaged bacilli proved to be more resistant than unpassaged bacilli to the action of streptomycin. These results contradict observations by Segal and Bloch (1957)who found that tubercle bacilli grown in uivo in lung tissue proved more virulent for mice than those grown in vitro and also exhibited marked differences in metabolic activity (Segal and Bloch, 1956).This difference is difficult to explain except for the fact that it might be due to the complexity of the in viuo environment. Multiple extracellular factors probably do not allow the demonstration of the described attenuation. Virulent smooth brucellae become more resistant to the bactericidal effect of bovine serum after intracellular residence or growth in MP from normal guinea pigs (Stinebring et al., 1960). These cell-grown bacteria are killed at a slower rate by the bovine serum than are bacteria grown on ordinary media. Serum resistance is lost upon cultivation in absence of cells. In contrast, brucellae grown in MP from immunized guinea pigs exhibited increased susceptibility to bovine serum.

F. TRANSFER OF CELLULAR IMMUNITY Transfer of resistance from immunized to normal animals by means of cells strongly suggests independence of cellular immunity from humoral factors. Experiments by several investigators indicate that cellular resistance can indeed be transferred. Sever (1960) using mice reported the successful transfer of a limited degree of increased resistance to tuberculosis by intravenous injection of peritoneal MP from animals immunized with live BCG. No protection was afforded by corresponding cells obtained from normal mice, or by transfer of plasma, spleen homogenate, or spleen filtrate obtained from either normal or immunized mice. Since the degree of protection seems dependent on the number of MP transferred, the limited success can be attributed to the small number of MP transferred in these experiments (three trans-

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fers of 4-6 x 106 cells, intravenously). In no case was prolongation of the survival time as high as after active immunization with BCG. Similar attempts at transfer of acquired resistance to tuberculosis were undertaken by Suter (1961). For the evaluation of the effectiveness of the transfer in vitro and in vivo techniques were employed. The protective effect was found to be limited to challenge infection with either a large dose of attenuated or a small dose of virulent tubercle bacilli. Using an in vttro test system, inhibition of intracellular multiplication of tubercle bacilli was observed by peritoneal MP from normal guinea pigs that had received intraperitoneally or intracardially 2-5 x lo7 lymph node cells from BCG-immunized but not from normal guinea pigs. In ~ i v o tests revealed that the tissue response to infection with tubercle bacilli was altered. Normal guinea pigs receiving 5 intraperitoneal injections of 1 . 5 3 x lo8 lymph node and spleen cells were found to respond to intradermal injections of BCG with the development of smaller skin lesions than guinea pigs receiving either saline or cells from normal donors. In none of the animals injected with cells from vaccinated animals could a Koch phenomenon be observed. Normal mice that had received spleen cells from normal donors responded after an intravenous challenge with virulent tubercle bacilli with a more severe reaction of lung tissue than mice receiving similar cells but from immunized donors. Likewise, the bacterial population in lung tissues of mice vaccinated with BCG or receiving 4 injections of 1.2-1.5 x lo7 monocytic peritoneal exudate cells from immunized donors was reduced after challenge with virulent tubercle bacilli as compared with that of control animals. Extent and severity of pulmonary lesions revealed a similar pattern. Two transfers of cells or a total of 2.9 x lo7 cells were found to have little if any effect in this system. On the other hand, and contrary to Sever's (1960) observations, the transfer of 3 x 108 peritoneal exudate cells obtained from immune donors did not interfere with the progress of a virulent infection. Transfer of BCG-induced resistance of peritoneal histiocytes infected in oitro with virulent tubercle bacilli could be achieved with either peritoneal histiocytes or lymph node cells (Fong et al., 1962). There was a latency between transfer and manifestation of cellular resistance of 11 to 13 days and the resistance remained over several weeks. Cellular resistance was demonstrable only if serum of an actively immunized animal was used in the in vitro test. The MP proved to be a more efficient cell type than the lymphocytes. Thus, 1 x lo7 MP gave maximum transfer, whereas 1 x lo8 lymphocytes were required for the same effect. The MP-transferred resistance persisted longer, i.e., at least 42,

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days compared with 20 days in the case of lymphocytes. Furthermore, lysates with distilled water of MP were effective for transfer, whereas lymphocyte lysates were not. Serial transfer of resistance by means of MP was possible at least through three recipients, whereas the factor was lost in the second recipient with lymphocytes. In further experiments the transfer factor was traced to ribosomal RNA susceptible to RNase (Fong et al., 1963a). The transfer factor proved to be nonspecific, i.e., resistance could be transferred from one host species to another, for example, from immunized rabbits to mice and guinea pigs or from mice to rabbits. Curiously enough, MP from immunized guinea pigs were unable to transfer resistance to a normal recipient (Fong et al., 1963b). All attempts to recover live bacteria from the transfer material, cells or extracts, were unsuccessful. Results similar to those of Sever (1960) and Suter (1961) were obtained by Saito d al. (1962) for Salmonella. It was found that peritoneal MP collected from mice that had received intravenously similar cells from mice immunized with live vaccine exerted in vitro inhibitory action against intracellularly located virulent Salmonella enteritidis. The inhibition, however, was found to be inferior to that exhibited by the donor MP. The fact that Pa*-labeled MP after intravenous injection could not be recovered from the peritoneum but were found to be 50 % in liver, spleen, and mesenteric lymph nodes, led the authors to believe that the inhibition is not due to the admixture of donor cells to the peritoneal cells of the recipient. Contrary to the findings of Fong et ul. ( 1962), sonically disintegrated cells were not effective in the transfer of cellular immunity to normal recipients. Furthermore, recipient cells were found to exhibit cellular resistant already from 3 to 4 days after the transfer of 0.Mx lo7 immune MP, findings which are in accord with those by Suter (1961). It was found that transferred cells always contained small numbers of viable organisms of the immunizing strain. Immunizing normal mice with such small numbers of in oitro grown vaccine strain, however, was found not to confer cellular resistance. Nevertheless, the possibility remains that bacteria transferred inside cells may have developed more rapidly, thereby inducing active immunity. As in Suter’s (1961) transfer experiments, the demonstration of transfer immunity depended on the use of small numbers of virulent organisms for the in vitro infection. Finally, in uitro transfer of cellular immunity has been reported by Mitsuhashi and Saito ( 1962). Tissue culture supernatant of MP obtained from mice immunized with viable vaccine of S. enteriditis seemed to contain the principle responsible for cellular immunity. It was found that

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peritoneal MP from normal mice maintained in uitro for three days in the presence of the tissue culture supernatant of immune cells acquired the ability to inhibit intracellular multiplication of virulent S. enteritidis. The supernatant of normal MP cultures or of cultures of MP derived from animals vaccinated with dead S. enteritidis did not confer cellular immunity to cells in uitro. Most experiments concerned with cellular transfer of resistance were done using allogenic cell systems. Therefore, it is to be expected that the life span of the transferred cells was limited and that the donor cells were not themselves involved in the expression of immunity by the recipient. This is borne out by experiments by Saito et al. (1962) who showed that the intravenously injected donor cells did not reappear in the peritoneal cavity and by the fact that tissue culture supernatant of immune donor cells conferred passive immunity to normal cells cultured with this supernatant ( Mitsuhashi and Saito, 1962). Furthermore, the success of cellular transfer to tuberculosis was not greater using the inbred C57 black strain than it was in the randomly bred Swiss white mouse ( Suter, 1961) . The results of Fong et al. (1963a) and Mitsuhashi and Saito (1962) suggest the importance of a transfer factor, possibly ribosomal selfreplicating RNA which carries information responsible for acquired cellular resistance. The implication of a transfer factor in cellular immunity suggests a resemblance to the transfer factor of delayed hypersensitivity described in man by Lawrence (1959). The active material appears to be a depolymerization product of cellular RNA (Lawrence, 1963). Final judgment as to the relations of these two phenomena and the significance of the findings has to be deferred until certain doubts have been satisfied by further experimental evidence. These doubts are concerned with the likelihood of transfer of antigen resulting in active immunization ( see induction period, serial transfer, and long persistence in Fong’s experiments) or the possibility of the activation of a latent virus inducing cellular changes responsible for increased resistance. Also, more information is needed in regard to the role of immune serum in Fong’s experiments and the molecular changes occurring within the immune cells. If possible, methods should be devised which permit the study of these reactions using single cells. Until such time it will remain an open question whether in transfer of cellular immunity or hypersensitivity the donor cells are involved in the reaction as advocated by Feldman and Najarian (1963) or whether a transfer factor released from the donor cells confers reactivity to cells of the recipient. It is of particular interest to note that MP form intercellular bridges in uitro. This allows the passage of

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cytoplasmic materials, such as RNA, from one cell to another (Aronson, 1963). The suggestion advanced by Nelson and Berk (1960) who visualize cellular immunity as a selection of naturally resistant cells rather than an alteration of individual cells, collides with the observation of serial transfer, Since no bacteria are transferred, as Fong et al. (1962) claim, or only small subvaccination doses are introduced in the recipient (Saito et d.,1962), the agent responsible for inducing selection is missing. Nevertheless, experiments on transfer of cellular immunity have substantiated the fact that the MP may be the carrier of infection immunity. G. RELATIONSHIP OF CELLULAR IMMUNITY AND DELAYED-TYPE HYPERSENSITIVITY

It is not possible to present any definite point of view in regard to the relationship of delayed-type hypersensitivity and cellular immunity. Presumably the same cell, namely, the monocyte or macrophage, is involved in both reactions, at least most phenomena ascribed to either state are closely linked with the presence or activity of this cell. The inflammatory reaction associated with delayed hypersensitivity has been implicated in immunity to reinfection with tubercle bacilli. The exaggerated and accelerated inflammation serves, according to this hypothesis, the rapid immobilization and possibly destruction of the pathogen (Krause, 1926). Current opinion describes delayed-type hypersensitivity as one of three forms of hypersensitivity comparable to, although distinguishable from, the other types, namely, anaphylaxis and Arthus reaction. It is proposed that delayed hypersensitivity, or better celluluar hypersensitivity, represents a unique and distinct reaction involving certain cells of the RES and requiring no mediator for its expression. The delayed inflammatory reaction, usually the measurable expression of the phenomenon, is secondary to the reaction occurring at the level of the sensitized cell and most frequently resulting in damage of the cell. It is suggested that the established techniques of determining delayed hypersensitivity either by skin reaction or cytotoxicity have a high threshold, and therefore, low levels of cellular hypersensitivity escape detection. For these reasons, claims that immunity is separable from delayed hypersensitivity may be true if presence or absence of a skin reaction serves as the measure for desensitization, but they may not be valid in terms of cellular hypersensitivity. These interpretations remain speculative as long as we do not have quantitative criteria for low levels of cellular immunity or hypersensitivity or with absence of a molecular substrate responsible for these

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reactions. At present they have to be described in operational terms. For both reactions, cellular immunity and hypersensitivity, the macrophage or monocyte appears to be the cell involved. I n uitro experiments on cytotoxicity and inhibition of intracellular multiplication as well as transfer experiments provide evidence for this. It is possible to speculate on how cellular hypersensitivity can be the effector of cellular immunity. Presence of the antigen within the phagocytic vacuole of a sensitive MP could lead to reactions resulting in a discharge of inhibitory material into the vacuole from lysosomes or the nucleus, or could on its own create conditions unfavorable for the pathogen. Of relevance may be the finding that MP from guinea pigs treated with meprabamate inhibit in vitro intracellular multiplication of Brucella suis in presence of 10 pg/ ml streptomycin. It is speculated that cellular permeability is altered allowing entry into the cell of components otherwise excluded, such as streptomycin or antibody (Kessel et al., 1961). Consequently, the immune MP could also be in the state of altered permeability permitting antibody action on intracellulary located pathogens which are protected from antibody within normal cells. Proof for these hypotheses requires work on single MP similar to the experiments by Nossal on single plasma cells (see Nossal and Makela, 1962). There are numerous questions which require experimental study before a theory of cellular immunity can be established. One ought to know whether all monocytes or macrophages are affected, as it seems is the case with increased acid phosphatase content. Also, a decision on the site of the action of the stimulating antigen or component is impossible. We may consider that a mediator with high affinity for MP similar to cytophilic antibody is involved ( Boyden and Sorkin, 1960), although attempts by Boyden (1963) to demonstrate such a function of cell-bound antibody proved negative. The further possibility that MP produce a sensitizing factor cannot be excluded since these cells probably represent an essential first station in antibody formation (Fishman, 1961). It is conceivable that MP play a rather crucial role in the over-all immune response. First, the MP which ingest foreign material and possibly dispose of it represent the site of contact between foreign material and the antibodyforming system. Second, their ability to destroy and contain microorganisms is part of their role in immunity to infection. Third, their ability to respond with changes to many stimuli adds flexibility to the adaptive power of a higher organism. Included here are hyperplasia, enzymatic changes, and ability to be mobilized. Fourth, the necessity of antigen to combine with RNA of these cells in order to elicit an antibody response would add further weight to their role, With the information at

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hand, the possible relation between the phenomenon of cellular immunity and hypersensitivity and the involvement of MP in antibody formation can only be surmised. H. THEIMMUNE CELLIN REACTIONSTO TISSUE AND CELLULAR HOMOGRAFTS

As a result of fairly recent investigations it has become increasingly clear that immunity to cellular grafts in all likelihood is mediated by a mechanism bound to cells of the lymphoid series. Several lines of evidence support this notion. Fundamental studies by Billingham et al. (1954, 1963) have shown that living, immunologically activated cells are capable of transferring sensitivity to skin homografts adoptively, whereas immune serum is quite ineffective (Brent and Medawar, 1961). Similarly, Mitchison ( 1955) succeeded in transferring immunity to transplantable tumors by means of immune lymph node cells but not by immune serum, and Weaver et al. (1955) and Algire et al. (1957) demonstrated the ineffectiveness of immune serum in the destruction of homografts. Homologous tissue enclosed in diffusion chambers, when placed into the peritoneal cavity of immune mice, would not undergo destruction unless the pores of the membrane were of a size that they allowed passage of host cells. Events taking place in the destruction of homografts resemble delayed-type hypersensitivity reactions (Medawar, 1959; Brent, 1958; Brent et aE., 1962; Ramseier and Billingham, 1963). Similarities include delay in onset, transferability by means of cells but not serum, histological picture of the lesions, and susceptibility to anti-inflammatory but not antihistaminic drugs. However striking this line of evidence is, the role of humoral antibodies in destruction of homografts is not easily assessable. In certain situations, especially with tumors of lymphoid origin, direct cytotoxic action has been demonstrated ( Algire, 1959; Winn, 1960; see also review by Amos, 1962). Studies on the mechanisms by which immune cells react against homologous cells have revealed interesting findings which also have shed light on quantitative aspects. Snell et al. (1961) and Winn (1961) studied quantitatively the reactions of immune lymphoid cells on homologous tumor cells. Various dilutions of lymphoid cells prepared from mice immunized with homologous tumor cells were mixed with a constant amount of donor strain type ascites tumor cells and the mixture was injected subcutaneously into recipient mice. At a time when control animals showed substantial growth of the tumor, the assay animals were

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killed and the tumors were removed and weighed. Immune cells obtained from spleen, draining lymph nodes, and contralateral lymph nodes were found to be effective suppressors. The inhibitory capacity reached a sharp peak at 5 or 6 days, but immune manifestations could be demonstrated up to day 31 when the experiment was terminated. At peak efficiency, approximately 2 x lo6 immune lymphoid cells reduced the growth of 3 X lo6 tumor cells by one-half. Not only was a clear numerical relationship between immune cells and target cells established, but the results also suggested that a local engagement of the cells was necessary. Injection of immune cells and target cells at different sites of the body led to a trivial depression of tumor growth. What appears to be the best evidence in demonstrating the reactions of immune cells against cells of homologous origin has been obtained from in vitro studies. Investigations in this area were conducted by Rosenau and Moon (1861, 1962), Koprowski and Fernandes (1962), Rosenau (1963), Wilson (1963), and Taylor and Culling (1963). These studies showed quite conclusively that target cells (usually a line of tumor cells) in tissue culture are destroyed by immune but not by normal lymphoid cells. The formation of clusters of immune cells around target cells followed by progressive injury of tumor cells was a typical observation. In some situations, the attacking lymphoid cell was found to die following the interaction. Contact between immune cells and target cells is of significance, for cells of both types, when enclosed in separate sections of a Millipore filter chamber, would not undergo cytolysis. Mixtures of different lymphoid cell types, originating from spleen, lymph nodes, and thoracic duct have been used as sources of immune cells. Convincing evidence is available pointing to the small lymphocyte as one of the effective cell types. The specificity of the reaction is given by the findings that lymphoid cells of animals sensitized to different antigens are ineffective. The mechanism responsible for killing of homologous cells is not well understood. The reaction leading to the death of target cells is independent of complement. Immune serum does not affect the system. On the other hand, sera of immunized animals are cytotoxic if guinea pig serum is present but this might represent a mechanism different from the one observed with lymphoid cells alone and in the absence of complement. Incubation of immune cells in the growth medium does not result in the extraction of a cytolytic factor nor does a Millipore filter allow passage of such a principle. Of importance is the observation that addition of hydrocortisone to the medium prevents cytolytic manifestations but not clustering of immune cells around target cells.

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It has become quite fashionable to explain mechanisms of cellular immunity in terms of “cell-bound antibodies” which are thought to be either tightly bound to the immune cell or released upon contact with the antigen. Unfortunately, nothing is known in physicochemical terms about the existence of a peculiar form of protein associated with the immune cell. It might also prove to be difficult to isolate a substance responsible for immune manifestations, for it could well be possible that a chain of reactions leads to the immune state. The interesting observation by Mannick and Egdahl (1962) suggesting that RNA extract obtained from sensitized lymph node cells is capable of rendering normal lymph node cells immunologically active is pertinent in this context and suggests that some form of protein synthesis is involved in the immune status. Further indication along this line is given by findings of Jankovib and Dvorak ( 1962) who showed that manifestations of delayed-type hypersensitivity (which result when sensitized lymph node cells are injected intradermally into normal rabbits) can be abolished by treating these cells with RNase. Observations on cellular reactions to homografts point to the importance of the viability of the competent cell. This would speak against the possibility that immune cells carry a preformed immune substance which is simply released upon contact with the antigen. It might be of significance that in tissue culture studies on homograft reactions several hours have to elapse before attachment of immune cells to target cells occurs with consequent destruction of the latter. Clustering of immune cells around target cells, a phenomenon termed “contactual agglutination” (Koprowski and Fernandes, 1962) could be due to a chemotaxic attraction. The following interaction leading to cytolysis is not understood. Apparently, clustering of normal lymphoid cells around homologous cells, when induced artificially, does by itself not lead to any deleterious reactions ( Rosenau, 1963). Although morphological observations, particularly the swelling of target cells or cell organelles with subseqent disruption of the cell’s membrane, indicate alterations of surface properties, it is not quite clear whether these are the primary cause of death or secondary manifestations. Likewise, the contribution of enzymes, either those of the attacking lymphoid cell or those of the target cell-possibly leading to autolysis after some primary alteration-is not known. Should such immune reactions be due to the presence of “cytophiIic antibodies” (Boyden, 1963) it would be interesting to investigate whether immune cells contain or elaborate during the immune interaction sufficient amounts of complement necessary for a cytolytic reaction. Results obtained by Rosenau (1963) not only indicate that the amount of

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complement present in sera of sensitized mice is insufficient to produce a cytolytic effect, but also show that normal lymphoid cells suspended in cytotoxic sera do not pick up enough serum and complement to cause cytolysis of target cells. A somewhat less complex situation exists in the reaction of immune cells against a number of homologous tumor cells. Here phagocytosis seems to play a major role as a defense mechanism, whereas this phenomenon can hardly be of significance in the rejection of solid, vascularized tissue homografts. Phagocytosis is of particular importance in the rejection of several ascites tumor cells, Certain aspects of this process are reminiscent of what was described for obligate extracellular parasites. Thus it was found that appreciable phagocytosis of ascites tumor cells coincided in time with the appearance of antibody in the serum (Amos, 1960).Phagocytosis of tumor cells in vitro by immune MP depended on specific opsonins (Bennet et al., 1963; Old et al., 1963). Studies by this group revealed the interesting finding that only MP from immune animals were able to phagocytize tumor cells in presence of antibody, whereas cells from normal animals were not. Immunity was found to be highly specific in that mice immunized against different tumors were not effective. Characteristics of cellular immunity were found by the observation that immunity to tumor cells could not be transferred passively by immune serum but by immune cells. Furthermore, immunity could not be reconstructed by addition of immune serum to normal cells, but rather the serum resulted in enhancement of tumor growth. Likewise, the supernatant of an immune population of MP resulted in enhancement when injected into normal mice with or without normal peritoneal MP. Tissue injuries as those which were briefly discussed in this section have to await their full understanding. It should be mentioned that most probably similar problems arise in a variety of autoimmune diseases. The role of antibody in transplantation immunity has been fully summarized by Stetson ( 1983). VI. Conclusion

The central position of the RES in reaction to infection is unquestioned in spite of the fact that PMN appear to be better equipped for intracellular kill. Longevity and functional potential of the RES cell both absent in the PMN, permit it to participate in adaptations resulting in fundamental changes of the host's reactive capacity. Stimulation by infection or immunization may result in changes of quantity and distribution of PMN, but has no influence on the functional capacity of the individual cell. By contrast, similar stimuli result in quantitative and

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qualitative changes of the RES cell. It is proposed that this responsiveness of the RES represents an essential part of the immunological response to infection and antigenic stimulation, expressed as cellular immunity, cellular hypersensitivity, and antibody formation. Although there is not enough information to present a rounded picture of the function of the RES cell, it seems justified to enumerate some of its properties which endow it with such a variety of functions. 1. It is likely that there exists interconvertibility between RES cells, plasma cells, and small lymphocytes, thus providing a link between cellular and humoral immunity. This is substantiated by the finding that complex formation between antigen and MP-RNA appears to be a first step in the process leading to antibody formation (Fishman and Adler, 1963). It is tempting to suggest that the RES cell, which has picked up and processed antigenic material, either passes the complex to other cells or is transformed into a cell with the capacity to produce antibody. The former notion is substantiated by the fact that a feeding mechanism between MP and lymphocytes is observable in tissue culture (Fishman, 1963). The very close relationship between the RES and antibody formation is further indicated by the effect of RES stimulation on antibody formation either by Freund's adjuvant, endotoxin (Johnson, 1964), or glucan ( Wooles and DiLiuzio, 1963). 2. Increased resistance to infection is dependent on RES function, which can be stimulated in a specific and nonspecific manner. The capacity of the RES for protein synthesis enables adjustment of enzyme levels and possibly of other specific proteins. Since the majority of RES cells appear to be participating in some of these changes, even upon local stimulation, the search for a regulatory substance which may belong to the category of antibodies, hormones, or others is indicated. 3. Cellular immunity exhibits similarity with cellular or delayed hypersensitivity. Although the identity between these two reactions has not been proven, the assumption of some connection or interaction seems justified. Until techniques are developed for the study of these phenomena with single cells, little progress can be expected. 4. Of great interest is the possible role of RNA in cellular reactions as demonstrated in the priming for antibody formation (Fishman and Adler, 1961), the transfer of cellular immunity by RNA derived from MP of immunized rabbits (Fong et al., 1963a), and the transfer factor for delayed hypersensitivity in man (Lawrence, 1963). In all instances, RNA from RES cells has been found to be the active fraction. 5. In spite of the considerable evidence for the importance of cellular immunity, in most instances circulating antibody has been found con-

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tributory. This is especially the case in transplantation immunity (Stetson, 1963). For each individual model the role of antibody has to be evaluated on its own merit. REFERENCES

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Michael, J. G., Whitby, J. L., and Landy, M,. (1962). J. Exptl. Med. 115, 131-146. Mika, L. A., Goodlow, R. J., Victor, J., and Braun, W. (1954). Proc. SOC. Exptl. Biol. Med. 87, 500-507. Miller, B. F., Abrams, R., Dorfman, A., and Klein, M. (1942). Science 96, 428-430. Mitchison, N. A. (1955). J . Exptl. Med. 102, 157-177. Mitsuhashi, S., and Saito, K. (1962). J . Bacteriol. 84, 592-593. Mitsuhashi, S., Kawakami, M., Yamaguchi, Y., and Nagai, M. (1958). Japan. J. Exptl. Med. 28, 249-258. Mitsuhashi, S., Sato, I., and Tanaka, T. (1961). I. Bacteriol. 81, 863-868. Morse, S. I. (1962). J . Exptl. Med. 115, 295-311. Mouton, D., Biozzi, G., Berthillier, Y., and Stiffel, C. (1963). Nature 197, 706. Munroe, J. F. (1963). Personal communication. Myrvik, Q., and Weiser, R. S. (1951). Am. Rev. Tuberc. 64, 669-674. Myrvik, Q. N., and Weiser, R. S. (1955). J. Immunol. 74, 9-16. Myrvik, Q. N., Leake, E. S., and Fariss, B. (1961). J. Immunal. 86, 133-136. Myrvik, Q. N., Leake, E. S., and Oshima, S. (1962). J. Immunol. 89, 745-751. Nelson, E. L., and Berk, R. S. (1960). Ann. N.Y.Acad. Scl. 88, 1246-1264. Njoku-Obi, A. N., and Osebold, J. W. (1962). J . Immunol. 89, 187-194. Noll, H. (1956). Adv. Tuberc. Res. 7, 149-183. S. Karger, Base1 and New York. North, R. J., and Mackaness, G. B. (1963a). Brit. J . Exptl. Path. 44,601-607. North, R. J., and Markaness, G. B. (196313). Brit. J. Exptl. Path. 44, 608-611. Nossal, G. J. V., and Ada, G. L. ( 1964). Nature 201, 580-582. Nossal, G. J. V., and MUelL, 0. (1962). Ann. Reu. Microbiol. 16, 53-74. Nyka, W. (1956). Am. Reu. Tuberc. Pulmonary Diseases 73, 251-265. Old, L. J., Clarke, D. A., Benacerraf, B., and Goldsmith, M. (1960). Ann. N.Y. Acad. S C ~ 88, . 264-280. Old, L. J., Boyse, E. A., and Bennett, B. (1963). In “Cell-bound Antibodies” (B. Amos and H. Koprowski, eds. ), pp. 89-98. Wistar Inst. Press, Philadelphia. , N., and Leake, E. (1961). Brit. J. Exptl. Puthol. 42, 138-144. Oshima, S., M y ~ i k Q. Pavillard, E. R. J. (1963). Australian J . Exptl. Biol. Med. Sci. 41, 265-274. Pearson, G. R., Freeman, B. A., and Hines, W. D. (1963). J. Bacteriol. 86, 1123-1125. Perkins, E. H., and Leonard, M. R. (1963). J. Immunol. 90, 228-237. Pomales-Lebrbn, A., and Stinebring, W. R. (1957). Proc. SOC.Exptl. Biol. Med. 94, 78-83. Potter, E. V., and Stollerman, G. H. (1961). J. Immunol. 87, 110-118. Puck, T. T. (1961). Haruey Lectures Ser. 55, 1-12. P u h g e r , E. J. (1936). I. Hyg.36, 456-466. Ralston, D. J., Baer, B. S., and Elberg, S. S. ( 1961). J. Bacteriol. 82, 342-353. Ramseier, H., and Billingham, R. E. ( 1963). Unpublished observation. Ramseier, R., and Suter, E. (1964). 1. Immuno2. In press. Repaske, R. (1956). Biochim. Biophys. Acta 22, 189-191. Ribble, J. C. (1961). Proc. SOC.Exptl. Biol. Med. 107, 597-600. Rogers, D. E., and Melly, M. A. (1960). J . Ex@. Med. 111, 533-558. Rogers, D. E., and Tompsett, R. (1952). J . Exptl. Med. 95, 209-230. Rosenau, W. ( 1963). In “Cell-bound Antibodies” (B. Amos and H. Koprowski, eds. ), pp. 75-83. Wistar Inst. Press, Philadelphia. Rosenau, W., and Moon, H. D. (1961). J . Natl. Cancer Inst. 27, 471-483. Rosenau, W., and Moon, H. D. (1962). 1. Immunol. 89, 422-426. Rous, P. (1925). J . Exptl. Med. 41, 399-411.

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Ultrastructure of Immunologic Processes JOSEPH D. FELDMAN Division of Experimental Pathology, Scrippr Clinic and Research Foundation, La lalla, California

I. Prologue ................................................... 11. Antibody .................................................. A. Formation ............................................. B. Storage and Release ..................................... C. Sensitized Cells ( Cell-Bound Antibody) .................... 111. Antigen ................................................... A. Exogenous ............................................. B. Endogenous ........................................... IV. Ultrastructure of Immunologic Reactions ........................ A. Antigen-Antibody Union ................................. B. Pathology ............................................. V. Epilogue .................................................. Acknowledgments ........................................... References .................................................

175 178 178 185 187 189 189 193 198 198 202 235 237 237

I. Prologue

The electron microscope was delivered upon the threshold of biology less than 25 years ago. With the advent of this instrument, it became possible to peer inside the cell, to see with clarity the organelles which constitute the cell, and to glimpse at individual molecules and molecular aggregates which are vital to cellular function. Within this same time span the discipline of immunology burgeoned and rooted itself in all fields of biology, including biochemistry, genetics, medicine, and biophysics. In its accelerated growth, immunology encompassed a wide range of phenomena from gross pathology to molecular structure and interaction. At the latter level, the electron microscope is the instrument par exceZZence for the visualization of subcellular events. The aim of this review is to examine the shape of immunology in the electron microscope. This is a propitious time, for it is still possible to read all the ultrastructural reports related to immunology. In a short while the task will be overwhelming. To focus the subject matter for the reader, I am defining ultrastructure as the form and relationship of cells, parts of cells, and tissues beyond the resolution of the light microscope. Such a definition excludes the observations obtained by ultracentrifugation, which is a form of ultradissection facilitating an examination of intracellular parts but without preserving the relationship of the parts to each other. Also 175

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excluded is histochemistry and immunohistochemistry ( fluorescence microscopy ) which uncover the position of specific cellular components but do not disclose structure. For all practical purposes, then, the ultrastructural image of immunologic processes and reactions is the image created by means of the electron microscope. Form by itself, however, is of little value without concomitant chemical or functional meaning. And so I shall use data from sources other than those directly concerned with electron microscopy when they are pertinent to and explanatory of ultrastructure. A complete bibliography of immunologic references is neither practical nor warranted, and citations, over and above those relating to ultrastructure, are limited to current relevant reports. II. Antibody

A. FORMATION An ultrastructural description of antibody formation requires the identification, first, of the cell type or types capable of synthesizing the new specific protein, and, second, of the locus of production within the cell. Neither of these two facets of the problem has been finally resolved. The plasma cell line, by several different kinds of demonstration, has been nominated as the series most likely to produce antibody. That plasma cells may contain specific antibody has been known ever since the immunohistochemical visualization by White (1954) and Coons et al. (1955) of antibody within their cytoplasm. That plasma cells or their precursors may synthesize specific antibody requires more evidence. It has been shown that the appearance of plasma cells in antigenically stimulated tissue is followed by detectable elevation of specific antibody in the circulation (Fagraeus, 1948; Neil and Dixon, 1959) and in the stimulated tissue (Kuge, 1957; Fuji, 1958); that the absence of plasma cells is accompanied by diminished levels or absence of circulating antibody (Good et al., 1960; Thorbecke, 1960; Sercarz and Coons, 1963); that plasma cell tumors make specific y-globulins (Potter and Fahey, 1960); that plasma cells possess the morphologic machinery capable of producing protein for export (Braunsteiner et al., 1953a); and finally, that this type of machinery, microsomes and ribosomes, are intimately and causally related to antibody synthesis (Kern et al., 1959; Eisen d al., 1961). The fine structure of the plasma cell was first described by Braunsteiner et al. (1953a,b,c) and has since been repeatedly redescribed by many others (Policard et ul., 1954, 1957c; Braunsteiner and Pakesch, 1955; Thiery, 1955, 1960; Dohi et al., 1956, 1957; Kautz et al., 1957;

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Wellensiek, 1957; Movat and Wilson, 1959; Movat and Fernando, 1962; W. Bernhard and Granboulan, 1960; Fruhling et al., 1980~;Stobbe, 1960; Welsh, 1960, 1962; Bessis, 1961; Dalton et al., 1961; Parsons et al., 1961a,b). The most characteristic morphologic feature is its extensive and elaborate rough-surfaced endoplasmic reticulum (Fig. 1 ) This organelle is composed of a congeries of parallel double membranes encrusted on their outer surfaces by a fairly regular spacing of ribonucleoprotein particles and it fills almost the entire cytoplasm of the cell. The significance of this type of intracellular machinery was immediately appreciated, since a similar organelle had been shown to be present in cells ( pancreatic acinar, thyroid epithelium, etc. ) which synthesized protein for export. Later, Palade and Siekevitz (1956) demonstrated the relationship of the rough-surfaced endoplasmic reticulum to the synthesis of protein in cells which export their proteins for use outside the cell. The other uhastructural components of the plasma cell, e.g., the Golgi element and mitochondria, are not distinctive and need not be described here. There is good evidence, then, morphologic and biochemical, that plasma cells and their precursors both contain and synthesize y-globulin and specific antibody. But the problem of the derivation of plasma cells remains unsettled and the morphologic recognition of their progenitors has not been satisfactorily reported. Ultrastructurally the plasma cell appears to be a type mi gene&, unrelated to the lymphocyte or the macrophage. There are those who believe it may be derived from a common stem cell through a transitional form (Fagraeus, 1948; Stoeckenius, 1957; Stoeckenius and Naumann, 1958; Granboulan, 1960) ; from perivascular adventitial cells ( Amano et al., 1951; Amano, 1958, 1962); and from mature or immature lymphoid elements (Sundberg, 1955). But the definitive ultrastructural observations on the development and maturation of the plasma cell have not been made. Such a study would require the use of a double label: one to tag the undifferentiated immature progenitor early in its life and a second to prove that such a tagged cell carries specific antibody when it matures. This has been accomplished at the light optical level by combining autoradiography and fluorescence microscopy of the same cells (Baney et al., 1962; Urso and Makinodan, 1963). Several investigators have pictured what they consider to be precursors of the plasma cell, but the presence of a primitive or even well-developed granule-encrusted ergastoplasm in a cell does not prove that such a cell is making either y-globulin or antibody, nor does the increase of a cell type in response to antigen indicate that the cell type is directly responsible for antibody synthesis.

.

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FIG. 1. Plasma cells ( P C ) from the spleen of a mink with Aleutian disease. The periphery of the cytoplasm is occupied by an extensive and complex roughsurfaced endoplasmic reticulum (er) which is composed of parallel lamellae encrusted on their outer surfaces with dense ribonucleoprotein granules (see inset). Within the space between the lamellae is a slightly dense material, possibly mink y-globulin. In the central cell a large dilated Golgi element ( G o ) is visible adjacent

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The plasma cell series synthesize y-globulin physiologically, under the stimulus of antigen, and pathologically, as in myeloma. To date no recognizable cytologic difference exists between normal and pathologic plasma cells. In mouse plasmacytoma, however, inclusions which resemble viral particles are visible in the cytoplasm of plasma cells (Dalton et al., 1961) (Fig. 2). Recent immunologic and morphologic evidence suggest there are at least two cell types, one associated with 7 s y-globulin and the other with 19 S y-macroglobulin. Cytologic and immunohistochemical examination of smears and tissues prepared from the blood and lymphoid tissues of some patients with Waldenstrom’s macroglobulinemia and of others with agammaglobulinemia and dysgammaglobulinemia have demonstrated 19 S y-globulin in the cytoplasm of “lymphocytoid or “reticular” plasma cells (Curtin and O’Dea, 1959; Fruhling et al., 1960a; Dutcher and Fahey, 1960; Parakevas et al., 1961; Cruchaud et al., 1962; Solomon et al., 1963). A marked increase of this cell type is also observed in the tissues and correlated with high levels of circulating 19 S y-globulin (Chadbourn and Zinneman, 1955; Braunsteiner et al., 1956a, 1957; Braunsteiner and Pakesch, 1980; Dutcher and Fahey, 1959). The few electron microscope studies of the “lymphocytoid” or “reticular” plasma cell portray a moderately developed granular endoplasmic reticulum which does not occupy the cytoplasm as fully as it does in the typical 7 S y-globulin-containing plasma cell (Braunsteiner et d.,1956a, 1957; Fruhling et al., 1960a,b; Zucker-Franklin et al., 1962; Zucker-Franklin, 1963) (Fig. 3). This is a minor morphologic variation and perhaps additional material will bring to light other differences. It is not known whether all 19 S y-globulin is synthesized in the “lymphocytoid or “reticular” plasma cell or in other cell types as well. Mellors and coworkers, with fluorescent stains (see Mellors et al., 1961a,b; Kritzman et al., 1961) have pictured rheumatoid factor (probably 19 S y ) in germinal center cells of lymph nodes, which may be another name for Braunsteiner’s “reticular” plasma cells. More recently, Mellors and Korngold (1963) have shown that the plasma cell line may carry a y2-, ylM-, or ylA-immunoglobulin but not more than one of these proteins in a cell. There are at least two alternatives that explain why such large quantities of y-globulin are synthesized in myeloma or Waldenstrom’s to the nucleus. Mitochondria ( M ) are few and dispersed throughout the cytoplasm. A lymphocyte (Ly) contrasts sharply with the plasma cells. Its cytoplasm is clear, has little or no endoplasmic reticulum, and several hardly visible mitochondria. Magdication: x 18,500; inset, x 35,500.

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FIG.2. A portion of a plasma cell, highly magnified, from a mouse plasma cell tumor. A ferritin antimouse myeloma globulin conjugate was reacted in uitro with tumor tissue. Ferritin particles are visible in the spaces between the lamellae of the endoplasmic reticulum (er ) indicating the presence of mouse y-globulin in these sites. Localization of ferritin was not found elsewhere and was specific. A viral-like particle ( v ) is present within an ergastoplasmic cistema. Magnification: x 98,000. (From Rifkind et al., 1962.)

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macroglobulinemia patients. The excessive amounts of abnormal protein may be merely an augmented end product of an increased number of cells multiplying without restraint, or they may be antibody to an unknown antigenic agent, such as virus. In murine plasmacytoma (Parsons et al., 1961a,b) and in one human case of multiple myeloma (Sorenson, 1961) structures resembling virus have been found in the plasma cells (Fig. 2 ) . In addition, Aleutian mink disease provides an excellent example of diffuse hyperplasia of plasma cells and hypergammaglobulinemia associated with a viral agent (Porter and Dixon, 1963).

FIG.3. A plasma cell from a case of Waldenstrom’s macroglobulinemia. Within the cytoplasm is a rough-surfaced endoplasmic reticulum much less developed and elaborate than that shown in Fig. 1. The nucleus occupies more relative volume than that of a typical 7 S plasma cell. This cell type has been shown to contain 19 S y-globulin in its cytoplasm and occasionally in its nucleus. Magnification: x 16,800. ( From Zucker-Franklin, personal communication. )

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Other cell types have been shown to contain either y-globulin or specific antibody. In thoracic duct lymph and in the population of spleen cells, antigenically stimulated, a very small number of cells, less than 1%, has been found which have antibody in their cytoplasm and which by all cytologic criteria resemble a small lymphocyte (Vazquez, in press). The ultrastructural design of this cell type would be most interesting to delineate. Would there be a well-developed and complex ergastoplasm? Or would there be another kind of intracellular machinery hitherto undescribed? To date, no one has pictured a cell which resembles a mature lymphocyte and which has any distinguishing cytoplasmic landmark to separate it from its confreres that do not carry antibody. Nor have there been any ultrastructural studies of the germinal center and primitive reticular cells which contain y2-, ylM-, and ylA-immunoglobulins in their cytoplasm (Mellors and Korngold, 1963). Recently, Schaffner and Popper (1962) described a cell, found in human liver, which shows y-globulin by fluorescence microscopy and ultrastructural features of a phagocyte by electron microscopy. One side of the cell cytoplasm is occupied by phagosomes and the other side by a small, moderately developed, rough-surfaced endoplasmic reticulum. The significance of this cell in antibody formation is unknown and it remains to be learned whether the y-globulin present in the cell is there because it is synthesized in situ or because it is engulfed. Solomon et d. (1963) have reported a whole spectrum of cells from patients with multiple myeloma and Waldenstrom’s macroglobulinemia which by fluorescence microscopy were shown to contain different kinds of immunoglobulins. Mention has been made also of germinal center elements that contain y-globulin in lymph nodes of patients with rheumatoid arthritis. The fine structure of these cell types and their synthetic apparatus, as well as their relationship to the production of the circulating globulins, await future clarification. The identification of the intracellular locus of antibody synthesis is beset with similar troubling problems. What can reasonably be considered synthesis and what storage or accumulation by engulfment? There is sufficient evidence now to conclude that the ribonucleoprotein particles of the cytoplasm, polymerized or otherwise, attached to parallel double membranes, are closely involved in the synthetic mechanism for protein export and, in the case of plasma cells, for antibody production. Eisen and his colleagues have shown that specific antibody is synthesized in a microsome fraction obtained from stimulated lymph nodes (Kern et al., 1959; Eisen et al., 1961). By using labeled amino acids and dissection by ultracentrifugation, they demonstrated new specific protein

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associated with microsomes, i.e., with rough-surfaced endoplasmic reticulum. With this technique it is not possible to pinpoint the cell type involved in synthesis of the labeled antibody. Askonas (1961a,b) has reported that the group of specific proteins produced by mouse myeloma cells is also associated with the microsomes which may be separated from these cells by ultracentrifugation. More recently, Wust and Novelli (1963) presented evidence that new antibody is intimately associated with ribosomes from stimulated lymph node cells. The actual morphologic demonstration of the site of intracellular y-globulin and antibody synthesis was achieved by Rifkind et al. (1962) and de Petris et d. (1963). The former group, using Singer’s ferritin protein conjugate technique, have illustrated the site of y-globulin in myeloma cells of the mouse. Ferritin label is found within the rough ergastoplasm, between the parallel double membranes, and not elsewhere (Fig. 2). Appropriate controls to eliminate nonspecific adsorption clearly display the specificity of the site of mouse y-globulin. It is proper to note that in this instance y-globulin is not antibody, although the new protein might be a response to unknown antigen. De Petris et al. (1963) have carried the observations one step further and show that antibody is present in similar sites. They use ferritin as an antigen to stimulate production of antibody and after a suitable interval of time remove the stimulated lymph nodes, react them in vitru with antigen, and find ferritin aggregates in the spaces between the encrusted parallel membranes of the endoplasmic reticulum (Fig. 4). Appropriate control manipulations eliminate the chance of nonspecific aggregation and adsorption in these intracellular loci. There is no evidence, morphologic, biochemical, or physiological, that any other organelles of the cytoplasm, e.g., Golgi element, mitochondria, or centrioles, might be involved directly in antibody synthesis. Hanaoka (1953), however, claims that mitochondria develop into Russell bodies following antigenic stimulation of mice. His micrographs are too inadequate to judge the validity of the statement and, further, the evidence is derived from light microscope observations which do not permit clear identification of these or other intracellular organelles of similar size. Antibody in nuclei of lymphoid cells from immunized mice was found by Coons et al. (1955) and others. Only a small proportion of the antibody-containing elements show the specific staining by fluorescence microscopy. The significance of intranuclear antibody has not been determined, nor is it known whether this might represent synthesis, storage after difFusion from the cytoplasm, or an artifact in which a small portion of antibody-containing cytoplasm is enclosed by nuclear material. There

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FIG.4. A portion of a plasma cell, highly magnified, from the spleen of a rabbit immunized with ferritin. The spleen tissue was reacted in vitro with ferritin which localizes within the spaces of the granular encloplasmic reticulum. The localization of ferritin in these sites is specific and indicates the presence of specific anti-ferritin. Nu, nucleus. Magnification: x 82,000. (From de Petris et al. ( 1963),)

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are no observations from electron microscopy concerning this light microscope finding (see, however, Section 11, B for intranuclear y-globulin in myeloma and Waldenstrom’s macroglobulinemia) . B. STORAGE AND RELEASE In the animal hyperimmunized by multiple injections of antigen, or in the human afflicted with an infectious process of long duration, tissue plasma cells are seen in which dense protein globules of various sizes appear in the cytoplasm. These are Russell bodies, and the electron microscope has shown them to be composed of dense material entrapped between the smooth surfaces of a pair of membranes forming the roughened endoplasmic reticulum. Indeed, a spectrum of plasma cells can be photographed depicting the earliest accumulation of a slightly dense material between slightly dilated endoplasmic reticulum membranes to a final concretion of dense spherical or ovoid masses so large that they destroy the enveloping endoplasmic reticulum membranes and even the cell itself. In laboratory models, when the antigen is known, these cytoplasmic accumulations of dense material can be identified, at least in part, as specific antibody, or otherwise as y-globulin. It is assumed that Russell bodies are the result of antibody storage, resulting from either excessive synthesis or diminished release from the cell. Intranuclear inclusions often resembling Russell bodies in texture and electron density, are occasionally present in plasma cells from patients with multiple myeloma and macroglobulinemia ( Thiery, 1957; Fruhling et aL, 1960b; Brittin et al., 1963). Brittin et al. (1963) believe that these inclusions, which contain, in part, y-globulin, and which histochemically resemble Russell bodies of the cytoplasm, arise in the nucleus and have no connection with cytoplasmic organelles. In one micrograph, however, a large complex inclusion is shown to be formed in part by invagination of the granular endoplasmic reticulum into the nucleus. Thiery (1957) illustrated by cinematography the appearance within and expulsion from the nucleus of intranuclear vacuoles. In the electron microscope they diger from those of Brittin et aZ. and are membrane-lined spaces. Antibody has not been shown to be present in them. Fruhling et aZ. (1960b) present several micrographs of plasma cells with intranuclear inclusions, also showing invagination of the perinuclear membrane into the nucleus. At present, the site of synthesis, the mode of formation, deposition and significance of intranuclear antibody-containing inclusions in plasma cells of myeloma and macroglobulinemia patients are unsolved problems. A number of authors have also illustrated protein crystals contained within the membranous boundaries of the rough endoplasmic reticulum

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of plasma cells (Thiery, 1957; J. Bernard et al. (1959); A, F. Goldberg, 1960). In most instances the observations have been made in cases of leukemia or lymphoma. Again, it is assumed that the crystals represent a storage phenomenon, The presence of protein crystals within the cytoplasm of a cell does not of necessity mean that they are synthesized by a rough-surfaced endoplasmic reticulum. Bessis ( 1961) and others have pictured such crystals in the cytoplasm of lymphocytes lacking this organelle, or at least an ergastoplasm that is highly developed; nor has the crystal been shown to be enveloped by endoplasmic reticulum membranes. Are these a geometric pattern of replicated virus rather than a synthetic product of the ergastoplasm? Or are they, perhaps, engulfed material arranged in crystal form? Charcot-Leyden crystals are also found in the cytoplasm of eosinophiles, most likely not as the product of the endoplasmic reticulum, but rather as phagocytized debris (Welsh, 1959; Fruhling et al., 1961a). Other than Russell bodies and the rare intracytoplasmic protein crystals, there seems to be no morphologic form of antibody storage, in the sense that thyroglobulin is retained within thyroid follicles or zymogen granules are packaged as droplets within a membrane-lined vacuole. When antigen stimulation is cut off, synthesis of antibody also declines and levels of antibody, at least in the circulation, rapidly fall. It would seem that the total quantity available at any specific time depends upon the number of plasma cells specifically producing that antibody, i.e., upon a dynamically changing population of lymphoid cells which can replicate and produce new protein on demand and which disappear when not needed. The opposite face of antibody storage is antibody release. At an ultrastructural level there is no acceptable illustration of this process. By light microscopy, cytoplasmic shedding by macrophages and perhaps other lymphoid elements has been an oft-repeated observation regarded as a type of secretion. This type of observation has been made only in vitro and the question always arises as to whether this is a phenomenon that might occur in vivo or whether it is an artifact of procedure. The electron microscope has not provided any clear answer. Often parts of cytoplasm, whether they are from plasma cells, macrophages, lymphocytes, or other cell types, are visualized completely contained within an intact cell membrane, apparently unrelated to the main body to the cell, and such parts look like shed cytoplasm. However, it is more than probable that these parts are connected to viable cells at other levels in the plastic block. It is certainly quite rare to find in tissues containing numerous plasma cells portions of plasma cell cytoplasm being pinched

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off in the manner of holocrine secretion, although Thiery (1957) has visualized such a process cinematographically and with electron micrographs in uitro. If the rough-surfaced endoplasmic reticulum is a branching complex of tubules and sacs in which new protein is made, it might be expected that such new protein would be discharged or released through this organelle. Ultrastructural evidence for this is lacking. Fusion of the ergastoplasmic membranes with the cell surface membrane to permit escape of contents from the endoplasmic reticulum has not been pictured in plasma cells. This may, of course, be an instantaneous event so that visualization of the act would be difficult to record. On the other hand, release of antibody may occur by simple diffusion of protein molecules across the membranes, or it may be the consequence of cell death. Helmreich et al. (1962) have indirect evidence that discharge of yglobulin is not the result of cell lysis. In all cases, the electron microscope has not yet demonstrated the pathway of antibody release. Caesar (1960) in a report on the formation of amyloid has stated that the plasma cell opens directly to the extracellular environment and that escape of parts of the roughened endoplasmic reticulum occurs into the surrounding milieu, By this means antibody is discharged and precipitates with mucopolysaccharides to form amyloid. Caesar does not indicate whether this implies cell disruption and death or whether there is re-formation of the cytoplasm and a return to complete cell integrity. (For additional discussion see Section IV, B, 1, d.) In any event, these are not the usual morphologic manifestations of antibody discharge. CELLS( CELL-BOUND ANTIBODY ) C. SENSITIZED The evidence is extremely persuasive that circulating antibody is derived from plasma cells, plasma cell precursors, or multipotential small lymphocytes closely related to the plasma cell. There is a group of classic immunologic processes, however, in which circulating antibody has not been prominent and which have been characterized by the fact that they can be transferred to a neutral host by transfer of sensitized lymphoid cells. Tuberculin and bacterial sensitivity, chemical sensitivity, and transplantation immunity have been placed in this category. Practically nothing else is known about these phenomena in terms of ultrastructural morphology. The passive transfer of specific immunity using lymphoid cells derived from spleen, lymph nodes, whole blood, peritoneal washings, or thoracic duct lymph implicates lymphocytes as the carriers of immunity. But the successful transfer is achieved by a melange of cells, including undifferentiated elements, macrophages, large and small lym-

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phocytes, and it is quite possible that a very minor population of cells, capable of proliferating often and rapidly, may carry the specific message of cell-bound antibody. Gowans et al. (1962) have indicated the immunologic competence of the small lymphocyte by transfer of thoracic duct contents containing 99.5 ”/. of this cell type. Such a pure population is capable of destroying homologous test skin grafts, of runting the recipient host, and of producing circulating antibody, thus pointing to the small lymphocyte as the key cell in immunologic processes. However, if immature cellular elements (the 0.5 k of the original population) could divide three times daily for 2 to 3 days (Vazquez, in press), they could proliferate to a number equal to the original total population. Since homograft rejection, runting of homologous host, and detection of antibody synthesis require more than 3 days from the initiation of each process, it is within the realm of the biologically possible that even a population of 99.5 ”/. small lymphocytes is insufficiently pure to prove undeniably Gowans’ thesis. If small lymphocytes are finally revealed as immunologically competent cells, the proportion of them which may carry the message of specific immunity and the fine structure of such competent lymphocytes, or their heirs, will remain to be solved. Several groups of investigators (Brieger and Glauert, 1954, 1956; Goodman et al., 1956; Goodman and Moore, 1956; Policard et al., 1957a,b; Yamamoto et al., 1958a,b, 1959; Roth et al., 1960; Imaeda and Convit, 1962) have illustrated cells containing tubercle bacilli, leprosy, and other bacterial agents which are commonly associated with delayed hypersensitivity. In most instances a phagocytic cell carries the whole or parts of the organism in a cytoplasmic space that may or may not be lined with a single membrane. With passage of time several vacuoles appear in the cytoplasm containing the indigestible remnants of the bacteria. After injection of tubercle bacilli Cedergren ( 1957) described mouse macrophages with three types of granules, seen rarely in uninfected mouse macrophages. The significance with respect to cellular immunity and delayed hypersensitivity of these granules and the vacuoles with bacteria or their remnants in the cytoplasm of phagocytes is unclear. It may be in the future that so-called “cellular immunity” is merely a quantitative difference of certain cell organelles. Recent reports have indicated that some immune reactions are accompanied by a loss of lysosomes from cell cytoplasm ( Cohn, 1963; Cohn and Hirsch, 1960; Hirsch and Cohn, 1960; Hirsch, 1962; Myrvik et al., 1962; Weissmann and Uhr, 1962; Uhr et al., 1963; Archer and Hirsch, 1963) and by elevation of protease concentration in the media containing the injured tissues (Ungar et d., 1961; Hayashi et al., 1960). By general agreement lysosomes are defined

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as membrane-lined droplets, within the cytoplasm of certain cells, whose contents are dense and inhomogeneous and which have been shown to contain hydrolytic enzymes. An increased number of lysosomes in an increased number of cells, representing an accumulation of greater than normal quantity of hydrolytic enzymes, may be the expression of delayed hypersensitivity, i.e., cells with delayed-type hypersensitivity are quantitatively but not qualitatively different from the same cell types that are not immune. At present there are no other straws to clutch at. 111. Antigen

Antigens may be broadly classed as particulate or as soluble and each type is treated by the animal host in a different fashion. Particulate antigen, after entry into the host, is usually rapidly removed from the circuIation, or from wherever it is desposited, by the reticuloendothelial (RE) system and circulating phagocytes. White and red blood cells, other tissues, bacteria, viruses, most macromolecules, and antigen-antibody complexes belong to this category. Soluble antigen, after injection, becomes distributed within a relatively brief period between intra- and extravascular spaces, then slowly disappears from the circulation at a rate equivalent to its catabolic degradation. With the advent of antibody synthesis, the circulating soluble antigen complexes with it and is eliminated rapidly from the blood. The rate and timing of these events is characteristic of both the soluble antigen and the recipient host. Native serum proteins and phage are representative of this category.

A. EXOGENOUS The route and distribution of antigen from the time it enters the host until it stimulates antibody is poorly comprehended and the electron microscope has not been of much help, so far, in casting light on the obscurities. The initial event, after introduction into the host of particulate antigen, is ingestion by cells of the RE system, either present locally at the site of deposit, or distantly in such organs as spleen and liver. The handling of soluble antigen by cells, after entry into a host, is unknown. With the arrival of the electron microscope in biology, cellular ingestion has been re-examined and several new concepts have appeared. As before, phagocytosis describes the uptake of particulate material ( i.e., particulate antigen) ; pinocytosis is reserved for the uptake of liquid droplets (is soluble antigen admitted into the cell in this fashion?). Both of these processes occur in vivo and in vitro, are easily examined in the light microscope, and are not immunoIogicaIly specific. More recently,

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rhopheocytosis has been added to the medical lexicon to describe cellular ingestion of small particles, of molecular size, a process which can be visualized only with the aid of the electron microscope (Policard and Bessis, 1958) . The terms micropinocytosis, cytopempsis, and potocytosis have also appeared to describe the presence of numerous intracellular vesicles which seem *to originate from the surface membrane of cells, particularly endothelial, and to traverse the cytoplasm ( Palade, 1953, 1960, 1961; Moore and Ruska, 1957; Wissig, 1958; Hampton, 1958; Brandt, 1960). It has been regarded as an intracellular transport mechanism, but proof of such a dynamic process by perusal of static micrographs is still not firm. The uptake and intracellular transport of colloidal gold, ferritin, mercuric chloride, etc., within such vesicles, as visualized in the electron microscope, may not be the same as that of proteins and antigens. Phagocytosis, pinocytosis, and rhopheocytosis appear to be related and to be achieved by similar cellular activity. In the electron microscope, foreign material, whether it is solid particle, cell or macromolecule, liquid droplets containing pinocytosis-stimulating solutes, or individual molecules of a single substance, first adheres to the surface of the ingesting cell (Odor, 1958; Brandt, 1958; Arai, 1960; Pappas and Tennyson, 1962). In some instances, ,the particles or molecules may also be concentrated at the cell surface (Parks and Chiquoine, 1957; Brandt and Pappas, 1960). Following adherence, the cell protrudes cytoplasmic processes which engulf the adherent material. In the case of phagocytosis, the cytoplasmic processes are usually long and often visible in the light microscope. With pinocytosis, the cytoplasmic folds are much shorter and cannot be seen in the light microscope. In rhopheocytosis, there is no protrusion of cell membrane, but rather an invagination with the formation of an intracellular vacuole. In all instances the engulfing cytoplasmic surface forms a cytoplasmic space containing the foreign material and is bounded by a thin single membrane. After their ingestion, virulent bacteria may destroy the host cell (Goodman et d,1956; Goodman and Moore, 1956). In other models of cellular ingestion the cytoplasmic vacuoles contain hydrolytic enzymes ( lysosomes ), and the foreign material is digested into unrecognizable fragments or amorphous substances (Essner, 1960). When lysosomes are formed, no regular and recognizable association between them and any cell organelle has been noted. What happens next to the lysosome with its antigenic contents remains undisclosed. And, of course, from this point on the whereabouts and form of antigen are hidden, The dynamic process of phagocytosis and pinocytosis can be observed

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in the light microscope by direct examination and by identification of soluble antigens with the fluorescent antibody technique. Rhopheocytosis can only be visualized when the molecules or particles of molecular size are electron dense and exhibit identifiable configuration, such as ferritin (see Fig. 8 ) or colloids of heavy metals. It is assumed that other molecular species, invisible because of their lack of density and identifiable form, behave in the same manner. There is no direct evidence for this, however, nor that the slow catabolic degradation of circulating soluble antigen is due to pinocytosis, rhopheocytosis, or any other process of cell ingestion. The phenomena of cytoplasmic ingestion are displayed not only by the cells comprising the RE system, but also by a variety of epithelial tissues, i.e., intestinal, tracheal, hepatic, renal, and tumor epithelia (Clark, 1959; Holtzer and Holtzer, 1960; Ryser et al., 1960; Miller, 1960; Thomason and Schofield, 1961). The process is similar in epithelia to that observed in the RE mesenchymal elements. It is also apparent that the quantity of antigen, particulate or soluble, taken up into the cytoplasm may vary according to the antigen used, to pinocytosis-stimulating agents, and to inert particles which enhance phagocytosis (ChapmanAndresen, 1957; Schumaker, 1958; Schoenberg et al., 1961; Sbarra et al., 1962). An interesting variant of phagocytosis was reported by Danon et al. (1961). They observed that influenza virus, in tissue culture, becomes fixed to the surface of erythrocytes. The erythrocyte plus virus is then engulfed by a leucocyte which promptly disgorges the red cell and retains the virus. Whether proteins and macromolecules other than virus may be processed by phagocytosis in a similar manner is not known. There has been no confirmation at an ultrastructural level that antigen reaches the nucleus of mammalian cells. Some years ago Coons and his colleagues (1951), in studying the fate of antigen with the fluorescent microscope technique, reported that antigen could be seen within the nuclei of a small number of cells, both mesenchymal and epithelial. In amebae, ferritin and colloids of heavy metals have been shown to reach the nucleus (Feldherr, 1962a,b). It would be of great significance to extend these observations and to determine whether antigen does arrive at the nucleus of ingesting cells as a general phenomenon, and if so, what part of the nucleus is involved. So far, the most refined methods have been incapable of following the intracellular sojourn and disposition of antigen after it has been taken up by the ingesting cells. Radioisotopic labeling, fluorescent tagging, and autoradiographic studies have indicated that antigen persists (McMaster and Kruse, 1951; Haurowitz et d.,1955; Campbell and

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Garvey, 1958; Speirs, 1963), but its form and cellular distribution elude the best techniques. Campbell and Garvey believe that the original antigen persists in liver cells as a specifically reactive fragment. Speirs (1963), more recently, feels that antigen persists as a complex with antibody and is distributed in macrophages. Very few investigators have sought to find antigen with the electron microscope. J. 0. Erickson d al. (1957) several years ago labeled tobacco mosaic virus (TMV) with C14 and tried to correlate the location of antigen after intravenous injection as revealed by isotope concentration in tissue fractions with the ultrastructural appearance of these tissue fractions and of thin sections of whole tissue. By radioactive counts, the antigen is found in the mitochondrial fraction, and micrographs of this fraction show TMV particles intimately associated with the mitochondria. However, micrographs of thin sections of the liver reveal TMV aggregates within cytoplasmic vacuoles of hepatic cells unrelated to mitochondria. Wellensiek ( 1961 ) injected ferritin intravenously into mice over a wide dosage range. Within 25 minutes the ferritin is visible in the endothelium of the vascular tree and also in the basement membrane and subendothelial space of glomerular capillaries. After 2 days the antigen is no longer present in the circulation but is visualized in the cytoplasm of RE cells, apparently incorporated by pinocytosis. A major unsolved problem concerning antigen distribution and localization is the connection between cellular uptake of the antigen and the cellular demonstration of antibody. At least for particulate antigens the RE system and circulating leucocytes ingest the foreign material, i.e., histiocytes, macrophages, endothelial cells, and leucocytes are the active participants. Several days to several weeks later antibody can be demonstrated in plasma cells or lymphoid cells closely related to plasma cells, and these elements are generally not phagocytic, although Thiery (1957) suggests that immature plasma cells exhibit pinocytic activity. Nor has there been any evidence that the phagocytic cells may evolve into plasma cells. The consensus of observations would, therefore, imply that the phagocytic cell transfers to the antibody-producing cells some specific information or specific molecular fragments that elicit the synthesis of specific antibody. Indeed, Fishman (1961; Fishman and Adler, 1963) have reported just such a cellular transfer in vitro, using a soluble antigen. Antigen, with macrophage ribonucleic acid (RNA), is capable of inducing antibody synthesis from lymphoid cell cultures, and the reaction is highly specific. Thiery ( 1957 ) has described a morphologic arrangement of plasma cells encircling a histiomonocytic cell which is compatible with such a transfer of information or of activated antigen, but similar rosettes

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of plasma cells occur around small blood vessels, and there is no conclusive evidence that this type of cellular islet is involved in the transfer of information or reagents for antibody formation. Coons et at. (1955) have reported that soluble antigen is found in the cytoplasm of lymphocytes of lymphoid organs. If it were true that lymphoid cells, or more particularly lymphocytes, are capable of converting to plasma cells or are major antibody producers, it would be an easy inference to state that some of these cells ingest antigen and are so modified by it that they become antibody producers and/or plasma cells. The work of Gowans et al. (1962) suggests, but does not directly or conclusively prove, that the small lymphocyte may modulate to a large pyroninophilic cell, capable of mitosis, which ultimately forms new small lymphocytes. Whether the large pyroninophilic cells may differentiate into plasma cells is not known. The subject of virus as antigen has been omitted from this review, for virus is a replicating antigen within a cell and thereby differs from other inanimate antigens. The details of mode of entry into the cell, its mechanism of replication and release, and of the pathology of the host environment, together provide enough data for a review of its own. Sufficeit to say that in this area the electron microscope is an invaluable instrument for the study of the life cycle and antigenic composition of viruses. B. ENDOGENOUS

There are numerous endogenous structural proteins which are a permanent part of the cell and which may act as antigens in immunologic processes and disease, It is with these that this review is concerned, in contradistinction to a host of proteins and polysaccharides which are secreted by the cell, e.g., zymogen granules of pancreatic acinar cells, thyroglobulin of follicular epithelium, and different mucins of the different gastrointestinal epithelia. R. E. Lee (1963) has tried to map the distribution of A substance in erythrocytes, using a ferritin-anti-A globulin conjugate. The antigen is dispersed over the red cell surface without any obvious pattern (Fig. 5 ) . It is not possible, with this technique, to learn the size and precise location of A substance nor its distribution within the erythrocyte as well as on the surface. With D erythrocytes and ferritin-anti-D conjugate, the number of reactive sites is much smaller than in A cells and there seem to be relatively large distances between antigen sites. There have been several reports on the distribution of blood group substances in tissues other than bIood, employing the fluorescence microscope tech-

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nique, but these have not disclosed the ultrastructural loci of such antigens. Easton et al. (1962) have also demonstrated the surface arrangement of certain antigens, apparently specific for ascites tumor cells. With ferritin-labeled, an titumor antibody they observe a concentration and adherence of ferritin to the cell membrane (Fig. 6 ) . In other experiments

FIG.5. Parts of two A erythrocytes agglutinated by anti-A globulin conjugated to ferritin. The clusters of ferritin indicate the surface sites of A antigens. No obvious pattern of distribution of A substance is discernible, although there is a suggestion of discrete loci. Magnification: x 66,000. Inset: A single molecule of ferritin illustrates the characteristic and recognizable arrangement and packing of iron in its core. Five micelles are visible. Magnification: x 950,000. (From Lee, R. E., personal communication. )

broken cells and the contents of disrupted elements are incubated with ferritin-antitumor antiserum. Ferritin is found either dispersed throughout the cytoplasm without aggregation on any organelle or the ferritin molecules are attached to fragments of smooth membranes which have been released from broken cells. The authors conclude that there are intracellular as well as surface antigens. However, there is no assuring evidence that the accumulation and distribution of ferritin intracellularly

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is qualitatively different in cells from specific tumor than in nonspecific cells. Nor have the antitumor antisera been sufficiently purified to eliminate soluble antigen-antibody ferritin complexes which might adhere to various structural cytoplasmic parts.

FIG.6. A portion of the surface of an ascites tumor cell showing the fimbriated cytoplasm characteristic of the cellular response to specific antibody. Ferritin was conjugated to rabbit antiserum raised against the ascites tumor and the conjupate was reacted in vitro with tumor cells. The aggregation of ferritin particles indicates the binding of the antiserum to the surface antigenic sites of the tumor cell. Magnification: x 68,000. (From Easton, J. bl. et nl., 1962.)

With a ferritin-conjugated antibody or ferritin anti-y-globulin it has been shown that the surface of the sea urchin egg differs antigenically from its surrounding jelly, but mapping or distribution of antigens has not been attempted (Baxandall at al., 1962). Pepe and co-workers have made several interesting attempts to

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identify voluntary muscle proteins using either specific antibody alone or specific antibody with a double fluorescent and ultrastructural tag (see Pepe, 1961; Pepe and Finch, 1961; Pepe et al., 1961). The A band is shown to contain myosin, but other muscle bands, as revealed by electron microscopy, are not correlated directly with other proteins. The technique of binding a fluor and an electron-dense stain to a specific antiprotein in order to follow structural proteins at both levels of magnification is alluring, but so far unsatisfactory. The electron microscope has not aided materially in uncovering the cellular sites of endogenous antigens, their distribution, or their configuration, if the latter is at all distinctive. There are numerous “titled antigens which may be of prime importance in immunologic systems and diseases, e.g., blood group antigens, transplantation antigen ( s ) , organ specific and species specific antigens. The presence and cellular distribution of such antigens have been disclosed by immunologic, biophysical, and ultracentrifugal methods while the electron microscope has been unable to make them visible. IV. Ultrastructure of Immunologic Reactions

A. ANTIGEN-ANTIBODYUNION 1. In Vitro Over 20 years ago some of the first efforts of biologists with electron microscopes were directed to the visualization of the effects of specific antiserum on viruses and bacteria. Among these early investigators, and later, there has been general agreement that the antigen (virus or bacterial flagella) becomes thicker, its edges fuzzier, and its surface denser, all probably as a result of antibody adhering to specific antigenic sites (Anderson and Stanley, 1941; Mudd and Anderson, 1941; Ardenne et al., 1941; Bloch et al., 1956; Bachrach and Breese, 1958). But precise spatial arrangement of antibody to antigen, actual numbers of antibodies linked to the organism, and the consequences of such union are not clearly portrayed. With improved resolution and techniques, especially with negative staining, the union between specific antiserum and virus, bacteria, and protein has been re-examined. Anderson et al. (1961) have reacted specific antisera against two types of phage and visualized the union with negative staining. In the presence of specific antibody the phage particles aggregate and become fuzzy. The fuzziness is apparently due to the attachment of thin fibers which bridge the particles and which are most likely antibody molecules. The fibers are 20-30 A. in width and 100-200 A. long and are not present

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when nonspecific antiserum is added to phage. In some instances the fibers form loops on the surface of individual phage particles. With similar methods, these same investigators are able to separate the antigenic specificity of poliovirus shells from the enveloped subunits (Hummeler et al., 1962). Wildy and Watson (1962) and D. H. Watson and Wildy ( 1963) have prepared specific antisera against herpes virus and against host cells to be infected by this virus. They, too, with negative staining, illustrate the specificity of the envelope covering the viral subunits and indicate that it is derived from host cells, whereas the subunits are strictly viral. Kleczkowski (1961) agglutinated tobacco mosaic virus at equivalence and in antibody excess. At equivalence, individual antibody molecules are seen with their long axes perpendicular to the surface of the virus and occasionally connecting pairs of virus. In antibody excess, the tobacco mosaic virus increases two times in diameter, and by computation Kleczkowski infers that single virus particles or side-to-side aggregates of the virus are covered on their surfaces by a layer of antibody molecules tangentially disposed. Recently, Almeida et al. (1963) elegantly illustrated the agglutination of viral antigen (wart and polyoma virus) by specific antibody, the arrangement of antibody to antigen through a range of antigen excess to antibody excess, and the appearance of single antibody molecules with reactive sites on each tip of the molecule (Fig. 7a,b, and c). Lafferty and Oertelis (1961, 1963) have also depicted the arrangement of antibody molecules to antigen (influenza virus) at varying concentrations of reactants. When antibody is in low concentration, single molecules form loops so that each end interacts with adjacent antigenic reactive sites on the virus. When antibody concentration is intermediate, virus particles are aggregated by antibody molecules bridging from one particle to another. When antibody is in high concentration, the antibody molecules are attached to virus at only one of their ends and radiate out from the surface. These observations are not so clearly illustrated by the electron micrographs as they are by the author’s diagrams (Lafferty, 1963). Similar interaction of purified protein and antibody has been studied by two groups of investigators. Easty and Mercer (1958) harvested antiserum to ferritin and pictured the antigen-antibody complex. Their micrographs show aggregated ferritin molecules, each surrounded by a halo of dense material, presumably antibody. Hall et al. (1959) isolated rabbit antibody to p-azobenzoate coupled to bovine y-globulin (BGG). The molecules are 30-40A. in diameter and vary from 100-800A. in length. They resemble very closely molecules of y-globulin from a normal rabbit. Attempts to produce specific agglutination with antiserum suggest

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end-to-end aggregation; there is, however, no surety that the aggregation is immunologic and not artifactual, nor are there any micrographs to illustrate protein-antiprotein union. After almost a quarter of a century of effort, sufficient resolution has been achieved to image individual antibody molecules and to observe,

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to some extent, their relationship to antigen. At a truly molecular level it may yet be possible to see the individual receptor sites of viral, bacterial, and protein antigens, and to correlate this union with inactivation, loss of specificity, etc. In vitro studies of specific antiserum directed against erythrocytes and nucleated cells have been attended with some success in penetrating the phenomena of agglutination and lysis at the ultrastructural level. Mazzella (1946) first described a wrinkling of the erythrocyte surface membrane exposed to specific agglutinins. Bessis (1950, 1955; Bessis and Bricka, 1950) and Ponder et al. (1952) later illustrated red cell spindling and filament formation accompanying agglutination. These alterations, however, are not specific and occur when agglutination of red cells is induced by different means. Rebuck (1953,1959; Rebuck and Hoff, 1954) described plateau formations of varying sizes, heights, and numbers when erythrocytes are agglutinated by specific antiserum directed against blood group antigens. He supposes that these protrusions represent actual reactive sites on the erythrocyte surface, but it is more likely that they are morphologic phenomena secondary to the unseen antigen-antibody union. Romanowski and Feltynowski ( 1955) described erythrocyte changes similar to those shown by Bessis and Rebuck and believe they are artifacts of specimen preparation. Latta ( 1952) investigated hemolysis

FIG.7. ( a ) Negatively stained preparation of wart virus in absence of antibody. Particles are evenly distributed over the field and their surfaces and substructures are clearly visible. Magnification: x 330,000. ( b ) Negatively stained preparation of wart virus in presence of antibody. The particles are clumped together and surrounded by antibody molecules which obscure their surfaces and substructures. Magnification: x 330,000. ( c ) Negatively stained preparation of wart virus in presence of antibody, near equivalence but in antigen excess. Two particles are linked by individual antibody molecules, apparently reactive at their ends. Magnification: x 600,000. (From Almeida, J, et al., 1963.)

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of red cells by specific hemolysin and illustrated cracks in the cell surface following union of antibody with its red cell antigen, a stigma not seen in osmotic hemolysis. Deutsch (1959) coated nonspecifically sheep red blood cells with BGG and reported that the erythrocyte surface becomes rougher and mottled, and its outline fuzzy. His micrographs do not illustrate these structural alterations. The results obtained by all these investigators demonstrate the difficulty of viewing a dynamic process by electron microscope techniques, and the actual morphologic events which take place between red cell antigen and its specific antibody continue to elude graphic demonstration. The ultrastructural process of agglutination and hemolysis by antibody, with or without complement, remains for future hands. Akin to this work is that done by Davis et ul. (1961) who examined the fine structure of red cell membranes used in antiglobulin tests. Red cell membranes are coated with incomplete anti-D antibodies, and, after washing, are reacted with antihuman globulin serum. Nonsensitized cells exhibit a typical plaque surface, with plaques ranging from 200-500 A. in diameter. After sensitization, the plaque diameters are enlarged to 500-800 A,, probably due to the adherence of antibody to the antigenic sites of the red cell membrane. The ultrastructural effects of specific antiserum directed against nucleated cells have been described by Latta and Kutsakis (1957), by B. Coldberg and Green (1959, 1960), and by Green and Goldberg ( 1 9 0 ) . The latter authors pictured a fimbriation of the cell surface of ascites tumor cells when these cells are incubated in the presence of specific antibodies. The fingerlike processes are focal and not distributed over the entire surface membrane (Fig. 6 ) . This phenomenon is regarded as a specific ultrastructural change induced by the antigenantibody union which causes the surface to become sticky and fixes the normal surface membrane movements in this fashion. The interior of the cell exposed to antibody remains intact and no obvious alteration of mitochondria, endoplasmic reticulum, Golgi element, nucleus, etc., is noted. The addition of complement to such a system of cells (antigen) and antibody effects the destruction of the cells. There is enlargement of the entire cell and all its components, apparently by imbibition of fluid. The surface membrane is stretched, in favorable preparations, without visible holes. The mitochondria, ergastoplasm, and nuclear membrane are all dilated. Latta (1959) depicted similar changes in chick embryo hearts grown in tissue culture and subjected to the action of specific antibody plus complement. Both these investigators and others have shown that the effect of antibody plus complement is to alter sur-

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face permeability, allow escape of electrolytes and small molecules, including protein, from the cell, and facilitate ingress of fluid into the cell. It seems, then, that the bloated cells elicited by antibody and complement are a relatively late reaction to changes which have occurred in or on the surface membrane but which remain invisible in the electron microscope. In tissue cultures of fibroblasts synthesizing collagen fibrils, specific anticollagen antiserum affects fibrogenesis adversely ( Robbins et d., 1955). Many of the collagen fibrils are small, lack external form, and internal periodicity. Alterations of the cells themselves are not described. It is interesting that in this in uitro model, antibody without complement is capable of influencing fibrogenesis.

2. In Viuo

The sequelae of antigen-antibody union in uiuo are complicated by host response, so that it is often difficult, if not impossible, to separate the primary direct effects of such union in tissues from secondary effects, e.g., liberation of pharmacologic agents and alterations of blood supply. The interaction of antigen and antibody may occur on or in a cell or tissue, where the antigen or antibody is part of the cell, or it may take place in the circulation and the complex carried to a distant site. Further, antibody may be free and circulating or cell-bound, as is postulated in delayed hypersensitivity. To simplify the situation on an immunologic basis, both the human afflictions and the experimental models of disease to be discussed have been classified into four categories. In the first category are those mediated by classic circulating antibody, usually raised against exogenous antigens, and appearing as antigen-antibody complexes or macromolecular aggregates. In the second category are those effected by a host immune response to endogenous antigens or to hapten bound to host protein (autoimmune). Diseases of the third class are mediated by cellular sensitivity ( delayed, bacterial) in the absence of circulating antibody. In the fourth category are those mediated by reaginic (atopic, skin sensitizing) antibodies. Of this last group there are no ultrastructural observations and there will be no comments about such diseases as asthma, hay fever, and eczema. As with other operational simplifications, this classification is quite imperfect and precise nosologic success is not always attainable. Several diseases cannot be fitted precisely to this scheme, or overlap in two or more classes.

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B. PATHOLOGY 1. Circulating Antigen-Antibody Complexes or Macromolecular Aggregates

Classic circulating antibody is a y-globulin of either 7 S or 19 S size. In uitro it is demonstrable by precipitation, agglutination, complement fixation, neutralization, and under some circumstances, by hemolysis and cytotoxicity. It is thermostable, being reactive after heating to 56°C. for 30 minutes. In viuo its synthesis is associated with plasma cells. It may fix reversibly in tissues, traverse the placenta ( 7 S only), and is transferable in serum to another host, Circulating antibody is generally the manifest response to a variety of antigens, either proteins, polysaccharides, synthetic macromolecules, or synthetic and natural haptens. The union of antigen and antibody by itself is probably not noxious. In the presence of complement, and perhaps other serum factors, this complex is capable of damaging cells and tissues. One secondary effect of antigen-antibody-complement union is the liberation of histamine or histamine-like material from tissue, and it is relatively easy to examine the ultrastructural changes induced by histamine. Alksne ( 1959), Majno et al. ( 1961; Majno and Palade, 1961), and Cochrane (1963) have traced the distribution of colloidal, electron-dense particles of macromolecular size in the vascular tree following injection of histamine. Histamine apparently causes swelling of endothelium, particularly in venules, and a separation of the interendothelial junctions. Through these spaces the particles pass and aggregate beneath the endothelium against the basement membrane, suggesting that while the particles are being concentrated in the wall, fluid is escaping across the basement membrane. It should be mentioned that although Alksne’s micrographs depict the same interendothelial gaps and concentration of material in a subendothelial position, he interprets these gaps as actual tears in the cytoplasm, an interpretation competently refuted by Majno et al. (1961; Majno and Palade, 1961) . Phagocytosis and pinocytosis by the injured endothelium is a late and slow process, appearing some 3 hours after histamine insult, and it is most likely a minor pathway for the escape of particles from the vascular lumen. Particles may also be found early in pericytes outside the basement membrane, and no picture has demonstrated clearly how they traverse the basement membrane to reach perivascular cells and spaces. Leucocytes take the same pathway through interendothelial gaps in inflamed connective tissue (Marchesi and Florey, 1960; Marchesi, 1961; Florey and Grant, 1961; Movat and Fernando, 1963a), although Williamson and Grisham ( 1961) believe they pass through endothelial cytoplasm

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via membrane-lined vacuoles. In addition to its vascular effects, histamine is reported to cause swelling and granulation of collagen fibrils, but the micrographs illustrating this are too inadequate to judge (Ishimoto, 1956). Movat et al. (1963) have examined connective tissues of the cornea and knee joint subjected to allergic inflammation (by complexes) and have not been able to observe any alterations of collagen. a. Anaphylaxis. Anaphylaxis is perhaps the simplest experimental immunologic disease available. In the rabbit which has been suitably immunized and has a high level of circulating antibody, the intravenous injection of antigen produces anaphylaxis, with the lung as target organ. The ultrastructural pathology of this lesion reveals the presence of relatively large aggregates of antigen and antibody within the alveolar capillaries (Feldman, 1963a). When ferritin is used as the immunizing and provocative antigen, large dense precipitates occupy almost the entire lumen of these fine vessels (Fig. 8). By staining for antigen (iron stain) and for antibody (fluorescent technique), the composition of the precipitates may be demonstrated. There is little else to observe in the electron microscope except for slight swelling of the endothelial cells lining the alveolar capillary wall. The information obtained is, therefore, no more than has been secured repeatedly previously with the light microscope, and it would seem that simple mechanical obstruction of blood flow by immunologic precipitates is one of the causes of symptoms and death. There are also phannacologic and physiologic events which develop in the lung and which are not readily examined in the electron microscope. Anaphylactic reactions in glomerular capillaries have been illustrated by Andres d al. (1962) and are essentially the same as those in the lungs. The symptomatology and pathology of anaphylaxis varies in different species as does the target organ. In man there is peripheral vascular collapse; in the dog the liver is the organ of principal importance; in the guinea pig, bronchial muscle spasm and peribronchial edema lead to death. The ultrastructure of anaphylaxis in species other than the rabbit has not been reported. In passive 'cutaneous anaphylaxis, Peterson and Good ( 1962) depicted the movement of thorotrast particles across venular and capillary walls which have been injured by the interaction of bovine serum albumin (BSA) and anti-BSA in skin. The timing, location, and passage of the electron-dense thorotrast in this situation are precisely the same as those illustrated by Majno et al. (1961; Majno and Palade, 1961) with histamine. b. Arthus Reaction. Another relatively simple model of immunologic reaction in vivo is the Arthus phenomenon, When either antigen or

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FIG.8. A capillary in the lung of a rabbit with pulmonary anaphylaxis. Ferritin was the sensitizing and provocative antigen. The lumen ( C L ) is almost completely occluded by precipitates of ferritin-antiferritin ( P ) , and by a polymorphonuclear leucocyte ( P M N ) which has precipitates of ferritin-antiferritin ( P l ) in its cytoplasm. Specific neutrophilic granules ( G ) are few in number, somewhat enlarged, and diminished in contents (degranulation). The endothelium ( E n ) is slightly swollen, contains no precipitates, and rests on an unaltered basement membrane ( B M ) . Magnification: x 18,500. Inset: Enlargement of area at x to show a swollen neutrophilic granule close to immunologic precipitates within an irregular cytoplasmic vacuole. Magnification: x 64,000.

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antibody is circulating in the blood and the other element is deposited in the skin, there is a union of the antigen and antibody as they diffuse toward each other, Precipitates of the reactants are present in the lumens of small vessels, in their walls, and in the spaces around the affected vessels. By fluorescence microscopy the precipitates contain antigen, y-globulin, and complement. There is accompanying edema at first and later a polymorphonuclear leucocytic infiltration into the areas with precipitates, which have been shown to be chemotactic for neutrophiles. The polymorphs phagocytize the complexes, catabolize them, and physically remove them from the lesion site. Concomitant with granulocytic infiltration and scavenging, there may be destruction of the vascular wall with resulting hemorrhage and tissue necrosis. If the polymorphs are prevented from reaching the site of immunologic precipitates (by parenteral nitrogen mustard, or by specific antipolymorph antiserum) , vascular damage, hemorrhage, and necrosis are minimal or absent. Two to 3 days after initiation of the reaction, mononuclear elements congregate in the lesion and plasma cells appear shortly after (Cochrane and Weigle, 1958; Cochrane et al., 1959). With the electron microscope, precipitates of antigen and presumably antibody are found within the vascular lumen, in the wall of small vessels between basement membrane and endothelium, and in the perivascular spaces (Sabesin and Banfield, 1961, 1963; Movat, 1962; Movat and Fernando, 1963b, Fernando and Movat, 1963). There are no additional insights into this immunologic process beyond what is revealed with the light microscope. Daems and Oort (1962) have also examined the ultrastructure of the Arthus reaction, but studied one of its secondary events, the phagocytosis of antigen-antibody complexes by neutrophiles. The precipitates within the cytoplasm are enveloped by a membrane. Degranulation of the polymorphs is striking (see Fig. 8). Antigen, and presumably antibody complexed with it, has also been found within the cytoplasm of eosinophiles, although no note is made about degranulation of the specific granules (Sabesin, 19f33a). The evidence today suggests that leucocytic granules are intimately related to the digestion of foreign substances such as bacteria and antigen-antibody complexes, i.e., this is not a specific immunologic response on the part of the neutrophiles, but a nonspecific process of catabolism by cell enzymes (Robineaux and Frbderic, 1955; Voisin et d.,1958-1959; Policard et al., 1959; Hirsch and Cohn, 1960; Cohn and Hirsch, 1960; Hirsch, 1962; Archer and Hirsch, 1963). Although there are no electron microscopic observations of the effects

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of complexes on mast cells, there are numerous morphologic, physiologic, and pharmacologic data to implicate the mast cell as the source of histamine, at least in the guinea pig. An ultrastructural description of mast cell degranulation elicited by a reagent (48/80) which liberates histamine from these cells is offered by Bloom et al. (1958) and by Orfanos and Stiittgen (1962). The latter authors and Bloom (1963) present a full photographic portrayal of degranulation and regranulation and the fine structure of the organelles presumed to contain histamine. By contrast, in basophiles of the blood and bone marrow, there is no degranulation when experimental manipulations to liberate histamine are made (Winquist, 1963). Related to the Arthus reaction is a study by Movat et al. (1963) who wished to explore the effects of antigen-antibody interaction on collagen fibrils. Into rabbits hyperimmunized with BSA or ferritin, specific antigen is injected into the cornea or knee joint space and tissues are examined in the electron microscope. Precipitates, presumably antigen-antibody complexes, are dispersed in the connective tissue and there is no alteration of the collagen fibrils, Aratake (1960) reported swelling of collagen fibrils and deposition of glycoproteins on them in Arthus lesions. His micrographs are inadequate to evaluate the claim. Steiner (1961) elicited liver injury in immunized rabbits by injecting antigen directly into the portal vein; he also administered by the same route antigen-antibody complexes in antigen excess, preformed in vitro. Extensive parenchymal necrosis occurs. In the electron microscope, hepatic cell microvilli become swollen and protrude into the space of Disse. According to Steiner, this type of cytologic response is seen only in livers damaged by antigen-antibody complexes and is considered to be a specific reaction to an immunologic precipitate. However, other possible pathways of injury have not been ruled out, e.g., by alteration of blood supply and local hypoxia, so that such villous swelling and protrusion may be secondary effects. Indeed, Sabesin (1963b) performed similar experiments using ferritin as antigen and concludes that the immunologic precipitates formed in the liver have no direct effect upon cells, but elicit necrosis by alteration of vascular supply. Complexes of antigen and antibody, in antigen excess, when injected intravenously into guinea pigs, localize in the walls of small pulmonary vessels between the endothelium and the restraining basement membrane (Cochrane, 1963). Since the precipitates of the immunologic reactants are not easily visible in the electron microscope, carbon particles are injected simultaneously or shortly after the parenteral administration of the preformed complexes. The size of the carbon particles is comparable

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with that of the complexes and presumably the distribution of the carbon particles is the same as that of the complexes. Within minutes of these manipulations, carbon particles traverse interendothelial gaps and accumulate against the basement membrane, a sequence of events reminiscent of those elicited by histamine. Antihistaminics prevent the deposition of particles in the vessel wall. c. Serum Sickness. Serum sickness is one of the earliest immunologic diseases described and has been frequently studied over the past 60 years. Recently, by combining the techniques of ultrastructural and fluorescent morphology and of immunology, a clearer understanding has been attained of the pathogenesis of this disease ( Dixon, 1963) . Acute serum sickness is elicited by the intravenous injection of a single large dose of heterologous antigen into a susceptible host. The antigen persists in the circulation, without detectable fixation to tissues, until antibody is formed, at which time soluble antigen-antibody complexes appear. Complement is bound, and perhaps other serum factors. The complex is an inflammatory agent per se and liberates other phlogogenic substances. The early formed complexes are small and soluble; later they become larger and insoluble and are removed rapidly from the circulation by the RE system and circulating leucocytes. It is during the early period of soluble complex development that pathology of blood vessels, heart, joints, and kidneys appears. By fluorescence microscopy the antigen, y-globulin (presumably antibody), and complement are visualized in the lesions, more particularly, in vessel walls. The factors which determine the site of localization in any particular vessel remain unknown. The lesions are reversible, and several days after all the antigen is eliminated from the circulation, there is healing and usually a return to normal morphology. The kidneys of rabbits with serum sickness have been examined with the electron microscope (Feldman, 1958; Robertson and More, 1961). At the time of antigen elimination, i.e., when complexes are being formed, and only at this time, there is marked proliferation and swelling of glomerular endothelium, often to the degree that the lumens of the glomerular capillaries are completely occluded (Fig. 9 ) . Often, too, circulating cells, either polymorphonuclear neutrophiles or mononuclear elements are trapped against the swollen cytoplasm. The glomerular basement membrane is for the most part intact, but there are small foci of segmental thickening or smudging of the lamina densa. The number and size of these foci do not correspond to the quantity and distribution of antigen and y-globulin (presumably antibody) which are seen by the fluorescence technique to be abundantly fixed to the basement membrane.

JOSEPH D. FELDMAN

FIG.9. A portion of two loops ( C L ) of a glomerulus from a rabbit with acute serum sickness. The basement membrane on the right (BM2) is somewhat thickened and frayed (broken arrows), whereas the one on the left (BM1) is apparently unaltered. In the latter loop ( C L 1 ) the endothelium ( E n ) is markedly swollen and fills the entire lumen. In CL2 the endothelium is only slightly swollen. Epithelial foot processes appear normal. CM, cell membrane. Magnification: x 15,000.

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Epithelial cells are generally unaltered and their foot processes are preserved in orderly arrangement. From 1 to 2 weeks after maximal development of the glomerular lesion there is complete restoration of ultrastructural morphology. In acute serum sickness, then, an antigen-antibody-complement complex, not positively visualized in the electron microscope, elicits a transient swelling and proliferation of glomerular endothelial cells. It should be emphasized that the kidney is not an immunologic target, but merely a chance receptacle for the deposition of phlogogenic immunologic reactants. Chronic serum sickness is elicited by repeated daily intravenous injections of a heterologous antigen into rabbits (Dixon et al., 1961). About 15 to 20 "/. of the animals are capable of producing antibody in small or moderate quantities. Consequently, soluble antigen-antibody complexes are formed daily and circulate for varying intervals of time each day. It is in these rabbits that a chronic proliferative, inflammatory or degenerative glomerulonephritis develops. Rabbits making a good antibody response produce sufficient antibody to combine with all the injected antigen at equivalence or in antibody excess, and these large complexes are quickly removed from the circulation. Rabbits making no antibody response do not have circulating complexes. These latter two groups of animals do not develop a chronic renal lesion. The ultrastructural stigmata of this laboratory renal disease are dense globular or ovoid deposits along the outer aspect of the glomerular basement membranes beneath the epithelial cells of all glomeruli ( Fig. 10). The deposits contain at least antigen, y-globulin (presumably antibody), and complement, revealed both by fluorescence and electron microscopy (Andres et al., 1963) (Fig. 11).In some manner, the precipitates interfere with basement membrane function, for proteinuria is markedly elevated at the time of their morphologic appearance. The deposits persist, in fact increase with time and continued injections, for at least a year. Even after cessation of antigen administration, the precipitates remain, although there is slow removal by epithelial cells (Feldman, 1963b). Associated with this structural change in the basement membrane are alterations of endothelium and epithelium. Endothelial cells increase somewhat in number, manifest local swelling, and develop a complex and extensive rough-surfaced endoplasmic reticulum. Epithelial cells show evidence of protein leak, i.e., smearing of foot processes or replacement by sheets of cytoplasm closely applied to basement membrane, protein droplets in the cytoplasm, perhaps Golgi enlargement. In time, as the basement membrane becomes more and more

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FIG. 10. A capillary loop of a glomerulus from a rabbit injected with BSA for 4 months. The basement membrane is distorted by the presence of numerous dense deposits ( D ) of varying size beneath the epithelium ( E p ) . In some places the lamina densa is still preserved (arrows), while elsewhere it is fused or lost in the deposits. By fluorescence microscopy the deposits contain BSA, host y-globulin, presumably antibody, and complement. Epithelial foot processes are absent and are replaced by extensive sheets of cytoplasm. The capillary lumen (CL ) is occupied by swollen endothelial ( E n ) cytoplasm. Nu, nucleus. Magnification: x 8600.

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FIG. 11. A deposit on the basement membrane (BM) of a glomerular capillary ( C L ) from a rabbit injected repeatedly with BSA. Ferritin was conjugated to antiBSA and aggregates specifically in the deposit (dep) and basement membrane indicating the presence of BSA in these sites. Ep, epithelium. Magnification: x 60,000. (From Andres, G . A. et al., 1963.)

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distorted by the dense deposits of immunologic precipitates, there is increasing alteration of glomerular architecture, i.e., narrowing of capillary lumens, enlargement of mesangial spaces with an increase of mesangial cells and of basement membranelike material, synechiae, collapse of a lobule, and finally, hyaline and scarred obsolescence. With this model of chronic serum sickness, it is possible to produce protracted degenerative lesions of the kidneys by the slow, continued deposition of antigen-antibody complexes into the basement membrane. The kidneys, again, are nonspecific nonimmunologic targets which by chance become the receptacles for these injurious macromolecules. The pathology of this renal model is quite similar to the pathology seen in some cases of human adult glomerulonephritis. d. Amyloidosis. The relationship of amyloidosis to antigen-antibody complexes remains obscure. In human beings, secondary amyloidosis, the most common variety, is associated with long-standing infections such as tuberculosis or osteomyelitis in which the level of circulating y-globulin is elevated. The other varieties, primary amyloidosis, amyloidosis of multiple myeloma, and localized amyloidosis associated rarely with tumors, on the surface have no apparent connection with immunologic reactants. In animals, amyloidosis can be produced by repeated injections of proteins, particularly casein, and its development is probably related to the increased y-globulin levels which appear in the circulation of the manipulated animals. The amyloid of primary and secondary human amyloidosis and of experimentally induced disease in rabbits and mice has been examined in the electron microscope and appears to be morphologically the same in all instances (Cohen and Calkins, 1959, 1960; Cohen et al., 1960, 1962,; Caesar, 1960, 1961; Hjort and Christensen, 1961; Fruhling et al., 1961b; Battaglia, 1962; Heefner and Sorenson, 1962; Thiery and Caroli, 1962). It is composed of a moderately homogeneous material in which is found a matlike arrangement of individual fibrils or swirling bundles of fibrils. The fibrils are about 100 A. in diameter; their lengths have not been accurately measured. No obvious intrafibrillar structure is recognizable, although several investigators have described occasional beading of single filaments. The origin and mode of dissemination of amyloid is still unknown. Its deposition is predominantly intercellular. However, Caesar ( 1960) and Heefner and Sorenson (1962) observed both the homogeneous and fibrillar components within cells and, in the opposite direction, have seen cell organelles such as mitochondria and ergastoplasmic vesicles surrounded by amyloid. Caesar (1960) is of the opinion that there is an

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unusual relationship between amyloid and plasma cells and he illustrates several instances of amyloid either entering into or being discharged from the cytoplasm of such cells. Since his micrographs also exhibit broken cellular membranes it is possible that he is describing an artifact. Heefner and Sorenson (1962) emphasize the intimate relationship of amyloid to reticulum cells and illustrate intracytoplasmic localization in such cells. As they themselves indicate, this may represent simple phagocytosis and not synthesis. It is obvious that ultrastructural studies have not yet discerned the source and pathogenesis of amyloid. The composition of amyloid or amyloids is still uncertain. One major protein component is glycoprotein. By fluorescence microscopy, y-globulin and complement have been shown to be present in amyloid deposits (Vazquez and Dixon, 1956; Vogt and Kochen, 1960; Lachmann et al., 1962). Whether the y-globulin is antibody, and antibody to what antigen, remains undisclosed. Paul and Cohen (1963) have used a ferritin-labeled anti-human y-globulin and reacted this ultrastructural reagent with a purified preparation of fibrils derived from human amyloid and have been unable to demonstrate a binding between these materials. In the electron microscopy of amyloidosis, the amyloid has been found extracellularly, e.g., between the loose elements of the spleen, or intimately associated with basement membranes of vessels (Fig. 12). In the spleen and liver, deposits are present between endothelium and basement membrane of sinusoidal vessels and compress the overlying cell cytoplasm. In the kidney there has not been unanimity of observation concerning the development of the glomerular lesion. From those reports in which the micrographs are technically illustrative and in which lesions have been taken serially, it seems there is a sequential series of events from initial basement membrane involvement to final obsolescence of the glomerulus (Miller and Bohle, 1956, 1957; Geer et al., 1958; Spiro, 1959; Cohen and Calkins, 1960; Movat, 1960b; Bergstrand and Bucht, 1981a,b; Sorenson, 1963). Early there is both diffuse and focal thickening of the basement membrane. The focal excrescences are usually present on the extracapillary side, although Movat ( 1960b) believes the first deposits are subendothelial, and they are evident before amyloid is identifiable. Later, deposits of dense material accumulate on both sides of the basement membrane, which remains intact, though thickened. Fibrils then appear within the deposits and by this time there are alterations of epithelial and endothelial cells. The latter cellular changes are most probably secondary to the previous basement membrane injury and perhaps to continued protein leak, since smearing of foot processes, accumulation of dense protein droplets, etc., are seen whenever there is protein leak from

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FIG. 12. Amyloid in wall of splenic sinus. An erythrocyte (Rbc) lies in the vascular space ( L ) , which is bounded by endothelial cytoplasm ( E n ) . Bundles of amyloid fibrils ( A ) are intimately associated with the endothelium or lie beneath it. Magnification: approximately x 37,000. (From Heefner, W. A., and Sorenson, G . D., 1962. )

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whatever etiology. Capillary lumens become narrowed by increasing deposits of amyloid and by enlargement of the mesangial zone with mesangial cells and amorphous material. The precise role of immunologic events in the pathogenesis of glomerular amyloidosis is undetermined. It is not known, for example, whether the early basement membrane excrescences are antigen-antibody-complement complexes or precipitated plasma proteins or even new basement membrane focally synthesized. Nor is it known what the composition of the amyloid deposits is, other than that they contain y-globulins, complement, and other unknown proteins and glycoproteins (Vazquez and Dixon, 1956; Vogt and Kochen, 1960; Lachmann et al., 1962). Finally there is no information concerning the derivation of amyloid, whether it represents a deposition of material from the blood stream, or a local product synthesized in situ. e. Human Diseases of Unknown Etiology. There is a heterogeneous group of human diseases whose nosological niches have not yet been found. At one time or another each has been considered to be immunologically based and somehow related to antigen-antibody interaction. In this review they all have one feature in common; the fine structure of the kidneys, biopsied from people with these afllictions, has been described. To date the electron microscope has provided a fine portrait of renal involvement in these diseases, unattainable with the light microscope, but it has not been of great help in unraveling the pathogenesis and etiology of the lesions. If these diseases are truly the result of immunologic reactions, then immunologic approaches will be needed to probe the origin and progression of the afflctions. Perhaps the disease which has the strongest immunologic background is lupus. This affliction may also be properly classifled in the “autoimmune” category, since host endogenous antibodies are present which react with endogenous tissue antigens. In some persons with lupus, antinuclear antibodies are found in the circulation and in a few, circulating complexes have been found (Dixon, 1963). Fluorescence microscopy of the kidney has demonstrated the presence of y-globulin and complement distributed in the glomerulus in the pattern of the capillary walls (Lachmann et al., 1962; Burkholder, 1963; Freedman and Markowitz, 1962a). Freedman and Markowitz ( 1962b) have extended their studies further and shown that antinuclear antibody can be recovered from lupus glomeruli. The lupus erythematosus (LX) body is also known to be a precipitate of y-globulin and nucleoprotein. Such an array of findings suggests that lupus may be immunologically based,

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although there still is no evidence that the immunologic reactants are the cause or the consequence of the disease. Glomerular ultrastructural pathology in lupus is not specific for this disease, but it is sufficiently characteristic to be helpful in diagnosis (Farquhar et al., 1957a,c; Farquhar, 1959, 1960; Vernier et al., 1958; Arnold and Spargo, 1959; Yardley and Walker, 1960; Pirani et al., 1961; Rallison et ai., 1961; Browne et al., 1963). In early and acute phases, the most prominent findings are endothelial proliferation and swelling, dense subendothelial deposits against the basement membrane, and diffusely thickened basement membranes. In later phases, dense masses protrude from the basement membrane on its extracapillary side, highly reminiscent of the precipitates which appear in the glomeruli of chronic serum sickness in rabbits (Fig. 13). With progression of renal involvement, the usual secondary alterations become manifest, i.e., smearing of foot processes, synechiae, narrowing of loops, general architectural distortion and finally, completely degenerate glomeruli. If by electron microscopic and immunologic techniques the subendothelial deposits and dense basement membrane excrescences can be shown to be composed of nucleoprotein (antigen), y-globulin (antibody), and complement, it will aid in understanding the pathogenesis of lupus as a disease mediated by circulating complexes. The LE cell and collagen from patients with lupus have also been studied ultrastructurally. The LE cell, a polymorphonuclear neutrophile, contains within its cytoplasm a membrane-enclosed dense mass, whose texture differs from that of nuclear material (Maldonado d al., 1963). Further information concerning its composition and derivation are not yet available in the electron microscope. Collagen fibrils from lupus patients are no different in size, shape, and intrafibrillar periodicity from those of normal people (Gale, 1951; Kajikawa and Sumita, 1953; Arakawa and Utsunomiya, 1957). C1omerulonephriti-s is another disease, or several diseases, belonging to this group of heterogeneous human afflictions. Currently, the term means all things to all people and encompasses a variety of clinical pictures and ultrastructural images which fit acute and chronic glomerulonephritis, proliferative and membranous forms, lipid nephrosis, and perhaps other more nebulous kinds of nephrotic syndrome, such as orthostatic proteinuria. Adult proliferative or membranous glomerulonephritis are assumed to be immunologic disorders. As in lupus, y-globulin and complement may be demonstrated in glomeruli (Mellors et al., 1955a, 1957; Taft d al., 1058; Freedman et al., 1960; Lachmann et al., 1962). Also, a high propor-

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tion of persons with the disease have raised antistreptolysin titers in their circulation, which bear no close relation to the severity of the disease. The presumed antigen is the streptococcus or derived products, but such antigens have not been found in the human kidney. Experimentally, it is

FIG. 13. A capillary loop of a glomerulus from a patient with lupus nephritis (compare with Fig. 10). The basement membrane (BM) is altered by a number of dense deposits ( D ) located on the epithelial side of the wall. These deposits are similar in texture, size, and distribution to those in the glomeruli of rabbits with chronic serum sickness. The lumen (CL) is patent and endothelium ( E n ) is focally swollen. Epithelial ( E p ) foot processes are absent and replaced by broad sheets of cytoplasm. Magnification: x 24,000.

possible to inject streptococci parenterally into mice and to demonstrate streptococcal antigens in glomeruli, but their stay there is short-lived (less than 16 days) and unrelated to the presence or absence of pathology (Kaplan, 1958; Seegal, 1959; Miller, 1961). Also, antibody raised in rabbits against Alz.41 streptococci causes slight glomerular damage when injected intravenously into rats (Markowitz et al., 1960). These observa-

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tions and those accumulated over the past 40 years on the clinical and epidemiological aspects of the disease point toward an immunologic background for glomerulonephritis. Numerous electron micrographs have been published to illustrate the ultrastructural morphology of adult glomerulonephritis ( Bergstrand and Bucht, 1957a; Farquhar et d.,1957a,b,c; Farquhar, 1959, 1960; Folli et al., 1958; Pollak, 1958; Arnold and Spargo, 1959; Fiaschi et al., 1959; Movat and McGregor, 1959; Movat, 1960a; Movat et al., 1961b, 1962; Spiro, 1959; Latta, 1960; Spargo and Arnold, 1960; Yardley and Walker, 1960; Fujisaki, 1961; Habib et al., 1961; Steiner et al., 1961, 1962a,b; Kimmelstiel et al., 1962b). But an intensive and extensive study of the fine structure of this disease, which portrays the pathogenesis of the lesion, has yet to be presented and this will require serial biopsies from the very earliest phases of the many clinical syndromes to the finally scarred kidney. In many cases of membranous glomerulonephritis the most obvious pathology is manifested by dense deposits within or on the epithelial side of the basement membrane, a picture reminiscent of the lesion found in rabbit kidneys with chronic serum sickness. In addition, there is generally a diffuse and irregular thickening of the basement membrane, smearing or loss of epithelial foot processes, and occasional endothelial swelling. With progression of clinical disease, the architecture of the glomerulus becomes effaced in a manner similar to that seen in the rabbit model of repeated antigen injections. There is wrinkling of the basement membrane, increase of mesangial and endothelial elements, formation of synechiae, diminution of the capillary lumen, with replacement by scar tissue. Proliferative glomerulonephritis presents a similar ultrastructural image, but lacks the dense deposits of the basement membrane. In addition, mesangial and endothelial cell increase are more obvious, and occasional circulating leucocytes are present in the loops. The end result is the same. Even less is known about the immunologic background, if any, of childhood lipid nephrosis. The fine structure of the glomerulus in this disease is characterized by thickened capillary basement membranes and extensive smearing or loss of epithelial foot processes (Farquhar et d., 195%; Vernier, 1961; Vernier et al., 1958, 1961a; Movat and McGregor, 1959; Movat, 1960a; Bouissou and Regnier, 1960; Regnier and Bouissou, 1960, 1962; Sore1 et al., 1960; Kobayashi et al., 1961a; Steiner et al., 1961). Significant alterations of endothelium, and of mesangium and its cells are minimal. In early phases, there are subendothelial deposits which apparently become incorporated into the basement membranes. In the laboratory a nephrotic syndrome and ultrastructural pathology

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similar to that of lipid nephrosis can be produced in rats by the administration of aminonucleoside (Feldman and Fisher, 1959; Vernier et d., 1959; Harkin and Recant, 1960; J. L. E. Ericsson and Andres, 1961; Lannigan et al., 1962), which is not the result of immunologic processes, and also under appropriate conditions by heterologous nephrotoxic serum (I. Watson et al., 1963). On the other hand, acute poststreptococcal glomerulonephritis, chiefly an aflliction of children, appears to be derived from an antigen-antibody interaction, although definitive proof of this is still lacking. The disease suddenly develops several weeks after streptococcal infection of the nasopharynges, persists for a period of several weeks, and in most instances, disappears. A small proportion of cases do not recover and die in renal insufficiency. The histology of the glomerulus is quite similar to that in rabbits with acute serum sickness (Earle and Jennings, 1961; Jennings and Earle, 1961). In the electron microscope, at the height of the clinical disease, the glomeruli are avascular consequent to a marked swelling and proliferation of endothelial cells and perhaps trapping of circulating leucocytes ( MacDonald et al., 1959; Kobayashi et al., 1960; Kobayashi and Wada, 1961; Strunk et al., 1963). The basement membranes exhibit scattered foci of thickening or fraying or small deposits protruding toward the epithelial side. The fine structural images of this human disease resemble indistinguishably those of acute serum sickness, induced either by one shot of a large quantity of antigen or by daily injections of smaller doses. AnaphyZactoid p u r p r a is another of the group of human diseases without known etiology, but is presumed to have an immunologic background. Renal lesions have been described at both the light and electron microscope levels without adding greatly to our comprehension of its cause and pathogenesis. The glomerular alterations are not at all characteristic except that they are patchy and focal, i.e., only a small number is involved and then only a part of the glomerulus is injured. The usual ultrastructural descriptions cite the thickening of basement membranes, increase of endothelial and mesangial cells, subendothelial deposits, etc., so that many micrographs of the glomerular pathology resemble those seen in lupus, or in adult glomerulonephritis, or in obsolescent kidneys (Bouissou et al., 1960; Kobayashi et d.,1959, 1961b; Vernier et al., 1961b). Sckrodemna is a disease of unknown etiology but is usually lumped with a group of human afaictions under the general phrase, “collagen diseases.” There is no good reason to believe that this disease is immunologically based other than that immunology is currently popular

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and that in some instances an antinuclear serum factor may be demonstrated (Bardawil et al., 1958). For the sake of completeness it is included here, since a renal biopsy of one case of scleroderma has been studied in the electron microscope (Hartley et al., 1963). Apparently the most striking changes are swelling of endothelial cytoplasm in glomerular capillaries and the occasional presence of fibrin thrombi. Epithelial cells contain dense droplets and fatty inclusions and the basement membrane is focally thickened, Collagen fibrils of the skin of patients with scleroderma have also been submitted to the electron beam (Scalabrino and Rossanda, 1956; Korting et al., 1959; Arakawa and Utsunomiya, 1957). No significant differences are visible between the collagen fibrils of scleroderma patients and of normal people. Scalabrino and Rossanda (1956) claim slight enlargement of the fibril diameter in the skin of one patient and slightly larger intrafibrillar period as compared with control fibrils. Their micrographs do not substantiate their claim. Recently there have been suggestions that diabetic glomerulosclerosis may be the result of antigen-antibody union. In some glomeruli studied by fluorescence microscopy, y-globulin is distributed along basement membranes, in intercapillary deposits, and in granules apparently within epithelial cells (Berm et al., 1962). At the ultrastructural level the diabetic glomerulus is characterized by abnormally wide basement membranes and by enlarged mesangial zones containing much basement membranelike material ( Bergstrand and Bucht, 1957b, 1959; Farquhar et al., 1959; Kimmelstiel et al., 1962a; Sabour et al., 1962). The older lesions exhibit all the stigmata of chronically injured glomeruli, i.e., narrowed lumens, encroachment of the mesangium upon the peripheral capillaries, angulation, distortion and final collapse of basement membranes, and terminal replacement of normal architecture by abundant amorphous material, It is not possible, from electron micrographs, to assess the immunologic nature of the glomerular alterations. In toxemia of pregnancy, renal injury frequently occurs. As with other afflictions of this group, the etiology and pathogenesis of toxemia is unknown although a current popular proposal favors slow intravascular clotting throughout the body triggered by uterine disturbances. There is no obvious antigen-antibody interaction operative in this complication of pregnancy. It has been suggested that the syndrome resembles a generalized Shwartzman reaction in which depression of the RE system seems to play a key role in its development. Recently, L. Lee (1963) has elicited the Shwartzman reaction in immunized rabbits by depressing the RE system with thorotrast and by injection of antigen intravenously. The interaction of antigen and antibody induces slow intravascular

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clotting and, in the Thorotrast-treated animals, eventual bilateral cortical necrosis. Biopsies of the kidney from women with toxemia have disclosed a marked swelling and perhaps proliferation of endothelial cells within glomerular capillaries, and extensive, coarse, dense, subendothelial deposits (Spargo et al., 1959; Altchek, 1961; Hopper et al., 1961; Mautner et al., 1962; Fiaschi et al., 1962). The deposits may or may not show fibrillar components with a periodicity of fibrin. Epithelial and basement membrane changes are minimal. In the laboratory the fine structural pathology of the glomerulus from animals in which the Shwartzman reaction has been elicited is quite similar to that observed in toxemia (Pappas et al., 1958; Bohle et al., 1959a). It is apparent, then, that the use of the electron microscope has delineated the anatomy of the glomerulus injured in a variety of human diseases and has disclosed similarities and dissimilarities between them and certain laboratory diseases of known pathogenesis. However, morphologic similitude does not establish etiology and pathogenesis, and though the images of the injured glomerulus from humans and animals may be strikingly alike, additional evidence is needed to prove that immunologic processes are, indeed, the basis for pathology in human nephropathy.

2. Host Immune Response to Endugenous Antigen (Autoimmunity) Despite the logarithmic increase in reports of autoimmune diseases, there is a dearth of information on the fine structure of the target tissue damaged by antibody. Perhaps this is so because there is no certainty that true autoimmune diseases exist, i.e., a pathologic lesion or lesions resulting from the union of endogenous antibody with target cellular antigens. There is no doubt about the existence of antibodies against tissue constituents or cellular antigens in a number of normal and afflicted people and animals, but there is doubt about their role in disease and whether they are cause or effect. So far, the succesful transfers of laboratory diseases of autoimmune nature have been achieved by transfusing lymphoid cells from diseased donors into normal hosts (Paterson, 1960; Felix-Davies and Waksman, 1961), but not by the use of serum antibodies alone. In human disease, antibodies capable of reacting with host tissue have been observed in some forms of hemolytic anemia, leukopenia, and thrombocytopenia, in lupus erythematosus, rheumatic heart disease, Hashimoto’s thyroiditis, ulcerative colitis, some forms of glomerulonephritis, lupoid hepatitis, Sjogren’s disease, macroglobulinemia, and a few cases of cancer. The list can be extended to include the “collagen

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diseases” such as dermatomyositis and scleroderma whose nexus with immunologic processes is still quite tenuous. In animals following suitable immunologic manipulation, antibodies against specific tissues can be raised against brain, peripheral nerve, thyroid, testis, skin, joint, adrenal, etc. Indeed, with persistence and diligence antibodies may probably be induced against any tissue. a. Kidney. There are more electron microscopic studies of the kidney, in health and disease, than of any other organ and it is appropriate to begin with this organ since it is one of the few whose ultrastructure has been profitably probed. In the category of pathology elicited by interaction af antibody with cellular antigens belong two experimental models of renal disease, an “autoimmune” type developed by Heymann and his colleagues (see Heymann et al., 1959a,b, 1962; Hunter, 1960; Hunter et al., 1960) and by Steblay (see Steblay and Lepper, 1961; Steblay, 1962,, 1963) and nephrotoxic serum nephritis. This latter disease necessitates the use of an exogenous heterologous antibody rather than an endogenous antibody, but the immunologic reaction occurs in the renal glomerulus and it is this model which has often been taken to explain human glomerulonephritis. With the recent resurgence of interest in nephrotoxic serum nephritis, both the immunologic and ultrastructural basis for this renal disease has been thoroughly described (Simer, 1954; Pie1 et al., 1955; Mellors et al., 1955a,b; Reid, 1956; Ortega and Mellors, 1956; Hasson et al., 1957; Sakaguchi and Suzuki, 1957; Miller et al., 1957; Bohle et al., 195913; Sitte, 1959; Calcagno and Rubin, 1960; Calcagno et al., 1963; Churg et al., 1960; Huhn et al., 1961; Arhelger et al., 1961a,b, 1963; Movat et al., 1961a; Winemiller et al., 1961; Burkholder, 1961; Jennings and Haber, 1961; Vogt and Kochen, 1961; Lange et al., 1961; Andres et al., 1962; Hammer et al., 1962; Hammer and Diron, 1963; Izumi, 1962; Seegal et al., 1962; Triedman et al., 1962; Feldman, 1963b; Feldman et al., 1963). The animal which is generally used in this model is the rat and the heterologous antiserum is usually derived from the rabbit. However, the disease has been produced in rabbits, dogs, and mice with nephrotoxic sera from sheep, horse, and ducks. In the rat nephrotoxic serum nephritis is a biphasic disease. Within hours of intravenous injection of nephrotoxic serum or y-globulin, the basement membrane (antigen) binds the heterologous nephrotoxin (antibody) and this union is maintained for months (Seegal et al., 1962). Complement is also deposited at the site of antigen-antibody complexing (Vogt and Kochen, 1961; Burkholder, 1961). Ultrastructurally, the glomerular basement membrane becomes thickened with a poorly defined

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wispy deposit, usually on the luminal aspect (Feldman, 1963b; Feldman et al., 1963) (Fig. 14).With a ferritin conjugate to rabbit y-globulin, this deposit has been shown to contain the heterologous nephrotoxin ( Andres et al., 1962) (Fig. 15). Accompanying the basement membrane alterations are swelling of endothelial cytoplasm and dilatation of the mesangial space with an increase of mesangial cells. Mitotic activity is noted in the light microscope and attributed to endothelial cells, which cannot surely be distinguished from mesangial elements (Jennings and Haber, 1961). Ultrastructural evidence of protein leakage past the basement membrane is manifested by the numerous droplets of varying size and density in epithelial cytoplasm and smearing of foot processes. It is not possible to ascertain conclusively whether the antigen-antibodycomplement complex damages only the glomerular basement membranes, or whether the cellular changes are also the result of an immunologic injury. From 5 to 7 days after the injection of rabbit nephrotoxin into rats, additional ultrastructural alterations appear and it is at this time that the host begins to make antibody to the foreign rabbit nephrotoxin bound to the basement membranes ( Feldman, 1963b; Feldman et al., 1963). Dense inhomogeneous deposits appear between the endothelium and basement membrane and increase in quantity and distribution for several weeks (Fig. 16). They are slowly incorporated into the basement membranes which become thickened up to twenty times normal. By fluorescence microscopy these new subendothelial deposits contain rabbit y-globulin (the nephrotoxin), host y-globulin (the antibody), and complement (Hammer and Dixon, 1963). The complement may be recently bound as a result of the second antigen-antibody reaction, or it may be the same complement retained from the original interaction of antigen and antibody. As in the early phase, there are endothelial swelling, dilatation of the mesangial zone now filled with cells and a network of basement membrane material, more extensive loss of epithelial foot processes, and gradual distortion and destruction of glomerular architecture. This model of renal disease is thus a biphasic immunologic phenomenon. The first phase is characterized by almost immediate injury to the glomerulus due to the complexing of fixed renal antigen, exogenous nephrotoxic antibody, and host complement. It is a reversible phase, since rats tolerant to rabbit y-globulin and incapable of producing antibodies against the bound nephrotoxin, recover morphologically and functionally within 4 weeks (Feldman et al., 1963). Similarly, in newborn rats, nephrotoxin elicits an immediate proteinuria and morphologic damage to the glomerulus, but recovery ensues within a few weeks as the

FIG. 14. A portion of two loops ( C L ) of a glomerulus from a rat injected 24 hours previously with rabbit nephrotoxin. The left basement membrane ( B M I ) is widened by an inhomogeneous deposit ( D ) which is intimately connected to the luminal side of the lamina densa and lifts off the overlying endothelium (En1), By fluorescence microscopy the deposit contains rabbit v-globulin ( nephrotoxin ) , and host complement. The right basement membrane ( BM2) is apparently unaltered. Endothelium ( E n ) here is slightly swollen. Capillary lumens ( C L ) are patent. Epithelial foot processes ( E p ) are of normal size, shape, and distribution. Magnification: x 35,000.

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FIG.15. A portion of a capillary loop of a glomerulus from a rat injected with rabbit nephrotoxic serum. Ferritin was conjugated to duck antibody against rabbit v-globulin. It specifically localizes in the basement membrane ( B M ) indicating the presence of the rabbit nephrotoxin. Ferritin particles in epithelium (Ep) and endothelium ( E n ) may or may not be specifically bound. Magnification: x 68,000. (From Andres, G . A. et al., 1962.)

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FIG. 16. Part of a loop ( C L ) of a glomerulus from a rat injected 4 weeks previously with rabbit nephrotoxin. An inhomogeneous deposit ( D ) is present between endothelium ( E n ) and basement membrane (BM ). By fluorescence microscopy this deposit contains rabbit y-globulin (nephrotoxin), host antibody, and complement. In time the material is incorporated into the basement membrane which then becomes widened and irregular. The lumen ( C L ) is widely patent and epithelial foot processes ( Ep) are preserved. Magnification: x 22,000.

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injured glomeruli are replaced by new functioning and unaffected glomeruli from the nephrogenic zone (Hammer et al., 1962; Calcagno el al., 1963). The fact that tolerant and neonatal rats recover from the nephrotoxic insult militates against altered host renal antigens as a cause for the continuation of the disease. The second phase is characterized by delayed injury to the glomerulus owing to host antibody reacting with the rabbit nephrotoxin fixed in basement membrane. This phase is irreversible and terminates with the eventual destruction of the glomerulus and uremia. Some of the ultrastructural lesions of some human diseases, e.g., lipid nephrosis, eclamptic nephropathy, and lupus nephritis, closely resemble the pathology of nephrotoxic serum nephritis, but there is no way at present to relate the human renal diseases to this model, nor, indeed, to be certain that human afflictions are immunologically based. It is obvious that people are not injected with anti-kidney antibodies, and there is little evidence that-such an antibody exists. It is possible, however, that a foreign antigen might lodge in the glomerular walls and excite a host antibody response to the foreign protein, thus simulating the second phase of nephrotoxic serum nephritis. Generally, most uncomplexed antigens do not become bound in the kidney. However, Kaplan (1958), Seegal (1959), and Miller (1961) have all reported that streptococcal M proteins are fixed in glomeruli of mice for from 8 to 16 days after parenteral injection, without the development of renal disease. On the other hand, in mice, following parenteral injection of Proteus mirubilk, antigen persists for weeks in the glomeruli, and glomerulitis develops (Wood and White, 1956). It is, therefore, possible that nephrotoxic serum nephritis might serve as a model to understand the pathogenesis of some human renal diseases. The “autoimmune” renal disease of Heymann in rats (see Heymann et ul., 1959a,b, 1962; Hunter, 1960; Hunter et al., 1960) and of Steblay ( see Steblay and Lepper, 1961; Steblay, 1982, 1963) in sheep and monkeys is another model to help uncover the pathogenesis of some kinds of human kidney pathology. Unfortunately, neither the immunology nor the fine structure of this laboratory experiment are completely elucidated. A glomerular lesion develops only after repeated intraperitoneal injections of homologous or autologous kidney in Freunds complete adjuvant. Heymann and his associates postulate that this is immunologically induced because precipitating antibodies against kidney antigens can be found in the circulation, complement is depressed during the period of proteinuria, kidney cortex is better than medulla for the production of

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glomerular lesions and both are better than any other tissue, and finally, because adrenocorticotropin ( ACTH ) and cortisone prevent the development of proteinuria and glomerular injury (Heymann et al., 1959a,b, 1962; Hunter, 1960; Hunter et al., 1960). A number of observations, however, suggest that an immunologic process may not be the etiology of renal disease in this model. First, adjuvant with Mycobacterium tuberculosis is essential, while talc, which produces a peritonitis when injected intraperitoneally, similar to that elicited by adjuvant, is ineffective. Further, only the intraperitoneal route of injection is successful and only rat kidney has so far been injured. Dog, guinea pig, and rabbit do not develop renal disease when treated in the same fashion as rats. A good correlation between circulating anti-kidney antibodies and the presence and severity of the disease is not obtainable. And most significantly, the disease has not been transferred by serum from nephrotic rats. Hess et al. ( 196Z) re-examined this “autoimmune” kidney disease and published several electron micrographs of the injured kidneys. Unfortunately, their pictures are poor and do not reveal much detail, but it is possible to see thickened glomerular basement membranes and protein droplets in epithelial cytoplasm. In addition, fluorescence microscopy discloses the presence of rat y-globulin in glomeruli. Even more important, these investigators claim to produce glomerular damage and proteinuria by transfer of lymphoid cells taken from nephrotic donors into recipients tolerant of the passaged cells. One electron micrograph from a recipient rat shows little or no alteration of glomerular structure. Blozis et al. ( 1962) have produced the nephrotic syndrome and glomerular alterations in rats by injecting (together) homologous kidney and Hemophilus pertussis vaccine. This model may belong to the same variety of “autoimmune” renal disease as does Heymann’s, but there are no immunologic data yet to judge. It is difficult to assess the immunologic basis of “autoimmune” renal disease. There is to date no solid evidence that an endogenous antikidney antibody is operating to produce disease, nor that any immunologic reaction is causally related to the renal injury and proteinuria. We have examined, in the electron microscope, a few kidneys from rats prepared according to Heymann’s recipe and have found thickening of the glomerular basement membrane and dense deposits on the epithelial side of the lamina densa. Such deposits have been seen in kidneys injured by antigen-antibody complexes. An alternative pathogenesis, then, might include the nonimmunologic deposition, from the circulation into the glomerulus, of anti-renal antibody complexed with the immunizing renal antigen, already suggested by Heymann and his associates. An additional

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interpretation is also available. It is possible that the adjuvant with its homogenized tubercle bacilli and renal tissue is disseminated to the kidney and may injure the glomerulus merely as a foreign body, or may incite a local reaction of delayed hypersensitivity. The “autoimmune” renal disease described by Steblay and Lepper (1961; Steblay, 1962, 1963) is also insufficiently studied to comprehend its pathogenesis. Heterologous glomerular basement membrane incorporated into Freund’s complete adjuvant and injected intramuscularly, subcutaneously, and intradermally produces fulminating and fatal renal disease in sheep, dogs, or monkeys. There are differences between the “autoimmune” disease so elicited and the “autoimmune” disease of Heymann et uZ. Steblay stresses that the intraperitoneal route of antigen injection is unnecessary, that a heterologous kidney antigen is used, that the recipients usually die of their renal disease within 90 days of the first injections. But it is still unknown whether in Steblay’s model the host produces an endogenous antibody which cross-reacts with its own kidney or with the foreign renal antigens trapped in adjuvant to yield circulating complexes, or whether the adjuvant damages the kidney nonspecifically and permits antibodies to injure the glomerulus. In rats, pretreated with complete adjuvant, a usually ineffective dose of anti-kidney antiserum readily damages glomeruli and produces proteinuria (I. Watson et al., 1963).We have examined, in the electron microscope, some kidneys from monkeys and sheep afflicted with this type of renal disease. In the glomeruli there are diffusely thickened basement membranes, some increase of mesangial and endothelial cells, and focal and inconstant blunting of foot processes. None of these alterations is characteristic or specific, and it is not yet possible to determine from the electron micrographs whether it may be an immunologic process which alters the glomerulus and, if so, its nature, or simply dissemination of adjuvant and tissue to the kidney where direct injury of a foreign body type or delayed hypersensitivity may occur. Rothbard and Watson (1959, 1961) have also produced a renal lesion in rats treated first with adjuvant and then with rabbit anti-rat-collagen antiserum. The renal lesion in this model is probably due to the delayed interaction of host antibody and the foreign anticollagen serum bound to glomeruli ( cf. nephrotoxic serum nephritis). The adjuvant most likely augments the host antibody response to produce manifest disease. The fixation in the glomeruli of anti-rat-collagen antiserum is specific and suggests an immunologic interaction between the antiserum and component antigens of the basement membrane. There are no collagen fibrils demonstrable in the normal rat glomerulus, and an altered or hidden

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form of collagen must be postulated for this reaction to occur. Ultrastructural studies of this laboratory renal disease are lacking. b. Formed Elements of the Blood. The best evidence for the existence of autoimmune disease is in the field of hematology. There have been numerous reports of human disease in which a circulating antibody has been found which could lyse, agglutinate, or alter in some fashion the erythrocytes, platelets, or white cells, and which could elicit disease by passive transfer into a recipient (Dixon, 1958). One group of investigators (Shulman et al., 1961; Shulman, 1963), however, feels that even in this collection of diseases it is doubtful that autoimmunity plays a role, but more likely that antigen-antibody complexes are the cause of destruction, at least of platelets, in some cases of thrombocytopenic purpura. Despite the ample supply of such cases and the relatively easy access to the blood, there is a scarcity of electron microscope studies of the ultrastructure of immunologic processes in or on the formed elements of the blood. Further, the few reports available do not aid significantly in an analysis or comprehension of the pathogenesis of disease. Bloch and colleagues (1956) some years ago described the surfaces of erythrocytes in health and disease. Red cells removed from healthy people or asymptomatic patients are sharp-edged in the electron microscope; red cells which have aggregated display fuzzy edges. Aggregation is seen only in patients suffering from one of a variety of diseases. Most likely the alteration of the erythrocyte surface is not the result of an immunologic process, nor is it due to coagulants or hemolysis. In paroxysmal nocturnal hemoglobinuria, the majority of the red cells has been reported to have a patchy or coarsely precipitated stromal pattern as compared with the smooth surface of normal cells (Braunsteiner et al., 1956b; Cecchi and Conestabile, 1957). In both reports the micrographs are inadequate so that the scriptural description cannot be visualized. It is, of course, still moot, that paroxysmal nocturnal hemoglobinuria is the result of antigen-antibody reactions. Davis et al. (1961) have described an increase in plaque diameter (500-800A , ) on the erythrocyte surface of 60 % of cancer patients as compared with 30 % of noncancer patients. The anti-globulin test is positive in 50 % of the cancer group and in 20 % of the noncancer group. Further, when hemoglobin-free red cell membranes from normal people are coated by an antiserum, the surface plaques increase in diameter to a range of 500-800 A. It is inferred from these observations that the plaques on the surface of red cells are enlarged when erythrocytes have been sensitized, presumably by globulins. Structural changes have not been described in erythrocytes from

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patients with acquired hemolytic anemia, either idiopathic or symptomatic, with warm or cold antibodies, with 7 S or 19 S globulins. It would seem that once appropriate methods are developed a host of observations might be forthcoming which will illustrate the cytologic alterations of erythrocytes in hemolytic disease and the processes of hemolysis, phagocytosis, agglutination, and spherocytosis. The white cells have been equally or more neglected than the erythrocytes by electron microscopists and ultracytologists. Of the many cases of immunologic neutropenia and agranulocytosis reported, in none have the granular leucocytes been studied for fine structure. The ultrastructure of normal platelets has been quite thoroughly described, but there are only a few observations of platelets altered by immunopathologic processes. The circulating normal platelet is round or oval. When it touches a wettable surface it forms numerous pseudopods. Shortly thereafter the hyalomer spreads as a thin film of cytoplasm and the granulomer condenses to a central pseudonucleus. In idiopathic thrombocytopenic purpura (ITP) (Braunsteiner et ul., 1954; Braunsteiner and Pakesch, 1959) and in Waldenstrom’s macroglobulinemia (Pachter et ul,, 1959), the platelets are atypical when observed through the electron microscope. Pseudopodia do not appear, the hyalomer does not spread, and there is no concentration of the granulomer. In addition, giant and atypical platelets are frequently seen. In one case of ITP the platelet agglutinin titer of the serum was 1:32. When this serum is incubated with normal platelets, the thrombocytes manifest the alterations listed above. Similarly, the serum of Waldenstrom’s macroglobulinemia causes normal platelets to appear pathologic, i.e., there is absence of pseudopods, hyalomer spreading, and granulomer condensation. When the thrombocytes of the macroglobulinemia cases are washed and incubated with normal serum, they exhibit normal structure. The structural alterations of thrombocytes from ITP and Waldenstrom’s macroglobulinemia cannot be considered the result of a specific immunologic event. Similar absence of pseudopods, granulomer condensation, and thin hyalomer may occur in defective platelets and also may be induced by several types of manipulation of the incubating medium. And thus, although platelet agglutinins or macroglobulins may distort the form of thrombocytes, other agents also do. Unfortunately, the electron microscope has not yet imaged the molecular events when immune globulins react with the surface membrane of thrombocytes. c. Central und Peripheral Nervous System. In the central and peripheral nervous system, only one immunologic model of demyelination has been studied-experimental allergic encephalomyelitis. The precise

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nosologic niche for this laboratory disease is still to be determined. The consensus of opinion has been that it belongs to the category of autoimmune disturbances. It may also be classified as a disease of delayed hypersensitivity. Lymphoid cells, removed from a donor with experimental allergic encephalomyelitis, will transfer the affliction to a new host (Paterson, 1960) and will also destroy glial elements in tissue culture (Berg and Kallen, 1963). Complement-fixing humoral antibodies appear late in the course of the disease, and Paterson and Martin (1963) have shown that they may suppress the encephalomyelitis. Bornstein and Appel (1961; Bornstein et aZ., 1962), in tissue culture of nerve cells, have also demonstrated a demyelinating effect of humoral antibodies harvested from rabbits with the disease. Ultrastructural examination of experimental allergic encephalomyelitis (Luse and McDougal, 1960; Bubis, 1963) reveal that Schwann cells and oligodendroglia may be the prime target in lesions of the peripheral and central nervous system, respectively. The cytoplasm of Schwann cells is swollen, contains many empty vacuoles, and may harbor inclusions of myelin remnants. Similar cytoplasmic changes are seen in oligodendroglia. Demyelination is focal and ranges from simple separation and disorderly arrangement of myelin lamellae to complete myelin destruction with denudation of the axon. In the central nervous system, lymphocytes, macrophages, plasma cells, and occasional neutrophiles accumulate in the Virchow-Robin spaces of larger vessels and in newly formed spaces around capillaries. Gliosis and remyelination are also observed, but are obviously late or secondary phenomena. Both Condie and Good (1959), and Luse and McDougal (1960) point out that there is an increase of mitochondria in the axon cytoplasm and distention of these organelles, However, these changes are probably nonspecific and are observable in wound-healing of the central nervous system (Vial, 1958). In experimental allergic encephalomyelitis, the earliest and most extensive injury occurs in Schwann and oligodendroglial cells and in myelin, while axons are preserved. In Wallerian degeneration, the axons are primarily injured and myelin alterations are secondary (Vial, 1958; Terry and Harkin, 1959; Honjin d aZ., 1959). The process of demyelination in experimental allergic encephalomyelitis is similar to that described in diphtheritic neuritis ( Webster et aE., 196l), but in the latter disease there are no inflammatory changes and lesions are most numerous near the nodes of Ranvier and are unrelated to the distribution of vessels. In addition, there is minimal, or absent, accumulation of mononuclear inflammatory cells in perivascular spaces. As in other pathologic processes, the electron microscope does not

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elucidate the pathogenesis of events at a molecular level. Unexplained still are the focal distribution of the lesion, the nature of the attack upon the myelin lamellae, and the relationship of sensitized lymphoid cells, as well as antibody, to the lesion. d. Bone and Joint. The last affliction which might be placed in the category of autoimmune diseases and for which there are electron microscopic observations is rheumatoid arthritis. There are probaly as many reasons to classify this illness as an autoimmune process, i.e., endogenous antibody interacting with host antigens, as there are to place it with the group of diseases elicited by circulating antigen-antibody complexes or aggregated y-globulins. Although several reports on the ultrastructure of synovial membrane are available, there is only one concerned with rheumatoid arthritis. Barland et al. (1963) have studied the fine structure of synovial membrane obtained at arthrotomy from patients suffering with rheumatoid arthritis. Two cell types line the human synovial membrane. Type “A” is characterized by large cytoplasmic vacuoles containing a granular material, by numerous villous extensions, and by many small pinocyticlike vesicles. Type “ B is characterized by abundant ergastoplasm and much less prominent villous extensions, vacuoles, and Golgi apparatus. In the synovia .of rheumatoid arthritis, only type A is altered. Most striking is the presence of large dark bodies ranging from 0.4 to 3 . 2 in ~ diameter, generally located close to the nucleus. The bodies are homogeneous in texture, occasionally vacuolated, sometimes granular, and at times assume the form of myelin figures. Some, but not all, are membrane lined. One biopsy specimen has exhibited acid phosphatase activity, following appropriate histochemical treatment of the tissue, in granules corresponding to those seen in the electron microscope. At present, the significance of these type-A cell dark bodies in rheumatoid synovia is obscure. 3. Cellular (Delayed) Sensitivity A large and poorly defined group of immunologic reactions is characterized by three features. First, the lesion which appears in the skin of a sensitized animal or human being at the site of antigen deposition develops slowly over a period of 24 to 72 hours. Second, this manifestation of sensitivity can be transferred passively by hypersensitive cells or cell fragments. And third, circulating antibody is neither necessary nor detectable in the blood when the host manifests delayed hypersensitivity. It is postulated, therefore, that this kind of immunologic reaction is mediated by an “antibody” which is intimately associated with mononuclear cells, a cell-bound antibody. In clinical medicine, cell-bound

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antibody is the key phenomenon in bacterial ( tuberculin) sensitivity, contact chemical sensitivity, and transplantation immunity. Despite the extensive and intensive work in this area of immunology there is little certain knowledge of the pathogenesis in sensitized hosts of delayed hypersensitive reactions. The interaction of antigen and cell-bound antibody has not been directly observed in uiuo; in uitro, conflicting results are reported. Rich and Lewis (1932) have stated that the antigen causes necrosis of the sensitized cells in tissue culture. Others have indicated that specific antigen stimulates sensitized cells to proliferate ( Waksman and Matoltsy, 1958; Pearmain et al., 1963). Indeed, the cell type which carries the specific message of hypersensitivity remains incognito, although selected candidates have been macrophages, ly tphocytes, immature lymphoid cells, and others. And there is mounting evidence that the cells which are specifically sensitized to bacterial antigens, chemical hapten, and transplantation antigens merely trigger the visible immunologic response which at its maximum is composed of cellular and fluid components nonspecifically assembled ( McCluskey et al., 1963; Najarian and Feldman, 1963; Feldman and Najarian, 1963). Certainly the effective number needed to manifest delayed hypersensitivity or transplantation immunity is very small. It is apparent that with such a paucity of information concerning this type of immunologic reaction at the cellular and histologic level, there would be a paucity also of ultrastructural informatior: B. Goldberg et al. (1962) have attempted to find and describe the cell type associated with delayed hypersensitivity. They use ferritin as antigen, immunize guinea pigs, and at the height of delayed skin hypersensitivity and before the appearance of circulating antibody, they biopsy the specific skin lesions elicited by ferritin. They find that the ferritin is ingested by skin macrophages, but they are unable to determine whether this is a specific or nonspecific response, i.e., immunologic. Norton and Ziff (1963) have tried to examine delayed ( tuberculin) hypersensitivity with the electron microscope using ferritin-labeled purified protein derivative (PPD ) and as a control a mixture of PPD and unconjugated ferritin. They, too, observe ingestion of the tagged antigen by macrophages within 15 minutes of injection; but ferritin alone, not fixed to PPD, is similarly handled by macrophages from control skin sites. Eight hours after initiation of the lesion, many macrophages are degenerate and after 24 hours ferritin is present in the test sites outside of and between host connective tissue cells. Transplantation immunity has generally been considered to belong to that part of immunology called “delayed hypersensitivity.” It can be

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transferred by adoptive immunity, i.e., by a transfusion of tissue-sensitized lymphoid cells to a neutral recipient, and the cells carrying the specific message of immunity are probably mononuclear. Binet and Math6 (1961, 1962a,b) have recently illustrated a new cell type which appears in the lymph nodes of a host draining a graft. This cell ( a hemocytoblast) looks like a young plasma cell in the light microscope, but ultrastructurally it lacks an extensive and elaborate rough-surfaced endoplasmic reticulum. Instead, its voluminous cytoplasm is rich in ribosomes, unattached to membranes. It is possible that a cell of this type may be the sensitized element of transplantation immunity. However, the crucial experiment has not been performed to demonstrate that a collection of such cells will actually destroy a homologous graft. The fact that there is an increase of such cells in the draining regional lymph node does not necessarily implicate it in graft rejection, for it may be a nonimmunologic proliferation in response to the injury elicited by transplantation antigen. Recently, Journey and Amos (1962) have presented electron micrographs of sensitized cells specifically attacking and destroying tumor elements. The killer cells are macrophages and the target cells are leukemia elements. It is difficult to assess at this time whether the host macrophages pictured are cells carrying the specific message of transplantation immunity. No controls are studied in this experiment, i.e., there are no nonsensitized macrophages mixed with the specific target cells, nor are there nonspecific target tumors (other leukemias) placed in the vicinity of the sensitized host’s macrophagic response. The elongation of cytoplasmic arms of the macrophages and their encirclement of the target tumor elements are not specific for transplantation immunity. This type of cytological “bear hug” is done by macrophages during any kind of phagocytosis. Finally, even if the illustrated macrophages are host cells carrying the specific message of sensitivity, they disclose no ultrastructural form that would distinguish them from normal macro, phages. Galle and de Montera (1962) present a series of electron micrographs of cells infiltrating a transplanted human kidney. From this report it is not possible to know whether the pictured elements are from the donor or the host and whether they have any relationship to graft immunity. V. Epilogue

One of the few pleasures in the preparation of a review is the traditional right of the author to inscribe forever a few platitudinous generalizations about his subject and I would like to enjoy this right.

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A panoramic view of the achievements of electron microscopy in immunology reveals two broad planes of operation. On one plane, the electron microscope is merely an extension of the light microscope and permits an examination of pathology in tissues and cells with greater sharpness and finer resolution .than heretofore attainable. With the help of other disciplines, specific stains, and well-planned experiments, relatively crude morphologic and chemical events can be visualized between cell and cell and part and part of tissues. The precise localization of antigen-antibody precipitates within the glomerulus of immunologically induced renal diseases is a representative example of success at the histologic level. The second plane of profitable endeavor is at the molecular level, and it is here that the electron microscope is capable of providing rewarding insight on its own. It is now possible to enter the confines of the cell and observe clearly its subcellular units, It is also feasible to examine the nexus of antibody molecule and antigenic reactive site, the locus of antibody production, perhaps even the configuration of injurious molecules as opposed to bland molecules. The interaction of specific antibody with antigenic sites on viruses is a representative achievement at this level. We may anticipate future insights into ultrastructural immune processes from at least three directions. First, additional refinements of the instrument may be expected. Such improvements will yield even more resolution than is currently available, more ease of operation, less specimen contamination, and the like, Second, intracellular histochemical and immunohistochemical reagents are just beginning to prove their value. Protein, carbohydrate, and lipid markers will be developed so that these basic components of an organism may be followed and accurately localized. Although ferritin is a recent and still unexploited tag, it may already be inadequate, because of its size, to delineate molecular interactions. Other stains are being developed. Methods are evolving to separate morphologically DNA from RNA, to distinguish surface antigens from each other, to find the reactive sites of antibody molecules. Last, electron autoradiography provides an opportunity to mark internally the molecular units of an organism. At present the resolution obtainable is quite coarse, perhaps in the neighborhood of a lOOOA., but it is conceivable that better emulsions and better techniques will yield better resolution. Both immunology and electron microscopy have reached a promising juncture. The expanding universe of morphologic immunology has now extended into the realm of the minute, where form and function blend

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in single molecules, and where the elemental comprehension of cellular health and disease lies hidden. ACKNOWLEDGMENTS I want to thank the following people who generously permitted me to use their electron micrographs: Drs. S. de Petris (Fig. 4 ) ; B. Goldberg (Fig. 6 ) ; A. F. Howatson (Fig. 7a,b,c); R. E. Lee (Fig. 5 ) ; R. Rifkind ( Fig. 2 ) ; B. C. Seegal ;Figs. 11 and 15); G. Sorenson (Fig. 12); and D. Zucker-Franklin (Fig. 3 ) . Figures 2, 4, 6, 7, 11 and 15 were first published in the Journal of Experimental Medicine, Figure 12 was published in Laboratoy Investigation. I am grateful to both of these journals for permission to republish these photos. I am indebted to Miss Ruth Sturrock for technical assistance of high quality.

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Cell Wall Antigens of Gram-Positive Bacteria MACLYN McCARTY AND STEPHEN 1. MORSE The Rockefeller Institute, N e w York, N e w York

............................................... .. Cell Wall Protein Antigens ................................... A. Protein Antigens of the Streptococcal Cell Wall .............. B. Protein Antigens in Cell Walls of Other Bacteria ............. Cell Wall Polysaccharide Antigens ............................. A. Streptococcal Cell Wall Carbohydrates ..................... B. Pneumococcal C and F Polysaccharides ..................... C. Cell Wall Carbohydrates of the Bacilli ...................... Cell Wall Teichoic Acids ..................................... A. Cell Wall Teichoic Acids of Staphylococci and Other Micrococci B. Cell Wall Teichoic Acids of Other Organisms ................ Intracellular, Capsular, and Diffusible Antigens Related to the Cell W d of Gram-Positive Bacteria ................................ A. Intracellular Glycerol Teichoic Acids ....................... B. Capsular and Diffusible Antigens .......................... References .................................................

I. Introduction

11. Isolation and Composition of Cell Walls of Gram-Positive Bacteria

111.

IV.

V. VI.

249 250 252 252 254 255 250 201 203 209 270 270 277 277 281 282

1. Introduction

Recent advances in techniques for immunochemical dissection have made it possible to localize bacterial antigens to discrete elements of microbial architecture. Whereas formerly all antigens firmly fixed to the bacterial cell were designated simply as “somatic” antigens, many of them have now been related to definitive loci such as the cytoplasm, cytoplasmic membrane, or cell wall. The improvements in fractionation and analytic methods have been applied in greatest extent to studies of the rigid bacterial cell wall, and abundant information has been obtained on both the chemical structure and immunological activity of cell walls of many microbial species. The biologically and serologically important cell wall antigens of gram-positive bacteria constitute the subject of this review. Despite certain basic similarities, there are sufficient differences between the cell walls of gram-positive and gram-negative organisms to warrant separate consideration. The cell wall of gram-positive organisms is clearly visible as a single layer distinct from the underlying cell membrane. In contrast, the walls of gram-negative organisms are complex, multilayered struc249

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tures. In addition, a large amount of lipid is present in the walls of gramnegative organisms, and in the case of the enterobacteria lipid is associated with both the “ 0antigen and the endotoxin moiety. Lipid and potent endotoxin activity are generally not present in the walls of grampositive bacteria. Mycobacterial cell walls, like those of gram-negative organisms, are multilayered and rich in lipid and have also been excluded from this discussion. Both protein and carbohydrate antigens have been found in the cell walls of gram-positive bacteria. Some of the antigens serve as serological determinants of group or type specificity; others such as streptococcal M protein also play important roles in microbial pathogenesis and virulence. The major portion of this article is devoted to a consideration of the structure and activity of these substances. Also included in this survey is a brief account of those antigens which, though not integral constituents of the cell wall, are nevertheless chemically or serologically related to known cell wall components. 11. Isolation and Composition of Cell Walls of Gram-Positive Bacteria

The isolation and analysis of intact, undegraded, homogeneous cell walls from disrupted bacteria by Salton and Horne (1951) provided the impetus which has led to an increasing fund of knowledge regarding their chemical nature, synthesis, function, and immunological properties. There is now no doubt that the cell wall is a distinct entity; that it is a rigid encasement around the protoplast; that it has at least the one function of maintaining structural integrity in the face of environmental vagaries; and that there is a basic unit common to all bacteria. Nevertheless, the use of operational definitions makes precise delineation of limits difficult. In general, cell walls are obtained by differential centrifugation of suspensions of disrupted bacteria prepared by subjecting the cells to mechanical, sonic, or shearing forces. The most commonly employed method of disruption is that of shaking a suspension of cells with glass beads in a high-speed vibrator (Dawson, 1949). These procedures avoid the extensive solubilization and degradation of wall components that occur when walls are isolated after exhaustive alkali extraction of intact organisms. Homogeneity of the preparations is ascertained by examination in the phase contrast or electron microscope; it is of interest, as well as of practical assistance, that the cell walls of gram-positive organisms do not retain gentian violet and are gram-negative. Cell walls of gram-positive bacteria contain, in common with all

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bacterial cell walls, a structured complex termed the mucopeptide.’ The mucopeptide is composed of a limited number of amino acids, some of which are in the Dconfiguration, and the amino sugars, N-acetylglucosamine and N-acetylmuramic acid. The amino acids most characteristically present are alanine, glutamic acid, lysine or diaminopimelic acid, and glycine; others, e.g., aspartic acid and serine, may also be found. It is the mucopeptide that confers spatial configuration to the cell of gram-positive organisms since isolated mucopeptide, free of other wall components, has the same shape as the microorganism from which it was isolated. Thus far, the mucopeptide has not been shown to be immunologically active. The fact that the mucopeptide as a unit, and muramic acid in particular, occurs exclusively in the cell wall is of great importance in determining the localization of antigens. Thus, the firm association of a mucopeptide moiety with an antigen isolated from intact bacteria or culture fluids strongly suggests that the antigen is related to the cell wall. In addition to the mucopeptide, both proteins and carbohydrates are found in the cell walls of gram-positive organisms. There is some evidence to indicate that, at least in certain instances, the carbohydrates are chemically linked to the mucopeptide. Small amounts of lipid are occasionally encountered which may represent contamination of the preparation with fragments of the cytoplasmic membrane. Elucidation of the protein components of the cell walls of gram-positive organisms has been restricted because of a procedural operation in the preparation of “purified cell walls. Following the work of Cummins and Harris (1956),many investigators utilize proteolytic enzymes, as well as nucleases, to free the isolated walls from adherent cytoplasmic remnants. It is apparent that protein constituents, as well as contaminants, are removed or destroyed under these circumstances. Because of the frequent use of proteolytic enzymes in the preparation of cell walls, the bulk of the available information on cell wall antigens concerns the serologically active polysaccharides. A variety of extractive and lytic agents have been used to solubilize wall components, and the polysaccharides are then recovered by fractional precipitation. The carbohydrates are rarely obtained free from mucopeptide, but the nature of the carbohydrate-mucopeptide linkages has not been established. Many different monosaccharides are represented in the cell wall polysac1 Detailed account of the structure of the mucopeptide of gram-positive organisms is beyond the scope of this discussion; the reader is referred to reviews by Salton (1961), Work ( 1981 ), Perkins (1963a), and Rogers ( 1963a).

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charides, and several, such as glucose, galactose, and rhamnose, are found in the walls of a number of bacterial species. The name “teichoic” acids has been given ,to a particular class of carbohydrate substances which are polymers of either ribitol phosphate or glycerol phosphate to which sugars and ester-linked alanine may be attached. The teichoic acids are widely distributed among gram-positive organisms both as cell wall components and as “intracellular” constituents; their immunological significance is only beginning to be explored. 111. Cell Wall Protein Antigens

For the reasons cited above, the demonstration of true protein components of the cell wall has not figured prominently in the studies of various gram-positive species. However, there is evidence that a variable amount of protein is present in the isolated cell walls of many organisms. With respect to their antigenic properties, the most extensive information comes from investigations of group A streptococci in which several protein antigens, identified prior to the development of methods for the isolation of cell walls, have subsequently been shown to be localized in this structure.

A. PROTEINANTIGENSOF THE STREPTOCOCCAL CELL WALL 1. M Proteins of Group A Streptococci Early in his work on the general properties of the bacterial cell wall, Salton (1953) found that the type-specific M protein is part of the isolated wall. This fact has been amply confirmed and extended to show that it holds in the case of the numerous different serological types of group A streptococci, The finding has been exploited in attempts to devise improved methods for isolation and purification of the protein antigen and to prepare nontoxic vaccines for the induction of typespecific immunity (Barkulis and Jones, 1957; Krause, 1958; Kantor and Cole, 1980). The widespread interest in the M proteins derives from their importance in determining the virulence of group A streptococci. These surface antigens have the property of preventing or greatly retarding phagocytosis of the organism, thus protecting it from destruction during the early phase of the infectious process. Antibodies to M protein can reverse this effect and they provide the primary basis for immunity in streptococcal infections, but this immunity is type specific, i.e., antibodies to the M protein of one type of group A streptococcus afford no significant protection against the other types. In effect, streptococci classiiied

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in group A represent a diverse group of closely related organisms which are divided into a large number of individual types on the basis of serologically distinct M protein antigens of the cell wall. Despite their serological diversity, the M proteins possess in common the biological property which results in an antiphagocytic effect. The present state of knowledge of the M antigen has recently been reviewed by Lancefield ( 1962). The manner in which the M protein is attached to the cell wall is not precisely known. However, it has proved possible to remove it from the wall in soluble form in significant amounts only by procedures such as mild acid hydrolysis or enzymatic dissolution of the wall ( Lancefield, 1928; Krause, 1958). This suggests that it is linked to underlying wall structures by primary chemical bonds. In this connection, it is of interest that protoplasts of group A streptococci, growing in agar medium after enzymatic removal of the cell wall, produce an antigen indistinguishable from M protein which is released into the medium (Freimer et al., 1959). Thus, in the absence of the mucopeptide-polysaccharide framework of the wall, synthesis of M protein continues but the antigen does not remain attached to a surface structure. The localization of M antigens in the most superficial layers of the cell wall is suggested by their prominent role in agglutination reactions and by the fact that proteolytic enzymes destroy them quantitatively without affecting the integrity or viability of the cell ( Lancefield, 1943). A direct demonstration of the superficial location of these antigens has been provided recently by the studies of Hahn and Cole (1963)using fluorescent antibody. These studies also reveal that the new synthesis of M protein, after removal of the antigen with trypsin, is associated solely with the production of new cell wall, and that none is laid down on that part of the wall which was subjected to proteolytic digestion. The isolation of M proteins in highly purified state has proved difficult. Analysis of the best preparations has not as yet revealed any unique property to account for their biological activity. Among the characteristics which the M proteins of various types possess in common are: alcohol solubility; exquisite susceptibility to proteolytic enzymes; and a rather inefficient capacity ,to induce precipitin formation in man and in laboratory animals. 2. Other Protein Antigem of the Group A Streptococcal Cell Wall

M protein does not appear to have sole possession of the surface of the group A streptococcal cell. At least two other classes of antigens, which were identified serologically by Lancefield and designated T and

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R antigens (Lancefield, 1940a,b; Lancefield and Perlmann, 1952), are also found in the isolated cell wall ( Lancefield, unpublished results). That these antigens are also localized at the cell surface is indicated by the vigorous agglutination of streptococci in the presence of specific antibodies directed against them. The T and R antigens have also been shown to be proteins, but they differ from M proteins in many of their properties, including their greater resistance to proteolytic digestion (Lancefield and Dole, 1946; Lancefield and Perlmann, 1952). The most significant biological difference is that they lack an antiphagocytic effect and play no demonstrable role in virulence. Identical or closely related T antigens have been shown to be shared by several different M types (Stewart et at., 1944), a fact which contributes to the complexity of the problem of serological typing of group A streptococcal strains. Similarly, an R antigen (designated 28R) originally mistaken for the Type 28 M antigen, occurs in Types 2,28, and 48 ( Lancefield, 1957). On the other hand, an antigen with somewhat similar general properties (designated 3R) has been found only in certain strains of Type 3 streptococci (Lancefield, 1958). The picture which emerges of the group A streptococcal cell wall is that of an intricate mosaic of a varying number of different proteins, and it is by no means certain that all the members of the mosaic have been serologically identified. An example of a possible new protein antigen of the cell wall comes from the studies of Kaplan (1963) on an antigen in certain streptococci that cross-reacts with antigens in mammalian heart. The findings are interpreted as indicating that this antigen is localized in the wall and that, despite certain similarities, it is distinguishable from the M protein.

B. PROTEIN ANTIGENSIN CELLWALLSOF OTHERBACTERIA Protein antigens of streptococci other than those of group A have not been intensively studied, and in no instance is there definitive evidence for their occurrence as part of the cell wall. Nevertheless, it is probable that antigens analogous to the surface proteins of group A streptococci are present on the walls of certain other groups. The strongest evidence for this view derives from the finding that a substance serologically indistinguishable from 28R antigen occurs in certain strains of groups B, C, and G streptococci (Maxted, 1949). Reports of the existence of protein antigens in the cell walls of other gram-positive bacteria are relatively sparse. Cummins ( 1954) has described the occurrence of a superficial, specific protein antigen in the walls of Coynebactedum diphthedae. This antigen, like the streptococcal

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surface antigens, is involved in agglutination reactions. Moreover, there is suggestive evidence that the antigen may be type-specific since the few strains of diphtheria bacillus examined, as well as other corynebacteria, showed no cross agglutination. The antigen is readily destroyed by pepsin but is relatively resistant to the action of trypsin and papain. More recently, Yoshida and Heden (1962) have found evidence for the presence of a protein agglutinogen in the walls of Staphylococcus aureus. Subsequent studies indicate that ,this is a common antigen in the numerous serotypes of S. aureus (Lenhart et al., 1963; Yoshida et al., 1963). Although the number of gram-positive bacteria in which protein wall components have been identified remains small, it seems clear that other examples of this class of antigen will be found in other species. IV. Cell Wall Polysaccharide Antigens

The cell walls of most gram-positive species contain carbohydrate components in addition to the glucosamine and muramic acid which are characteristic of the mucopeptide portion of all bacterial walls. Cummins and Harris (1956) have shown that the monosaccharide composition of the cell wall carbohydrate provides useful additional information in the classification of bacterial species. On general grounds, it is probable that these carbohydrates are in most instances antigenic components of the cell, but direct evidence of their antigenicity and extensive studies of the carbohydrates as antigens are limited to a relatively few species: most notably, streptococci, pneumococci, and lactobacilli. It is important to note that their ability to induce the formation of specific antibodies is demonstrable only by the injection of whole cells or isolated cell walls, and soluble cell wall polysaccharides have usually not proved to be antigenic regardless of the method used to release them from the wall. Strictly speaking, therefore, the soluble carbohydrates are haptens, although their in vitro reactivity with antisera is typical of polysaccharide antigens. The hemolytic streptococci were divided into several distinct serological groups by Lancefield (1941) on the basis of antigens which were shown to possess the properties of carbohydrate. Subsequently, additional streptococcal groups were added to the list by other workers. In certain of the more common groups, the antigen upon which group classification is based has now been identified as the cell wall carbohydrate. This is not universally true, however, and in the case of the group D streptococci the antigen originally selected as expressing group specificity is an “intracellular” glycerol teichoic acid (vide infra), whereas the cell wall

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carbohydrates appear to be the basis for type differentiation within the group. This fact introduces an element of inconsistency into the classification scheme but does not detract from the importance of the cell wall carbohydrates as cellular antigens. Rhamnose is present in the walls of nearly all the recognized groups and may be considered the characteristic monosaccharide component of these streptococcal carbohydrates. The other monosaccharide constituents vary from group to group.

A. STREPTOCOCCALCELLWAJ~L CARBOHYDRATES 1. Group A Streptococcal Cell Wall Carbohydrate The group-specific antigen of group A streptococci was the first polysaccharide antigen to be clearly identified as a cell wall component (McCarty, 1952; Salton, 1953). The carbohydrate can be released from the cell wall in a soluble, serologically active form by a variety of procedures. When cell wall-dissolving enzymes ( muralytic enzymes ) are used for this purpose, the isolated polysaccharide always contains small amounts of muramic acid and the amino acids characteristic of the mucopeptide portion of the cell wall. On the other hand, extraction of the carbohydrate by mild acid hydrolysis or by formamide yields preparations that are essentially free of elements of the mucopeptide (Krause and McCarty, 1961 ). The relationships between the carbohydrate and mucopeptide moieties of the cell wall are best demonstrated by formamide extraction, since nearly all the carbohydrate is removed, leaving an insoluble structure composed of mucopeptide which retains the appearance of cell walls on electron microscopic examination. Although it is likely that the carbohydrate is linked to the mucopeptide through primary chemical bonds in the intact cell wall, it is clear that the two polymers have separate backbones. The serological reactivity of purified preparations of group A carbohydrate obtained by different extraction procedures is essentially the same, even in the quantitative precipitin test, and there is no evidence that the mucopeptide present in enzyme-extracted carbohydrates contributes to their behavior as antigens. The group A streptococcal carbohydrate is composed of only two monosaccharide constituents-L-rhamnose and N-acetylglucosamine-in a ratio of approximately 2:l (Schmidt, 1952; McCarty, 1952). One mutation involving the synthesis of the carbohydrate has been recognized which results in the production of a serologically distinct carbohydrate (Wilson, 1945). Strains of this type of mutant have now been isolated from many different serological types of group A streptococci, and in each case the chemical and serological properties of the cell wall carbohydrate have proved to be identical (McCarty and Lancefield,

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1955). Rhamnose is the predominant constituent of A-variant carbohydrate, and N-acetylglucosamine is present only in small amounts. For example, in formamide-extracted preparations which lack mucopeptide hexosamine, the ratio of rhamnose to glucosamine exceeds 20:l. The group A and A-variant carbohydrates are serologically distinct in their reactions with rabbit antisera, although minor cross reactivity is demonstrable with some specimens of antiserum. Some aspects of the structure and the chemical basis for serological specificity of the group A and A-variant carbohydrates have been worked out. The largest increment of information came from an analysis of the action of induced enzymes from soil bacilli capable of degrading the carbbhydrates ( McCarty, 1956).In the case of A-variant carbohydrate, a soluble enzyme ( V enzyme) was obtained which catalyzes extensive hydrolysis of the antigen so that it is reduced almost completely to dialyzable split-products. The split-products, consisting primarily of rhamnose oligosaccharides, are potent specific inhibitors of the reaction between the intact carbohydrate and rabbit antibody. Neither the monosaccharide unit isolated from the split-products nor commercial rhamnose monohydrate (at a final concentration of 1 %) shows significant inhibition of the quantitative precipitin test. However, a component with the properties of a rhamnose disaccharide is strongly inhibitory at a concentration of 0.1 %, and an unresolved mixture of tri- and tetrasaccharides is even more effective. The enzyme directed against the group A carbohydrate destroys its ability to precipitate with group A antisera without such extensive degradation of the molecule. The only dialyzable split-product is Nacetylglucosamine, amounting to 60-75 "/. of the total glucosamine of the carbohydrate. The enzyme has been identified as a P-N-acetylglucosaminidase with the ability to split terminal nonreducing N-acetylglucosaminide residues present in a macromolecule. The most reasonable interpretation of the data is that the group A carbohydrate is a highly branched structure with P-linked N-acetylglucosamine as the terminal unit on the branches or side chains. The primary importance of these terminal units is clearly indicated by the loss of serological activity resulting from their enzymatic removal. Confirmatory evidence that N acetylglucosamine is the major determinant of group A specificity is provided by the demonstration that the amino sugar itself as well as synthetic products, such as P-phenyl-N-acetylglucosaminide,are effective inhibitors of the precipitin reaction. In addition, synthetic antigens prepared by coupling p-aminophenyl-P-N-acetylglucosaminidewith proteins cross-react with group A antibody (McCarty, 1958).

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The nondialyzable residue remaining after enzymatic treatment of group A carbohydrate cross-reacts strongly with A-variant antisera and precipitates 90 ”/. as much antibody as the homologous antigen. Thus, the removal of terminal N-acetylglucosamine residues, in addition to eliminating group A specificity, unmasks determinants with A-variant specificity. The deglucosaminated carbohydrate is now susceptible to complete destruction by V enzyme, The basic structure of the rhamnose polysaccharide appears to be the same in the two carbohydrates, and they differ principally in the presence or absence of terminal N-acetylglucosaminide residues on the side chains ( McCarty, 1958). The presence of 1,Srhamnose linkages in the group A and A-variant carbohydrates is indicated by immunochemical studies of the cross reaction with horse antisera to Type I1 pneumococcal capsular polysaccharide, an antigen which is known to have this type of linkage (Heidelberger and McCarty, 1959). The slight cross reaction observed with group A carbohydrate is greatly enhanced by enzymatic removal of the terminal N-acetylglucosaminide residues so that it becomes nearly equivalent to that of A-variant Carbohydrate. More direct chemical evidence for the occurrence of 1,Srhamnose linkages in the streptococcal carbohydrate was subsequently obtained by periodate oxidation ( EstradaParra et al., 1963) and by methylation studies (Heymann et al., 1963). Preparations of group A carbohydrate show varying degrees of cross reactivity with A-variant antisera, and this cross reactivity is completely eliminated without loss of group A reactivity by treating the carbohydrate with V enzyme. It would appear that a small and variable number of the side chains on some molecules of the carbohydrate are uncapped with terminal N-acetylglucosaminide residues, and that the exposed rhamnosyl units are responsible for precipitation with A-variant antiserum. One strain of streptococcus, designated group A intermediate, has been encountered which consistently yields carbohydrate preparations reacting almost equally well with group A and A-variant antisera ( McCarty and Lancefield, 1955). Chemical and enzymatic analysis indicates that both types of side chain are present in approximately equal numbers. The synthetic mechanism for placing terminal N-acetylglucosaminide units on the rhamnose side chains does not always appear to be effective, and in the group A to A-variant mutation the mechanism is completely lost. In summary, both the group A and A-variant carbohydrate are multibranched structures with a small amount of glucosamine in the backbone. The oligosaccharide side chains in A-variant are composed solely of rhamnose with 1,Slinkages dominating, and similar side chains in

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group A are for the most part capped with terminal p-linked N-acetylglucosamine residues. The possibility exists that the rhamnose may have minor additional substituents, for example, O-acetyls, as indicated by the chromatographic behavior of the enzymatically released monosaccharide unit from A-variant and the failure of authentic rhamnose to act as an effective inhibitor ( McCarty, 1956). 2. Group C Streptococcal Cell Wall Carbohydrate The group C streptococcus is in many ways biologically similar to group A, e.g., hyaluronic acid capsules are produced by both groups and the range of extracellular products is similar. It is, therefore, of interest to compare the cell wall carbohydrates of the two groups. The group C cell wall carbohydrate has also been identified as the groupspecific antigen, and its relationship to the mucopeptide portion and the methods for obtaining it in soluble form are identical to those described for group A carbohydrate. The group C carbohydrate also contains rhamnose as a major constituent, but it differs from group A in that N-acetylgalactosamine is the dominant amino sugar and that glucosamine is present in only small amounts (Krause and McCarty, 1962a). N-acetylgalactosamine strongly inhibits the precipitin reaction between group C carbohydrate and its antibody, whereas N-acetylglucosamine is completely without effect. The evidence suggests that terminal N-acetylgalactosaminide residues play the same dominant role in serological specificity that has been demonstrated for N-acetylglucosamine in group A. Further evidence for the close relationship between the structure of group A and group C carbohydrates has been obtained. A number of strains of group C streptococci have been encountered which yield carbohydrates that react both with group C and with A-variant antisera (Krause and McCarty, 1962b). These have been termed group C intermediate, and it has been shown that the A-variant reactivity of these carbohydrates is completely abolished by treatment with V enzyme without loss of group C reactivity. More recently, strains have been isolated from phage-resistant mutants of group C streptococci which react only with A-variant antisera and not with group C antisera ( Araujo and Krause, 1963). This finding is of interest from the point of view of the biological significance of the carbohydrate, since group C carbohydrate has been shown to inactivate group C bacteriophage and thus to be the probable phage receptor (Krause, 1957). The carbohydrate of group C variant strains contains only small amounts of hexosamine, and it is similar in chemical constitution, serological reactivity, and susceptibility to V enzyme, to group A-variant carbohydrate. These facts indicate that

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the structure of the rhamnose portion of group A and group C carbohydrates are essentially identical and that the two carbohydrates differ primarily in the nature of the terminal hexosamine units. The anomeric configuration of the N-acetylgalactosamine end groups of group C carbohydrate has not been determined, The (3-N-acetylglucosaminidase ( from the soil bacillus) which attacks group A carbohydrate is without effect on group C carbohydrate, although it is able to split synthetic (3-N-acetylgalactosaminides at a slow rate. 3. Cell Wall Carbohydrates of Other Streptococcal Groups The cell wall carbohydrate of group G streptococci has also been isolated and shown to represent the group-specific carbohydrate (Krause, 1963). In addition to hexosamine and rhamnose, the carbohydrate contains galactose. Quantitative precipitin studies have recently shown that L-rhamnose, but not hexosamine, galactose, or galactose oligosaccharides, is a strong specific inhibitor of the reaction of group G carbohydrate with rabbit antiserum, suggesting that rhamnose represents an important antigenic determinant. This is in contrast to the ineffectiveness of rhamnose as an inhibitor of the group A-variant reaction and further substantiates the suggestion that minor substituents may be involved in A-variant specificity. Moreover, no cross reaction between group G and A-variant carbohydrates has been detected. The streptococci which comprise group D represent a diverse group of organisms which are serologically defined on a different basis than groups A, C, and G. It is now clear that the common antigen responsible for classifying these organisms in group D belongs to the class of substances which are currently called “intracellular teichoic acids,” as discussed below. It was noted by Cummins and Harris (1956) that there are differences in the monosaccharide composition of the cell wall of different group D strains in that glucose is present in some and galactose in others. The subsequent work of Elliott (1960) showed that the cell wall carbohydrates prepared from representative type strains of group D were distinct serologically and were identifiable as the type-specific antigens. These carbohydrates contain rhamnose, glucosamine, and a hexose, most commonly glucose; and it is thus evident that they are analogous to the group-specific antigens of groups A, C, and G. There is no information on the chemical basis for the differences in serological specificity displayed by the several closely related cell wall carbohydrates of group D. With regard to the other groups of streptococci, the localization of the group and type-specific antigens remains to be clearly defined. There

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is evidence that in some, for example, group E, the cell wall carbohydrate serves as the group-specific antigen; but in at least one other instance, group N, the relationships simulate those of group D (Elliott, 1963).

B. F’NEUMOCOCCAL C AND F POLYSA~ZIARIDES Tillett et d. (1930) discovered a species-specific pneumococcal antigen, the C polysaccharide, which was distinct from the capsular polysaccharides and common to all pneumococci. The C polysaccharide is present in the cell in relatively large amounts and is released in soluble form upon autolysis. Analysis of purified preparations of C polysaccharide supplied some general information on its chemical constitution, the most striking findings being the presence of 4-5 % phosphorus and 5-6 % nitrogen (Heidelberger and Kendall, 1931; Goebel et d., 1943). The possibility that C polysaccharide represents the cell wall carbohydrate of the pneumococcus was entertained soon after the demonstration of the localization of the group A streptococcal carbohydrate, but the autolytic properties of the organism complicate the isolation of intact cell wall preparations and direct experimental test of this possibility. Although cell walls obtained under conditions designed to minimize autolysis contained large amounts of C polysaccharide ( McCarty, ISSO), an unequivocal demonstration of the cell wall nature of this antigen awaited the detailed chemical studies of Liu and Gotschlich (1963). These workers found that the characteristic components of bacterial walI mucopeptide-muramic acid, alanine, lysine, and glutamic acid-are consistently present in the carbohydrate. Small amounts of serine and glycine are also found. One-half of the alanine is in the n-form, and the ratio of alanine to lysine and glutamic acid is approximately 2:l. In addition, glucosamine is present in amounts which suggest that it is referable to the mucopeptide fraction. These findings establish the cell wall origin of C polysaccharide, and they suggest that the preparations are analogous to group A streptococcal carbohydrate released from the wall by muralytic enzymes. In this instance the cell wall is presumably dissolved by an endogenous enzyme that is part of the autolytic system. The dominant single constituent (35 % ) of the C polysaccharide is galactosamine &phosphate, identified for the first time as a component of bacterial wall carbohydrate. This substance, together with a small amount of muramic acid phosphate, accounts for all the phosphorus present in the polysaccharide. All the components of the antigen have not yet been idenaed, the known hydrolytic components accounting for only about 80 % of the weight, and the evidence suggests the presence of

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an additional nitrogenous substance which decomposes during hydrolysis. However, initial studies indicate that galactosamine 6-phosphate is the important determinant in the reaction of the polysaccharide with rabbit antisera. Hornung and Berenson ( 1963) have reported somewhat similar findings on the chemistry of C polysaccharide, although they suggest that a uridine nucleotide is an integral part of the molecule. It is of interest that their preparations contain diaminopimelic acid rather than lysine. By a special procedure involving extraction of the insoluble residue remaining after autolysis of pneumococci, the C polysaccharide has also been obtained in combination with a lipid component (Goebel et al., 1943). This substance, designated F polysaccharide, is of interest, because it appears to represent the Forssman-like heterophile antigen present in pneumococcus (Goebel and Adams, 1943). The lipid content of F polysaccharide is only 6 % and the analytic values are surprisingly similar to that of C polysaccharide. However, in contrast to the C polysaccharide, F polysaccharide acts as an antigen in rabbits when injected intravenously in soluble form. It induces the formation not only of sheep cell hemolysins but also of precipitins which react with F and to a lesser degree with C polysaccharide. F polysaccharide strongly inhibits the lysis of sheep erythrocytes by pneumococcal heterophile antisera, and the cross-reactive relationships are further demonstrated by absorption of pneumococcal antisera with boiled sheep erythrocyte stromata, which removes one-third of antibody precipitable by F polysaccharide but none of the antibody precipitable by C polysaccharide. The nature of the lipid component has not been determined, nor has its relationship to the cell wall been established. Another notable property of the pneumococcal C polysaccharide is its ability to react with an abnormal protein which appears in the blood of man during the acute phase of a wide variety of pathological conditions (Tillett and Francis, 1930). The precipitation of this protein, designated C-reactive protein ( MacLeod and Avery, 1941), by the carbohydrate is dependent on the presence of calcium ion, and the phenomenon appears to be quite distinct from that of antigen-antibody interaction. Further discussion of C-reactive protein is not pertinent to this review, although it should be noted that interest in this protein led to the demonstration of subtle differences in C polysaccharide, depending on the method of isolation employed, A fully analogous acute-phase protein is produced by the rabbit but is precipitable only by certain preparations of C polysaccharide which have been prepared by modified procedures (Anderson

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and McCarty, 1951). The chemical difference between these special preparations ( Cx polysaccharide ) and the classic polysaccharide remain obscure, but there is evidence that the two forms of the hapten possess significant though minor differences in their reactivity with pneumococcal antisera in addition to the sharper distinctions in their reactivity with rabbit and human C-reactive proteins. The heterogeneity of C polysaccharide is now becoming evident on other grounds, since continuation of the studies of Liu and Gotschlich (1963) discussed above indicates that typical preparations of the carbohydrate can be fractionated into several chemically differentiable, though serologically similar, components. Hornung and Berenson (1963) believe that the uridine nucleotide found in their preparations is important in the interaction of the carbohydrate with C-reactive protein. C. CELLWALLCAREOHYDRATES OF THE BACILLI 1. Coynebacteriurn diphtheriae

Cell walls of Coynebacterium diphtheriae contain arabinose, mannose, and galactose in addition to the structural elements of the mucopeptide (Holdsworth, 1952a; Cummins and Harris, 1956)- Isolation of a compound containing these sugars by Holdsworth ( 1952b) probably represents the first separation of a polysaccharide from cell walls prepared by present-day methods. The organisms were disrupted by shaking with glass beads, and small amounts of residual protein were extracted from the walls with warm phenol. The oligosaccharide released from the phenol-treated walls by hot picric acid contained D-galactose, D-mannose, and D-arabinose in a molar ratio 2:1:3. Only galactose was destroyed by periodate, and since the polysaccharide had no inherent reducing power, it was deduced that all terminal units were nonreducing galactose groups. The carbohydrate remained as a homogeneous boundary in free electrophoresis and the molecular weight calculated from the diffusion coe5cient was approximately 1200 (Bowen, 1952). This value approximated that derived from oxidation studies and was in close agreement to the theoretical minimal molecular weight. Material with identical chemical composition and molecular weight was isolated from baryta extracts of the walls suggesting that the carbohydrate may indeed exist as a low molecular weight compound in the intact wall. Orlova (1950) isolated a carbohydrate from C . diphtheriae after extracting the organisms with 10 N NaOH. Galactose, mannose, and arabinose were found in a molar ratio of 4:1:3 rather than 2:1:3. The

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molecular weight was 4050, and the polysaccharide did not react with homologous antisera. It is not clear whether the vigorous method of extraction resulted in alteration of the proportions of the monosaccharide constituents and precluded potential serological reactivity. The carbohydrate isolated by Holdsworth was not tested for immunological properties. C u m i n s ( 1954), however, has demonstrated that polysaccharide components of the cell walls of C. diphtheria are of immunological significance. Intact cells of C. diphthmiue are agglutinated only by homologous antisera, whereas cell walls free from the specific protein antigen agglutinate in the presence of heterologous diphtheria antisera. Thus, antisera prepared against a prototype mitis strain of C. diphthe& agglutinate protein-free cell walls of i n t e m d i u s and gravis strains as well as cell walls of mitis organisms. Moreover, there is reciprocal cross reactivity. Coynebacterium diphtheriae cell wall agglutinins are not removed by absorption with walls of Cuynebacterium xerosk, Coynebactedum renale, or Coywbucterium hofmannfi. Cell walls of C. xero& are known to contain arabinose, galactose, and mannose in common with the diphtheria bacillus; those of C. rende contain in addition glucose and galactosamine; walls of C. hofmunnii also contain glucose and galactosamine but no mannose. Walls of Coynebacterium oob absorb a portion of the antibody, and there is other evidence that C. ovis and C. ddphtheriue may be related since certain strains of corynebacteria produce both diphtheria toxin and a toxin related to that of C. ouis (Jebb, 1948). The monosaccharide cell wall components of C . oois include glucose as well as arabinose, mannose, and galactose. The cell wall-agglutinating antigen is heat stable, rapidly inactivated by periodate, and, therefore, presumably a carbohydrate. Curiously, there have been no subsequent reports of studies designed to relate cell wall agglutination to the polysaccharide isolated by Holdsworth or to the group reactive carbohydrate antigen of C. diphtheria described by Wong and Tung (1939). 2. Anthrax Bacillus Although earlier workers described carbohydrate substances of the anthrax bacillus (Krambr, 1921; Tomcsik and Szongott, 1932, 1933), the first extensive chemical and serological studies of the anthrax polysaccharide were those of Ivbnovics ( 1940a,b,c). The carbohydrate was isolated from both virulent and avirulent organisms after cell lysis with acriflavine, and was primarily composed of equimolar amounts of D-

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galactose and N-acetylglucosamine. Horse serum containing antibodies against the anthrax polysaccharide also reacted both with Type 14 pneumococcus polysaccharide ( S 14) and partially hydrolyzed blood group A substance (Ivhnovics, 1940b). When the polysaccharide was subjected to partial acid hydrolysis, four fractions were isolated which still precipitated with horse, but not with rabbit antiserum; the fractions did, however, inhibit the reaction between rabbit antiserum and the reactive polysaccharide, whereas the monosaccharide substituents were ineffective ( Ivhnovics, 1940~). An identical polysaccharide was also isolated from ammonium carbonate lysates of anthrax bacilli obtained from infective exudates of the guinea pig (Smith et d.,1953a,b; Smith and Zwartouw, 1954, 1956). The polysaccharide contained 38-43 % galactose; 38-43 "/. glucosamine; 14.5 "/. acetyl; 4.0 "/. nitrogen; and 0.3 "/. a-carboxylamino nitrogen. The molecular weight was estimated at 27,000. The excess of nitrogen could not be accounted for as glucosamine, and the a-carboxylamino nitrogen also indicated the presence of other nitrogen-containing compounds; glycine, aspartic acid, alanine, and glutamic acid were subsequently detected (Smith and Zwartouw, 1956). Further studies also revealed that a,&-diaminopimelicand muramic acids were present (Smith et al., 1956). Two to 3 "/. of the compound was composed of these mucopeptide elements which could not be separated from the polysaccharide. Moreover, the compound was homogeneous in electrophoresis and ultracentrifugation (Record and Wallis, 1956). The firm union of the mucopeptide unit with the polysaccharide suggests that the anthrax polysaccharide is a cell wall component. In support of this thesis it has been shown that anthrax antiserum containing polysaccharide antibody reacts with cell walls but not with the capsule of anthrax bacilli (Tomcsik and Grace, 1955). Nevertheless, anthrax polysaccharide also has been isolated from culture filtrates of organisms grown for 22 hours (Strange and Belton, 1954), a period of growth that seems too short for extensive bacterial lysis. The polysaccharide obtained from the supernatant fluids was of the same composition and contained mucopeptide units. It would be of significance to know whether the extrusion of anthrax polysaccharide represents extraneous synthesis, or turnover of cell wall substance. Utilizing the polysaccharide preparation of Smith et al. (1956), Heidelberger et al. ( 1958) demonstrated that the anthrax carbohydrate was precipitated by Type 14 pneumococcal antiserum. Absorption of Type 14 antiserum with anthrax polysaccharide reduced the antibody nitrogen precipitated by S 14 from 1010 pg./ml. to 867 pg./ml. The

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amount of nitrogen precipitated from Type 14 antiserum by the anthrax polysaccharide was 219 pg./ml.; prior absorption with carob mucilage in which all the galactose occurs as nonreducing end groups, reduced this by about 70 %. Moreover, a reciprocal relationship occurred; i.e., absorption with anthrax polysaccharide decreased the antibody nitrogen precipitable with carob mucilage by approximately 74 %. However, the cross reaction between S 14 and anthrax polysaccharide cannot be accounted for solely by the common presence of terminal nonreducing galactose units. Absorption of Type 14 antiserum with anthrax polysaccharide reduced by 60 % the amount of antibody precipitated by the arabogalactan of Jeffrey pine in which very little of the galactose is present as end groups. Moreover, absorption of antiserum with anthrax carbohydrate reduces the amount of antibody precipitated by periodate-oxidized S 14, in which terminal nonreducing galactose units should have been destroyed, Mester and IvPnovics (1957) prepared the formazan from periodate-treated anthrax polysaccharide. The compound still contained 30 % glucosamine, but no galactose. They suggested that the majority of galactose units were l,&linked, but this seems unlikely since 1,6-linked galactose units are not present in S 14. Another serologically active polysaccharide prepared from both culture supernatants and baryta lysates of anthrax bacilli contains D-galactose and N-acetylglucosamine in a molar ratio of 2:1, rather than 1:l ( Cave-Browne-Cave et al., 1954). This material, however, does not crossreact with Type 14 pneumococci (Heidelberger et al., 1958). Although excess nitrogen was present, the nature of the nitrogen-containing compounds was not described. Since anthrax polysaccharide is found in both virulent and avirulent organisms, does not act as an antiphagocytic agent, is nontoxic, and does not enhance in viuo infection, it apparently plays no role in virulence (Smith and Zwartouw, 1956). 3. Bacillus megaterium Phase contrast examination of the interaction between antisera and Bacillus megderium has provided evidence for serological activity of cell walls of this species (Tomcsik, 1951; Tomcsik and Guex-Holzer, 1954a,b; reviewed by Tomcsik, 1956). The prototype strain studied was designated BmilZus M; originally thought to be a variant of the anthrax bacillus, it was later classified as a true strain of B. megaterium. Bacillus M produces capsular glutamyl polypeptide, but antibodies to this compound are not induced by injection of encapsulated Bacillus M. Polyglutamate antibody can be produced by immunization with en-

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capsulated anthrax bacilli, and addition of anthrax polyglutamate antiserum to a suspension of BaciZlus M results in a typical quellung reaction. After addition of homologous B . megaterium antiserum, transverse septa appear at the peripheral edge of the capsule as well as indefinite striations. When homologous antibody is added during lysozyme dissolution of Bacillus M the transverse septa then become visible in full length from capsular surface to cell wall, and the cell walls and cross walls become distinct. Similar observations were noted after trypsin treatment. Polyglutamate antibody does not produce these refractive changes. The capsular material of Bacillus M was extracted with boiling water, and after removal of polyglutamate by fractional precipitation an immunologically active polysaccharide containing galactosamine, glucosamine, and an unknown nonreducing amino sugar ( Guex-Holzer and Tomcsik, 1956) was isolated from the extract. Serologically active material was also prepared from nonencapsulated Bacillus M after partial lysozyme digestion. This preparation contained 7.6 % nitrogen and 4.7 "/. phosphorus; galactosamine, glucosamine, diaminopimelic acid, alanine, and glutamic acid were detected in acid hydrolyzates. A similar substance was isolated from the underlying cell of encapsulated organisms after prior extraction of the capsule with boiling water. All three preparations reacted only with homologous antisera. Moreover, absorption of antisera with any one preparation removed antibody which produced the cell wall reaction and rendered the transverse septa and the capsular striation visible. The absorbed sera did not agglutinate cell walls of Bacillus M. Tomcsik and Guex-Holzer ( 1954c) also demonstrated that antibody produced against nonencapsulated, trypsin-digested Bacillus M precipitated the polysaccharides prepared both from capsular material and from cell walls to the same extent. On the basis of these observations, Tomcsik (1956)postulated that carbohydrates with identical serological properties are present both in the cell walls and capsular framework of B. megaterium. This situation is not analogous to the occurrence of anthrax polysaccharide in culture supernatant fluids since the anthrax carbohydrate is not present in the capsule as evidenced by the fact that antibody to the-anthrax carbohydrate reacts only with cell walls. Moreover, changes resembling the capsular transverse septa appearing in Bacillus M do not occur (Tomcsik, 1956). Cell walls of B. megaterium probably also contain a ribitol teichoic acid with glucosyl residues (Baddiley, 1961; Ghuysen, 1961). This may,

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in part, account for the phosphorus content of the polysaccharide prepared from Bacillus M walls. Serological activity of the teichoic acid has not been studied. 4. Lactobacillus Utilizing hot dilute acid extracts of intact organisms as antigen, Sharpe (1955)classified 70 % of 442 strains of lactobacilli into six major serological groups and one subgroup. These serological groups conformed closely to conventional taxonomy based upon biochemical and physiological characteristics (Briggs, 1953). The antigens specific for Sharpe’s groups B and C have recently been identified as polysaccharide components of lactobacillus cell walls (Knox, 1963; Glastonbury and Knox, 1983). Cell walls of group B strains of Lactobacillus cmei var. casei contain rhamnose as the predominant carbohydrate together with lesser amounts of glucose and, in some instances, galactose. Walls of three strains were dissolved by the muralytic enzymes of Streptomyces albus, and group B reactive material was isolated. In addition to considerable amounts of mucopeptide the antigens contained rhamnose (39-48% ), glucose (3.&17 %), end galactose (0-16 %). Precipitation of all three preparations with group B-reactive sera was significantly inhibited by L-rhamnose; the hexoses were less potent and hexosamines were inactive. Periodate treatment of the antigen destroyed 54 % of the rhamnose and reduced serological activity by 74 %. In double diffusion tests only one serologically active component, which was identical for the three antigens, was demonstrated. An identical group B antigen is also released in soluble form from cell walls of strains which possess potent autolytic enzymes (Knox and Brandsen, 1962). The cell walls of group C strains of L. casei var. cmei contain little or no rhamnose, and approximately equal amounts of glucose and galactose together constitute 31-45 % of the wall substance. Group C-reactive antigen containing small amounts of rhamnose (1.7 %), and large amounts both of glucose (27.5%) and of galactose (23 % ) has been isolated after lysis of the walls of one strain with Streprnyces albus enzymes. The remainder of the antigen was composed of mucopeptide components. Glucose was the most potent monosaccharide inhibitor of precipitation and (hy$ucosides were more effective than a-derivatives. Of the diglucosides containing p-linkages tested, gentibiose ( 1,6-) was the most inhibitory. Antigenic analysis of cell walls of L. casei var. rhamnosus presents a more complicated problem. These organisms are also in Sharpe’s group

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C. However, in contrast to group C strains of L. cmei var. casei, the cell walls of L. casei var. rhamnoszrs usually contain 16-29 % of rhamnose in addition to 12-23 % of glucose and 12-33 % galactose; variable amounts of mannose may also be present. Cell walls of two strains were lysed, and in both instances group C specificity was present in a minor fraction soluble in 70 % (NH4)2S04.This material was low in rhamnose, similar in chemical composition, and serologically identical to the group C antigen isolated from L. cusei var. casei. The bulk of the cell wall rhamnose together with glucose was found in a major fraction of the lysate which was insoluble in 70 ”/. (NH4)2SO,; galactose was also present in one preparation and mannose in the other. Only minor amounts of mucopeptide were found. This fraction did not react with different group C antisera but reacted only with homologous antisera; preparations from both strains were serologically identical. Although the precipitin reaction was difficult to inhibit, it appeared that p-D-ghcoside was the antigenic determinant of this fraction as well as of the group C antigen. On the basis of differences in chemical, solubility, and serological properties, Kmox (1963) suggested that the two fractions from L. casei var. rharnnosus walls represented two distinct antigens: one, which cross reacted with the cell wall carbohydrate of group C strains of L. casei var. casei and, therefore, was group-specific and another, rich in rhamnose, which was type-specific for L. casei var. rhamnosus. Although the evidence for this thesis is suggestive, the antigenic determinants for both the proposed group and type-specific compounds are similar if not identical, and further quantitative studies on serological cross reactivity are required for corroboration. It has also been postulated that the cell walls of a group B lactobacihs strain contain both group and type antigens, but, again, detailed serological investigations have not been reported. V. Cell Wall Teichoic Acids

Linked to the mucopeptide of the cell walls of many gram-positive bacteria are substituted polymers of ribitol phosphate or glycerol phosphate which have been termed “teichoic” acids from the Greek teichos = wall (reviewed by Baddiley, 1962). Early evidence that such compounds might be present in bacteria was derived from the work of Mitchell and Moyle (1951a,b) who found an excess of phosphate in staphylococcal cells which could not be accounted for by nucleic acids, phospholipids, or other known phosphorus-containing compounds and which were thought to be derived from glycerol phosphate. Subsequently both cytidine diphosphoribitol ( CDP-ribitol) and CDP-glycerol were identified

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in extracts of certain gram-positive organisms (Baddiley and Mathias. 1954; Baddiley et d.,1956), and in view of the known role of nucleotidesugar complexes in polysaccharide synthesis it was postulated that polymers of glycerol and ribitol phosphate might exist. Polyribitol phosphate was then isolated from the cell walls of several species of gram-positive organisms ( Armstrong et al., 1958). Later polyglycerol phosphate was also found to be a cell wall component of some species of gram-positive organisms (Armstrong et al., 1959). The structural attributes of these polymers have been extensively studied by Baddiley and co-workers who found that both walanine in labile ester linkage and different monosaccharide substituents were attached to the polyolphosphate backbone. The designation “teichoic” acid was shown to be a misnomer when serologically active polyglycerol phosphate was isolated from streptococcal cells and found to be absent from the cell walls (McCarty, 1959). Cross-reacting “intracellular” glycerol teichoic acids have been found in a number of gram-positive bacteria including those that contain cell wall teichoic acids; thus far “intracellular” ribitol teichoic acids have not been described. The function and interrelationships of the cell wall teichoic acids and the intracellular teichoic acids are completely unknown. The wall teichoic acids, like the other wall polysaccharides, are apparently not involved in structural rigidity since their complete removal leaves behind the mucopeptide residue which has the shape of the original cell wall. It is possible, however, that these substances play some role in the synthetic activity of the cell. Of interest in terms of bacterial taxonomy is the fact that, with one possible exception (Clarke and Lilly, 1962), the polyolphosphate compounds have not been found in gram-negative organisms either as intracellular or as cell wall components. Reports of direct assay of the serological activity of the teichoic acids are comparatively few. The cell wall teichoic acids of the staphylococci are now known to have group-specific activity, but virtually nothing is known of the immunological reactivity of the many other wall teichoic acids which have been isolated. Comparatively more is known of the serology of the intracellular glycerol teichoic acids.

A. CELLWALLTEICHOIC ACIDSOF STAPHYLOCOCCI AND OTHERMICROCOCCI

1. Staphylococcus aureu+Stnrcture of the Cell Wall Teichoic Acid Thirty years ago Julianelle and Wieghard (1935; Wieghard and Julianelle, 1935) isolated a carbohydrate antigen from extracts of viru-

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lent staphylococci which precipitated Staphybcoccus aureus, but not Staphylococcus epidermidis ( Stuphylococcus albus), antisera. The antigen, polysaccharide A, was rich in phosphorus but constituent chemical units were not defined and the nature and reactivity of the carbohydrate received little further attention. More recent investigations indicate that the group reactive polysaccharide A and the cell wall teichoic acid of S. uureus represent the same serological entity (Haukenes et ul., 1961; Haukenes, 1962). The cell wall teichoic acid of S. uureus is a polymer of D-ribitol linked 1,5 by phosphate, to which N-acetylglucosamine and alanine residues are attached. Teichoic acid isolated from cell walls of S. uureus strain H has a phosph0rus:amino sugar:alanine ratio of 1:1:0.66. One residue of acetylglucosamine is in glycosidic linkage with the hydroxyl at C-4 of each ribitol. Alanine is in the D-configuration and is ester-linked to either C-2 or C-3 of ribitol; the ester bond is labile to acid and alkali, and it is likely that some of the amino acid is lost during isolation and purification (Baddiley et al., 1961, 1962b). 4-0- ( N-Acetylglucosaminyl ) -D-ribitol has been prepared from the teichoic acid of strain H by alkaline hydrolysis followed by treatment with phosphomonoesterase. P-N-Acetylglucosaminidaseremoved 85 "/. of the amino sugar from one preparation of the disaccharide. However, differences in optical rotation of various batches of the intact teichoic acid suggest that the proportion of a- and P-linked acetylglucosamine residues is not constant (Baddiley et al., 196%). The cell wall teichoic acid of S. uureus strain Copenhagen is similar to that from strain H, and contains 1 mole of acetylglucosamine and 0.5 mole of alanine per mole of phosphorus (Sanderson et ul., 1962). On the basis of periodate oxidation and phosphomonoesterase analyses, the compound has an average chain length of 12-15, assuming no branch points; there are 8 units in the teichoic acid of strain H. The molecular weight, calculated from ultracentrifuge data, is 6000, compared with a theoretical value of 5400-7300. As in the case of the teichoic acid from strain H, the majority of the acetylglucosamine residues in the preparation from strain Copenhagen are in P-glycosydic linkage; 83 "/. of the amino sugar is removed from the intact teichoic acid by P-N-acetylglucosaminidasefrom pig epididymis; the remaining amino sugar units are hydrolyzed by the mixed aand P-acetylglucosaminidases from rat epididymis. Teichoic acid preparations from strain Cophenhagen show no variation in the proportion of a- and p-linked amino sugar, nor is there evidence for preferential location of a particular configuration at one end of the chain.

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Most of the studies on the ribitol teichoic acids of S . aureus cell walls have been performed on material isolated after extraction of the walls with cold 10 % trichloroacetic acid (TCA) for 4&72 hours. However, it is not clear which bonds are cleaved by TCA. The teichoic acid is not extracted by reagents such as urea and strong salt, suggesting that hydrogen bonds and/or ionic forces are not involved. The hypothesis that covalent bonds between the free amino groups of alanine and the mucopeptide link the teichoic acid to the wall is no longer tenable (Mandelstam and Strominger, 1961). Ester-linked alanine can be removed in sltu from the teichoic acid by treating the walls with dilute alkali and no teichoic acid is released (Archibald et d,,1961a). Frequent covalent linkages between ribitol and/or acetylglucosamine and the mucopeptide also do not occur. After removal of ester-linked alanine, exposure of the walls to periodate releases 80 % of the wall phosphorus; a situation that would not occur if there were frequent covalent linkages between the sugar moieties and the mucopeptide (Rogers, 196313). Strominger and Ghuysen (1963; Ghuysen and Strominger, 1963) presented evidence for terminal covalent linkage of the teichoic acid of S. uureus to the mucopeptide. After dissolution of S. uureus cell walls with an acetylhexosaminidase, a nondissociable teichoic acid-glycopeptide complex was isolated. One disaccharide of acetylglucosamine and acetylmuramic acid, and 1 peptide subunit composed of 5 glycine, 2 alanine, 1 glutamic acid, and 1 lysine residues were present for each 8-10 repeating units of the teichoic acid. Less than 1 phosphomonoester group per lo00 phosphates was released by phosphomonoesterase as compared with 1 phosphomonoester end group per 14 phosphates in preparations isolated from TCA extracts of cell walls. Formaldehyde end groups, however, were obtained after periodate oxidation. The peptide components and the bulk of the amino sugars were removed from the teichoic acid-glycopeptide complex by an amidase without an increase in the number of phosphomonoester end groups. Strominger and Ghuysen suggest that the teichoic acid is terminally linked by a phosphodiester bond to a sugar fragment of the mucopeptide, but thus far the units blocking the terminal phosphomonoester groups are unknown. Alternative explanations include attachment of the terminal phosphate to a substance such as methanol, or the possibility that naturally occurring teichoic acid does not contain phosphomonoester end groups and that these appear only after cleavage of phosphodiester bonds by TCA. It is of interest that the material released from cell walls after 16 hours of cold TCA extraction contains many fewer phosphate end groups than the teichoic acid isolated after more prolonged extrac-

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tion; the number of formaldehyde groups liberated by periodate oxidation is the same in both (Sanderson et d.,1962). The synthetic process in the formation of the wall teichoic acid of S . aureus is not completely known. The N-acetylglucosamine residues can be attached to polyribitol phosphate in vitro by incubation with uridine &phosphate (UDP) N-acetylglucosamine in the presence of sonic extracts of staphylococci (Nathenson and Strominger, 1962). The enzymes are in a particulate bacterial fraction which sediments at 105,OOO g. There is both an a-UDP-N-acetylglucosamine-polyribitolphosphate transferase and a (3-transferase. The relative activity of the two enzymes is reflected in the configuration of the acetyIglucosamine in the teichoic acid. Thus, there is a preponderance of P-transferase activity in the sonic extracts of organisms with primarily (3-linked amino sugar in the teichoic acid. The converse is true for strains in which a-linked acetylglucosamine residues are most prevalent. Teichoic acid synthesis in sensitive staphylococci is inhibited by both penicillin and oxamycin with a concomitant accumulation of CDP-ribitol (Clarke et al., 1959; Saukkonen, 1961). Teichoic acid synthesis is far less sensitive to antibiotic action than mucopeptide formation and is not completely blocked even in high concentrations of penicillin (Rogers and Garrett, 1963). The product formed under these circumstances, in contrast to that formed in the absence of penicillin, is readily extracted by dilute alkali and hydroxylamine.

2. Staphylococcus aureus-Serological Reactioity of the Cell Wall Teichvic Acid Although there are many unanswered questions regarding the chemistry of teichoic acid and its relationship t o the mucopeptide, it is also true that the information available far exceeds the amount of immunological data at hand. Cell walls of S . aurms are agglutinated by S. aureus antisera and the reaction is inhibited by isolated teichoic acid (Juergens et al., 1960, 1983; Sanderson et al., 1961; Strominger, 1962). N-Acetylglucosaminides also block cell wall agglutination, and, in general, the most effective configuration for hapten inhibition by the amino sugar is that predominating in the wall teichoic acid (Nathenson and Strominger, 1902). In the case of cell walls of strain Copenhagen, in which 83 % of the acetylglucosamine residues in the teichoic acid are in the (3-configuration,it is of note that homologous antibody has primarily a-specificity. However, the walls are also agglutinated by heterologous S. aureus antiserum with P-acetylglu-

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cosamine specificity. Thus both the proportions of a- and 0-linked amino sugar and their relative immunogenicity play a role in the reaction. Cell walls of an 80/81 strain of S . uureus were agglutinated by a variety of S . uureus but not by Staphylococcus epidermidis antisera, and the agglutinin was absorbed out only by homologous and heterologous S. uureus walls (Morse, 1962b). There was no cross reaction with the mucopeptide prepared by treating walls with hot TCA and thereby preferentially removing 95 % of the cell wall phosphorw and teichoic acid. The teichoic acid from the 80/81 strain, unlike other preparations, absorbs onto tanned sheep erythrocytes, and the sensitized cells are agglutinated by S . uureus antisera; P-N-acetylglucosamine derivatives inhibit hemagglutination. Hapten inhibition of quantitative precipitin tests also demonstrates that P-linked acetylglucosamine is an antigenic determinant of this S. uureus teichoic acid. The occurrence of acetylglucosamine in different immunologically active linkages on the ribitol phosphate backbone of S . uureus cell wall teichoic acids presents several immunochemical questions and interposes a note of caution in assessing serological data, It is thus far uncertain whether individual polyribitol phosphate units contain glucosamine in only one configuration or whether the heterogeneity is based upon the presence of mixed configurations on each unit. Serological evidence on this point utilizing specific hapten inhibition could only be obtained after knowing the number of glucosamine residues on each chain available for combination with antibody, Although it is clear that the wall teichoic acid of S. uureus is the group antigen, at this time it would be well to state only that the determinant is N-acetylglucosamine in either a- or 0-glycosidic linkage to ribitol phosphate. Serum antibody reactions will depend upon both the amino sugar configurations in the antigen tested and the ability of the individual animal or human subject to produce antibodies specific for a particular linkage. Cross reactions between S . uureus teichoic acids and other compounds which induce antibodies with acetylglucosamine specificity have not been fully explored. It is known that antisera against the C carbohydrate of group A streptococci, which has p-glucosamine specificity, and antibody reacting with horse serum conjugated with azophenyl-P-acetylglucosamine agglutinate S. uureus Cophenhagen cell walls (Juergens et ul., 1963). Moreover, serum of rabbits hyperimmune to Escherichiu coli crossreact with teichoic acids of S. uureus (Saukkonen et ul., 1963). In the earlier studies with polysaccharide A, Julianelle and Hartmann (1936) found that 65-70 % of normal adults and 12 % of normal children had positive skin reactions; virtually all patients with staphylococcal

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disease had positive reactions. Normal sera did not precipitate the antigen, but a few sera from patients with severe, deep-seated staphylococcal disease contained precipitating antibodies. Although data are meager, it is known that intracutaneous injection of teichoic acid produces a wheal and flare reaction in adults (Strominger, 1962); and more recent studies indicate that the sera of most normal adults also contain a low level of circulating precipitating antibody (Allen et al., 1964). It remains to be seen whether the teichoic acid of S . uureus plays a role in staphylococcal disease, but it has been suggested that the compound is a determinant for opsonization (Mudd et al., 1963). 3. Staphylococcus epidermidis and Other Micrococci Wieghard and Julianelle (1935) also described a group reactive polysaccharide, polysaccharide B, isolated from acid extracts of avirulent staphylococci. The antigen contained phosphorus and a reducing sugar which reacted to form an osazone identical with glucosazone. Morse (1963) isolated a polymer of glycerophosphate and glucose from cell walls of a strain of Staphylococcus epidemnidis (Staphylococcus albus). A small amount of contaminating mucopeptide was also found and no ester-linked D-alanine was present. However, the extraction procedure utilized would have removed the labile amino acid. The g1ucose:phosphorus ratio was 0.6: 1.0, and on acid hydrolysis only monoglucosides were detected. Although structural analyses were not performed, it is likely that the glucose residues are attached to C-2 of glycerol units which are linked 1,3 by phosphodiester bridges. The compound reacts with homologous and heterologous S . epidermidis antisera but not with S . aureus antisera. Acid extracts of cell walls of four other strains of S . epiderrnidis from human sources yielded lines of identity with the prototype antigen in gel-diffusion studies. The antigen did not cross-react with streptococcal polyglycerophosphate nor with group D streptococcus antigen. In hapten-inhibition studies, a-linked glucosides were the most effective compounds in reducing immune precipitation. Nonspecific inhibition by phosphate radicals also occurred, but polyglycerophosphate was no more effective than monomers. The presence of immunologically inactive p-glucosyl residues was not excluded. Davison and Baddiley (1963) studied the composition of the cell wall teichoic acids of several strains of coagulase-negative micrococci. They utilized the classification system of Shaw et al. (1951) in which fermentative organisms which produce a positive Voges-Proskauer test are designated as Staphylococcus saprophyticus; this group is synonymous

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with S. epidemzidis as defined by Breed (1957). In support of Morse’s findings they found glucose and glycerol phosphate in the wall teichoic acids of four such strains. Glucosamine and ribitol were found in a fifth strain, but other authors have pointed out that this organism is probably not a true S . epldermidis (Jones et aL, 1983). The other strains studied were designated Staphylococcus Zactis, a species known to be heterogeneous and discarded in current taxonomy. One strain, probably Cuffkyu tetragew, contained cell wall glycerol teichoic acid with glucosamine substituents; this compound was structurally different from the usually occurring polymers since saccharinic acid was formed on alkali hydrolysis and no glycerol was produced. Two of the strains had wall teichoic acids which contained glycerol and galactosamine. One glycerol-galactosamine compound was studied in detail ( Ellwood et aZ., 1963).N-Acetylgalactosamine was present on C-2 of approximately one-third of the glycerol residues; the remaining glycerols had labile alanine units in ester linkage on C-2. Most of the galactosamine was in a-linkage, but p-acetylhexosaminidase removed ca. 20 % of the amino sugar. The teichoic acid reacted with a few antisera prepared against coagulase-negative staphylococci ( Haukenes et aZ., 1961). It has been stated that the “micrococcus provides what is probably the worst example in bacteriology of uncritical systematic work” (Abd-elMalek and Gibson, 1948). At present, in the family Micrococcaceae, most organisms in the genus StuphyZococcus can be separated into the two species, aureus and epidemidis, on the basis of their cell wall teichoic acids as well as on physiological and biochemical grounds. It is hoped that further chemical and immunological analyses of cell wall teichoic acids, such as the glycerophosphate-galactosamine polymer, may provide a solid basis for taxonomic separation of other genera and species in the micrococcus family. However, recent studies indicate that complexities may arise since some strains of coagulase-negative staphylococci have been found to possess a polyribitol phosphate-glucosamine antigen and a few others may possibly contain both the polyglycerol phosphate glucose compound and a polyribitol phosphatsglucosamine teichoic acid (Losnegard and Oeding, 1963a,b).

B. CELLWALLTEICHOIC ACIDSOF OTHERORGANISMS Group-specific carbohydrate antigens occur in cell walls of groups B and C lactobacilli (vide supra). Cell walls of these organisms and those of group F contain varying amounts of phosphorus, but teichoic acid breakdown products have not been detected in acid hydrolyzates

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(Ikawa and Snell, 1960; Baddiley and Davison, 1961). Teichoic acid is, however, present in the walls of other groups. Walls of group D strains contain a ribitol teichoic acid and in the polymer isolated from Lactobacillus urubinosus 17-5, a-glucosyl residues occur at C-4, or C-3 and C-4, of many of the ribitol units; ester-linked D-alanine is at C-2or C-3 (Archibald et d.,1961b; Sargent et d.,1962). Glycerol teichoic acids are present in the walls of strains in groups A and E (Ikawa and Snell, 1980; Baddiley and Davison, 196l), but the intact compounds have not been studied in detail. On the basis of differences in cell wall polyol phosphates and possible variations in their monosaccharide substituents, taxonomically useful distinctions may be made between lactobacillus groups. However, direct serological studies of the cell wall teichoic acids of lactobacilli, as well as those in other gram-positive species such as BQeillus subtilis, are required before it is certain that they have the same significance as the wall teichoic acids of the micrococci. Antigenic analysis of B. subtilis cell walls may prove to be even more complex since the walls contain a mucopolysaccharide composed of equimolar portions of glucuronic acid and N-acetylglucosamine (“teichuronic” acid) (Janczura et d.,1961), as well as a ribitol teichoic acid with p-glucosyl residues (Armstrong d d.,1960, lssl). VI. Intracellular, Capsular, and Diffusible Antigens Related to the Cell Wall of Gram-Positive Bacteria

A. INTRACELLULAR GLYCEROL TEIMOICACIDS

The relationship of the substances now commonly designated as intracellular glycerol teichoic acids to the cell wall is not yet fully known. However, because of their serological reactivity and the evidence that relates them to known wall components, it is important that they be included in a discussion of cell wall antigens. The term “intracellular” derives from the fact that following disruption and fractionation of the bacterial cell, these glycerol teichoic acids are predominantly four1 in the soluble supernatant fraction and are usually present in no more than trace amounts in the isolated cell wall. Clearly, therefore, they are not firmly attached to the wall structure as are, for example, the streptococcal carbohydrates and the staphylococcal ribitol teichoic acids. Nonetheless, it is probably misleading to call them “intracellular,” with the implication of residence in the cytoplasm, since their behavior suggests they have a quite superficial localization. In many instances, they may be obtained in amounts representing a substantial proportion of the total present in the cell by simple extraction of intact cells with buffer solutions or saline

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under conditions which do not release significant amounts of other cellular components. They may also be released into the medium during growth of the cells. These facts, together with their close relationship to wall-bound teichoic acids and the occurrence of typical wall constituents such as D-alanine in ester linkage, suggest that they are involved in some way in the synthesis or function of the cell wall. It has been suggested that they are localized between the cell wall and the cell membrane (Hay et al., 1983), but it seems equally likely that they are synthesized in the cell membrane and move out into wall substance to serve their as yet undiscovered function. The serological properties of these glycerol teichoic acids indicate that they are important both as heterophile antigens, possessed in common or in closely related form by a variety of gram-positive species, and as species-specific antigens in certain instances. At present, the evidence indicates that they induce the formation of antibodies only when the intact cell is used as antigen-a situation similar to that observed in the case of the cell wall carbohydrates. The first observations on the antigenicity of the glycerol teichoic acids were, in retrospect, the hemagglutination studies in which unknown substances produced by a variety of gram-positive microorgranisms were used as coating antigens (Rantz d al., 1956),since it has now been shown that substances of this class were at least in part responsible for the reactions observed (e.g., Gorzynski et al., 1960). Their capacity to act as precipitating haptens was demonstrated by the reactions of certain antistreptococcal rabbit sera with a substance identified as polyglycerophosphate, the basic structural unit of the glycerol teichoic acids ( McCarty, 1959). 1. Composition of the Glycerol Teichoic Acids The studies of Baddiley’s group (see review, Baddiley, 1962) indicate that the backbone of the glycerol teichoic acid is a chain of glycerol molecules linked 1,3 by phosphodiester bridges, or in effect a poly-aglycerophosphate. The second hydroxyl of each glycerol is free for substitution, and it is the substituents at this point which account for the diversity in serological specificity. The most common of these substituents is ester-linked D-alanine which appears to be present to some extent in all preparations that have been handled with s d c i e n t care to avoid splitting this readily hydrolyzed linkage. The other type of substituent is represented by monosaccharide or short oligosaccharide chains attached at this point through a glycosidic bond. The molecular size of the serologically active glycerol teichoic acids has not been established, and conflicting data on this point have been obtained in attempts to

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estimate size by different indirect approaches. It seems probable, however, that they are relatively small with molecular weights below lo4. 2. Serological Specificity of Glycerol Teichoic Acids The purified polyglycerophosphate obtained from group A streptococci in attempts to determine the nature of the antigen was nitrogenfree ( McCarty, 1959). The procedures employed to purify the substance are sufficient to remove the ester-linked alanine now known to be present. However, polyglycerophosphate in this form is an excellent precipitating antigen with appropriate antistreptococcal sera, and inhibition studies demonstrate that specificity is dependent on the glycerophosphate configuration. Sodium a-glycerophosphate causes definite, though relatively weak, inhibition of the precipitin test; but synthetic polyglycerophosphate with an average chain length of 6 units prepared by Michelson (1958) is highly effective and results in almost complete suppression of precipitation at moderate concentrations. Antigens released from a variety of gram-positive organisms either by acid (pH 2) hydrolysis or by disruption in the Mickle disintegrator give strong precipitin reactions with the streptococcal antisera selected for their reactivity with purified polyglycerophosphate. Thus, streptococci of most serological groups, staphylococci, and aerobic sporulating bacilli possess antigens of this class. On the other hand, similar substances have not been detected in pneumococci, corynebacteria, clostridia, or in any of the gram-negative species examined. It has not been determined to what extent the cross-reactive antigens represent substances which are identical in structure to the polyglycerophosphate of group A streptococci. Alternatively, they may represent members of the glycerol teichoic acid family which possess the same basic polyglycerophosphate structure but with substituent sugars not found in the group A streptococcal material. In any event, it is clear that the cross reaction is dependent on glycerophosphate specificity. For example, the precipitin reaction between extracts of Stuphylococcus aweus and the streptococcal antisera is almost completely inhibited by Michelson’s synthetic polyglycerophosphate. As noted previously, the group-specific antigen of group D streptococci belongs in the family of intracellular glycerol teichoic acids (Elliott, 1962; Wicken et al., 1963). The antigen is readily extracted at pH 9 at room temperature and is found in the soluble fraction after disruption in the Mickle disintegrator. The distinctive feature is the occurrence of 30 to 40 % glucose which is present in the form of glucosyl (and, possibly, in some instances di- or trisaccharides of D - ~ ~ U C residues OS~) attached to C-2 of the glycerol units. In preparations not exposed to

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alkaline conditions, D-alanine is found in ester linkage. The data suggest, however, that in these antigens the amino acid residues are linked to glucose hydroxyl groups rather than to glycerol. The close relationship in the composition of the group D antigen to that of the cell wall glycerol teichoic acid of Staphylococcus epidermidis discussed above is evident, and this represents one piece of evidence which suggests that the distinction between cell wall and intracellular teichoic acids is artificial. It is pertinent, however, that there is no serological cross reaction between the group D streptococcal and the staphylococcal antigen, indicating that the configuration of the glucosyl residues is different. Furthermore, neither of these antigens show appreciable cross reactivity with antisera to group A streptococcal polyglycerophosphate. Since the basic glycerophosphate chains are similar in constitution, it would appear that the presence of the glucosyl residues in some way renders the glycerophosphate determinants unavailable for interaction with antibody. Additional evidence for the effect of substituent groups on the serological reactivity of the basic polyglycerophosphate chain has recently been obtained. Wilson and Wiley (1963) have demonstrated the presence of an antigen, designated E4, in certain extracts of group A streptococci which is distinct from polyglycerophosphate but clearly related to it. The distinction is illustrated by the fact that many sera with excellent E4 antibody give no reaction whatever with polyglycerophosphate, and the relatedness is demonstrated by immunoelectrophoresis and double-diffusion precipitin analysis. Subsequent work ( McCarty, 1963) indicates that E4 represents polyglycerophosphate with a heavy complement of ester-linked alanine. Purified preparations obtained from group A streptococci under conditions which minimize the loss of alanine esters have a phosph0rus:alanine ratio of not greater than 2:l. These preparations react strongly with E4 antisera but give no more than weak reactions with polyglycerophosphate antisera. Treatment under mild alkaline conditions with loss of alanine causes a progressive decrease in reactivity with E4 antisera and progressive increase in reactivity with polyglycerophosphate antisera. At intermediate stages of the process the material reacts well with both antisera, but the fully dealaninated product is indistinguishable from the polyglycerophosphate originally described. In quantitative precipitin tests, specific inhibition is observed with alanine methyl esters but not with free alanine. These experiments indicate that, contrary to the findings with ribitol teichoic acid (Haukenes et d.,lWl),ester-linked alanine can contribute serological specacity to glycerol teichoic acid. The alanine not only

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adds a new specificity but is able to suppress any expression of the basic glycerophosphate specificity. 3. Intracellular Teichoic Acids in Other Organisms In addition to the studies of the individual intracellular teichoic acids discussed above and the demonstration of antigens in a variety of grampositive species that cross-react serologically with streptococcal p l y glycerophosphate, Baddiley’s group has carried out detailed chemical investigations of other representatives of this class of compounds. Although no information is presented on the serological activity of these preparations, their relation to the other members of the group is obvious. An intracellular teichoic acid of Staphylococcus aureus H isolated from a ribosomal fraction ( RajBhandary and Baddiley, 1963) is a glycerol 1,3phosphate polymer with D-alanine ester residues at C-2 of most of the glycerol units. A small amount of gentiobiosyl residues in p-linkage and a lesser amount of N-acetylglucosamine units are on C-2 of some of the glycerol elements. Neither the homogeneity nor the serological activity of this preparation has been established. Intracellular polyglycerophosphate has also been detected in all strains of lactobacilli examined, irrespective of cell wall composition (Baddiley and Davison, 1961); that from Lactobacillus casei contains only D-alanine residues on C-2 of each glycerol residue (Kelemen and Baddiley, 1961). The polymer from the cell contents of LuctobaciUus urabinosus 17-5 consists of 18 glycerol units joined through 1,Sphosphodiester linkage. On two glycerols, a-glucopyranosyl residues occur at (2-2; D-alanine esters are on most of the remaining glycerols (Critchley et al., 1962). Whether the glucoside imparts special serological significance is not clear. AND DIFFUSIBLE ANTIGENS B. CAPSULAR In addition to the intracellular teichoic acids, other antigenic substances chemically or immunologically related to the bacterial cell wall have been isolated from bacterial fractions free of wall material. As noted previously, the cell wall polysaccharide of the anthrax bacillus can be obtained from culture supernatant fluids under circumstances in which bacterial lysis should not be a factor, and polysaccharides with identical serological activity have been isolated from both the capsule and cell wall of Bacillus meguterium. Certain other immunologically reactive substances from extra-wall material bear chemical hallmarks which indicate a relationship to the wall. D-Glutamic acid is a characteristic mucopeptide component, yet

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poly-D-glutamyl polypeptide is a capsular substance of anthrax and other bacilli and the polymer does not occur in the cell wall (Thorne, 1956; Tomcsik, 1956). Other capsular substances have been shown to contain firmly associated mucopeptide units. Alanine, aspartic acid, glutamic acid, lysine, and glucosamine were found in an immunologically reactive polysaccharide isolated from culture supernatants of the distinctive encapsulated Smith strain of StuphyZucuccus aureus ( Morse, 1962a). The mucopeptide elements were not separable from the major portion of the antigen which was composed of glucosaminuronic acid ( Perkins, 1963b). It is not known whether the cell walls of this strain of S. aureus contain aminohexuronic acid, but this class of compounds does occur’in microbial cell walls (Czerkawski et al., 1963; Perkins, 19631,). Many pneumococcal capsular antigens contain polyol phosphates despite the fact that pneumococci contain neither intracellular nor cell wall teichoic acids. Of 2,l phosphorus-containing type-specific capsular polysaccharides examined, 8 gave ribitol or anhydroribitol as major hydrolytic products; 7 others yielded glycerol derivatives ( Shabarova et aZ., 1962). Type 6 pneumococcus polysaccharide ( S 6 ) is known to have a repeating unit of galactopyranosyl-glucopyranosyl-rhamnopyranosyl-ribitol joined by phosphodiester linkages ( Rebers and Heidelberger, 1959, 1961); S 34(41) has a repeating unit of galactofuranosylglucopyranosyl-galactofuranosyl-galactopyranosyl-ribitolalso linked by phosphodiester bonds (Roberts et al., 1963). As more fundamental information accrues, it is entirely likely that the bacterial cell wall will be found to participate directly or indirectly in the production of compounds such as these which are not localized to the wall. The cell wall may serve as a template or matrix upon which certain bacterial components are synthesized. Alternatively, synthetic machinery responsible for the production and incorporation of cell wall and mucopeptide components may be partially or completely diverted to the manufacture of non-cell wall substances. Demonstration of such events would explain the presence of “typical” cell wall components in immunological entities isolated from anatomic sites other than the cell wall.

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Lancefield, R. C. (1982). J. Imrnunol. 89, 307-313. Lancefield, R. C., and Dole, V. P. (1946). J. Exptl. Med. 84, 449-471. Lancefield, R. C., and Perlmann, G. (1952). J. Exptl. Med. 98, 83-97. Lenhart, N. A., Mudd, S., Yoshida, A., and Li, I. W. (1983). 1. Immunol. 91, 771778. Liu, T. Y., and Gotschlich, E. C. (1983). J. BioZ. Chern. 238, 1928-1934. Losnegard, N., and Oeding, P. (1963a). Acta Pathol. Microbid. Scand. 58, 482-492. Losnegard, N., and Oeding, P. ( 1983b). Acta Pathol. Microbb2. Scand. 58, 493500. McCarty, M. (1952). J. Exptl. Med. 98, 569-580. McCarty, M. (1958). J. Exptl. Med. 104, 829-843. McCarty, M. (1958). J. Exptl. Med. 108, 311-323. McCarty, M. (1959). J. Exptl. Med. 109, 381-378. McCarty, M. ( 1960). Unpublished observations. McCarty, M. ( 1983). Unpublished observations. McCarty, M., and Lancefield, R. C. (1955). J . ExptZ. Med. 102, 11-28. MacLeod, C. M., and Avery, 0. T. ( 1941). J . Exptl. Med. 73, 183-190. Mandelstam, M. H., and Strominger,-J. L. ( 1961). Bbchern. Biophys. Res. Commun. 5, 468-471. Maxted, W. R. (1949). J. Gen. Microbid. 3, 1-8. Mester, L., and Ivhovics, G. (1957). Chern. Znd. (London) p. 493. Michelson, A. M. ( 1958). Chern. Znd. (London) p. 1147. Mitchell, P., and Moyle, J. (195la). J. Gen. Microbiol. 5, 988-980. Mitchell, P., and Moyle, J. (1951b). J. Gen. Microbiol. 5, 981-992. Morse, S. I. (1982a). J. Exptl. Med. 115, 295-311. Morse, S. I. (1982b). J. Exptl. Med. 116, 229-245. Morse, S. I. (1963). J. Exptl. Med. 117, 19-28. Mudd, S., Yoshida, A., Li, I. W., and Lenhart, N. A. (1983). Nature 199, 1200-1201. Nathenson, S. G., and Strominger, J. L. (1982). J . BloZ. Chem. 237, 3839-3841. Orlova, 0. K. (1950). Biokhimiya 15, 382-367. Perkins, H. R. (1983a). Bacteriol. Rev. 27, 18-55. Perkins, H.R. (1983b). Biochem. J. 86, 475-483. RajBhandary, U. L., and Baddiley, J. (1963). Biochem. J. 87, 429-435. Rantz, L. A., Randall, E., and Zuckerman, A. (1958). J. Infectious Diseases 98, 211-222. Rebers, P. A., and Heidelberger, M. (1959). J. Am. Chem. SOC. 81, 2415-2419. Rebers, P. A,, and Heidelberger, M. (1981). J . Am. Chem. SOC. 83, 3056-3059. Record, B. R., and Wallis, R. G. (1958). Biochem. J. 03, 453-454. Roberts, W. K., Buchanan, J. G., and Baddiley, J. (19e3). Biochem. J . 88, 1-7. Rogers, H. J. ( 1983a). In “The Structure and Function of the Membranes and Surfaces of Cells” (0. J. Bell and J. K. Grant, eds.), pp. 55-105. Cambridge Univ.Press, London and New York. Rogers, H. J. (198313). J. Gen. Microbiol. 32, 19-24. Rogers, H. J., and Garrett, A. J. (1983). Biochem. J. 86, p. 8. Salton, M. R. J. (1953). Biochirn. Biophys. Acta 10, 512-523. Salton, M. R. J., ( 1981). “Microbial Cell Walls.” Wiley, New York. Salton, M. R. J., and Home, R. W. ( 1951). Biochim. Biophys. Acta 7, 177-197. Sanderson, A. R., Juergens, W. G., and Strominger, J. L. (1981). Biochem. B40phys. Res. Commun. 5, 472-478.

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Sanderson, A. R., Strominger, J. L., and Nathenson, S. G. (1982).J. Biol. Chem. 237. 3803-3813. Sargent, L. J., Buchanan, J. G., and Baddiley, J. (1982).J . Chem. SOC. pp. 21842187. Saukkonen, J, J. (1981).Nature 192, 818. Saukkonen, J. J., Lahti, A., and Vainio, T. (1963).Biochem. Biophys. Res. Commun 12, 128-131. Schmidt, W. C. (1952).J . Exptl. Med. 95, 105-118. Shabarova, 2.A., Buchanan, J. G., and Baddiley, J. (1982).Blochim. Biophys. Acta 57, 148-148. Sharpe, M. E. (1955).J . Gen. Mlcrobiol. 12, 107-122. Shaw, C.,Stitt, J. M.,and Cowan, S . T. (1951).J. Gen. Mlcrobiol. 5, 1010-1023. Smith, H., and Zwartouw, H. T. (1954).Biochem. J . 56,viii. Smith, H., and Zwartouw, H. J. (1958).Biochem. J . 63, 447-453. Smith, H., Keppie, J., and Stanley, J. L. (1953a).Bdt. J. Exptl. Puthol. 34,471-478. Smith, H.,Keppie, J., and Stanley, J. L. (1953b).Brit. J . Exptl. Pathol. 34,477-483. Smith, H.,Strange, R. E., and Zwartouw, H. T. (1958).Nature 178, 885-888. Stewart, W. A., Lancefield, R. C., Wilson, A. T., and Swift, H. F. (1944).J. Exptl. Med. 79, 99-114. Strange, R. E., and Belton, F. C. (1954).Brit. J. Exptl. Puthol. 35, 153-185. Strominger, J. L. (1982).Federation Proc. 21, 134-143. Strominger, J. L., and Ghuysen, J, M. (1983).Biochem. Biophys. Res. Commun. 12, 418-424. Thome, C. B. (1958). In “Bacterial Anatomy,” 8th Symp. SOC.Gen. Microbiol. (E.T. C. Spooner and B. A. D. Stocker, eds.), pp. 88-80.Cambridge Univ. Press, London and New York. Tillett, W. S., and Francis, T., Jr. (1930).J . Exptl. Med. 52, 581-571. Tillett, W. S., Goebel, W. F., and Avery, 0. T. (1930).J . Exptl. Med. 52, 895-900. Tomcsik, J. ( 1951).Experknth 7, 459-480. Tomcsik, J, ( 1958).In “Bacterial Anatomy,” 8th Symp. SOC.Gen. Microbiol. (E.T. C. Spooner and B. A. D. Stocker, eds.), pp. 41-87.Cambridge Univ. Press, London and New York. Tomcsik, J., and Grace, J. B. (1955).J. Gen. Microblol. 13, 105-110. Tomcsik, J., and Guex-Holzer, S. (1954a).J. Gen. Microbiol. 10, 317-324. Tomcsik, J., and Guex-Holzer, S. ( 1954b).Schweiz. 2.Allgem. Puthol. Eakterfol. 17, 221-240. Tomcsik, J., and Guex-Holzer, S. (1954~). Experientia 10, 484-485. Tomcsik, J., and Szongott, H. (1932).2. Immunltuetsforsch. 76, 214-234. Tomcsik, J., and Szongott, H. ( 1933).2. Immunltuetsforsch. 78, 88-99. Wicken, A. J., Elliott, S . D., and Baddiley, J. (1983).J . Gen. Mlcrobiol. 31, 231-239. Wieghard, C. W.,and Julianelle, L. A. ( 1935).J. Exptl. Med. 62,23-30. Wilson, A. T. (1945).J. Exptl. Med. 81, 593-598. Wilson, A. T., and Wiley, G. G. (1983).J. Exptl. Med. 118, 527-558. Wong, S. C., and T’ung, T. (1939).Proc. SOC. Exptl. Biol. Med. 42, 824-828. Work, E. ( 1981). J . Gen. Mtcrobiol. 25, 187-189. Yoshida, A., and Heden, C.-G. (1982).J. Immunol. 88, 389-393. Yoshida, A., Mudd, S., and Lenhart, N. A. (1983).1. Immunol. 91, 777-782.

Structure and Biological Activity of Immunoglobulins

.

SYDNEY COHEN AND RODNEY R PORTER

.

Department of Immunology. St Mary’s Hospital Medical School. London. Fngland

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I I1.

111.

Introduction ............................................... Physical Studies ............................................. A . Molecular Weight ...................................... B. Electron Microscope Studies ............................. C . Tertiary Structure ...................................... Chemical Properties ........................................ A . Amino Acid Analysis ................................... B Peptide Patterns ....................................... C. Enzymatic Splitting of Immunoglobulins .................... D. Reduction of Immunoglobulins ........................... E. Stability of the Disulfide Bonds in Rabbit IgG .............. F. Structural Relationships of IgG. IgM. and IgA .............. G. Carbohydrate Content of Immunoglobulins ................. H Possibility of Three Types of Peptide Chains ............... I Position of the Antibody-Combining Site ................... J. Heterogeneity of Immunoglobulins ......................... Biological Properties of Immunoglobulins ...................... A. Antigenic Properties .................................... B. Allotypes of Immunoglobulins ............................ C Urinary Excretion of Immunoglobulin Fragments ............ D. Transfer of Antibodies from Mother to Fetus ............... E . Fixation of Antibody to Skin .............................. F. Complement Fixation ................................... G Distribution and Turnover of Immunoglobulins ............. H. Synthesis of Antibodies ................................. Comments ............................................... References ................................................

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IV.

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V.

287 289 289 290 290 291 291 294 295 297 302 303 30.5 308 309 316 319 319 325 331 332 334 336 338 339 341 342

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1 Introduction

It seems clear that antibody activity is present in three classes of serum proteins . The major component. y2 or 7s y. comprises some 85430% of the total. whereas the second. Y1M. PzM. or 19 S y. has a much higher molecular weight. a higher electrophoretic mobility at pH 8.6, and contains about five times as much carbohydrate as the major component. The third protein in the group. y l A or &A. was not detected until the technique of immunoelectrophoresis was introduced by Grabar and Williams (1953) when it was realized that there was another protein which was antigenically related to 7 S y and 19 S y (Grabar et d.,1956; Heremans et al., 1959) It has been suggested that these proteins be col-

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lectively known as immunoglobulins (Heremans, 1960) and, by analogy with the hemoglobin nomenclature, the 7S, 19s and Y ~ Afractions be referred to as IgG, IgM, and IgA, respectively. Indication of the nature of the constituent peptide chains by a subscript may soon be possible, This terminology will be used throughout this article. The association of antibody activity with IgG and IgM has been recognized for many years (Tiselius and Kabat, 1939; Heidelberger and Pedersen, 1937), but there was some doubt as to whether antibodies were present in IgA. It now seems probable that reagins-the skin-sensitizing antibodies-in human sera are associated with this fraction (Heremans and Vaennan, 1962,; Fireman et al., 1963; Yagi et al., 1963), and Heremans et al. (1963) and Vaerman et al. (1963) showed that IgA prepared from the serum of patients recovering from infection with Brucellu abortus contained antibody activity. IgG and IgM are readily recognized in sera from all species examined, but IgA has only been clearly identified in human serum. However, Schultze (1959) and Heremans (1959) have suggested that the T- or p2-globulin which contains most of the antitoxic activity of serum from a horse strongly immunized with diphtheria or tetanus toxoid (Kekwick and Record, 1941; Van der Scheer et al., 1941) may be the equine equivalent to IgA. This component has a molecular weight of about 150,000, a higher electrophoretic mobility at pH 8.6, and a higher carbohydrate content than IgG from the same serum. Antigenically it is related to IgG and IgM, but is not identical with it and, hence, in all its properties the T-or &!-globulin conforms with IgA. A rather similar protein has been reported present in guinea pig serum after prolonged immunization (Benacerraf et al., 1963; White et d.,1963). No carbohydrate analysis has been made, but from other properties this antibody-containing component also seems likely to be IgA. Rabbit colostrum shows the presence of a component which probably corresponds with IgA, but it was not detected in rabbit serum (Feinstein, 1963). Schwick and Schultze (1961) have drawn attention to the presence, detected by immunoelectrophoresis, of other components in horse antiserum which are antigenically related to IgG. Kunkel and Rockey (1963) have found from ultracentrifuge studies that there are globulins containing anti-red-cell antibodies with sedimentation coefficient &15 S in some human sera and that these may be related to the IgA known to be present. There is, therefore, every prospect of further subdivision of the three main types of immunoglobulin.

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II. Physical Studies

A. MOLECULAR WEIGHT The reported values for the molecular weight of IgG from different species have ranged from 136,000 to 190,000 (Porter, 1960a), but recent measurements favor the lower values. Cammack (1962) estimated the molecular weight of rabbit IgG from measurements of sedimentation rate and diffusion coefficient and found a negative concentration dependence extrapolating to give a value of 137,300. Pain (1963), using similar methods for horse IgG, found no concentration dependence and arrived at a value of 151,000, whereas Marler et aZ. (1964) give 145,000 2 5000 as the molecular weight of rabbit IgG. Small et al. (1963), however, found the molecular weight of rabbit IgG to be 170,000 but this was estimated in solutions containing 6 M guanidine HC1. It was possible that IgG contained a mixture of molecules of differing sizes and that part of the difficulty arose from selection during preparation. However, Pain (1964) could not confirm this. He found that if horse IgG was fractionated on a long Sephadex G-200 column it gave a remarkably symmetrical elution peak except for a barely detectable aggregated component which appeared with the void volume of the column. The molecular weights of the material in the leading and trailing edge of the main peak were the same and both were close to 150,000. Similar results were obtained with rabbit IgG, but the average molecular weight was somewhat lower (140,000), more aggregate was present, and it re-formed when solutions of the main component were allowed to stand for several days at 2°C. It is possible that these small amounts of aggregated material are responsible for the higher molecular weight of IgG reported in earlier papers. Few critical studies have been made of the molecular weight of IgA from normal human serum, as it is very difficult to isolate in appreciable amounts, free of contaminants. Pathological IgA proteins prepared from the serum of patients with myelomatosis may have sedimentation coefficients in the range 7-15 S (Laurell, 1961). Horse IgA (T) has a molecular weight very close to that of IgG (Largier, 1958; Smith and Brown, 1950; Pappenheimer et al., 1940). IgM from horse or human serum has a sedimentation coefficient of 18-20 S and a molecular weight of about 1,OOO,OOO, but smaller amounts of material with a higher sedimentation coefficient are present in most preparations and probably also in the serum from which they were isolated (Kunkel, 1960).

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B. ELECIXON MICROSCOPE STUDIES The shape of antibody molecules has been calculated from hydrodynamic data to be asymmetrical, with a ratio of the long to short axis of 8 or 9 to 1, the dimensions being about 250-300 and 40 A. (Neurath, 1939). Some assumptions have to be made in these calculations and several laboratories have attempted to obtain more direct data from electron microscopy studies. Hall et al. (1959) and Hoglund (1964) used shadow-casting techniques and obtained pictures showing granular moleclues with average dimensions close to those calculated by Neurath. Hoglunds work suggested a dimerlike structure for IgG, whereas IgM appeared as a roughly spherical molecule of diameter about 300 A. Valentine ( 1959), using negative staining, obtained pictures suggesting that rabbit y-globulin molecules were spherical with a diameter of about 70 A., but he now considers that the objects visualized may have been incompletely dissolved particles and that the individual molecules may have been invisible with this technique (Valentine, 1964). Others have photographed antibody bound to tobacco mosaic virus ( Kleczkowski, 1961), human wart virus (Almeida et al., 1963), influenza virus (Lafferty and Oertelis, 1961), and bacterial flagella (Elek et al., 1964). The antibody molecules, in most cases, appeared more elongated than when photographed alone, but the dimensions measured by Almeida et d. (1963) were again close to those calculated by Neurath ( 1939). C. TERTIARY STRUC~URE Jirgensons ( 1958) reported optical rotatory dispersion measurements which suggested that y-globulin contains little or no peptide in a-helical formation. Winkler and Doty (1961) have confirmed this from studies with rabbit y-globulin and the papain digest pieces and have suggested that this may be due to the high content of proline. In an attempt to define the molecular properties of IgG more closely, Edelhoch and collaborators have investigated the changes in a variety of physical parameters brought about by low and high pH and high concentrations of urea or guanidine. The methods used included velocity sedimentation, viscosity, optical rotation, solubility, fluorescence, and ultraviolet spectrophotometry. It was found that, while 8 M urea caused swelling of the molecule, some organization remained and the effect was reversible. Complete disorganization was achieved only in solution of IgG in 8 M urea at pH 11-12 or in solutions of detergent ( trimethyl dodecyl ammonium chloride) at the same pH (Edelhoch et al., 1962; Steiner and Edelhoch, 1962), Similarly, 20 of the 58 tyrosine residues would not

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react with iodine in aqueous solution or in solution in 8.5 M urea at pH 9. If 5.3 M guanidine was used, less than 10 residues were unreactive and there was some evidence that at higher iodine concentration this number could be reduced further (Edelhoch and Schlaff, 1963). Iodination of the easily reactive tyrosine in aqueous solution destroyed the specific affinity of rabbit antithyroglobulin for its antigen as expected from earlier work (Johnson et al., 1960; Grossberg et al., 1962). The unusual stability of IgG in 8 M urea agrees with other observations that, although antibody activity cannot be demonstrated in such a solution, no loss can be demonstrated after the urea has been dialyzed away (Karush, 1958; Nisonoff and Pressman, 1959; Winkler and Doty, 1961). If the antibody is kept in solution in 10 M urea for 5 days, activity is not recovered after dialysis ( Winkler and Doty, 1961), but under such conditions it is possible that reactions, such as that of cyanate with amino groups, occur. More remarkable is the recent demonstration (Buckley et aL, 1963) that after standing in solution of 7.5 M guanidine for 12 hours, papain piece I of rabbit anti-bovine serum albumin will regain its specific affinity for the antigen. Maximum unfolding of the molecule was demonstrated by optical rotation, values being obtained for (m-)365 which were close to those for other completely unfolded molecules. As the authors point out, this ability to regain activity when dialyzed in the absence of antigen is strong evidence that the specific affinity is determined by the covalent structure of the molecule. They argue on statistical grounds that the disuEde bonds present cannot play a major role and, hence, conclude that amino acid sequence must determine antibody specificity. 111. Chemical Properties

A. AMINOACIDANALYSIS Introduction of the automatic amino acid analyzer has led to the publication of accurate analyses of the immunoglobulins from several species, and these are summarized in Table I. The characteristic features that distinguish these figures from those of other proteins are the high values for the hydroxy and dicarboxylic amino acids and, particularly, the proline content which is higher than that of any other globular protein. Analyses of several purified rabbit antibodies have been published (Smith et al., 1955;Fleischer et al., 1961; Askonas et al., 1960), and their amino acid contents were all very close to the figures quoted above for rabbit IgG (Crumpton and Wilkinson, 1963). Since only about 1% of the total molecule may be concerned in the antibody site (Karush, 1962),

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it was not surprising that no convincing differences were found even if amino acid sequence does vary with antibody specificity. However, Koshland and Englberger (1963) have now found small differences between rabbit antibodies to an acid hapten (p-azobenzenearsonic acid) and to a basic hapten (pazophenyltrimethylammonium). The antibodies were prepared by dissociation of a specific precipitate and their purity measTABLE I AMINO ACIDANALYSISOF IMMUNOGLOBULINS Amino acid residue (gm.)/lOOgm.protein Rabbit Human Human Horse Horse Amino acid IgGo 1gCa IgMb IgGo IgA (T)o Lysine 5.76 7.06 4.91 6.77 6.50 Histidine 1.73 2.44 1.98 2.58 2.57 3.02 4.75 3.34 4.02 Arginine 4.42 Aspartic acid 8.08 7.25 7.31 7.77 6.95 Threonine 10.37 8.31 7.13 7.04 6.17 Serine 9.29 8.32 9.13 6.58 9.88 Clutamic acid 11.05 10.27 9.36 11.18 9.92 Proline 6.79 6.40 4.95 6.02 6.52 Glycine 3.68 3.53 3.98 3.37 2.91 Alanine 3.71 3.60 3.28 3.29 3.12 Valine 8.36 5.77 8.14 7.74 7.92 Methionine 1.13 0.78 0.59 0.93 1.02 Isoleucine 3.49 3.14 2.70 2.83 2.16 Leucine 6.73 6.51 6.09 6.63 7.40 5.55 4.93 Tyrosine 6.17 5.44 5.76 Phenylalanine 4.15 3.79 3.47 4.07 3.85 Cystine 2.08 1.90 2.63 2.07 2.30 Tryptophan 2.90 2.63 2.47 2.57 2.47 2.40 2.80 12.30 2.40 4.90 Carbohydrate 101.6 97.4 95.70 97.80 94.80 0 Crumpton and Willrinson (1963). b Chaplin et al. (1965). 0 Weir ( 1964).

-

wed by their hapten binding power. Some preparations were from pooled antisera and some from the serum of a single rabbit immunized with both haptens. In the last case the chances of individual variations influencing the result would be reduced, though not necessarily eliminated, as antibodies may be associated with different allotypic groups in one individual (Cell and Kelus, 1962). Multiple analyses were carried out and careful corrections made for all the experimental variables. The results from analysis of the two antibodies prepared from the serum of a single rabbit are given in Table 11. There is close agreement in the

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

293

number of residues per molecule for each amino acid except aspartic acid and arginine. The differences of +4 aspartic acid and -2 arginine for the antibody to the basic hapten are in the direction expected if there are salt linkages in the hapten-antibody combination, as suggested by Grossberg and Pressman ( 1960). In view of the care with which the work was carried out, these results are of great interest, but the authors are cautious about drawing TABLE I1

AMINOAcm ANALYSISOF Two PURIFIED ANTIBODIESISOLATED FROM THE

Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine 0

SAME RABBIT" Residues/160,000 p. Arsonic Ammonium antibody antibody 69.8 69.4 16.4 16.6 44.7 42.5 106 110 162 162 151 151 125 127 109 110 110 110 81.1 81.4 128 128 13.8 13.5 48.4 46.4 89 91 56.1 56.2 44.3 44.9

Koshland and Englberger (1983).

definite conclusions as to their significance. The difficulty of interpreting the data is shown by the observation that antibodies of a given specificity may have any mobility in the IgG range and yet papain pieces I and I1 have different amino acid compositions related to their mobility (Mandy et UI!., 1963). There is also a variation of the amide content (Feinstein, 1962) and the sialic acid content of whole IgG with charge (Schultze, 1982). Thus, there is a variation in composition unrelated to specificity which is difficult to control. These underlying complexities make it very difficult to get a convincing relation between the properties of the whole molecule and its antibody activity.

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R. PORTER

PEPIlDEPAWS

The demonstration by Ingram (1956) that small differences in amino acid sequence of abnormal human hemoglobins could be detected and identified by the relatively rapid technique of comparing peptide patterns suggested that this method might offer a short cut to deciding whether antibodies of different specificity had the same amino acid sequence or not. With this method the protein is denatured and the disulfide bonds are reduced or oxidized in order to destroy all tertiary structure. An enzymatic digest is prepared (often using trypsin because of the welldefmed specificity) in which all the potentially susceptible bonds are broken. This is difficult to achieve but is essential if reproducible results are to be obtained. The complex mixture of peptides is spread on a sheet of filter paper by electrophoresis in one direction and by chromatography in the other. Such a pattern of peptides can then be revealed by spraying with ninhydrin and other reagents, but Ingram (1961) has stressed the considerable technical difficulties in obtaining satisfactorily clear and reproducible results. With the abnormal hemoglobins it was apparent that, although the majority of the peptides were common to all, one or two were unique to each type. When rabbit antibodies with different specificities were examined, no clear-cut differences could be distinguished (Gitlin and Merler, 1961; Gourvitch et al., 1961b). Reasonably clear patterns were obtained from a tryptic digest of oxidized papain pieces from inert rabbit y-globulin (Seijen and Gruber, 1963) and the number of peptides detected in piece I11 was half that expected, suggesting a duplicate structure. Givol and Sela (1964a) studied digests of reduced rabbit antibodies using several enzymes and reported that after tryptic and chymotryptic digestion of papain pieces I and 11, insoluble, large molecular weight material remained. Pepsin and nagarse gave more complete digestion, and the resulting peptide maps from the digest of two different antibodies were very similar but with differences detectable in a few peptides. Peptide maps of piece I11 were clearer and appeared to be identical whether derived from inert y-globulin or antibody. The most surprising finding to be made with this method has come from a comparison of the peptide patterns of Bence-Jones proteins of different antigenic types. These urinary proteins are excreted by some patients with myelomatosis and appear to correspond with the smaller peptide chain of the abnormal immunoglobulins in the serum of the same patients (Edelman and Gally, 1962). Putnam (1962) also suggested that Bence-Jones protein was a peptide chain of myeloma globulin, on the grounds that there were tryptic peptides common to both. The Bence-

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295

Jones proteins occur in two antigenic types, and Putnam et al. (1963) found that the peptide patterns of these two types contained very few common peptides, with the implication that they had a different amino acid sequence. Schwartz and Edelman ( 1963) isolated the corresponding B ( L ) chains from myeloma proteins of different antigenic types and found, as expected, the same difference in peptide pattern. The normal human immunoglobulins also occur in these two antigenic types in the ratio of about 2:1, and, if the B chains from these also show the same big digerences, then any preparation of normal human y-globulin will contain equivalent peptide chains with very different sequences. This work has been carried out so far only with pathological human immunoglobulins, but it is possible that a similar complexity occurs in normal immunoglobulin from humans, rabbits, and other species. This might vitiate attempts to relate amino acid sequence to antibody specificity. Such evidence as is available suggests, in agreement with other data, that all antibodies from the same species have a very similar structure, but the position seems to be too complex for any definite conclusion to be drawn about the sequence of amino acids at the combining site. OF IMMUNOGLOBULINS C. ENZYMATIC SPLITTING The size and heterogeneity of immunoglobulins made it unlikely that correlation of structure and function could be achieved from studies of the whole molecule. As it was known from much earlier work that the specific affinity for antigens survived some enzymatic digestion, this method of reducing the size without loss of activity has been studied in some detail. The results of enzymatic digestion of IgG have been reviewed recently (Porter and Press, 1962) and may be summarized briefly as follows. IgG from all species examined is split by papain into approximately thirds with very little production of small peptides. Two of these pieces contain an antibody site and are identical, their range of electrophoretic mobility being related to the IgG from which they were prepared (Palmer et d., 1962). From the rabbit IgG these pieces are known as I, if of higher mobility at neutral pH, and 11, if of lower mobility. From IgG of human and other serum these pieces are known as S (Edelman et al., 19eO), or A (high mobility) and C (low mobility) (Franklin, 1960). The third piece, which contains no antibody site and is of quite different character in all properties, can be crystallized easily from rabbbit IgG and from guinea pig IgG and, with difficulty, if prepared from human IgG (Hershgold et al., 1963). In the rabbit this piece is named I11 and in most other species F or B.

296

SYDNEY COHEN AND RODNEY R. PORTER

If, instead of papain, pepsin at pH 5 is used to digest IgG, then one piece of molecular weight approximately 100,000 is obtained, together with smaller peptides. The large fragment, usually referred to as the 5 s fragment, will precipitate with antigerl and can be split into two equal halves by reduction with thiol (Nisonoff et d.,1960). Each half possesses one antibody-combining site and is very similar to papain pieces I and 11. (During peptic digestion the part of the molecule equivalent to I11 is degraded to smaller peptides.) It follows that the two pieces I or I1 obtained by papain digestion were also probably linked by a disulfide bond which is split by the 0.01 M cysteine added to the digestion medium to activate the enzyme. That this was so, was proved by Cebra et al. (1961) who used papain made insoluble by coupling to an amino acid polymer. If this enzyme was activated, washed free of thiol, and used to digest rabbit IgG, 3-4 peptide bonds were split, but there was no reduction of molecular weight and no loss of precipitating power if antibody was used. If cysteine was now added, in the absence of papain, the molecule broke to give three pieces, identical with those prepared with soluble papain in the presence of cysteine. The presumption is that the two pieces, I and 11, are held together by a disulfide bond and that I11 is held to both by noncovalent bonds that break when the other two pieces separate. Putnam et al. ( 1962) examined the action of a variety of other enzymes on human IgG; generally, cysteine was necessary to get high yields of 3.5s components. Rather surprisingly, trypsin was remarkably effective in the presence of only 0.001 M cysteine. Both Skvafil (1960) and Hanson and Johansson (1962) reported considerable degradation of human IgG by trypsin in the absence of cysteine, though no sedimentation studies of the products were made. Schrohenloher (1963) has found that trypsin will split human IgG to give about 50% 3.5 S pieces, the remainder being apparently undigested 7 S globulin. Addition of 0.01M cysteine raised the amount of 3.5S products to about 60%. This would seem to suggest that trypsin can digest half of a preparation of human IgG to 3.5s products without any reducing agent being present and that, even in the presence of cysteine, most of the other material is resistant to digestion. These results are not easily explicable in terms of the rather simple structure for rabbit IgG of two antibody site containing pieces held together by a disulfide bond and attached to the third piece by peptide and noncovalent bonds. There may be a species difference in the placing of the disulfide bonds and possibly differences in individual molecules in this respect. Attempts to split pieces I or I1 further by enzymatic digestion without loss of the power to combine with antigen were unsuccessful (Porter, 1960b), but

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

297

Kulberg and Tarkhanova (1962) reported that this can be achieved using papain in 6 M urea. The molecular weight of the active fragment was given as 13,000 k 1800. Investigations of the biological activities of the papain digest pieces are discussed later, but perhaps the most surprising result is that all the activities investigated survive this drastic splitting of the molecule into thirds. This suggests that the original molecule may have a tripartite structure and that the enzyme is acting at one or two limited sites without affecting the steric structure of the three main sections of the molecule. Support for this idea is given by the observation of Goodman and Gross (1963) that no new antigenic sites are exposed by papain digestion.

D. REDUCTIONOF IMMUNOGLOBULINS Edelman ( 1959) and Edelman and Poulik (1961) showed that human and rabbit IgG could be split into smaller components by reduction in urea solution and, hence, demonstrated that both proteins were probably composed of several polypeptide chains. Equine (Fran&k, 1961) and bovine (Ramel et al., 1961) IgG behaved similarly. These results proved that the IgG of several species had a similar number of peptide chains, contrary to results with N-terminal amino acid assay where in several species the total gave less than one molecule of N-terminal amino acid per molecule of I@. It is clear that several N-terminal acids must be substituted on the amino group or be unreactive for other reasons. Cterminal amino acids, however, may be free, as a total of 4 moles ( 2 glycine, 1 serine, and 0.5 threonine and 0.5 alanine) per mole of rabbit IgG was estimated (Silman et al., 1962). The method used (hydrazinolysis) does, however, require correction for loss which exceeds 50%. Difficulties were met in the fractionation of the products of reduction in urea, as they were insoluble except in urea solution, and the recoveries from chromatographic columns were poor. Since all biological activity was lost, Fleischman et al. (1962; cf. Porter, 1962) re-examined the conditions of reduction in the absence of denaturing agent and found, as previously reported (Porter, 1950), that, although several disulfide bonds were split, there was no apparent change in molecular weight nor, if antibody was used, in the power to precipitate with the antigen. If, after reduction, the ten sulfhydryl groups liberated were reacted with iodoacetamide and the reduction mixture dialyzed against 1 N acetic or 1 N propionic acid, dissociation into two components resulted and these could be separated either by zone electrophoresis or, more simply, on a Sephadex column (Fig. 1). The larger component was named A and the smaller B (Porter, 1962). When examined on starch-gel electrophoresis in 8 M urea

298

SYDNEY COHEN AND RODNEY R. PORTER

and pH 3.5 (Fig. 2), conditions used by Edelman and Poulik (1961), it was apparent that these components were equivalent to those obtained by more drastic reduction and named H and L, respectively, by Edelman and Benacerraf ( 1962). It seemed likely that these two components were the peptide chains of IgG, and an investigation of their properties led to the postulation of a diagrammatic structure for the molecule (Fig.

0.6

04

0.2

0

FIG. 1. Fractionation of reduced human IgG on a column (2.5 Sephadex G-75 in 1 N acetic acid (Cohen, 198313).

x

70 cm. ) of

3) (Porter, 1962). More detailed studies have given support to this structure, and the evidence from rabbit IgG (Fleischman et al., 1963; Crumpton and Wilkinson, 1963; Pain, 1963) and human IgG (Cohen, 1963b) may be summarized as follows 1. Complete reduction of all the disulfide bonds of either A or B in 6 M guanidine caused no further reduction in molecular weight. Conditions expected to split esterlike bonds also had no effect, and, hence, it is probable that IgG consists of only two types of peptide chains, unless either A or B is two chains held by an unknown type of linkage. 2. The molecular weights (A, 50,000 and By 20,000) and yields

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

299

(70-75% A and 25-30% B ) of these chains are consistent with a fourchain structure and agree with the weight of the whole molecule (140,OOO-150,000). Small et al. ( 1963) reduced rabbit IgG in 6 M guanidine and separated the A and B chains in 6 M guanidine solution and got

FIG.2. Electrophoresis in 8 M urea, formic acid, starch gel of Iiuman JgC (U), reduced IgC ( R ) , A chain ( A ) , and B chain ( B ) (Cohen, 1963a).

300

SYDNEY COHEN AND RODNEY

R. PORTER

higher yields (about 33%) of B. They estimated the molecular weights to be 50,000 for A and 25,000 for B, in agreement with the higher yield of B. Marler et al. (1964) determined the molecular weights of y-globulin reduced in 6 M guanidine and also found values of 50,000 and 25,000 for A and B, close to the values of Small et al. 3. The amino acids of the whole molecule are accounted for by the amino acids present in two A and two B chains. Papain-digestion piece I or II

Papain-dlgertion piece 111 Site of papain digestion

J-

:i; 7 B Chain

I

I

s

I

I

I

I

t

I

I

S

I s

C

I I

I

I S I 5

Chain A

I

5

I

Carbohydrate

5

I

I 5 II

Chain A

I I I

FIG.3. Diagrammatic structure of rabbit IgC (Porter, 1962).

The carbohydrate content of the rabbit IgG A chain equals that of the whole molecule, although there appear to be traces on the B chain. The N-terminal amino acids of rabbit IgG are found almost entirely on the B chain-the small amounts on the A probably arise from contamination with B. In human IgG the N-terminal amino acids are present in both chains. 4. There are four S-carboxymethylcysteine residues per mole of A chain and one per mole of B chain. No dissociation occurs without reduction and it is, therefore, likely that B is held to A by one disulfide bond and, if it is assumed that only interchain bonds have broken, A to A by three disulfide bonds.

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

301

The relation of the peptide chains to the pieces produced by papain was determined simply by testing the reaction of A and B with goat antiserum to rabbit pieces I and 111. Chain A reacted with both antisera and B reacted only with anti4 and, hence, I contained B and part of A, whereas I11 contained only A (Fleischman et ul., 1962). From the known molecular weights of the pieces, the papain must hydrolyze the A chain approximately at the midpoint as shown in Fig. 3. It follows that, if this is true, the molecular weight of 111 should halve on reduction, and this appears to be true (Marler et ul., 1964). Pieces I or 11, on reduction and alkylation, should give B chains, as obtained from the whole molecule, together with the N-terminal half of A named A piece. This has been confirmed, and B from whole IgC and papain piece have been shown to be identical in size, amino acid analysis, antigenic specificity, N-terminal amino acids, and carbohydrate cbntent. The A piece was separated from B by its ability to form a dimer under acid conditions. It differed in amino acid content, antigenic specificity, and in having very little Nterminal amino acid, as does the whole A chain. There were two S-carboxymethylcysteine residues per molecule of A piece, in agreement with the suggested position of the disul6de bonds, it being assumed that one links A piece and B and the other A piece to A piece. The investigation of the antigenic relation of the papain piece to the peptide chain was repeated using human IgG and rabbit antisera (Olins and Edelman, 1962; Cohen, 1963a,b). The results with rabbit IgG were confirmed in that anti-F reacted with A and anti8 with B and, similarly, a n t i d reacted with F and anti-B with S. This suggests that F, as 111, contains A chain, whereas S, as I or 11, contains B, but neither laboratory showed a reaction between anti-S and A or between anti-A and S. There is thus no direct evidence that S contains A piece as well as B, although if the structure shown is correct for human as well as rabbit I@, it must be present. Clearly, investigation and isolation of the components of the reduced S pieces will be necessary. Another observation that does not fit the proposed structure was made by Cebra (1964). He found that if rabbit IgG was digested by insoluble papain in the absence of cysteine, there was no change of molecular weight, although 3 4 peptide bonds were split. If sodium dodecyl sulfate was now added, I11 could be dissociated leaving I and I1 joined by a disuEde bond. However, the release of I11 was slow and could be inhibited by thiol reagents, such as N-ethyl maleimide, suggesting that disuMde interchange was necessary. This finding might be reconciled with the proposed structure if it is assumed that insoluble papain has split a peptide bond within an intrachain disulfide bond of the A chain

302

SYDNEY COHEN AND RODNEY R. PORTER

and that with soluble papain this whole sequence is hydrolyzed. Comparative analysis of the products of the two methods of hydrolysis should clarify this discrepancy.

E. STABILITYOF THE DISULFIDE BONDSIN RABBIT IgG The evidence obtained so far is most easily explained on the assumption that the interchain disulfide bonds are much more labile than the intrachain bonds, in agreement with the observation of Cecil and Wake (1962) on a number of other proteins. There are also, however, differences in stability within the two types of bonds. Mandy and Nisonoff (1983) showed that the 5 S pepsin fragment is split into halves by standing in 0.008 M 2-mercaptoethylamine for 1 hour at 37"C., pH 5, with the reduction of 1 disulfide bond, but that under more drastic conditions 5 more disulfide bonds were split without any further change in molecular weight at neutrality. The labile bond is presumably that shown between A piece and A piece (Fig. 3).When reoxidation is allowed to take place, even after breaking 6 disulfide bonds, only a 5 S product results. The other SH groups must be held close to each other so that the original disulfide bonds are re-formed; random formation of disulfide bonds would lead to aggregation. Palmer and Nisonoff (1963) showed that this disulfide bond between the N-terminal sections of A chains is equally labile in the whole molecule, since gentle reduction followed by pepsin digestion also gives 3.5 S fragments. If gentle reduction in 0.03 M mercaptoethanol was followed, not by peptic digestion, but only by acidification, the sedimentation value at pH 2.5 fell to 3.5s and was believed to be due to the formation of half-molecules. Under these conditions, 2-3 disulfide bonds were split with very little release of B chains, and it seems likely that the disulfide bonds between A chains are more labile than those between A and B chains. Further work (Palmer and Nisonoff, 1964) has now shown that when whole rabbit y-globulin is reduced at pH 5 in 0.015M mercaptoethylanime, about two-thirds of the molecule is split into halves and can be separated from undissociated material on a Sephadex G-200 column. If the halfmolecules are reacted with iodoacetamide, analysis shows that there is one S-carboxymethylcysteine and, therefore, one SH group per 75,000. This is strong evidence that in at least some of the molecules only 1 disulfide bond holds the A chains together, not 3, as shown in Fig. 3. This suggests that the 2 interchain disulfide bonds in the C-terminal end of the A chain may, in fact, be intrachain, though, if true, it is not clear why piece I11 cannot be split into halves without prior reduction (Marler et al., 1964). Clearly, it is too early to draw firm conclusions as to the

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

303

position of the interchain disulfides. These discrepancies in the apparent number of interchain disul6de bonds may result from the occurrence of disulfide interchange during fractionation, or may reflect the heterogeneity of any preparation in this respect, as well as many others. When horse IgA (T) is examined (Weir, 1964), quite a different situation is found. Papain digestion in the presence of 0.01 M cysteine gives a divalent 5 s antibody together with smaller peptides. The 5 S antibody can be split into halves but 0.2 M mercaptoethanol is necessary to break this disulfide bond completely. Since horse IgG gives (like rabbit IgG) only 3.5 S pieces on papain digestion, it is clear that the differences in structure between these two immunoglobulins from the same species include the interchain disulfide bonds. In horse IgA present evidence suggests that there are 2 disulfide bonds between the A piece, rather than 1, as in IgG, and this may explain their greater resistance to reduction. It should be stressed that in all the discussion of the position of the disulfide bonds it is assumed that the thiol groups formed on reduction come only from disulfide bonds. Though probable, this is not necessarily correct and, indeed, Karush et d.(1964) have developed a novel technique for the assay of thiol groups in proteins which gives nearly 25% higher values in rabbit IgG than those expected from the cystine content estimated after oxidation (Smith et al., 1955; Crumpton and Wilkinson, 1963), or after reduction (Koshland and Englberger, 1963). As this discrepancy between the different methods is not found in other proteins, it is of considerable interest and it may reflect some peculiar structural features which have not yet been recognized.

F. STRUCTURAL RELATIONSHIPS OF IgG, IgM, AND IgA In the human immunoglobulins, IgG, IgM, and IgA all across react with antisera to any one (see p. 319), but much of the antibody is specific to the homologous globulin. This suggests that parts of the structure are common to all three types and parts are distinct. In agreement with this, one allotypic antigenic marker of the human immunoglobulins, InV, is common to all three types, but the other, Gm, is unique to IgG. It would be expected that one chain, carrying the InV marker, was common to all, and the other, carrying the Gm in IgG, was not (Edelman and Benacerraf, 1962). As the InV factor is also found on papain piece S and the Gm on F, the presence of InV on the B chain and Gm on the A chain was the likely distribution, and this was demonstrated by Lawler and Cohen (1965) who found a clean separation of the two antigenic markers in the two chains. Cohen (1963a,b) also showed that the common B and distinct A chains were apparent when the reduced immunoglobulins were

304

SYDNEY COHEN AND RODNEY R. PORTER

electrophoresed in starch gel in 8 M urea at pH 3.5 (Fig. 4) (see also Carbonara and Heremans, 1963). The B chains of the three types have the same mobility, whereas the A chains have different mobilities. The identity of the B chains is more striking when electrophoresed at alkaline pH

FIG.4. Electrophoresis in 8 M urea, formic acid, starch gel of A chain ( 1 ) and B chain ( 2 ) of normal IgC, of A chain ( 3 ) and B chain ( 4 ) of normal IgM, and of whole, reduced IgC ( 5 ) (Cohen, 1963b).

when the same complex pattern is obtained from the B chain of either IgG or IgM (Cohen and Porter, 1964). Amino acid analysis of the separated chains of human IgG and IgM also shows near identity for the B chains and an obvious difference in the A chains (see Tables 111 and

305

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

N )(Chaplin et al., 1964). Thus it is clear that human immunoglobulins carry a common B chain and a distinct A chain. Similarly, the B chains of horse IgG and IgA ( T ) are identical in all chemical and physical properties, whereas the A chains show differences (Weir, 1964). TABLE 111 AMINO ACID COMPOSITION OF A CHAINS Residues/50,000 gm.

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Half-cystine S-Carboxymethylcysteine Tryptophan Hexose Hexosamine

Rabbit IgG 23 7 16 33 49 49 40 36 33 24 41 5 15 30 17 14 10

Human IgG 29 10 12 33 34 50 39 33 28 19 41 4 8 31 17 13 7

4 8 4.5 4

4 8 4 4.5

Human IgM 20 7 18 34 38 42 40 29 28 26 36 5 13 30 12 16 7 3(74) 7 20 12

Horse IgG 26 10 10 30 37 45 38 31 28 22 46 4 13 28 16 13 3 3(74) 8

5 4

Horse IgA(T) 27 12 10 34 33 46 38 38 28 22 45 3 11 31 16 12 6 4 7 10 7

Rabbit IgG carries allotypic antigenic markers controlled by two independent loci (Oudin, 1956), again suggesting that two peptide chains are present, but both sets of antigenic specificities are present on IgG and IgM (Todd, 1963). The b locus determines the specificity of the B chain and identity between IgG and IgM was expected here. The fact that the A chains of IgG and IgM also carry the same allotypic specificity suggests a partial identity of sequence which could arise if, as discussed below, A was in fact two peptide chains. G. CARBOHYDRATE CONTENT OF IMMUNOGLOBULINS The immunoglobulins of all types and all species contain carbohydrate. It seems unlikely that it plays any part in the antibody-combining

306

SYDNEY COHEN AND RODNEY R. PORTER

site, as papain pieces I and I1 can be obtained almost free of carbohydrate, yet retaining their power to combine specifically with antigen. The carbohydrate may well be an important factor in other biological activities and, clearly, is an important structural feature of, for example, the TABLE IV AMINOAcm COMPOSITION OF B CHAINS Residues/20,000 gm. Rabbit IgG Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Pheny1a1anine Half-cystine S-Carboxymethylcysteine Tryptophan Hexose Hexosamine

8 2

3 16 25

19 18 10 15 13 18 0.9 6 11 10 5 6

1 4

0.3 0.2

Human IgG

Human IgM

Horse IgG

10 3 6 13 15

10

10

2

2

5 13 15

4 12 17

24 19

28

24 20 11

11 12 13 0.6 5 13 8 6 3 1 2 0.25 0.2

11 12 12 13 0.7 5 13 8

16 11 16 12 14 0.8 10 11 0

5

4

3

3

1 3

1 3 0.4 0.1

0

0.3

Horse IgA(T) 10 2

4 13 19 32 16 12 18 13 14 0.4 7

12 6 4 3

1 3 0.6 0.1

A chain of human IgM where it comprises some 15% of the total weight. Analytical procedures for the estimation of carbohydrate are not yet as satisfactory as those used for amino acids, but the data available for the three immunoglobulins from different species are summarized in Table V. In all cases examined sqfar (human IgG and IgM, rabbit IgG, and horse IgG and IgA) the carbohydrate is almost entirely confined to the A chains. The traces present in the B chains may be due to contamination by A, but they are remarkably difficult to remove and might be an integral part of a small percentage of the B chain molecules. When the carbohydrate content of the enzymatic pieces is estimated, the position is not so uniform. In rabbit papain pieces, about two-thirds of the carbo-

307

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

hydrate is firmly bound to piece I11 and the remainder is present in a glycopeptide which adsorbs onto piece I but which can be digsociated by acid (Fleischman et all, 1983). It seems likely that a similar distribution of the carbohydrate occurs in human IgG (Dische and Franklin, 1964) and horse IgG (Weir, 1964). With horse IgA (T) one-third is bound to the nonantibody-containing part equivalent to piece I11 and two-thirds to the equivalent of pieces I or 11, and none of this material can be dissociated from the protein by acid (Weir, 1964). Schultze ( 1963) who used peptic digestion also found that two-thirds of the carbohydrate of horse IgA ( T ) was in the 5 S fragment which, by analogy with rabbit, is TABLE V CARBOHYDRATE COMPOSITION OF IMMUNOGLOBULINS ( %) Carbohydrate Hexose Fucose Hexosamine Sialic acid

b 0

d f

Rabbit IgcD

1.2 1.0 0.2 2.4

Human

Horse

IgGb

IgMC

IgAd

1.2 0.3 1.1 0.2 2.8

6.2 0.7 3.3 2.0 12.2

4.8 0.2 3.8 1.7 10.5

IgCe 1.1

IgA(T)e

1.1 0.2 2.4

1.9 0.9 4.9

2.1

Bovine Igcl

0.9 0.2 1.5 0.3 2.9

Fleischman et al. ( 1963). Miiller-Eberhard et al. (1956). Miiller-Eberhard and Kunkel ( 1959). Heremans ( 1959 ) . Schultze ( 1959). Nolan and Smith (1962a).

equivalent to pieces I and 11. It is clear, therefore, that in IgA there are two distinct fractions of the carbohydrate attached to the A chain. In IgG there is probably only one carbohydrate fraction. Smith and collaborators (Rosevear and Smith, 1961; Nolan and Smith, 1962a,b) have isolated the glycopeptides obtained from a papain digest of heatdenatured IgG from human, bovine, and rabbit serum and concluded that there is only one carbohydrate moiety which is bound to an aspartic acid residue present in the following sequence: Human: Asp NH, Tyr Glu Asp Carbohydrate Bovine: Glu Glu NH, Phe Asp Carbohydrate Rabbit: Glu NH, Glu NH, Phe Asp Carbohydrate

This is not easy to reconcile with the presence of carbohydrate in both a glycopeptide and piece I11 from rabbit and human IgG. In both cases analysis shows significant differences between the two portions, but it is possible that these conflicting results could arise from the heterogeneity of molecules which might be such that different molecules contain

308

SYDNEY COHEN AND RODNEY R. PORTER

slightly different carbohydrate moieties and were digested by papain in different ways. A fuller discussion of glycoprotein aspects of immunoglobulin structure is given by Press and Porter (1964).

H. POSSIBILITY OF THREE TYPES OF PEPTIDECHAINS The evidence above suggests that there are two pairs of peptide chains in all the immunoglobulins, but some biological evidence would be more easily explained if there were three pairs of chains. First, it is claimed that the antibody site is on the A piece and it is presumably similar in IgG, IgM, and IgA, yet the A chains of the three

s

I

S

s

I S

S

s

S

S

I

I

FIG.5. Possible diagrammatic structure of a six-chain IgG molecule.

types differ. If A were two chains, A and C, as shown in Fig. 5, then both A and B might be common to the three types and only C different. However, the position of the antibody site is still in dispute and, further, the stability of antibody acitvity to gentle reduction digers in IgG and IgM (Jacot-Guillarmod and Isliker, 1962). The latter suggests that the antibody sites of these two types of globulin may not be formed on identical structures. Second, when the allotypic antigenic specificities of the different fractions of rabbit IgG were examined, it was found that A l , A2, and A3, controlled by the a locus, were on the A chain, and A4, A5, and A6, controlled by the b locus, were on the B chain (Stemke, 1964; see also Feinstein et al., 1963). Both antigenic sites are on papain pieces I or I1 and, hence, the a locus must be on the gene controlling the synthesis of

STRUClWRE AND ACTMTY OF IMMUNOGLOBULINS

309

the A piece. Todd (1963) has now found that the a locus phenotypes are present on rabbit IgM as well as I@, and Feinstein (1963) has made a similar finding with what is believed to be rabbit IgA. It, therefore, seems likely that part of A piece is common to IgG, IgA, and IgM, although the whole A chain differs. Again, if only a .C chain differed, this would fit well. Third, there may be three independent loci involved in the synthesis of human IgG: InV, associated with the B chain, Gm, associated with the A chain, and a third sex-linked locus which appears to be concerned in some cases of agammaglobulinemia. Similarly, in rabbit IgG there has been a report of a third locus, P, which may be independent of a and b (Dray et al., 1963b). Although other explanations are possible this evidence could be used as support for a six-chain structure. None of these arguments can be considered conclusive at present, but taken together, suggest that the immunoglobulins may consist of three, rather than two, pairs of peptide chains and that A is really two chains. However, there is good evidence that there is no disulfide interchain bond present within a single A chain and some evidence that there is no ester interchain bond. Further, it would follow that there must be two blocked N-terminal amino acids on rabbit IgG A chain and, so far, none have been identified. Hence, there is at present no chemical evidence in support of a six-chain structure.

I. POSITION OF THE ANTIBODY-COMBINING SITE Several attempts have been reported to place the position of the antibody-combining site in terms of the peptide chains of IgG. Conflicting results have been obtained but, from examination of the papain pieces, it is certain that the combining site is on piece I or piece I1 and, hence, must be on the B chain, the A piece, or be formed jointly by these chains. Edelman et al. (1961, 1963a) found that if guinea pig antibodies prepared by dissociation of specific precipitates were reduced and a b l a t e d and then electrophoresed in starch gel in 8 M urea at pH 3.5, the A chain gave a relatively compact band identical with that from reduced inert y-globulin. The B chain from reduced antibody globulins, however, gave a series of sharp lines, in contrast to the broad smudge shown by reduced inert IgG. Several antihapten and antiprotein antibodies were examined and a correlation was found between the pattern of the banding and the specificity of the antibody used. If guinea pig antidinitrophenyl protein antibodies were fractionated by dissociation of the specific precipitate with dinitrophenol, followed by dinitrophenyl-

310

SYDNEY COHEN AND RODNEY R. PORTER

lysine, two fractions were obtained specific for either one or the other hapten (Fig. 6 ) . It could be shown that some bands were present in the B chain of one, but not of the other, again giving a correlation between the type of banding, under these conditions of electrophoresis, and the specificity of the antibody. The papain fraction S containing the antibody activity was also reduced and alkylated and this showed a series of rather indistinct bands overlapping but slightly faster than the B bands of reduced whole antibody. This correlation between electrophoretic patterns of the reduced B chain with antibody specificity suggested that the B chain contains or contributes to the antibody-combining site. This phenomenon is not observed with rabbit or horse antibodies where the B chains from antibody or inert y-globulin are equally diffuse when electrophoresed under the same conditions (Fleischman et al., 1963). Further, if the B chain from inert IgG of any species is electrophoresed at neutral pH and 8 M urea in starch gel, than all give some ten widely separated bands (Cohen, 1963~;Cohen and Porter, 1964). If B chains from guinea pig antibodies were used, there was a difference in relative intensity of staining of some of the bands, but all were present. As discussed later, there is slight evidence that the bands formed under these conditions are related to the cell type making the IgG molecules. If true, this would imply that different cell types were contributing unequally to the different kinds of antibody, but that there was unlikely to be any direct relation of B chain pattern to the antibody specificity under these conditions of electrophoresis. When a direct attempt to measure antibody activity in the separated chains was made (Fleischman et a,?., 1963), different results were obtained. If reduction in the absence of urea was used, about 20% of the activity survived with rabbit antiprotein antibody as judged by specific coprecipitation. If horse antirabbit IgG was used, about 70% activity was recovered, again measured by coprecipitation, and the same recovery was found with horse antidiphtheria toxoid, but with this flocculating system, measurement was by specific inhibition of precipitation. With these methods, the activity was found to be entirely on the A chain, absent from the B chain, and not significantly increased by addition of B to A. The conclusion was drawn, therefore, that the antibody site, at least in horse antibodies, was situated in the A chain. It presumably was, therefore, in the A piece and, in fact, activity could be demonstrated in this fraction (Weir, 1964). Utsumi and Karush (1963) used rabbit antihapten antibody reduced in aqueous solution and separated the A and B chains on a Sephadex

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

311

FIG. 6. Specifically isolated fractions of dinitrophenyl (DNP) guinea pig albumin ( GPA) antibodies and picryl (Pic) guinea pig albumin antibodies compared after dissociation, reduction, and electrophoresis in starch gel containing 8 M urea and formic acid, pH 3. 1. DNP-lysine fraction, DNP-GPA antibodies, animal 8; 2. DNPOH fraction, 3. DNP-lysine fraction, DNP-GPA antibodies, DNP-GPA antibodies, animal 8; animal 1-3; 4. DNPOH fraction, DNP-GPA antibodies, animal 1-3; 5. DNPlysine fraction, Pic-GPA antibodies, animal 2-1; 6. DNPOH fraction, Pic-GPA antibodies, animal 2-1 (Edelman et al., 1963a).

312

SYDNEY COHEN AND RODNEY R. PORTER

column in 0.03M sodium decyl sulfate. Under these conditions they found a much higher recovery of the activity (measured by equilibrium dialysis) than had been found using 1 N propionic acid as dissociating agent, and it was again entirely in the A chain (Fig. 7).

? 2 x

8-

732

7-

- 28

6-

- 24

5-

.- 20

+

1'

0

A-PLAC K ~ 2.1 . x to4

4-

-16 x

KA. 18 x lo4

u

\ L

V

\

-0

3-

-12

2

k-pLA - 8

+ \O

I- 4

0

0.2

1

I

0.4

0.6

I

0.8

1.0

1.2

\ 1.4

4

1

1.6

I

1.8

2.0

FIG. 7. Binding curves obtained by equilibrium dialysis at 25°C. of Lac dye

[p-( p-dimethylaminobenzeneazo)phenyl-f3-lactoside]with mildly reduced and alkylated rabbit anti-Lac antibody ( MRA-Plac) and component A ( A-Plac) derived from it. The haptenic group (Lac) is p-azophenyl-8-lactoside. The plots are r/c versus r, where r is the average number of dye molecules bound per antibody molecule at the free equilibrium dye concentration c. The unitary free energy (AF,) for the binding of Lac dye by A-Plac is - 8 . 2 8 kcal per mole of hapten compared to -9.55 kcal for MRA-Plac. No binding was shown by component B (+) from anti-Lac nor by component A (A) prepared from normal rabbit y-globulin. ( Utsumi and Karush, 1964).

Cebra et al. (1963) also used detergent but the reduction with mercaptoethanol was carried out in the sodium dodecyl sulfate and, hence, more disulfide bonds would be split (Karush, 1957). When rabbit antilysozyme or rabbit antibacteriophage antibody was reduced under these conditions, there was little or no loss of antibody activity, even though the drop in sedimentation coefficient showed that dissociation had

STRUCTURE AND A.(;TMTY OF IMMUNOGLOBULINS

313

occurred. On removal of the detergent with ion-exchange resin, 10-2070 of the reduced antibody remains in solution, the molecular weight is of the order of 50,000, and the activity, measured as inhibition of enzyme, is close to that of native antibody (Cebra et al., 1963). There is some doubt as to the nature of this material but the most recent evidence suggests that it consists of B chain and probably also material equivalent to A piece (Jaquet et al., 1964). Frandk and Nezlin (1963) separated the peptide chains of horse antibodies to diphtheria and tetanus toxoid after splitting the disulfide bonds with sodium sulfite and cupric nitrate to give S sulfo derivatives and fractionation on Sephadex G-100 in 0.05 M formic a c i d 4 M urea. Measurement of activity was made by estimation of the proteins bound to toxoid coupled to cellulose (Gourvitch et al., 1961a), and it was found that under these conditions of reduction and isolation 9%97% of the antibody activity was lost. There appeared to be some activity in the A chains which was increased by addition of the nonspecific B chain and more by addition of the specific B chain. It was concluded that the combining site was on the A chain, but that the B chain was necessary for full activity. In view of the very low recovery of activity, some uncertainty remains as to the significance of these results. Edelman and colleagues (1963b) had similar results using guinea pig antibodies to two bacteriophages and a hapten. In this work the A and B chains were separated according to Fleischman et al. (1962), and there was a heavy loss (80-95%) of activity with the phage antibodies and less ( 5 0 % ) loss with antidinitrophenol antibodies. What activity survived was in the A chain, but it was increased from 1.5- to 10-fold by the addition of the B chain. An indirect approach is to label part of the antibody molecule which is concerned in the combination with antigen, prepare the peptide chains, and find which contains the label. This has been done in two ways. Pressman and Roholt (1961) and Roholt et d. (1963) iodinated a rabbit antihapten antibody with I1sl in the presence of the hapten, and with 1125 in the absence of the hapten. The two preparations were mixed and digested with pepsin, the peptides separated by high-voltage electrophoresis on paper, and the relative labeling with the two isotopes measured. Most of the peptides had the same relative labeling as expected, but three fractions showed preferential labeling with 1126, suggesting that the tyrosyl residues were partly protected from reaction with Ilsl when hapten was bound to antibody. When this was repeated, but the A and B chains separated (according to Fleischman et al., 1962) before digestion, then the unequally labeled peptides were entirely in

314

SYDNEY COHEN AND RODNEY R. PORTER

the B chain, suggesting that these parts of the B chain are closely associated with the combining site or, possibly, are protected in some less direct manner. The second possibility is suggested by the observation that the binding of 2 moles of fatty acid to 1 mole of serum albumin alters considerably the susceptibility of the tyrosine residues to iodination, though direct protection is impossible on steric grounds (Glazer and Sanger, 1963). In the second method, Metzger et al. (1963) used a technique which they named affinity labeling. A hapten is substituted to give a reactive group, and when it reacts with antihapten antibody the specific association is followed by covalent bonding with a reactive amino acid residue in, or close to, the combining site. Thus, rabbit antibenzene arsonic acid was mixed with p-( arsonic acid)benzene diazonium fluoroborate, and it was shown that 2 moles were taken up per mole antibody at a rate some 100 times greater than the nonspecific reaction with other sites in the molecule. With antibody specifically and covalently labeled, A and B chains could be separated and now the label was found to be in both, but about twice as much was in A as in B. Further evidence has been obtained by Metzger and Singer (1963) who used rabbit antibody to the hapten dinitrophenyllysine. The antibody was reduced and fractionated in 1 N propionic acid columns according to the method of Fleischman et al. (1962) and gave the expected yield as judged by absorption at 280 mp of 25% B and 75% A. If the hapten was added before acidification of the reduced antibody, there was almost complete retention of hapten-binding affinity and, when run on the Sephadex column, all the hapten was associated with the A peak. However, the yield of B chain fell from 25 to 16%, i.e., in the presence of hapten, one-third of the B chain was not dissociated from the A chain by acid (Fig. 8). Either some of the B chains were directly involved in the A chain-hapten linkage, or the presence of hapten restricted the unfolding of the A chain by propionic acid and hence was indirectly responsible for the persistent affinity of the A chain for the B chain. Givol and Sela (1964b) have used yet another method based on their observation that if polytyrosyl gelatin forms a specific precipitate with its antisera and this is digested with collagenase, the gelatin is hydrolyzed to peptides and leaves antibody with polytyrosyl groups bound firmly to it. These are the haptenic groups of the original antigen and can only be fully dissociated at pH 1.8. Givol and Sela prepared such antibody with bound C14-polytyrosine and made the A and B chains according to Fleischman et al. (1962) by reduction and separation on a Sephadex column in 1 N propionic acid. Two peaks of radioactivity were

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

315

1.6 1.4

-

c -

12

11 / \

1.0:

3 08 n 0 06: 0.4

I

-

0.2-

\

t

- - % - . O

+++tt+

' ' . -02 80 IOU 120 140 160 180 200 220 240 260 I

I

I

'

'

'

1

FIG.8. ( A ) Protein elution patterns of reduced, alkylated normal rabbit IgG (open circles) and anti-DNP antibodies (closed circles) from the same Sephadex G-75 column equilibrated with 1 M propionic acid; ( B ) protein elution patterns and ( C ) DNP lysine elution patterns, of reduced, alkylated rabbit IgG (open circles, solid line) and anti-DNP antibodies (closed circles, dashed line) containing excess DNPlysine from a Sephadex G-75 column equilibrated with 1 M propionic acid containing 6 x 10-6 M DNP-lysine (Metzger and Singer, 1963).

316

SYDNEY COHEN A N D RODNEY R. PORTER

found, the largest running very slowly and corresponding to dissociated free polytyrosine and a smaller peak moving at the front of the A chain peak. The B peak was inactive. As 80% of the hapten was dissociated from reduced or unreduced antibody in 1 N propionic acid, the results are not decisive, but it was suggested that the polytyrosine was bound either to an A dimer or possibly to undissociated antibody. Clearly, with this series of conflicting results, firm conclusions are impossible. Direct evidence with high over-all recovery favors A as the location of the antibody-combining site, but this cannot be accepted without reserve until some explanation of the other results has been found. J. HETEROGENEITY OF IMMUNOGLOBULINS The physical, chemical, and biological heterogeneity of immunoglobulins has often been discussed (see Fahey, 1962). The resolution of the three types of immunoglobulins and the definition of the relation between them has helped to reduce the confusion, but it remains abundantly clear that there is a complexity of structure in any one type which is unique in protein chemistry. It is highly probable that this complexity is related to biological function, but there is little convincing evidence as to what the connection is. More detail of the heterogeneity has been obtained by studies of the isolated chains (Cohen and Porter, 1964). The A chain, when electrophoresed in starch gel in 8 M urea at neutral pH, shows a diffuse band whose mobility is related to the mobility of the immunoglobulin from which it was prepared. At acid pH, A appears less heterogeneous than the whole IgG. The B chain gives a broad smudge when electrophoresed in 8 M urea at pH 3.5, except when derived from guinea pig antibodies, as discussed above, or mouse antihapten antibodies (Merryman and Benacerraf, 1963). However, when electrophoresed under the same conditions, but at pH 7-8, the B chain from IgG of all species examined gives about ten well-separated components (Fig. 9) and the mobility of these fractions is unrelated to the mobility of the whole molecule. Similarly, the B chains from IgG from all individuals of one species are indistinguishable and the complexity does not, therefore, seem to arise from genetic variations, as in the haptoglobins (Smithies, 1959). It was possible that this multiplicity arose as an artifact during handling but no support could be found for this and perhaps the best evidence that this was a genuine phenomenon came from examination of the B chains of human myeloma globulins. These globulins have long been known to be much more homogeneous by charge (Putnam, 1960), and Edelman and

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

317

FIG. 9. Electrophoresis in 8 M urea, glycine, starch gel of IgG B chains of rabbit ( 1 ), guinea pig ( 2 ) , bovine ( 3 ) , horse ( 4 ) , baboon ( 5 ) , and human ( 6 ) (Cohen and Porter, 1964).

318

SYDNEY COHEN AND RODNEY R. PORTER

colleagues showed that the R chain was a sharp band when electrophoresed in 8 M urea at pH 3.5 (Edelman and Poulik, 1961). At p H 7.5 myeloma B chains again gave a single main component, but sometimes with a second minor component (Fig. 10). As this material was prepared

FIG. 10. Electrophoresis in 8 A l urea, glycine, starch gel of the B chains of normal human IgC ( 1) and of five myeloma imrnuno~lol~ulins(2-6) ( Cohen ant1 Porter, 1964).

in exactly the same conditions as the B chain of normal :,-globulins, it is unlikely that complexity of the latter is an artifact. The myeloma globulins are thought to be synthesized by a relatively homogenous group of cells, possibly deriving from a single clone, and this raises the possibility

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

319

that the ten forms of B may be derived from a corresponding number of different cell types. Differentiation of the cells would be expected to occur soon after birth, when immunoglobulin synthesis increases very rapidly; hence, the B chain was prepared from the IgG of a colostrum-deprived calf at different time intervals after birth. A progressive increase in complexity was anticipated but, in fact, although there was an obvious change in the relative intensity of the bands from 3 to 6 weeks after birth, there was no increase in complexity. Much more convincing evidence will be necessary to relate cell and B chain types. Further, it is apparent that even the single bands of B chains are not homogeneous since bands of identical mobility prepared from myeloma IgG may be either of antigenic Type 1 or Type 2,' and presumably both are present in the normal subfractions of B. There is already evidence that the A chain may show the same type of multiplicity if handled suitably, and it is probable that both chains exist in many different molecular forms. Until that complexity, which in many cases is unrelated to antibody specificity, is resolved, correlation between specificity and structure will be very difficult. IV. Biological Properties of immunoglobulins

A. ANTIGENIC PROPERTIES It has often been remarked that the antigenic specificity of IgG, in contrast to its many other variable properties, shows a surprising homogeneity in any one species. Thus, analyses using anti-y-globulin sera in gel-diffusion tests usually reveal only a single precipitation line. Recent investigations showed, however, that the antigenic structure of immunoglobulins is far more complex than was originally appreciated. The concept of the immunoglobulins as a family of related proteins arose from the observation that several serum globulins which appear to possess antibody activity also carry common antigenic determinants; the same determinants are present on myeloma proteins, pathological macroglobulins, and Bence-Jones proteins ( Heremans, 1960). In addition, each of the three main classes of immunoglobulins has specific antigenic determinants which can be revealed by immunoelectrophoresis and gel diffusion (Korngold and Lipari, 1956a; Franklin and Kunkel, 1958; Grabar and Burtin, 1960). Recent studies have shown that the common determinants present on all immunoglobulins are of two distinct types. It has been known for some time that Bence-Jones proteins can be divided into two mutually exclusive antigenic types which were called Groups A and B by Korngold and Lipari (1956b) and Groups I1 and I, respectively, by Burtin

320

SYDNEY COHEN AND RODNEY R. PORTER

et al. (1956). In the same way myeloma proteins were differentiated into three antigenic groups termed Group 1 and Group 2, which are present on IgG type proteins, and Group 3, which has since been shown to consist of IgA myeloma proteins (Korngold and Lipari, 1956a). Later studies have shown that the antigenic determinants that differentiate the two types of IgG myeloma proteins are the same as those present on the two types of Bence-Jones proteins (Mannik and Kunkel, 1962; Franklin, 1962); for this reason the latter are now referred to as Types I and I1 Bence-Jones proteins, instead of Groups B and A, respectively, as originally suggested by Korngold and Lipari. On the basis of the same determinants it is possible to divide myeloma proteins belonging to both IgA and IgM fractions into the two distinct types, I and I1 (Franklin, 1962; Mannik and Kunkel, 1962; Fahey and Solomon, 1963). Approximately two-thirds of myeloma proteins have Type I and about one-third, Type I1 determinants (Fahey and Solomon, 1963). In these studies the antigenic classification of human myeloma proteins has been carried out using antisera to isolated Bence-Jones proteins or antisera to normal human IgG absorbed with a myeloma protein of the appropriate group. With the use of the same methods, the Type I and I1 determinants have been demonstrated on normal IgG, IgA, and IgM fractions of human serum (Fig. 11) (Franklin, 1962; Mannik and Kunkel, 1963a; Fahey, 1963a), as well as on the low molecular weight fragments of immunoglobulin present in normal human urine ( Stevenson, 1962; Fahey, 1963a) , Precipitation of immunoglobulin preparations with type-specific antisera has shown that the determinants are carried on separate molecules; approximately 60% of normal IgG molecules have Type I determinants and about 30% carry Type I1 specificity (Mannik and Kunkel, 1963a). The proportion of Type I and I1 molecules appears to be similar in the three normal subfractions of immunoglobulins ( Fahey, 1963a) , All antibody activities investigated have been associated with both antigenic types of immunoglobulin (Mannik and Kunkel, 1963b). In different individuals there is considerable variation in the proportion of Type I and Type I1 molecules present in antibodies of a given specificity. In a single individual the ratio of the two antigenic types varies considerably in different specific antibodies, and these ratios are different from that observed in the total Ig (Mannik and Kunkel, 1963b). Antisera to human IgG produced in rhesus monkeys and absorbed separately with two myeloma sera have been reported to show three distinct antigenic determinants in normal 7 S y-globulin (Dray, 1980). Molecules carrying these determinants have somewhat different mobilities

STRUCTURE AND ACTIVITY OF I M m O G L O B U L I N S

321

on immunoelectrophoresis and cannot, therefore, be correlated with Type I and Type I1 molecules which have the same electrophoretic distribution (Mannik and Kunkel, 1963a; Fahey, 1963a). Moreover, both myeloma proteins used for absorption of the antiserum were Type I which indicates that the monkey antisera are revealing an additional antigenic heterogeneity within the Ig system. In the case of IgM, antigenic heterogeneity has been demonstrated by immunoelectrophoresis using rabbit antisera to human macroglobulin (Fessel, 1963).

FIG.11. Demonstration of the presence of

two antigenic types of normal human

I@. Purified, Types I and 11, IgG myeloma ( y mp) proteins were placed in wells of an Ouchterlony plate adjacent to chromatographically prepared normal IgG (6.0 S y ). Pooled antiserum against Types I and I1 Bence-Jones proteins was placed in the lower well ( A S ) ( Fahey, 1963a).

Recent investigations have established the location of the common and specific antigenic determinants on the fragments of immunoglobulin molecules produced by reduction or enzymatic digestion. The enzyme papain separates parts of the molecule which carry the common and specific determinants. The fragment designated F (Edelman et al., 1960) is associated with the specific determinants of human IgG, whereas the S fragment carries determinants common to normal and pathological IgG, IgA, and IgM, as well as Bence-Jones proteins (Heremans, 1960; Franklin and Stanworth, 1961; Migita and Putnam, 1963). Similar findings have been reported for the immunoglobulins of mouse (Askonas and Fahey, 1962), rabbit (Thorbecke and Franklin, 196l), and guinea pig (Thorbecke et al., 1963). The F fragment obtained by papain digestion is made up of part of the A (or H ) polypeptide chains, whereas the S fragment contains the B chain (Olins and Edelman, 1962). The isolated A chain has now been shown to carry the type-specific, antigenic determinants of normal and pathological immunoglobulin subfractions, whereas the common determinants of Types I and I1 have been identified

322

SYDNEY COHEN AND RODNEY R. PORTER

FIG. 12. Reaction of rabbit anti-( B c h i n of Iiuman IgC) with Rcnce-Jones protein Types I (1) and 11 ( 2 ) , and with B c h i n of 1gG ( B ) , S fr'igmmt of IgC ( S ) , and a mixture of Types 1 und 11 Bencc-Jonc\ protc4n\ ( 1 2 ) . 13otli tlic S fragment and the B chain form slight spurs with the miuture of Typca 1 und 11 Bence-Jones proteins (Cohen, 1963h).

+

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

323

on B chains (Fig. 12) (Cohen, 1963a,b; Fahey, 1963b). Thus, antisera against purified immunoglobulin fractions can be made specific for the corresponding fraction by absorption with B chain. Similarly, rabbit antisera against the human B chain of IgG give reactions of identity with other normal immunoglobulin fractions, whereas antisera against the A chain react only with IgG (Fig. 13). Certain antigenic determinants of immunoglobulins appear to be concealed in the whole molecule and may be revealed by enzymatic digestion. Osterland et al. (1963) used anti-Rh antibodies digested with pepsin or with papain at pH 4.1 to coat Rh-positive red cells and showed that these were agglutinated by the 7 S globulin fraction from normal sera and from sera of some patients with rheumatoid arthritis and bacterial endocarditis. These same sera did not, however, agglutinate cells coated with the whole antibody; in addition, the agglutination could be inhibited by fragments obtained by enzymatic digestion at acid pH, but not by native y-globulin or by the isolated S fragment obtained by papain digestion at pH 7.4. Goodman and Gross (1963) also found that no new antigenic determinants were exposed by papain digestion of rabbit IgG at neutral pH. These results indicate that hidden antigenic determinants of the S fragment of IgG are revealed by enzymatic digestion only when this is caried out at acid pH under conditions that destroy the F fragment. Repeated attempts to show that isolated antibodies possess individual antigenic specificity have been unsuccessful (see Kabat and Mayer, 1961). However, such individual specificity has recently been demonstrated in the case of several antibodies. Kunkel et al. (1963) isolated four human antibodies of the 7 S class from specific precipitates and used these with Freund's adjuvant for immunization of rabbits. The rabbit antisera after absorption with normal human IgG or normal human serum reacted with the corresponding individual antibody, but not with antibodies of the same combining specificity obtained from other subjects. Oudin and Michel ( 1963) obtained antisera specific for individual antibody preparations by immunizing rabbits with Salmonella typhosa agglutinated by the antiserum of another rabbit. In some instances antibodies were produced which precipitated with the immunizing antiserum but failed to precipitate with the serum of the same rabbit before immunization with S . typhosu or, subsequently, after antipneumococcal immunization; nor did such antisera precipitate with the immune or nonimmune sera of other rabbits. Deutsch and MacKenzie ( 1964) have shown that individual monkey antisera to Rh saline agglutinins (IgM antibodies) can inhibit the serological activity of some Rh saline agglutinins but not others.

324

SYDNEY COHEN AND RODNEY R. PORTER

FIG.13. Reaction of normal human IgC ( 1) and normal human IgM ( 2 ) in ( a ) , left, with rabbit antihuman IgC ( 7 ) and, right, with the same antiserum after absorption with IgC B chain, in ( b ) , left, with rabbit antihuman IgM (19) and, right, with the same antiserum after absorption with IgC B chain (19); in ( c ) , left, with rabbit anti-IgC B chain ( B ) and, right, with rabbit anti-IgC A chain ( A ) (Cohen, 1963b).

STRUCTURE AND ACTIVITY OF IMMVNOGLOBULINS

325

The fact that some antisera used in these studies reacted only with the antibodies of single individuals suggests that their specificity is not directed against the combining sites of the antibodies used for immunization. Kunkel et al. (1963) found that the individual specificity of the human antibodies was associated with the S fragment isolated after papain digestion; in addition, when the antibodies with individual specificity were reduced, they showed particularly sharp banding of B chains on starch-gel electrophoresis (Kunkel et al., 1963). These findings suggest that the determinants responsible for the observed individual specificity may be localized on B chains, which, as shown above, form part of the S fragment. It is of interest that individual specificity has previously been found on myeloma proteins (Slater et al., 1955), macroglobulins (Habich and Hassig, 1953; Korngold and van Leeuwen, 1957), and “monoclonal y-globulins” ( Mannik and Kunkel, 1963c), all of which are known to have characteristically homogeneous B chains (Poulik and Edelman, 1961; Cohen and Porter, 1964). In considering the implications of these experiments which demonstrate individual specificity on antibodies and pathological immunoglobulins, it is worth bearing in mind that studies on insulin (Berson and Yalow, 1961, 1963) and ribonuclease (Mills and Haber, 1963) have shown that proteins, which are apparently identical in chemical structure, may yet have distinct antigenic properties. It seems likely from the known heterogeneity of the constituent peptide chains that the antigenic structure of all antibody preparations will prove to be extremely complex. The experiments outlined above provide evidence for the antigenic complexity of human B chains which are known to occur in two distinct antigenic forms. In several species, B chains are separable by electrophoresis on urea-glycine starch gels into multiple components which probably differ in amino acid composition and can be expected to have distinct antigenic properties; several BenceJones proteins have recently been shown to possess such antigenic individuality (Stein et al., 1963). The significance of the multiple forms of the B chain is not understood, and it will be a matter of great interest to establish whether the B chain occurs in two fundamentally distinct antigenic forms in species other than man.

B. ALLOTYPESOF IMMUNOGLOBULINS During recent years it has become apparent that in several species, including man, rabbit, mouse, guinea pig (Benacerraf and Gell, 1961 ), baboon (Kelus and Moor-Jankowski, 1962), and chimpanzee (Podliachouk, 1959; Boyer and Young, 196l), there occur individual variants of

326

SYDNEY COHEN AM) RODNEY R. PORTER

immunoglobulin which can be differentiated on the basis of serological differences. These variants, which Oudin ( 1956) named allotypes, were first recognized by injection of specific precipitates of rabbit antibody together with Freund’s adjuvant into other rabbits. When the recipients carried a different allotypic specificity from that of the donor, they responded by producing isoantibodies which precipitated with immunoglobulin from the donor rabbit. With the use of gel-diffusion techniques it has been possible to demonstrate six allotypic specificities in rabbit IgG and, according to an agreed terminology (Dray et al., 1962), these are now referred to as A1 to A6 (Table VI).The specificities Al, A2,and A3 appear to be controlled TABLE VI NOMENCLATURE FOR ALLOTYPIC SPECIFICITIES IN RABBITIgG Previous nomenclature Present nomenclature A1 A2 A3 A4 A5 A6

Oudin b C

d a

g f

Dubiski and Kelus

Dray and Young

E B D F

J

A

c

L K I1 I

-

by three allelic genes at one locus ( a ) , and A4, A5, and A6 by three allelic genes at a second locus (b). Specificities which are systematically found together and are, therefore, probably controlled by the same allele or by closely linked genes, are designated with the superscripts “prime” or “double prime,” e.g., Al, Al’, and Al” (Oudin, 1960a,b; Dray and Young, 1961; Dray et a,?.,1963b). The available data indicate that the a and b loci are not closely linked and are not sex-linked (Dray and Young, 1960, 1961; Dubiski et al., 1962; Dray et al., 1963b). Individual rabbit sera always contain at least two allotypes, but animals heterozygous at one or both loci may have three or four allotypes. Dray et al. (1963b) have recently shown that rabbit IgC carries two additional allotypic specificities, P and T. The genetic control of the former was investigated and evidence obtained for the presence of a single gene at a third locus distinct from a and b. The distribution of allotypic specificities on various purified antibodies was studied by Gel1 and Kelus (1962). In the case of a heterozygous rabbit (Al-A3, A4-A5), the specificity A5, which is determined by the b locus, appeared to be eliminated from an antihapten antibody, while

STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS

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A3, determined by the a locus, was also probably absent. A similar absence of some allotypic determinants from specific antibodies is indicated by the fact that anti-T2 phage antisera from heterozygous rabbits (A1,4,5) retain phage-neutralizing activity after addition of excess anti-A5 antiserum (Stemke, 1963), but are inactivated by whole anti-B chain antiserum. Rieder and Oudin (1963), on the other hand, found that in heterozygous rabbits purified antibodies against ovalbumin, dinitrophenol, and Type I1 pneumococcal polysaccharide contained all the allotypic specificities of the animal; however, the relative concentrations of the allotypes varied considerably in different antibodies produced by the same rabbits. Oudin (1961, 1962) showed by agar tube analysis of individual sera that the nonallelic A l and A6 were present on the same molecules, whereas the allelic forms, A1 and A3, were on different molecules. The distribution of allotypic specificities on individual IgG molecules has been further clarified by the development of a method of analysis which is based upon the successive precipitation of specific allotypes from Ilal-labeled IgG with monospecific antiallotype sera ( Dray and Nisonoff, 1963). This method was used to study the contribution of allelic genes At,* and Ab6 to the formation of IgG in heterozygous rabbits. In these animals about 65% of 1131-labeled IgG was precipitated by anti-A4 sera and about 25% by anti-A5; since the observed proportions were independent of the order of precipitation it is clear that A4 and AS were carried on separate molecules (Dray and Nisonoff, 1963). About 1040% of I'*l-labeled IgG was not precipitable by either antid4 or anti-AS; from subsequent experiments (Dray et al., 1963a,b) it seems probable that such molecules carry one or more of the allotypic specificities defined at other genetic loci. The contribution that genes at the a and b loci make to the formation of IgG was studied by the same method of immune precipitation and also by gel diffusion (Dray et al., 1963a). From these studies it is clear that in double homozygotes (Al, A5) as well as in double heterozygotes ( Al-A3, A4-A5 ) the two nonallelic, allotypic specificities, A1 and A5, occur on separate molecules as well as on the same molecule. The proportions of A1 molecules associated with A4 and AS, respectively, were found to be sufficiently close to the proportions of A4 and A5 in the total population to suggest that the formation of individual molecules involves a random association of nonallelic specificities. In cases in which A1 and A4 were under the control of genes derived from different parents, the hybrid molecules of the offspring have an antigenic specificity not present in either parent; results previously obtained by Oudin (1962) had suggested this possibility. It is clear from

328

SYDNEY COHEN AND RODNEY R. PORTER

these studies in the rabbit that nonallelic genes contribute to the formation of single antibody molecules, whereas allelic genes do not. Among laboratory animals, mice, which exist in a variety of inbred strains, are likely to prove particularly valuable for studies on the genetic control of antibody synthesis. Kelus and Moor-Jankowski (1961) first demonstrated the presence of an allotypic form of Ig in BALB/c mice; in these experiments antisera were obtained by immunization of C57BL recipients with P r o t m oulgaris coated with BALB/c antibody. The isoantigen present in BALB/c mice was subsequently designated MuAl (Dubiski and Cinander, 1963); this was found to be absent from the immunized strain (C57BL), but was identified in three other strains. By immunizing BALB/c mice, another isoantigen (MuA2) has been found in C57BL and SJLmice (Dubiski and Cinander, 1963). What is probably the same isoantigen (referred to as Gg-2), has been identified by Wunderlich and Hertzenberg (1962) in all the C57BL strains tested, as well as in the sera of several other strains; the study of segregation ratios indicated that this specificity is under the control of a single gene. The specificities MuAl and MuA2 have been identified by Dray et a2. (1963~) using isoantibodies harvested from peritoneal fluid exudates after comparatively brief periods of immunization. A single allotypic specificity was present in each of forty-two inbred strains studied; the inheritance of these specificities appeared to be determined by codominant, autosomal alleles. Individual variants of human Ig have been distinguished from one another by differences in their ability to inhibit the agglutination of sensitized cells by sera containing substances serologically related to rheumatoid factors. Since the time this technique was introduced by Grubb (1956), several genetically determined types of human Ig have been described. Of these Gm( a ) (Grubb and Laurell, 1956), Gm( b ) (Harboe, 1959), Gm(x) (Harboe and Lundevall, 1959), Gm( r ) (Brandtzaeg et al., 1961), and probably also Gm( p ) ( Waller et al., 1963) are determined by genes at one locus (Gm). In Caucasians, genes controlling the production of Gm(a) and Gm(b) behave as alternate alleles (Harboe, 1959), whereas among Negroes, these factors appear to be produced by a single allele ( Gmab) (Steinberg et al., 1960a). Two alleles at an independent locus ( InV) determine the factors InV( a ) and InV( b ) (Ropartz et al., 1961; Steinberg et d.,1962). The seventh factor, Gm-like (Steinberg et al., 1960b) is found in Negroes, but is extremely rare in other racial groups; this locus is independent of the InV locus in population studies (Steinberg, 1962), but its relationship to the Gm locus is uncertain because all Negroes are Gm (a+ b+). The work on human

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allotypes has been impeded by the fact that some typing sera have been derived from individual patients and have, therefore, had a limited and transient availability; interest, therefore, attaches to the attempts which are being made to use experimental animals to raise antisera which will distinguish allotypic variants of human immunoglobulins. There is some evidence that isolated human antibodies may not contain all the genetic characters of the total Ig fraction of the individual; thus, Harboe ( 1 9 0 ) found that many human anti-D sera from Gm( b+) individuals did not appear to carry Gm( b ) specificity. Studies of myeloma proteins which are generally regarded as products of individual cell clones, have provided further information about the probable distribution of allotypic specificities on separate human IgG molecules (Fahey and Lawler, 1961; Franklin et d.,1962; Mlrtensson, 1961; Harboe et al., 1962a). It has been shown that myeloma proteins belonging to the IgG fraction may have both Gm and InV specificities, but in heterozygous individuals these proteins do not carry more than a single allelic form of either specificity (Mlrtensson, 1961; Harboe et al., 1962a); the Gm (a+ b+) myeloma proteins found by Fahey and Lawler (1961) were apparently contaminated with small amounts of normal immunoglobulin present in the serum. Myeloma proteins may be Gm (a+ x+) (Mhtensson, 1961; Harboe et al., 1962a), but this is to be expected since the genetic studies have shown that Gm(a) and Gm(x) are controlled by a single allele or by two closely linked genes. These findings indicate that, as in the case of the rabbit, nonallelic genes contribute to the formation of individual human antibody molecules, whereas allelic genes do not. The structural basis of allotypic specificity is unknown, but in the case of the B chain this specificity is probably not associated with a carbohydrate moiety. The distribution of distinct specificities on different types of immunoglobulin and on molecular subunits has been investigated. Studies on myeloma proteins have shown that the Gm factor is associated only with human I@ and is not found on IgA or IgM, whereas InV specificity occurs on all types of immunoglobulin (Fahey and Lawler, 1961; Mlrtensson, 1961; Franklin et al., 1962; Harboe et al., 1962a). The Gm specificity of IgG is localized on the F fragment of the molecule obtained by papain digestion (see above) and the S fragment carries the InV determinants (Franklin et al., 1962; Harboe et al., 1962b). Fleischman et al. (1962) showed that papain piece I11 of rabbit IgG, which is equivalent to human F, consists only of A chain; this suggested that Gm determinants must be carried by A, and InV specificity by B chains. This distribution of specificities was subsequently confirmed by Lawler and

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SYDNEY COHEN AM) RODNEY R. PORTER

Cohen (1965) who found that Gm and InV specificities of normal human IgG are confined to A and B chains, respectively (Cohen, 1963a). The distribution of InV specificity on the two distinct antigenic types of normal human B chain has not been established. Studies on Bence-Jones proteins, which appear to be composed of B chains (Edelman and Gally, 1962; Schwartz and Edelman, 1963), showed that InV specificity is frequently present on proteins of Type I, but is very rarely found on Type I1 Bence-Jones proteins (Franklin et al., 1982; Harboe et al., 1962a). However, Harboe et al. (1962a) found that InV factors occur in both Group I and Group I1 myeloma proteins. This indicates that InV specificity is associated with B chains of both antigenic types and that Bence-Jones proteins, in some cases, are not identical with the corresponding myeloma B chains. It is evident from the above discussion that the autosomal locus, InV, determines the synthesis of the polypeptide chain ( B ) which appears to be common to all types of immunoglobulins, whereas the second autosomal locus, Gm, controls the synthesis of the polypeptide chain ( A ) which is specific for IgG. The IgA and IgM fractions also have specific A chains and are presumably determined by two additional genetic loci; polymorphisms corresponding to these have not been identified; however, their separate genetic control is suggested by familial cases of agammaglobulinemia having normal or increased levels of IgM (Burtin, 1961; Rosen et al., 1961; Fudenberg et al., 1963), by the absence of IgA in the relatives of some agammaglobulinemics ( Fudenberg et al., 1963) , and also by cases in which IgA and IgM are absent but IgG is present in normal or increased amount (Barandun et al., 1959; Giedion and Scheidegger, 1957). In contrast to results obtained with human allotypes, the rabbit specificities determined by the two genetic loci, a and b, appear to be present on all types of immunoglobulin. Todd (1963) fractionated rabbit serum by gel filtration on Sephadex G-200and showed that the macroglobulin peak contained A1 and A4 specificities; this peak failed to react with antisera to piece I11 and was, therefore, not significantly contaminated with IgG (Fig. 14). On the other hand, Feinstein et al. (1963) using a gel-diffusion method, were unable to detect A3 determinants and obtained doubtful reactions for A1 and A2 in macroglobulin preparations from rabbit sera which contained all these specificities. An immunoglobulin fraction, which appeared to have a faster electrophoretic mobility than IgM and was antigenically distinct from I@, has been isolated from rabbit colostrum; this fraction, which may correspond to human IgA, was shown to carry allotypic specificities determined by

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allelic genes at both the a and b loci (Feinstein, 1963). After papain digestion of rabbit I@, the allotypes determined by both genetic loci are found on pieces I and I1 (Kelus et al., 1960; Marrack et al., 1962; Feinstein et al., 1963). The isolated rabbit B chain is associated only with allotypes determined by the b locus (Kelus, 1963; Feinstein et d.,1963). Allotypic specificities determined by the a locus would, therefore, be expected to occur on the A chain; it seems likely that these determinants are on that portion of the A chain ( A piece) which is present in papain 3.5. 61%

2 3.0. 0

-:

2

0 ._ c

f

2.52.0

I .o

0.5

0

400

BOO

1200

1600

2000

2400

Volume of eluate(mls.)

FIG.14. Optical density at 280 mv of the effluent from the G-200 filtration of 15 ml. of A1,4 rabbit serum on a Sephadex G-200 column; bed volume, 2100 ml. Shaded areas indicate fractions that gave positive tests for both allotypic specificities. Percentages at the top of each peak represent the per cent of the total optical density units (OD x volume) found in each peak. The fraction at the center of the first peak give a positive test for A1 at a dilution of 1/4 and for A4 at a dilution of 1/8.It was negative for 111 specificity. The fraction at the center of the second peak gave a positive test for A1 and A4 at a dilution of 1/64 and for 111 specificity at a dilution of 1/256 (Todd, 1963).

pieces I and I1 and may be common to IgG, IgA, and IgM. Experiments reported to date have shown, however, that the A chain contains specscities determined by both a and b loci and this may indicate that preparations of rabbit A are contaminated by B chain (Kelus, 1963; Feinstein et al., 1963). C. URINARYEXCRETION OF IMMUNOGLOBULIN FRAGMENTS The urine of normal subjects contains small amounts of relatively low molecular weight proteins which have antigenic determinants in common with serum immunoglobulins (Webb et d.,1958; Franklin, 1959; Stevenson, 1960; Berggard, 1961; Rowe and Soothill, 1961; Cornillot et ul., 1963). These proteins are antigenically related to the S fragment of IgG

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SYDNEY COHEN AND RODNEY R. PORTER

and carry determinants corresponding to Types I and I1 Bence-Jones proteins (Hanson and Berggard, 1962; Stevenson, 1962; Fahey, 1963a). These findings suggest that urinary immunoglobulin fragments may be composed of B chains. Berggard and Edelman (1963) have demonstrated the close similarity between immunoglobulin fragments present in normal urine and B chains in regard to antigenic structure and thermosolubility; in addition, the molecular weight of the urinary immunoglobulin fragment determined by equilibrium sedimentation was 25,000, which agrees approximately with the value for the B chain monomer. Using antisera specific for A and B chains of IgG, Cohen (1964) found that a normal subject excreted daily 5-10 mg. of a protein which carried only B chain determinants. What appears to be an unrelated antibody fragment has been recovered from the urine of subjects immunized with poliovirus vaccines or with tetanus toxoid (Remington et d.,1962; Merler et al., 1963). Concentrates of normal urine were fractionated by chromatography on diethylaminoethyl cellulose columns followed by centrifugation in a sucrose gradient. The top fraction of the sucrose gradient contained a protein of molecular weight about 13,000 which was antigenically related to serum IgG and was able to precipitate with the appropriate antigen. This fragment gave a pattern of tryptic peptides which showed little overlap with those of serum IgG, and its relationship to circulating antibody is not clear. Rowe (1963) was unable to confirm these findings in studies on the urine of a normal subject immunized with typhoid vaccine; in this instance fractionation on a Sephadex G-200 column showed that urinary antibody activity was apparently confined to whole immunoglobulin and could not be detected in smaller fragments. The origin of urinary immunoglobulin fragments has been studied in radioactive-labeling experiments. The results of Franklin ( 1959) and Webb et d. (1958) indicated that these fragments were derived from the degradation of normal immunoglobulin. However, other studies (Stevenson, 1962) have shown that the urinary proteins which probably correspond to B chains arise as precursor or by-products of immunoglobulin synthesis and correspond in this respect to Bence-Jones proteins.

D. TRANSFER OF ANTIB~DIES FROM MOTHERTO FETUS In a paper written in 1892, Ehrlich provided detailed experimental evidence for the occurrence of a passive transfer of antibodies from mother to offspring both in utero and during suckling. These early experiments were carried out in mice, and it has since become apparent that the mechanism of antibody transfer from mother to her offspring

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varies considerably in different species. In the pig, horse, and goat, immunoglobulin is present only in trace amounts at birth and is absorbed from the colostrum during the first 2 days of life. Human subjects, on the other hand, as well as rabbits, guinea pigs, and monkeys, acquire maternal antibodies only during prenatal development ( Hemmings and Brambell, 1961). Immunoglobulin begins to appear in the human fetal circulation at about the fourth month of pregnancy and the level rises progressively until it reaches that of the mother at term. The transfer of IgG takes place across the pIacenta in the rhesus monkey and probably also in human subjects (Bangham, 1960); in the rabbit, transfer is effected by way of the uterine cavity and the vascular, fetal, yolk sac splanchnopleure. Transmission across the fetal membranes is a highly selective process since IgG is transmitted preferentially to the other serum proteins, homologous IgG is transferred more readily than heterologous protein (BrambeIl, 1958) and human IgG is freeIy transferred, whereas IgA and IgM are not (Hitzig, 1957; Franklin and Kunkel, 1958; Gitlin et d., 1963). This implies that transmission is dependent on specific structural features of the molecule. Investigations using the papain fragments of rabbit IgG have shown that piece 111, isotopically labeled with 1131, is transmitted across the membranes of the fetal circulation of the rabbit eleven times more rapidly than piece I and six times more rapidly than piece 11; piece I11 was transmitted at 70% the rate of labeled IgG (Brambell et d.,1960). Experiments in suckling mice and rats similarly indicate that piece I11 is involved in the transmission of antibodies across the gastrointestinal mucosa during neonatal life (Morris, 1963). The human placenta during the last month of pregnancy is freely permeable to the F fragment, but evidence was also obtained for transmission of the S fragment of IgG (Gitlin et al., 1964). The chemical configuration required for the transmission of intact human antibodies across fetal membranes may be associated primarily with the portion of the molecule equivalent to rabbit piece 111. If this were so, the observation made by Hartley (1951) that peptic digestion of horse diphtheria antiserum destroys its ability to pass from mother to fetus, would be explained, since pepsin destroys piece I11 and leaves an active fragment consisting of I and 11. As mentioned above, piece I11 is composed of part of the A chain which has different properties in the case of IgG, IgA, and IgM. Failure of the latter two human immunoglobulins to cross the fetal membranes may, therefore, be due to the absence of the necessary transmission site from their respective A chains. In the case of the rabbit, the transfer of IgM agglutinins from mother to fetus has been reported

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S M N E Y COHEN AND RODNEY R. PORTER

(Hemmings and Jones, 1962); this suggests that the transmission site may be present on both IgG and IgM molecules in this species.

E. FIXATION OF ANTIBODYTO SKIN The antibodies that mediate certain hypersensitivity reactions have the property of fixing to skin and other tissues. The technique of passive cutaneous anaphylaxis (PCA) has been used to investigate the skinattaching properties of various antibodies in the guinea pig (Ovary, 1958). In the PCA reaction, antiserum is injected intradermally and is followed by intravenous injection of antigen together with a blue dye; a positive reaction is shown by diffusion of dye through a localized area of increased permeability. Ovary et al. (1980) showed that human 7 S antibodies are able to elicit a positive PCA test in guinea pigs, but 19 S antibodies of the same specificity gave a negative response, apparently owing to an inability to fix to guinea pig tissue. Subsequent studies have shown that normal human IgA and nine different IgA myeloma proteins, belonging to the two major antigenic types, were all unable to sensitize guinea pig tissues (Franklin and Ovary, 1983). These studies indicate that of the human immunoglobulins only IgG can sensitize the skin of the guinea pig. This suggests that the attachment site for guinea pig skin is carried on the IgG A chain, since as shown above, this chain is associated with the antigenic and allotypic determinants specific for IgG. This idea is supported by experiments using the fragments of rabbit antibodies; it was shown by the reverse PCA reaction that the site responsible for attachment to guinea pig skin is localized on piece I11 (Ovary and Karush, 1961) and, as shown above, this part of the molecule consists only of a part of the A chain. Similarly, Liacopoulos et al. (1963) showed that the Schultz-Dale reaction of guinea pig tissues to rabbit antibody can be inhibited by piece I11 of nonimmune IgG, whereas piece I has no inhibitory effect. Thus, as pointed out by Brambell (1983), the same section of the IgG molecule is concerned in skin fixation and in the passive transfer of immunity from mother to offspring. In the case of human IgG the site necessary for skin sensitization appears to be lost during papain digestion, although the in vivo antibody activity of S fragments separated by the same procedure was retained (Franklin and Ovary, 1983). Antibodies that occur spontaneously in the sera of allergic subjects with immediate-type hypersensitivity are distinguished by their apparent ability to become rapidly and firmly attached to human epithelial tissues. These antibodies, which are referred to as reagins, are detected by the passive transfer of wheal and erythema reactivity (the P-K reaction). No

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satisfactory means is available for their quantitative estimation since specific antigens (allergens) do not produce any detectable in uitm interaction with reaginic sera, probably as a result of the extremely low levels of antibody present. Chan (1963) has developed a method for estimating the concentration of reaginic antibody in sera from subjects showing immediate-type hypersensitivity to horse serum albumin. The capacity of reaginic sera to bind highly purified samples of horse albumin labeled with 1131 was determined by electrophoresis on cellulose acetate membranes. The total concentration of albumin-binding antibody was calculated from the weight of antigen bound per unit volume of serum assuming an antibody-antigen weight-combining ratio of 10:1. The concentration of heat-labile (reaginic) antibodies was less than 30 pg./ml. in the subjects tested so that the total circulating plasma always contained less than 65 mg. of antibody; the difficulty of obtaining sufficient human reaginic antibody for detailed structural studies is apparent from these results. In addition, it was found that the degree of sensitivity shown by subjects reacting to horse albumin was unrelated to the concentration of reaginic antibody and was apparently determined by the relative proportions of heat-labile (reaginic ) and heat-stable (blocking) antibodies. For example, two subjects whose sera were equally active, as judged by P-K testing, had concentrations of 0.8 and 29 pg. of reaginic antibody per milliliter serum; the levels of blocking antibody were 1.5 and 66 pg./ml., respectively, so that the ratio of blocking to reaginic antibody was about 2 : l in each case. It will be a matter of great interest to establish whether a similar relationship between clinical reactivity and the ratio of blocking-to-reaginic antibody concentration is observed with other purified allergens. Human reaginic antibodies do not have the ability to sensitize the guinea pig for the PCA reaction (Augustin, 1955) and they do not cross the placental barrier in allergic mothers (Bell and Eriksson, 1931; Sherman et al., 1940). Since human IgG is able to sensitize guinea pig skin and is transmitted freely into the fetal circulation, these findings suggest that reaginic antibodies do not belong to the IgG fraction. Evidence which suggested that reaginic antibody is associated with the IgA fraction of serum (Augustin, 1961; Heremans and Vaerman, 1962) could not be regarded as conclusive because of the difficulty of isolating IgA in pure and uncontaminated form. More recently, however, Fireman et al. (1963) showed that the specific removal of IgA by immune absorption eliminated all detectable skin-sensitizing activity from the sera of three ragweed-sensitive individuals, whereas similar absorption of IgG had no detectable effect. Antibodies to I13'-labeled ragweed

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SYDNEY WHEN AND RODNEY R. PORTER

antigen have been shown by immunoelectrophoresis to be associated with IgG in the sera of eight ragweed-sensitive subjects. Antibody activity was also present in the IgA fraction of seven cases and in the IgM of two subjects; the biological activity of these various antibody fractions was not determined (Yagi et al., 1963). In view of the controversy which exists in regard to the molecular weight and electrophoretic behavior of reagins, it seems probable that the distribution of these antibodies among the immunoglobulin subfractions may vary in different sera. Their association with IgA is of particular interest since this would account for the fact that reagins which often appear to have a sedimentation coefficient of 7 (Stanworth, 1963) fail to cross the placenta and cannot sensitize guinea pig skin for the PCA reaction. The presence in IgA of reagins that sensitize human skin suggests that different molecular sites are involved in the attachment of human antibodies to the tissues of different species. In this connection it is of interest that a study of guinea pig immunoglobulins has shown that a 7 S yz-globulin fraction was unable to elicit the PCA reaction in guinea pigs, whereas a 7 S yl-globulin which may be analogous to IgA of other species effectively sensitized guinea pig skin (Ovary et d.,1963).

F.

COMPLEMENT

FIXATION

Complement fixation is known to occur with antibodies belonging to both IgG and IgM fractions, but the few IgA antibodies which have been studied have been inactive in this respect. Thus, antibrucella agglutinins in human IgG and IgM were able to fix complement, but IgA antibodies of the same specificity lacked this capacity (Heremans et al., 1963). Similarly, complement-fixing activity has been demonstrated on guinea pig 7 S yz-globulins but not on 7 S yl-globulins which may be analogous to the human IgA fraction (Bloch et al., 1963). Complement is fixed by IgG molecules not only upon reaction with antigen, but also after nonspecific aggregation by heat (Taranta and Franklin, 1961; Amiraian and Leikhim, 1961; Ishizaka et al., 1962). IgG treated with 0.1 M mercaptoethanol and subsequently dialyzed against iodoacetamide can still combine with antigen and is aggregated by heat, but cannot fix complement ( Wiedermann et al., 1963). These results indicate the importance of disulfide bonds in the complement fixation reaction; it is also apparent that loss of complement-fixing ability after treatment with sulfhydryl reagents cannot be regarded as a valid means of identifying antibodies as belonging to the IgM fraction. Several attempts have been made to establish which part of the

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immunoglobulin molecule is involved in complement fixation. Taranta and Franklin (1961) found that a peptic digest of rabbit antiovalbumin or anti-bovine serum albumin would not bind complement when precipitated by addition of antigen in the presence of fresh guinea pig serum. Since peptic digestion destroys a part of the molecule equivalent to piece 111, the result suggested that this piece carried complement-fixing sites. In agreement with this is the demonstration that piece 111 aggregated by coupling with bisdiazotized benzidene or by heating is able to bind complement (Ishizaka et aZ., 1962). Similarly, piece I11 has been shown to inhibit immune hemolysis of sheep erythrocytes primarily by interacting with guinea pig complement, whereas peptic digests of normal and antipertussis IgG are not inhibiting ( Amiraian and Leikhim, 1961); however, in the latter experiments some complement fixation occurred with peptic digests of rabbit anti-sheep erythrocyte IgG in the presence of the antigen. Further evidence that the pieces ( I and 11) that survive peptic digestion may be involved in complement fixation comes from the experiments of Schur and Becker (1983a,b); washed specific precipitates obtained by adding antigen to peptic digests of rabbit or sheep antibodies fixed complement when added to fresh guinea pig serum. The 5 S component precipitated with antigen absorbed up to 40% of the complement which was fixed by a precipitate of whole IgG and antigen. However, the absorption of complement did not occur if the precipitation of the 5 S antibody fragments was carried out in the presence of guinea pig serum, and no explanation of this anomalous behavior was found, Reiss and Plescia (1963) provided evidence that complement was present on the papain fragments I and I1 but not on piece I11 when these were separated from ovalbumin antiovalbumin precipitates which had fixed human serum complement prior to enzymatic digestion; localization of complement was based upon the interaction of the papain pieces with a rabbit antiserum to human serum. Direct evidence that all three pieces of the antibody molecule may be involved in complement fixation was provided by Cebra (1963). He showed that rabbit antibody which has been split by insoluble papain, but not dissociated by thiol, precipitates with antigen and binds complement as effectively as the native antibody. When thiol is added to the specific precipitate carrying bound complement, only a partial dissociation occurs suggesting that the antibody is held together by components of complement bound to all parts of the molecule. The conclusion at present, therefore, seems to be that piece 111 is predominantly concerned in the binding of complement to antibody reacted with antigen, but that other parts of the molecule are also involved in this process.

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SYDNEY COHEN A N D RODNEY R. PORTER

G. DISTRIBUTION AND TURNOVER OF IMMUNOGLOBULINS

Estimations based on immunological techniques have shown that the immunoglobulins comprise about 20% of the total protein in human serum. The average normal concentrations of IgG and IgM are about 1250 and 125 mg./100 ml. serum, respectively (Soothill, 1962; Fahey and Lawrence, 1963; Chodirker and Tomasi, 1963); estimates of the mean normal level of IgA have varied from about 150 (Heremans, 1960; Chodirker and Tomasi, 1963) to 400 mg./100 ml. serum (Fahey and Lawrence, 1963). The total IgG of the body (about 80 gm.) in healthy adults is distributed equally between the circulating plasma and the interstitial fluids. About 25% of the circulating IgG fraction passes across capillaries into the extravascular fluids each day, and a similar amount is returned to the bloodstream through the main lymphatic ducts (Cohen, 1963d). A fluorescent antibody technique has shown that extravascular immunoglobulin is present in all extracellular fluids as well as in the ground substance of connective tissue (Gitlin et al., 1953). The concentration of immunoglobulin in the interstitial fluids varies considerably in different sites, being highest in the hepatic lymph (about 1 gm./100 ml.) and relatively low (200 mg./100 ml.) in the interstitial fluids of muscle and subcutaneous tissue (Gitlin and Janeway, 1954). The distribution of IgM differs from that of IgG in that only a small proportion of the total pool is present in extravascular tissues (Cohen and Freeman, 1960; Wochner et al., 1963). The IgA fraction appears to be present in relatively high concentration in human milk (Hanson, 196l), as well as in saliva and tears (Tomasi and Zigelbaum, 1963; Chodirker and Tomasi, 1963), suggesting that this protein may have a specific transmission site responsible for secretion into these biological fluids. All plasma protein fractions, including the immunoglobulins, are in a state of dynamic equilibrium undergoing constant degradation and replacement by newly synthesized molecules. A homogeneous protein, such as human albumin, when labeled with 1131has a constant rate of breakdown (measured by urinary excretion of label) over a period of several weeks. On the other hand, the fractional catabolic rate of P31labeled human immunoglobulin prepared by zone electrophoresis falls progressively during the first 1 or 2 weeks after injection, suggesting the presence of a mixed population of molecules having different breakdown rates. This metabolic heterogeneity appears to be attributable mainly to differences in the turnover rates of 19 and 7 S fractions. Thus, human IgM isolated by ultracentrifugation and zone electrophoresis (Cohen and Freeman, 1960) or by electrophoresis and gel filtration (Wochner et al.,

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1963) has a relatively high turnover rate, whereas IgG is catabolized at a slower rate; a similar digerence in the half-lives of small and large molecular weight antibodies has been reported in rabbits (Taliaferro and Talmage, 1956). Little is known about the site and mechanism of immunoglobulin catabolism. On the basis of labeled protein studies it has been suggested that plasma cells which are known to be involved in antibody synthesis may also be responsible for immunoglobulin breakdown (Soons and Westenbrink, 1958).However, perfusion experiments in which biologically screened proteins are used show that the normal rat liver, which is not a site of antibody synthesis, catabolizes IgG at a rate equivalent to 30% of the total breakdown in vivo (Cohen et al., 1962). The fractional breakdown rate of IgG can be increased in the mouse by infusing large amounts of either IgG or piece I11 derived from it, but not by injecting IgA or IgM; the removal of circulating protein by a process such as pinocytosis cannot easily account for such selectivity, and the presence of specific mechanisms controlling immunoglobulin breakdown appears likely (Fahey and Robinson, 1963). Normal subjects synthesize and break down about 2 gm. of IgG and 0.5 gm. of IgM per day, but in pathological conditions associated with hypergammaglobulinemia the absolute rate of immunoglobulin turnover may be increased as much as sevenfold (Cohen, 1963d; Birke et al., 1963; Solomon et al., 1963; Wochner et al., 1963). Increased rates of immunoglobulin formation presumably result from the replication of antibodyproducing cells but are not necessarily associated with an enhanced response to antigenic stimulation; for example, African children with hypergammaglobulinemia often show a relatively poor response to immunization with tetanus toxoid ( McGregor and Barr, 1962).

H. SYNTHESIS OF ANTIBODIES The structural studies described above indicate that the chains of immunoglobulin molecules are extremely heterogeneous. In particular, the polypeptide chain which carries the antibody-combining sites must occur in a very large number of different forms. The question of how many of these immunoglobulin chains can be synthesized by individuaI cells cannot be answered at present. The in vitro production of antibody by singIe lymph node cells indicates that a single cell can synthesize both A and B chains and assemble these into the complete antibody molecule. The possibility that some cells may synthesize B chain only i s suggested by the observation that Bence-Jones proteins sometimes have a difFerent InV speciscity from the corresponding myeloma protein; this

340

SYDNEY COHEN AND RODNEY R. PORTER

suggests that at least under pathological conditions the two proteins may originate in different cell clones (Harboe et d.,1962a). Myeloma proteins always belong to one or other of the three immunoglobulin types; since each type has a distinctive A chain, this suggests that the A chains of IgG, IgA, and IgM are synthesized in separate cells. Similarly, the relative homogeneity of myeloma B chains described above (Fig. 10) supports the idea that different forms of the normal B chains are derived from distinct cell types. In addition, analysis of the allotypic specificities of myeloma proteins suggests that individual cells synthesize molecules which have only a single form of allelic specificity (Harboe et al., 1962a). A similar conclusion is suggested by the finding that young rabbits whose mothers were immunized against a given allotypic specificity showed a diminution in molecules of that specificity, but no decrease in the molecules of an allelic specificity (Dray, 1962). On the other hand, immunofluorescent studies of intracellular y-globulin in the rabbit have shown that two allelic forms of the allotypic specificity determined by the b locus (A4 and A5) may be present in the same cell (Colberg and Dray, 1983). Since these allelic forms do not occur on the same molecule, Dray and Nisonoff (1963) suggested that chains controlled by a single gene form pairs immediately after synthesis. There is now a considerable amount of information about the antibody-forming capacity of individual cells, but it has not been possible to reconcile the different results which have been obtained. Nossal and collaborators (Nossal, 1962) found that cultures of single cells taken from rats immunized with a mixture of antigens almost invariably form detectable amounts of only one antibody; less than 2% of the cells tested were found to be doubly active. Attardi et al. (1959, 1984) also found that the majority of individual rabbit lymph node cells produced antibacteriophage antibody of a single specificity. However, in these experiments 10% of the cells studied were shown to form two antibodies; this incidence is considerably greater than would be expected if each diploid cell formed only two different antibodies. Thus, Attardi et al. (1964) point out that if an animal can make a total number of antibodies, S, and each cell can synthesize only two randomly distributed specificities, then the expected ratio of cells responding to one antigen, as compared to those responding to two antigens, would be 1/S. If, as is generally assumed, S > lo4 then 1/S < whereas the observed ratio was l O - l , i.e., the incidence of doubly active cells was at least a thousand times greater than expected. Unless immunization is associated with a selective proliferation of doubly active cells, then these results lend support to the view that individual antibody-forming cells are pluripotential.

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At present, therefore, it is not known whether every antibody-forming cell carries a very large number of genes controlling immunoglobulin synthesis, or has a limited genetic potential. Smithies (1963) has pointed out that a considerable degree of variability in the expression of the immunoglobulin genetic loci could arise by chromosomal rearrangements between sister chromatids or between single chromatids occurring during mitotic divisions of antibody-forming cells. There is evidence that such chromosomal rearrangements have given rise to variants of human haptoglobins (Smithies et al., 1962) and hemoglobins (Baglioni, 1962; Nance, 1963), but it should be pointed out that the complexity of antibodies is of a different order of magnitude from that observed in the case of these proteins. An analysis of the amino acid sequences of individual variants of antibody chains will be required in order to show whether their structural variations could have arisen from somatic rearrangements of genes occurring during the differentiation of immunoglobulin-forming cells. V. Comments

The multichain structure of antibody molecules has been established during the past few years. Correlation of the properties of the separated polypeptide chains with those of fractions obtained by enzymatic digestion has provided a diagrammatic picture of the basic immunoglobulin molecule consisting of two A ( H ) chains and two B ( L) chains. However, certain biological considerations suggest that A is two separate chains, so that the molecule may consist of six, rather than four, peptide chains. The structural relationship between the three types of immunoglobulins has been clarified by the identification of a common pair of peptide chains; if the six-chain structure is correct, then there may, in fact, be two pairs of peptide chains common to all immunoglobulins. There has been a satisfactory allocation of antigenic and genetic markers and of many other biological activities among the different parts of the molecule. However, the position of antibody-combining sites is still in doubt, and no clear concept of the structural basis of any biological activity has been suggested, The function of the carbohydrate of immunoglobulins is entirely unknown as, indeed, is true of most other glycoproteins. The structural complexity of all the immunoglobulins becomes increasingly apparent as further information is gained. The resolution of these multiple forms and the characterization of their chemical variations may be required before the structure of antibodies can be correlated with their activity. An understanding of the biological origin of this complexity

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may well be an essential preliminary to a solution of the mechanism of antibody function. In spite of the great complexity of the problem, it is encouraging that, at last, the accumulation of factual information has become more rewarding than the rephrasing of old theories.

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44. Steinberg, A. G., Stauffer, R., and Boyer, S. H. (1960b).Nature 188, 160. Steinberg, A. G., Wilson, J. A,, and Lanset, S. (1962).Voz Sangutnts 7, 151. Steiner, R. F.,and Edelhoch, H. (1962).J. Am. Chem. SOC. 84, 2139. Stemke, G.W. (1963).Ph.D. thesis, Univ. Illinois, Urbana, Illinois. Stemke, G. W. (1964).Personal communication. Stevenson, G. T. (1960).J. Clfn. Invest. 39, 1192. Stevenson, G. T. (1962).J. C h . Invest. 41, 1190. Taliaferro, W. H.,and Talmage, D. W. (1958).J. Infect. Dtseuses 99,21. Taranta, A,, and Franklin, E. C. (1961).Sctence 134, 1981. Thorbecke, G. J., and Franklin, E. C. ( 1961). J. Immunol. 87, 753. Thorbecke, G. J., Benacerraf, B., and Ovary, Z. (1963).J. Immunol. 91, 670. Tiselius, A., and Kabat, E. A. (1939).J . Exptl. Med. 69, 119. Todd, C. W. (1963).Btochem. Biophys. Res. Commun. 11, 170. Tomasi, T.B., and Zigelbaum, S. (1983).1. Clin. Invest. 42, 1552.

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Autoa ntibodies and Disease H. G. KUNKEL AND E. M. TAN The Rockefeller Institute, New York, New York

I. Introduction ............................................... 11. Widely Prevalent Autoantibodies .............................. A. Antinuclear Antibodies .................................. B. Anticytoplasmic Antibodies .............................. C. Anti-y-Globulin Antibodies .............................. 111. Specific Diseases Associated with Autoantibodies . . . . . . . . . . . . . . . . A. Rheumatoid Arthritis ................................... B. Systemic Lupus Erythematosus ........................... C. Thyroiditis ............................................ D. Pernicious Anemia ..................................... E. Myasthenia Cravis ...................................... F. Scleroderma and Dermatomyositis ........................ G. Pancreatic Disease ..................................... H. Addison’s Disease ...................................... I. Ulcerative Colitis ...................................... References ................................................

351 352 352 357 359 388 388 373 378 380 382 384 386 387 388 389

1. Introduction

An increasing body of evidence indicates that the occurrence of autoantibodies is by no means a rare phenomenon and that these may be found in the sera of patients with a wide variety of disorders. Nor is their distribution limited to disease; many and perhaps all normal individuals appear to possess such antibodies. It is becoming increasingly clear that the “horror autotoxicus” concept with its dire implications as elaborated by Ehrlich requires considerable revision. Many autoantibodies appear to be without harmful effects and the strong possibility exists, at least in certain instances, that they may actually be of benefit to the organism. Perhaps autoantibodies should be divided into two major groups, the rare pathogenic types and the more common ‘physiogenic autoantibodies.” The latter term has been applied to immunoconglutinin which appears to be an autoantibody to complement. Beneficial effects in the removal of bacteria have been demonstrated for immunoconglutinin in experimental animals (1).The same may well be true of the many types of anti-y-globulins. Their fixation to y-globulin as it coats organisms in the form of antibody could conceivably aid in the phagocytosis and removal of such organisms in a fashion similar to complement. Anti-y-globulins are found in all normal sera and reach extreme levels 351

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in the sera of patients with rheumatoid arthritis. In certain instances some secondary harmful effects may be produced, but in the vast majority of cases this is not apparent and consideration should be given to the possibility that they may be beneficial or at least represent a normal physiological response. Disagreement continues to exist concerning the use of the term autoantibody for many of the so-called lupus, rheumatoid, and other factors found in human sera. A major objective of this review will be the presentation of evidence for and against this concept. It should be pointed out that much of the argument against the autoantibody concept appeared before various classes of antibodies were clearly delineated. The fact that most of the anti-y-globulins are 19 S class proteins was initially used as a point against their being antibodies. Now, with the clear recognition that 19 S antibodies are an integral part of the immunological response to all antigenic stimuli, it favors the antibody hypothesis. Interest in these human factors actually played an important role in the elucidation of the entire immunoglobulin concept. The major portion of this review will be divided into two parts, with a broad discussion first of a number of widely distributed autoantibodies or presumed autoantibodies and a limited discussion of various specific diseases associated with such antibodies. The evidence for a possible relationship to the basic mechanism of the diseases will be presented. The field of autoantibodies to red cells and other blood cell elements will be omitted because of the breadth and highly specialized character of this work. In addition discussion of certain relevant disorders such as liver diseases has been omitted. II. Widely Prevalent Autoantibodies

A. ANTINUCLEAR ANTIBODIES Studies on the mechanism of the lupus erythematosus (LE) cell phenomenon have uncovered a whole spectrum of antinuclear factors in the sera of patients with systemic lupus erythematosus and a variety of other disorders (24). A number of these have been isolated and the evidence is overwhelming that these are antibodies; they will be termed such in this review. The following nuclear antigens have been clearly identified as reacting with separate antinuclear antibodies: (1)Deoxyribonucleic acid (DNA); ( 2 ) histone; (3) nucleoprotein; and ( 4 ) phosphate extract. This is a minimal number because additional evidence exists for several types of nucleoprotein antibodies, and the multiple components in the phosphate extract can be separated to reveal distinct

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antigens. Methods of complement fixation have proved the most useful for showing the multiplicity of antinuclear components but precipitin and fluorescent antibody methods have also been helpful. Table I illustrates the reaction of various sera with the different antigens. A specific profile of reactivity is obtained that is characteristic of individual patients and, to some extent, of their disease. The vast majority of lupus sera react with isolated nucleoprotein as demonstrated by complement fixation ( 4 ) and by fluorescent antibody studies on smears of nucleoprotein on glass slides ( 5 ) . A smaller number react with DNA, particularly after therapy with steroids. The reaction with isolated histone is unusual but definite in a few instances. The antibodies to phosphate extracts are widely prevalent in lupus sera and certain other conditions. They were first observed with complement fixation methods but recent evidence indicates that they are responsible for the speckled type of nuclear fluorescence (6, 7). This type of fluorescence is usually obscured by the diffuse nucleoprotein fluorescence of lupus sera. Absorption with nucleoprotein removes this interference and the speckled type remains in many sera (7) (Fig. 1).Some evidence is available from the fluorescent antibody method that specific antibodies for the nuclear membrane exist. The best evidence for the multiplicity of antigenic components in nuclei comes from the selective destruction of the antigens with enzymes (8). DNase, for example, destroys the complement fixation and precipitin reaction of DNA with lupus sera but has no effect on the phosphate extract reaction, The latter is specifically inhibited by treatment with periodate suggesting that a carbohydrate antigen is involved. The primary precipitin reaction with nuclear material is the wellstudied DNA reaction (3, 9). However, additional reactions in agar plates have been described ( 10-13). Ordinary isolated nucleoprotein has not been shown to give precipitins but disrupted nuclei have shown multiple lines which appear to be caused in part by nucleoprotein (11, 13).Recently, precipitin lines have been obtained with calf thymus nucleoprotein preparations in the author’s laboratory. A major problem is to get sufficient nucleoprotein in solution and in a form that will diffuse through the agar under pH and salt conditions which will still give antigen-antibody interaction. Isotonic saline and the usual buffered saline solutions used in immunological studies represent very poor solutions for nucleoprotein. The nuclear antigen which is found in isolated nucleoprotein and in most whole tissue and nuclear extracts is completely resistant to DNase but is somewhat trypsin-sensitive. Its exact nature is currently under study. The precipitin reaction with this antigen occurs much more frequently than that with DNA and the reaction is considerably more

TABLE I

COMPLEMENT FIXATION REACTIONSBETWEEN VARIOUS CELLFRACTIONS AND Two LUPUSSERAAND Two BILIARYCIRRHOSIS SERA Serum dilutions Serum SLE ( A.M. )

SLE (L.B.)

Biliary cirrhosis

LE cells

++++

+++

2

4

8

16

32

64

4 4 4 4 4

4 4 4 4 4

4 4 4 4 4

4 4 4

4 4

2 4

0 4

4 4 0 0

Nuclei

4 4 4 4 4

4 4 4

4 4

Nucleoprotein DNA Cytoplasm

4 4 4 4 4

2

0 4

Nuclei PO, extract Nucleoprotein DNA Cytoplasm

4 0 0 0 4

4 0 0 0 4

4

Nuclei PO, extract Nucleoprotein DNA Cytoplasm

4

4 0

4 0

4

0

0

4

4

3

0

0 4

0

3 0

4

4

PO, extract

0

( R.G.)

Biliary cirrhosis (L.S.)

Antigen used Nuclei PO, extract Nucleoprotein DNA Cytoplasm

++

4

4 4 0 0 0

3

3

0 0 0 4

256

512 0 0

0 0

0 0 0 0

4

4

3

2

4 4 0 0 4

4 4

2 3

0 0 4

0 0 4

0 0 0 0

o 0 o 0

5

2

1 0 0 0 4

0 0 0 0

0 0 0 0

0 0 0 0

O O

m Z

4 1

3

1

4

4 3

0

0

0

0

0

0

1

0

0

0

0 0

0 4

0 4

0

3

128 3 3

3

0 2

O O

F P

j r

0

-

o

g

0

0 2

FIG. I . Various types of fluorescent staining with different lupus sera. Indirect immunofluorescent technique using mouse kidney section as substrate, patient’s serum, and fluorescein-conjugated anti-human y-globulin. The center picture illustrates homogeneous nuclear staining; upper left, nuclear membrane; and upper right, speckled or patchy stains. Lower left picture shows staining of nuclei and peritubular connective tissue; and lower right, nuclear and cytoplasmic staining.

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intense. Figure 2 illustrates the strong band between a leucocyte nuclear antigen and the serum of a patient with lupus erythematosus compared to the negative reaction with various control sera. Approximately twothirds of the lupus sera give the reaction with this antigen to various degrees. Other precipitin reactions with cell extracts also may be due to nuclear antigens but some are certainly cytoplasmic (12, 13). The LE cell factor appears to be most closely related to the antibodies reacting with nucleoprotein. Absorption of lupus sera with either DNA or histone alone does not remove the LE cell factor, but absorption with the nucleohistone combination is effective (8, 14). It would appear that

FIG.2. Precipitin line between the serum of a patient with SLE (bottom well) and a glycine buffer extract of lymphocytes. Various control sera are shown in the other outside wells.

the reaction requires both constituents of the nucleoprotein, and, therefore, perhaps represents an antibody directed against the point of union. Nucleoprotein absorbs considerable protein from certain lupus sera and current evidence indicates that the LE cell factor and the DNA and histone antibodies represent only a portion of the nucleoprotein antibody. These antibodies are readily isolated through the action of DNase on the nucleoprotein antigen. The isolated DNA antibody fails to produce LE cells even in high concentration ( 9 ) .

DNA Antibodies The initial finding that DNA antibodies occurred in some lupus sera was made independently in three laboratories: Seligmann ( 3 ) , Celada and Ceppellini ( 15), and Holman and associates (16, 17) all reported in 1957 immunological reactions with DNA. The previous complete lack of success in producing such antibodies in experimental animals made

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H. G. KUNKEL AND E. M.TAN

these observations all the more significant. These were demonstrated by precipitin and complement fixation reactions and later by tanned cell agglutination (18),complement consumption tests ( 19), and coated particle agglutination (20). One major problem in this work is that the acidic DNA molecule will not only precipitate but also fix complement with normal y-globulin under certain pH conditions. However, in alkaline buffers, above the isoelectric point of the y-globulins, precipitins develop only with y-globulin from lupus serum and similarly specific complement fixation reactions are observed (9). Numerous precipitin curves have been published of the reaction of DNA and lupus sera. The curves differ considerably for various sera, but a constant finding is the very high ratio of protein to DNA. Figures as high as 5000 for the molar ratio of protein to DNA have been obtained, This is very difficult to understand and raises the possibility of coprecipitation by nonspecific y-globulin. However, this has not been demonstrated and the alternative explanation of a vast number of antigenic sites on the large DNA molecule is not unreasonable. Precipitin curves have failed to distinguish different types of DNA, and single-stranded heat-denatured DNA gives the same results as native DNA. Strong confirmatory evidence that the sera of patients with lupus erythematosus do, indeed, possess antibodies to DNA has arisen from the recent work of Levene and associates (21, 22). Quantitative complement fixation curves at various concentrations of DNA have furnished many new observations that were not apparent in the earlier precipitin and complement fixation studies. Most striking was the great superiority of denatured single-stranded DNA as antigen as compared to native DNA. In addition, inhibition of complement hation was obtained with a wide variety of nucleotides and purine derivatives. Strong inhibition was obtained with oligonucleotides isolated from DNase digests of calf thymus DNA. Inhibition was also obtained with compounds such as theobromine, f3-mercaptopurine7isoguanine, purine, adenine, xanthine, and chloroquin. Certain lupus sera showed antibodies which were directed primarily toward adenine whereas others were more specific for thymine. It appeared clear that the antibodies were specifically directed against the purine and pyrimidine bases in DNA. This explains the greater reactivity with single-stranded DNA because of the exposure of these groups which are buried in the ordinary double-stranded helical configuration. It seems probable that the early precipitin work dealt with antibodies against a different portion of the DNA molecule. Some support for this concept arises from the strong inhibition of precipitation by means of substances binding the phosphate groups of DNA such as histones and certain basic

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dyes (9). The accumulated evidence indicates that lupus sera contain a variety of antibodies directed at a wide variety of sites on the DNA molecule, also against the histone moiety of the nucleoprotein, and even some antibodies that require the intact nucleoprotein. It is of special interest that the DNA antibodies are highly specific for the sera of patients with lupus erythematosus, and all workers agree that they have not been found in other conditions with the possible exception of rare cases of rheumatoid arthritis with other manifestations of systemic lupus. These antibodies do not seem to be a by-product of hyperimmunization or to be nonspecifically associated with hypergammaglobulinemia. In this respect they differ from the antinuclear antibodies detected by fluorescent antibody methods which are very broadly distributed. The recent report of DNA antibodies in rabbits after long immunization with bacteria may prove of interest in this connection (23).

B. ANTICYTOPLASMIC ANTIBODIES The distinction of nuclear and cytoplasmic antibodies in the sera of patients with systemic lupus and other disorders is not an easy one, primarily because there are some very reactive antigens that are readily extracted from nuclei by ordinary buffers. The phosphate extract antigens are typical examples, and these contaminate most cytoplasmic fractions. However, it is now clear that a number of different cytoplasmic antibodies do exist. Figure 1 illustrates one type which reacts with the basement membrane of certain cells and is found in occasional lupus sera. Another type shows diffuse fluorescence of the cytoplasm of a variety of cells. This group has proved very difficult to pin down, primarily because of some background fluorescence of cytoplasm with normal serum. Complement fixation reactions with various cytoplasmic cell fractions have indicated the presence of y-globulins reacting with microsomes, mitochondria, and other elements (24, 25). Antigens can also be extracted with lipid solvents which react with certain lupus sera (25). These reactions are considerably less specific for systemic lupus than certain of the nuclear reactions and are found in many disorders such as liver disease, Sjogren’s syndrome, scleroderma, and a variety of hyperglobulinemic states. The widest experience has been gained with a complement fixation reaction with whole saline extracts of liver, kidney, or spleen tissues. It has been known for many years that a variety of animal and human sera fix complement with such extracts (26, 27). Normal rabbit sera and some normal human sera also show similar reactions. FIocculation reactions

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H.C. KUNKEL AND E. M.TAN

have also been described, particularly with chicken sera (28). These are increased with immunization and can be absorbed out with the respective antigens involved in the immunization. Considerable emphasis has been placed on the complement fixation reaction, in particular in respect to the proposal of an autoimmune concept of liver disease (29). The reaction has been termed “autoimmune complement fixation” ( AICF). High titers have been observed in certain patients with liver disease. Biliary cirrhosis cases have shown particularly high titers (25, 29) (Table I ) . It has been reported that this reaction shows isospecificity and the factors react poorly or not at all with the patient’s own tissues (30). This may apply in some instances, but studies in the authors’ laboratory have shown that it is by no means the usual phenomenon. A number of investigators have been concerned with the possibility that the AICF reaction may not represent a reaction of tissue antigen with antibody. Other standard immunological procedures have not detected antibodies which run parallel. Recent work with precipitin reactions described above have shown a number of precipitating systems, but the most prevalent of these, at least in lupus sera, has turned out to be nuclear in origin. Some of these do appear to be cytoplasmic, but they do not follow the serum distribution of the complement fixation reactions. The complement fixation reactions also show a general correlation with elevated y-globulin levels regardless of the disease involved. Certain sera from patients with Waldenstrom’s macroglobulinemia show extreme titers in the complement fixation reactions (29). One such serum has been studied in the authors’ laboratory which fixed complement with a wide variety of tissues and tissue fractions; this appeared to be completely nonspecific and there was much to suggest that this did not represent a true antigen-antibody reaction. The suggestion has been put forward that there is a y-globulin-like component in tissues that aggregates in the presence of certain sera and fixes complement like y-globulin aggregates ( 31) A somewhat more plausible hypothesis is that there are certain yglobulins that show a strong tendency to react with tissues. These have been encountered in fluorescent antibody studies and have led to the standard absorption with tissue powders. Recent studies have clearly defined such cell-binding y-globulins and antibodies ( 32). They appear in rabbit sera following simple immunization and can be detected by the adsorption of labeled antigens, such as albumin, to cells that have absorbed the antibodies. The cell-binding antibodies represent only a small fraction of the total antibodies and therefore have distinct characteristics. However, it is clear that these antibodies do not bind tissues

.

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359

because of an antigen located there. No studies have been made to see if these systems fix complement. It is well known that certain y-globulins

show a great tendency to aggregate to a state where they fix complement; others have been found that bind lipids readily. Such proteins might well react with tissues in a manner which causes complement to be fixed. Consideration should be given to the possibility that the so-called autoimmune complement fixation reaction with whole saline extracts of tissues may well represent the measurement of a special property of certain y-globulins rather than a reaction with an antigen in these tissues.

C. ANTI-Y-GLOBULIN ANTIBODIES Interest in the rheumatoid factors which are found in high concentration in the sera of many patients with rheumatoid arthritis stimulated considerable work on a variety of anti-y-globulins, which now clearly appear to be antibodies directed against y-globulin. As in the case of the lupus factors, the evidence that they are antibodies is now very considerable (33-35). A number have been isolated and they show all the physical, chemical, and antigenic properties of classic 19 S antibodies; the characteristic sedimentation rate, the dissociation with mercaptoethanol, the high carbohydrate content, the cross reaction with 7 S y-globulin in the L chains, the antigenic individuality in the H chains, the Groups I and I1 antigens, and many other properties. Their fine specificity for a variety of determinants on the y-globulin molecule, particularly the Gm genetic factors, answers the essential specificity criterion of antibodies. The early belief still held in some quarters (36) that they represent a complementlike component which fixes to y-globulin, appears increasingly improbable. The recent work on the ready production of similar antibodies following reinjection of slightly altered autologous y-globulin in rabbits has aided considerably in understanding the human antibodies ( 37-39). The following is a list of the various main types found in human sera: 1. Antibodies to y-globulin genetic characters: isospecific and autospecific 2. Rheumatoid factors with primary specificity for antigenantibody complexes and aggregates of y-globulin 3. “Anti-antibodies’’ or “Milgrom factors” 4. Antibodies to buried determinants revealed by enzymatic splitting

1. Anti-y-Globulins with Gm and 1nV Specificity Following the initial demonstration of Grubb (40) on the detection of genetic differences between the y-globulins of different individuals

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through the use of naturally occurring anti-y-globulins, great progress has been made in the elucidation of the genetic systems involved. Much of this work has been reviewed recently by Steinberg (41). Groups of alleles at two genetic loci (the Gm and InV loci) have been delineated. Relatively little attention has been paid to the significance of the occurrence of the anti-y-globulins which have proved so useful for these genetic studies. The vast majority of the work has been carried out with the human antibodies, although it is now clear that similar antibodies can be produced in primates, which should prove of considerable value in future work (42, 43). Rabbit antisera have thus far failed to differentiate any of the genetic types of human y-globulin. The anti-y-globulin sera which were used as reagents in the initial studies came primarily from rheumatoid arthritis sera. In the last few years, however, it has become clear that anti-y-globulins, found in a small percentage of normal individuals, possess many advantages (44, 41). The two types appear- quite different in a number of properties, and it now appears probable that the mechanism by which they are produced is also different; they have been termed “serum normal agglutinator” ( SNagg ) and “rheumatoid agglutinator” ( Ragg) , The various anti-Gm factors have been found in both groups but the anti-InV antibodies have not thus far been found in rheumatoid arthritis sera. The primary differences (45, 48) between the two types of anti-Gm factors are the following: 1. The system employing the SNagg reagents shows much greater specificity for Gm(a+) y-globulin, whereas in the Ragg system some inhibition is obtained by Gm( a-) y-globulin. 2. Aggregation of Gm( a-) y-globulin causes it to become considerably more inhibitory in the Ragg system, whereas this treatment has relatively little effect on the inhibition capacity in the SNagg system. Gm( a+) y-globulin also becomes more inhibitory after aggregation in the Ragg system. 3. The difference between unmodified Gm(a+) and Gm(a-) yglobulin is much greater in the SNagg system, being of the order of 500to 1000-fold compared to 32- to 64-fold for the Ragg system. 4. The SNagg type of anti-y-globulin is always isospecific, whereas the Ragg type may be either auto- or isospecific. The last point concerning iso- and autospecificity is of considerable interest and of particular relevance to this review. Several major developments in the last year have thrown considerable light on the mechanism of development of the SNagg-type anti-y-globulins. It has become clear

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that these arise from immunization with foreign y-globulin either from external sources, as in y-globulin injections and transfusions, or by the mother’s y-globulin. This readily explains the clear isospecificity that is always found, and these anti-y-globulins are analogous to the allotype antibodies produced by Oudin (47) by the injection of y-globulin from one rabbit into another. The study of children who have received multiple transfusions has indicated that isospecific anti-y-globulins develop in a high proportion of Gm(a-) recipients (48). Anti-Gm(a), antiGm(b), and anti-Gm(x) antibodies have been found in an incidence as high as 70 % of negative recipients. Titers as high as lj4080 were obtained but no precipitating antibodies were detected. Several unexplained features concerning these results with multiple transfusions have become apparent (49). First, not all children develop these antibodies despite many injections of the foreign y-globulin. Second, no anti-InV antibodies have been encountered. Third, adults appear to be much more refractory to the formation of these antibodies, Recent studies of older patients with hemophilia and leukemia have revealed a much lower incidence of anti-Gm factors than in children with thalassemia. The question of whether the disease plays any role in the latter incidence is not clear. Certainly they are found in children receiving transfusion €or other causes. Serial studies have shown that the antibodies do develop in recipients who are initially negative or weakly positive and then develop significant titers after further transfusions. The problem is somewhat complicated by the effects of Gm( a + ) transfused blood on the antibodies where transfusions are given at close intervals. Undoubtedly, some of the SNagg reagents utilized in the past arose through the injection of foreign y-globulin; one widely used reagent came from a patient with leukemia who received multiple transfusions (50). However, in many instances it is not possible to obtain a history of injection of foreign y-globulin despite the existence of these antibodies. The obvious alternative source of the foreign y-globulin that might have elicited these antibodies is maternal y-globulin. Evidence is now available that this is indeed involved. In a recent study (51) of the mothers of individuals possessing SNagg-type antibodies, a statistically significant occurrence of the corresponding Gm-positive y-globulin was found. These observations have been confirmed in a direct study of Gm-positive mothers with Gm-negative offspring (49). A very high incidence of anti-Gm antibodies are found, These usually appear in the first year of life, some months after the maternal y-globulin has disappeared from the circulation. These observations raise many important questions. Why are these

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offspring who are exposed to maternal y-globulin for a prolonged period in utero not tolerant to this type of y-globulin? Instead, a high incidence of antibodies occurs. Why do these antibodies persist for such prolonged periods without restimulation? Part of the answer to these questions derives from the fact that these antibodies are primarily of the 19 S type which are known to develop relatively early in life. However, these antibodies require persistent antigenic stimulation for the maintenance of continued levels and this cannot be explained in the present state of our knowledge. Isospecificity of anti-Gm factors is also frequently found for the factors in rheumatoid arthritis sera (50, 52). No evidence is available regarding a possible maternal role in their development, but this possibility remains. The fact that these isospecific anti-Gm factors differ considerably, as described above, from those encountered in normal sera suggests a different mechanism of production. In addition, it is now clear that autospecificity is frequently found in rheumatoid arthritis (50, 52). It has been known for a number of years that certain rheumatoid arthritis sera showed marked prozone reactions in the Gm system (53). This was demonstrated to be due to inhibition by the patient’s own y-globulin. Isolation of the rheumatoid factors (a), or simple purification by euglobulin precipitation (50), showed the anti-Gm specificity which was frequently obscured by the inhibiting y-globulin in whole serum. Following such procedures an incidence of approximately 50 % was found for specific anti-Gm agglutinators in Gm-positive individuals. Thus, clear autospecificity even in the Gm system has been shown for rheumatoid factors. The accumulated evidence indicates that anti-Gm factors are found frequently in rheumatoid arthritis sera which possess either iso- or autospecificity. These results, which appeared so mysterious a few years ago, are now more readily understood with the elucidation of the normal isospecific agglutinators and their clear differentiation from both types of rheumatoid arthritis agglutinators. The latter with their markedly lower degree of anti-Gm specificity are probably produced against autologous y-globulin which is altered in some fashion. They may be directed against a site close to the Gm site which is influenced by the Gm character of the y-globulin and thus dserentiate in a partial fashion between Gmpositive and Gm-negative y-globulin. Evidence for a concept of this sort stems from the finding that the vast bulk of nonspecific rheumatoid factors are directed against the F fragment of human y-globulin produced by papain (55, 56). This is also the site of the Gm factors. In addition, recent experiments in rabbits indicate that the injection of altered autol-

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ogous y-globulin produce antibodies which appear to distinguish genetic types of y-globulin (57). 2. Rheumtoid Factors The vast bulk of the anti-y-globulins found in the sera of patients with rheumatoid arthritis do not show specificity for genetic factors. It is possible that some of these in the future will prove specific for new genetic determinants, but almost certainly many will remain which are directed against other sites. Figure 3 is a diagrammatic sketch of the 7 S y-globulin molecule which encompasses a possible three-type chain structure. It has become clear that the F fragment produced by papain con-

B

a I

A A

B

f

7 ?

e I

I

S I

f t

d c b

s

e

S I S

d c b

L

Q

S

F

FIG.3. Tentative diagrammatic representation of the y-globulin molecule showing the A and B chains linked by disulfide bonds and the S and F fragments produced by papain. Sites a-f illustrate the approximate areas of the molecule with which the different anti-y-globulins react.

tains the primary determinants with which the rheumatoid factors react (55, 5 6 ) . This is indicated by site c on this chain which is close to site b, the Gm site discussed above. This corresponds to fragment 3 in rabbit y-globulin in the Porter nomenclature, and rheumatoid factors directed against rabbit y-globulin have been found to react with fragment 3 (58). A certain amount of evidence suggests that the F component and fragment 3 may represent independent polypeptide chains although thus far they have not been separated from the rest of the A chain without enzymatic splitting. Observations with whole A chains put on tanned cells indicated strong agglutination with rheumatoid arthritis sera (59). Inhibition of the reaction with whole y-globulin is marked with H chains and also F fragment with very little inhibition with B chains; B chains put on tanned cells gave some agglutination with rheumatoid arthritis sera which was specifically inhibited with B chains. Anti-InV factors that react with the B chains have not been found in rheumatoid arthritis sera.

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Numerous experiments have demonstrated that a number of different rheumatoid factors can be separated by various column procedures. The primary separation in all this work represents factors reacting primarily with human y-globulin from those reacting primarily with rabbit y-globulin. Early work with DEAE chromatography separated one factor which reacted only with human y-globulin and another which reacted with both human and rabbit y-globulin (60, 61). Later work with polystyrene columns to which human Qr rabbit y-globulin were coupled indicated clearly that rheumatoid factors with at least three different specificities could be separated (62). Two of these are indicated in Fig. 3. Particularly striking was the recovery of a fraction that showed no reaction with human y-globulin when it coated cells or latex particles but reacted strongly with rabbit y-globulin on cells. It might be thought that such a finding is hard to reconcile with an autoantibody hypothesis and that it should react with human y-globulin if this represents the antigen. Careful inhibition experiments, however, with aggregated and otherwise altered human y-globulin indicated that there was inhibition with this material in the rabbit y-globulin system but not with native human y-globulin. Thus it was apparent that the strong reactivity with rabbit y-globulin could be explained on the basis of cross reactivity with buried determinants in human y-globulin which represented more exposed codgurations on rabbit y-globulin. Other workers (63, 64) have reached similar conclusions through the use of gel-diffusion experiments with y-globulin aggregates and absorption techniques with human antigen-antibody complexes. In addition, earlier studies (33) had demonstrated the isolation of rheumatoid factors reacting with rabbit y-globulin through the use of aggregates of human y-globulin. The experiments in rabbits (37, 39) where, following the administration of altered autologous y-globulin, strong reactivity with human y-globulin was obtained represent just the reverse of the situation in regard to the human rheumatoid factors. They add considerable validity to the interpretation that at least some of the rheumatoid factors represent antibodies to relatively buried antigens in autologous y-globulin. The question of just what form the autologous y-globulin is in when it acts as antigen in the production of rheumatoid factors has intrigued a number of workers. The strong reactivity of rheumatoid factors with aggregated y-globulin in inhibition experiments and in precipitin reactions suggests that this form which is closely analogous to antigen-antibody complexes might be involved. This is very possible. However, the observations in the rabbit that enzymatically split, autologous, y-globulin is equally effective in eliciting similar antibodies raises new possibilities

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(39). Unfortunately it has not been possible to decide which procedure, aggregation or splitting of autologous y-globulin in the rabbit, gives rise to antibodies closest to those in rheumatoid arthritis sera. The two results are very similar. Long-term intravenous administration of killed Escherichia coli in rabbits has also given rise to 19 S y-globulins which are closely analogous to rheumatoid factors (38). It seems highly likely that these result from autologous antigen-antibody complexes which produce secondary antibodies. The greater predominance of 19 S antibodies following this procedure, as compared to the findings after altered autologous y-globulin in Freunds adjuvant, may well result from the different route of administration. Despite the primary reactivity with aggregated y-globulin and antigen-antibody complexes, ultracentrifuge evidence indicates that there is also some reactivity of the vast majority of rheumatoid factors with native y-globulin. This is strikingly demonstrated by the 22s complex which is characteristic of sera with large amounts of rheumatoid factor (65,67). This indicates reactivity with autologous y-globulin. Isolated preparations of rheumatoid factor when added to y-globulin also form similar complexes (52). These complexes appear quite labile as if the affinity is relatively weak, and certain evidence with labeled y-globulin indicates an equilibrium reaction. A few Waldenstrom macroglobulins that fail to complex with y-globulin have been encountered with rheumatoid factor activity (68). However, even some of these show complexing if the pH is optimal. It seems probable that some rheumatoid factors will be encountered that fail to show this apparent nonspecific complexing, but what is surprising is the constancy of this finding and the apparent nonspecificity of the reaction in regard to genetic type and other characteristics of y-globulin. Complexes have also been obtained with the F fragment of y-globulin ( 5 6 ) . Complement is not fixed in these reactions. The vast majority of the rheumatoid factors that are measured by the various test procedures are 19 S y-globulins. However, considerable evidence indicates that there is also a spectrum of 7 S type factors in rheumatoid arthritis sera that complex with y-globulin in a fashion similar to that described above (66, 69, 70). “Intermediate complexes,” as these have been termed, are characteristic of rheumatoid arthritis sera where high titers of rheumatoid factors exist. These may reach extreme levels in certain sera and, in general, represent the dominant abnormality in the gross protein pattern of such sera. The major problem in studying these apparent 7s factors which form complexes is that they do not react in the ordinary serological tests. Recently, latex fixation activity has been found for such 7 S rheumatoid factors (6Q),but this experience

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is not universal. It is possible that these factors are much more readily inhibited by the serum y-globulin than is the case with the 19 S factors.

3. Similar Factors in N mml S e w and Those of Other Diseases The initial impression that rheumatoid factors occur at very low incidence in normal individuals and patients with diseases other than rheumatoid arthritis has been modified considerably by recent investigations. It is now clear that disorders such as liver disease (71), subacute bacterial endocarditis (72), parasitic disorders, and certain other diseases (73) may have an incidence as high as 50 %. In some instances in these and other conditions extreme levels are sometimes found. However, as a group the rheumatoid arthritis patients still stand out in respect to incidence and titer over all other conditions thus far studied. There is one striking difference between the type of rheumatoid factors observed in the rheumatoid arthritis group as compared to other diseases: the factors that react with human y-globulin show a marked preponderance in the nonrheumatoid states and those reacting with rabbit y-globulin are much more characteristic of rheumatoid arthritis (73). There are many exceptions to this generalization, but it suggests that there are basic differences in the mechanism of antigenic stimulation. The studies in bacterial endocarditis have been particularly revealing in regard to the production of rheumatoid factors. Here, chronic bacterial infection in the presence of bacterial antibodies gives rise to high levels of rheumatoid factor for prolonged periods-a picture in many ways analogous to the situation in rheumatoid arthritis. However, following specific antibacterial therapy, this level falls gradually in the majority of patients and frequently reverts to normal (72). It appears that the antigenic stimulus of the organism is essential for the persistent levels and where this is removed the level falls. In striking contrast, the patient with rheumatoid arthritis keeps a persistent high level for many years. Similar antibacterial therapy has no effect on these titers. This type of indirect evidence suggests that there is a very persistent antigenic stimulus in these patients in order to sustain such levels. It has long been known that certain normal sera contain rheumatoid factors in an incidence of as high as 5 %. Recently, it has been shown that this incidence increases considerably in older age groups (74). Also, certain populations have a higher incidence although the role of parasitic disease and other factors in such groups is difficult to evaluate. Of particular importance is the observation that all normal individuals possess definite rheumatoid factors that can be isolated very readily. In a study on the isolation of the 11S component of complement by pre-

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cipitation with soluble aggregates, it was found that a 1 9 s y-globulin always occurred in those precipitates which could be isolated by acid elution (75). This material gave strong reactions in latex fixation tests and in the agglutination of cells coated with incomplete Rh antibodies (76). It is of special interest that these were all directed solely against human y-globulin with no evidence of reactivity toward rabbit y-globulin. They thus resembled the anti-y-globulins found in nonrheumatoid sera and one of the factors found in the sera of patients with rheumatoid arthritis. 4. Anti-y-Globulins of the Nonrheumatoid Type ( Milgrom Factors) One of the major characteristics of rheumatoid arthritis sera and rheumatoid factors is the great variation in the agglutination obtained with difTerent incomplete Rh coats attached to red cells. Rh antibodies from one serum may serve as excellent coats for one rheumatoid arthritis serum but as a very weak coat for another serum of similar titer in respect to other rheumatoid factor tests. There is great selectivity of reaction which extends well beyond the anti-Gm character of these anti-y-globulins (52). Some coats, like Ripley and Murphy which are widely used in studies of rheumatoid factors, react with almost all rheumatoid arthritis sera in striking contrast to certain other anti-Rh sera of similar titer in the Coombs reaction. No clear explanation for this reactivity is at present available. It was thought initially that it might be because these sera would be heterozygous for all the genetic factors. However, this is not entirely the case. It may be related to long-term immunization of these individuals with a greater variety of antibodies for the different rheumatoid factors to combine with. One clue may come from the fact that anti-Rh antibody Ripley shows the unique ability to fix complement when attached to cells (77). In striking contrast to this selectivity of reactivity with different Rh coats for rheumatoid factors, a number of other factors originally described by Milgrom (78) have been encountered in normal sera which react with all coats nonselectively like a rabbit Coombs serum. These are not inhibited by pooled y-globulin but only by y-globulin in the aggregated form or complexed to antigen. Studies with individuals receiving multiple transfusions have revealed a high incidence of these factors (49). Recent evidence suggests that these anti-y-globulins owe their unusual characteristic to the fact that they are directed against a different portion of the y-globulin molecule than the rheumatoid factors (79). This is illustrated in Fig. 3 by site f. Some points regarding this localization require further clarification because these factors do not react with the S frag-

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ment of Rh antibodies (80) as would be expected from the reported localization (79). 5. Antibodies to Buried Determinants of y-Globulin It appears clear that many of the rheumatoid factors represent antibodies to buried determinants in the autologous y-globulin which react better with the y-globulin of other individuals and of other species. However, another anti-y-globulin has been encountered in some normal sera and also in the sera of patients with other diseases, which only reacts with enzyme-split y-globulin (55). There is no reactivity with whole y-globulin regardless of the source. This factor is brought out through the use of pepsin digests of incomplete Rh antibodies. This treatment with or without cysteine gives 5 S or 3.5 S fragments which contain the antibody site. The S fragment produced by papain under acid conditions also gives rise to similarly active fragments. Both of these types on red cells are agglutinated by certain sera but, since the F fragment is lacking, these are not agglutinated by the bulk or rheumatoid factors. The specificity of the anti-y-globulin directed against the S fragment was brought out by inhibition experiments. There was no inhibition by whole y-globulin but strong inhibition by the S fragment of y-globulin. The incidence of this anti-y-globulin was approximately 20 % in normal sera, 43 % in bacterial endocarditis sera, and 57 % in rheumatoid arthritis sera. The highest titers, up to 1/640, were encountered in the disease groups. Similar factors have been described in the sera of rabbits (59). The most characteristic feature of these anti-y-globulins was that they were almost entirely 7 S in character in striking contrast to all the other anti-yglobulins described above. Recently Williams ( 59) has demonstrated other factors reacting only with isolated L chains. 111. Specific Diseases Associated with Autoantibodies

A. RHEUMATOIDARTHRITIS The large amount of evidence indicating that the rheumatoid factors represent autoantibodies to y-globulin might appear to strengthen the hypothesis that rheumatoid arthritis represents some type of autoimmune disorder. However, this is scarcely the case and it has become clear that the rheumatoid factors could readily develop as a response to some type of foreign organism. Antibodies to the organism, following reaction with the particular antigen or fragments thereof, would form secondary anti-yglobulins to these antigen-antibody complexes. This possibility has been strengthened by the experimental production of anti-y-globulins in rabbits following injection of coliform organisms (38), as well as their

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appearance in human sera following various types of infection. It appears very possible that there is some type of antigen-antibody complex which develops in rheumatoid arthritis, which acts as a constant stimulus to the production of the various anti-y-globulins, The nature of such a hypothetical antigen, whether it is a foreign organism or an autologous tissue constituent, represents a most important unanswered question. The study of the rheumatoid factors, particularly their production in experimental animals with various types of y-globulin and y-globulin complexes, represents a possible avenue of investigation to get to the nature of the possible antigen involved. The demonstration that antibodies develop readily to autologous yglobulin if this is split enzymatically (39), raises the possibility of a totally different mechanism for the development of the rheumatoid factors. Here a completely nonimmunological process such as increased proteolytic activity, perhaps in the joint space, might produce y-globulin fragments which in turn would be antigenic and produce the anti-yglobulin. It is evident that a wide variety of mechanisms could conceivably be involved in altering the autologous y-globulin. An overwhelming body of evidence is now available suggesting that the rheumatoid factors themselves do not play a direct role in the joint lesions of rheumatoid arthritis. They have been found at high levels in other disorders without arthritis and even in certain normal individuals. One normal individual has been studied particularly who has had an extreme titer of rheumatoid factors of both human and rabbit specscity for a number of years (81). The serum which has also been studied in the authors' laboratory shows a 22 S component in the analytical ultracentrifuge, and in all respects the serological studies resemble those found in many cases of rheumatoid arthritis. Yet this individual shows no disease. A picture closely resembling rheumatoid arthritis has also been described in patients with agammaglobulinemia where no rheumatoid factor is detectable in the serum (82). In addition, infusion of serum containing a high content of rheumatoid factor has failed to produce symptoms (35). Despite this cumulative evidence against a direct role of the rheumatoid factors there exist some findings which require further consideration. Recent work indicates that peripheral blood leucocytes may carry rheumatoid factors which are readily detectable by fluorescent antibody techniques when they are not detectable in the serum (83). There is also considerable evidence for local rheumatoid factor production in the joints themselves, Certain patients have higher titers in the joint fluid than in the serum (84, &S), and fluorescent antibody studies of plasma cells

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in synovial membranes indicate production of rheumatoid factors (86). Recently, an unusual cell, termed the “R.A.” cell, has been consistently observed in the joint fluid of rheumatoid arthritis patients (87). This appears to be a phagocytic cell that has engulfed rheumatoid factor complexes. Disruption of this cell has shown rheumatoid factor activity even when it is absent in the joint fluid or serum (88). There also is evidence for local complement consumption because of low levels in the joint fluid of rheumatoid arthritis patients as compared with serum and the joint fluid of patients with other diseases (89). It has become clear that future studies will need to be directed more toward the findings in the joints with less emphasis on the peripheral blood. A possible hypothesis that might be considered is that with the strong stimulation to the production of various anti-y-globulins some are produced that might precipitate with autologous y-globulin and fix complement and cause considerable local injury. The anti-y-globulins that are found in the peripheral blood are the unimportant ones which either form benign soluble complexes or are directed against buried determinants of y-globulin and therefore have little or no affinity for the native molecule. Local phagocytosis of y-globulin-anti-y-globulin complexes would stimulate the further production of anti-y-globulins leading to a type of vicious cycle. It also seems possible that perhaps the various polysaccharides in joint fluids might, under certain conditions, complex or alter y-globulin enough to expose antigenic determinants which might initiate the above cycle of events. A certain amount of evidence is available suggesting a more direct role of the rheumatoid factors in some of the secondary manifestations of rheumatoid arthritis. Numerous observers have demonstrated close correlation of subcutaneous nodules and high titers of rheumatoid factors (90, 91). In fact, the finding of nodules almost guarantees high levels in the serological reactions. However, the association does not necessarily hold in the other direction; many individuals have high titers without nodules. Recent observations in children with rheumatoid arthritis have again brought forth this relationship; both findings are relatively uncommon but they occur together in striking fashion. Some evidence has been obtained recently by fluorescent antibody studies for rheumatoid factor deposits within the nodules (92). Another association of a somewhat similar type is the almost invariable finding of very high levels of rheumatoid factors in patients with rheumatoid arthritis who show manifestations of Felty’s syndrome (91). Here again the correlation is unidirectional, with many individuals having high serological reactions without the splenomegaly, leukopenia, and anemia characteristic of this syn-

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drome. It seems possible that the splenomegaly here is a direct result of the marked proliferation in the spleen of plasma cells which produce the large amount of rheumatoid factor, and the manifestations of hypersplenism are simply the result of this enlargement. Recently, there has been considerable interest in the possibility that some of the serious generalized vascular manifestations found in certain patients with rheumatoid arthritis may be related to rheumatoid factor or its complexes with the patient’s y-globulin (93). The majority of these patients show extreme changes in the serological reactions and many have very obvious alterations in their ultracentrifuge patterns. Certain patients studied in the authors’ laboratory have had high levels of “intermediate complexes” as well as “22 S complexes.” However, again, some patients show similar ultracentrifugal findings without the severe vascular alterations. Long-term followup of such patients is required in view of several recent experiences with such patients who developed vascular and pulmonary complications several years after the initial finding of many complexes in the serum. Actually, it is surprising that they do not have more difficulty with such relatively insoluble complexes constantly in their circulation; the absence of renal manifestations is particularly evident, It seems probable that most of these complexes fail to fix complement as indicated by the normal or elevated levels in the serum of these individuals and this may be the reason for their relatively benign character most of the time, The impression of numerous workers that steroid administration also plays a role in the severe vascular changes (94) is difficult to fit into the picture with regard to the high levels of complexes. Pulmonary alterations also have been described in connection with high levels of rheumatoid factors and intermediate complexes (95). Perhaps the most suggestive evidence for a derangement in rheumatoid arthritis that might be immunological in character stems from its association with various other disorders where an immunological basis is more apparent, This is particularly true of systemic lupus erythematosus. Manifestations of this disease have been reported frequently in patients with rheumatoid arthritis, In addition, serological reactions with nuclear antigens and antinuclear antibodies detected by immunofluorescence have an impressive incidence in these patients. Several reports have appeared recently describing an incidence of 35-50 % for antinuclear factors (96, 97). In the authors’ laboratory this incidence is approximately 40 %, with some patients with severe rheumatoid arthritis showing extremely high titers. Complement fixation reactions have given falsely low values in the past because of inhibition by rheumatoid factors (98, 99). Some evidence indicates that 19 S antinuclear factors are more prevalent

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in rheumatoid arthritis than in systemic lupus erythematosus, and their detection depends on a fluorescent anti-y-globulin which shows up the 19s antibodies (100). Another disorder that has aroused considerable interest in view of its association with arthritis is Sjogren’s syndrome. In many ways it serves as a connecting link between rheumatoid arthritis and systemic lupus erythematosus and other more clearly delineated conditions with immunological alterations. These patients usually show extreme elevation of y-globulin similar to systemic lupus erythematosus patients and contain many similar antibodies, much more so than the bulk of rheumatoid

/

Lupus

Rheumatoid Arthritis

Erythematosus

\

‘Acquired Agamrnaglobulinernia /

Myasthenia Gravis /

\ Derrnat om yosit is’

FIG.4. Diagram illustrating disorders linked through clinical, laboratory, and genetic studies to systemic lupus erythematosus.

arthritis patients (25,101). In addition, the vast majority of these patients have rheumatoid factors often at extreme levels (101). Serologically they show the findings of both rheumatoid arthritis and systemic lupus erythematosus patients. An immunological mechanism for the lesions of the lacrimal, salivary, and other glands appears highly likely in view of the character of the cellular response in these organs. It resembles that seen in chronic thyroiditis where an immunological alteration has been clearly demonstrated. Figure 4 illustrates to some extent the overlap between these disorders as brought forward through serological and genetic studies. A third condition that aids in linking rheumatoid arthritis with immunological alterations is acquired agammaglobulinemia. A number of studies emphasized the occurrence of arthritis in this latter condition (102,103).Recently it has become apparent through family studies that

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both rheumatoid arthritis and systemic lupus erythematosus are found in increased frequency in the relatives. A number of striking pedigrees have been published (104, 105). High titers of rheumatoid factors were also encountered in some family members with and without overt joint disease. The relationship is not a simple genetic one and many more studies are necessary to establish it. Particularly convincing have been two families with systemic lupus in close relatives of the propositi with agammaglobulinemia ( 104). It appears possible that the mysterious arthritis of agammaglobulinemia may be clarified in its relation to rheumatoid arthritis through further genetic studies.

B. SYSTEMICLUPUSERYTHEMATOSUS The chief evidence that systemic lupus erythematosus belongs in the autoimmune disease category stems from the great multiplicity of autoantibodies that have been found in the sera of these patients. The surmise seems difficult to escape that in some fashion one or several of the described or undescribed autoimmune phenomena are involved in the causation of the disease. It seems unlikely that an antibody occurs which specifically attacks a target organ to produce lesions. No evidence has been obtained of antibodies specifically directed against kidney cells that might be harmful to this organ which is the dominant one involved. A more reasonable hypothesis is that certain of the multiple antibodies found in those patients react with tissue breakdown products to produce antigen-antibody complexes. Such complexes would be highly likely to localize in the renal glomeruli as has been demonstrated so strikingly in experimental animals following injection of antigen-antibody complexes (106).Complement would be bound and this might secondarily affect regional cells. Studies in a number of laboratories have clearly shown the localization of y-globulin in the glomeruli of these patients (107,108). This is in general more striking than in any other renal disorder. In addition, complement is also readily localized in similar regions by the fluorescent antibody technique, Antisera specific for plo, part of the C’3 system, and for C’4 have both shown specific localization of these complement constituents (108). It has been known that total complement and several of the individual components of complement are very low in the sera of patients with systemic lupus (109, 110). Recent studies have indicated that the complement level is a good index for gauging the degree of disease activity and the effect of various types of therapy. Striking curves have been obtained in some patients showing the return of complement levels to normal after steroid therapy (110,111). Attempts have been

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made to relate these levels to individual antibodies in order to see if any one of the group would be more involved in the complement fixation. In general, there appears to be some correlation with DNA and nucleoprotein antibodies, at least better than with certain others. The DNA antibodies have thus far only been found in patients with systemic lupus and usually only in the acute stages of the disease. They fall rapidly after therapy. There is thus some suggestive evidence that these may be more directly concerned in the renal lesions. This has beeen stressed by several observers (3, 112). However, these antibodies are also usually at the lowest titers and therefore might be expected to fall most readily on treatment. Thus far no one has clearly determined the antigen in the kidney to which the y-globulin and complement are bound, although some attempts in this direction have been made. Another possibility is that perhaps no specific antigen is involved but that with the extreme hypergammaglobulinemia in these patients certain rather insoluble y-globulins are produced that behave as aggregates, fixing complement and localizing in the kidney. Some possible evidence for such a concept has been obtained from studies indicating a high incidence of cryoglobulins in the sera of these patients (113). These cryoglobulins fix complement as they precipitate ( 113). Analyses of these precipitates have thus far failed to demonstrate any antigen, only yglobulin and complement. Peripheral vascular disturbances are seen in some patients with systemic lupus, and these could well be related to proteins of this type. A possible role for some type of cellular immunity in the manifestations of systemic lupus erythematosus has been postulated by several observers. No real evidence for such a concept has been forthcoming. Possible evidence of cellular autoimmunity was thought to have been obtained from the finding of delayed-type skin reactions following injection of autologous cell extracts. Certainly, the lupus erythematosus patients exhibited hyper-reactivity, but similar patterns were also obtained in certain controls (114). Recent observers have felt that these reactions are nonimmunological in type and are more related to the enzymes in the tissue extracts (115). Lymphocyte culture studies have indicated in some cases many cells in the peripheral blood which divide and progress to cells resembling plasma cells. Thus far no specific autoantigen has been found to stimulate the lymphocytes of these patients. The skin lesions of patients with systemic lupus erythematosus have always been a subject of intense speculation concerning their origin, particularly in regard to the effect of sunlight in initiating their develop-

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ment. A clear answer to this question remains unavailable. However, recent studies of the skin lesions by the fluorescent antibody technique have sliown considerable y-globulin deposition. In addition to the bandlike layer of y-globulin at the dermal-epidermal junction (Fig. 5a),

FIG.5. Distribution of y-globulin in skin of patients with SLE. Direct immunofluorescent staining for bound y-globulin in skin biopsies. Bandlike deposits along the junctional region between epidermis and dermis ( a ) . Large lumpy deposits in this region ( b ) . y-Globulin in the epidermal cell nuclei ( c ) and in nuclei of cells in wall of arteriole and dermal connective tissue ( d ) . (Figs. a, c, cl: x 250; b: x 450)

which has been reported (116), studies from this laboratory (117) have shown the presence of large lumpy deposits which appear to be in the lumens of the capillaries subjacent to the basement membrane of the epidermis (Fig. 5b). It is not clear at this time whether the deposits of y-globulin in these areas are antibodies directed against some specific component of skin. Further findings with the fluorescent technique show

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the presence of y-globulin in the nuclei of epidermal cells (117). Direct staining of skin biopsies with fluorescein-tagged antihuman y-globulin show the specific localization of y-globulin in the epidermal cell nuclei and no staining of cell cytoplasm or intercellular processes (Fig. 5c). There is evidence that this fixation of antinuclear antibody in d u o is not due to nonspecific localization in necrotic or altered epithelial cells. In biopsies taken from hyperkeratotic and nonkeratotic skin areas of a patient with lupus skin rash, y-globulin was present in epidermal nuclei of nonkeratotic skin but not present in hyperkeratotic seminecrotic skin. In addition, in some patients the nuclei of infiltrating cells in the dermis contain y-globulin, as do the nuclei of cells in the muscular wall of some arterioles (Fig. 5d). Further evidence in support of an antigen-antibody reaction is the demonstration of fllo component of complement in the epidermal cell nuclei staining for y-globulin and in the deposits of yglobulin at the dermal-epidermal junction. It is evident from these studies that considerable antinuclear antibody is taken up in viuo by skin tissue and that this may be a factor in the skin sensitivity and the development of lesions. Although antinuclear antibody fixation in viuo has not thus far been reported in other tissues, it would not be surprising if a thorough study demonstrated its presence in cell nuclei and tissue structures of other diseased organs where substances may be released locally which alter the permeability of such cells.

C. THYROJDITIS Hashimoto’s disease, also called ‘lymphadenoid goiter” and “chronic thyroiditis,” has been regarded as the exemplary form of autoimmune disease in man. The laboratory and clinical investigations that led to this concept were pioneered largely by the work of Witebsky and his colleagues in America and by Roitt and Doniach in England. The former investigators reported in 1958 that autoantibodies in rabbits could be produced by immunization with saline extracts of homologous and autologous thyroid (118) and that histological changes were present in the thyroid glands of immunized animals (119). In the same year, Roitt et al. (120) reported the finding of thyroid autoantibodies in patients with Hashimoto’s disease. These antibodies were demonstrated by precipitin reactions in agar between serum and saline extracts of thyroid and human thyroglobulin. Similar findings in human disease were also obtained by Witebsky and associates (121). Since the initial discovery of autoantibody to thyroglobulin in human thyroid disease, there has been great interest in the role these antibodies might play in the pathogenesis of Hashimoto’s and other thyroid diseases.

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The standard immunological methods that have been employed by investigators for detecting thyroid antibodies in human sera have included precipitin, tanned red cell agglutination, complement fixation, and fluorescent antibody techniques. The antigen or group of antigens in thyroid extracts which react in the precipitin or tanned cell agglutination tests have been shown to reside in thyroglobulin (122-125). Precipitin reactions in agar with purified thyroglobulin and Hashimoto sera have given as many as three lines (126, 127). In quantitative precipitin studies, some sera gave curves typical of the rabbit precipitin system, whereas others gave curves which resembled the horse flocculating system in which antigen-antibody complexes were soluble in the presence of excess antibody (126). These two types of quantitative precipitin curves exhibited by Hashimoto sera and thyroglobulin have also been reported by others (128), but the reason for the different precipitating systems remains unexplained. It was observed that although some thyroid autoantibodies of human origin could be easily demonstrated to form precipitin reactions with purified thyroglobulin, these same human sera did not react or reacted weakly in complement fixation tests with thyroglobulin (126, 129). However, complement-fixing activity in a high percentage of Hashimoto sera was obtained when saline extracts of thyrotoxic thyroid were used (122). It was shown that the antigen was probably related to a particulate component of the extracts since activity could be taken down by highspeed centrifugation. Cell fractionation studies showed that the reactive antigen was in the microsomal fraction and was present in both thyrotoxic and normal thyroids ( 130, 131 ) , With the use of the fluorescent antibody technique, histological localization of the thyroid antigens reacting with serum antibodies was possible, with the result that the distinctness of the precipitin and complement-fixing antibody systems was further demonstrated. Antibodies of the precipitin type were shown to stain the follicular colloid, and complement-fixing antibodies stained the cytoplasm of acinar cells ( 132). These studies have been confirmed recently ( 133). The cytotoxic factor reported in Hashimoto serum (134) has been shown to be related to antibodies giving cytoplasmic staining of thyroid cells ( 135). A third type of autoantibody has been described (123). With the fluorescent antibody technique a small percentage of Hashimoto sera, which showed no antibodies to thyroglobulin or microsomal fraction of cells, stained thyroid colloid. The pattern of staining which was uniform or homogeneous, differed from that given by thyroglobulin antibodies which gave a “floccular” stain. The homogeneous type of stain was

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apparently obscured by the floccular stain since, in the majority of Hashimoto sera, absorption with thyroglobulin revealed the homogeneous staining antibody. This appears to be similar to the situation in systemic lupus erythematosus mentioned previously, in which the speckled nuclear stain was obscured by homogeneous nuclear stain and could be revealed by specific absorption with nucleoprotein. In addition to these thyroid autoantibodies, there have been isolated reports of antinuclear antibodies in the sera of patients with Hashimoto’s disease ( 133, 136). However, the incidence of antinuclear factors in thyroiditis appeared to be of a low order, and autoantibodies in this disease seemed to be directed primarily against the thyroid gland (137). The autoreactivity of the thyroglobulin and thyroid cell cytoplasmic antibodies has been demonstrated by many workers. Some interesting observations were made with respect to thyroglobulin antibodies ( 138). Extracts of thyroids removed during surgery were studied for precipitin reactions to autologous and nonautologous Hashimoto sera. In six of these extracts precipitin reactions were shown with the patients’ own sera and with other Hashimoto sera. Another five extracts showed no reactivity with either autologous or other sera, and in one thyroid extract there was no precipitation with the patient’s own serum but positive reaction with other sera. This last thyroid extract, therefore, appeared to react in isoprecipitating systems but not in the autoprecipitating system. Further studies of this type have not been done and it cannot be ascertained at this time whether there are, indeed, thyroid antibodies with isoreactivity, but without autoreactivity, similar to what has been described (139) for certain sera with “autoimmune complement-fixing antibodies. A large body of information has been accumulated regarding the incidence of thyroid autoantibodies in various types of thyroid disorders. With tissue culture cytotoxicity and fluorescent antibody tests, the incidence of thyroid antibody in Hashimoto’s disease is higher than 90 %, whereas with the precipitin and tanned cell agglutination tests, the incidence is about 50 % (140). However, when these tests were combined, the reported incidence was over 99 % in Hashimoto’s disease and extremely high in myxedema and thyrotoxicosis (141). Thyroid autoantibodies have been reported to be higher than controls in systemic lupus erythematosus (137), rheumatoid arthritis ( 137, 142), d’iseases with hyperglobulinemia (143), leprosy (144), in parents of patients with Hashimoto’s disease (145), and in pernicious anemia ( 146). In view of the apparent prevalence of thyroid autoantibodies in a wide variety of disease states, it is difficult to assess the significance of control studies which have been carried out in hospital patients without overt thyroid

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disease (147, 148). In one such study, out of eighteen patients who subsequently died and at autopsy showed lymphocytic infiltration and Askanazy cell changes in their thyroids, the complement fixation test was positive in seven ( 149). The evidence is impressive that Hashimoto’s disease or chronic thyroiditis is associated with an unusual immunological response, resulting in the production of circulating autoantibodies with specificity for the individual’s own thyroid. However, the relationship of these autoantibodies to the pathogenesis of the disease has not been demonstrated. There is much evidence against circulating antibodies alone being responsible. Passive transfer of these antibodies into animals in large quantities has failed to produce the disease (130), and attempts to relate cretinism to mothers with thyroid antibodies have been largely unsuccessful (135, 150). Perhaps of greater weight against the implication of these autoantibodies in pathogenicity is the observation of the high incidence of thyroid antibodies in thyrotoxicosis, a disease which rarely results in hypothyroidism. On the other hand, there is much experimental and indirect evidence in favor of a relationship. Animals immunized with autologous thyroid in adjuvant develop a disease histologically similar to the human disease, and circulating antithyroid antibodies are present in the serum. Inflammatory reactions in the thyroid gland of rats could be produced by passively transferred heterologous antibodies if the recipient thyroids were first damaged by irradiation or radio-iodine (151). Initial damage to some structure of the gland was apparently necessary before thyroiditis could be produced, There was evidence for this in other experimental systems in that cytotoxic antibodies were not injurious to thyroid cells in tissue culture unless the cells were first exposed to proteolytic enzymes (135). In this context the significance of basement membrane changes in thyroiditis has been emphasized ( 152). In correlating pathological and serological studies, a close relationship between fragmented thyroid follicular basement membrane and the presence of thyroglobulin antibodies in the serum was demonstrated. All these experimental and histological observations appear to point to the loss of integrity of the protective membranes of the gland as an important feature associated with the presence of circulating thyroid antibodies. There are some data which suggest that the infiltrating mononuclear cells in chronic thyroiditis might play a role in this process. In tissue cultures of Hashimoto glands, lymphocytes were observed to penetrate the thyroid acinar cells and move freely within the cytoplasm ( 153). In immunofluorescent studies of Hashimoto’s disease (154, 155), thyroglobulin was detected in the

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mononuclear cells of the adjacent lymph nodes and in the cells infiltrating the thyroid, suggesting that these cells had phagocytized thyroglobulin. Specific antibody to thyroglobulin was also present in these cells. Factors initiating the disease and the role of delayed hypersensitivity are not yet understood, and the meaning and significance of circulating thyroid autoantibodies must await clarification from these studies.

D. PERNICIOUS ANEMIA In recent years, interest in a possible immunological mechanism in pernicious anemia arose as a result of certain experimental observations and clinical studies. Rabbits immunized with extracts of human and hog gastric mucosa incorporated in Freund's adjuvant produced antisera reacting with the extracts in precipitin and complement fixation systems and with intrinsic factor in hemagglutination tests (156, 157). The antisera were tested in pernicious anemia patients and found to inhibit the ability of human gastric juice or hog intrinsic factor to promote absorption of vitamin B12. This latter method was employed in the study of pernicious anemia patients who had been treated with hog intrinsic factor (158). It had been observed that a certain number of these patients had become refractory to treatment after some time, and the question arose whether this could be due to the development of antibody to hog intrinsic factor. In a significant number of the patients, inhibitor of hog intrinsic factor activity was present in the serum. However, a discrepancy was noted in that some pernicious anemia patients not refractory to hog intrinsic factor showed the presence of inhibitor in the serum, and it soon became apparent that multiple factors were involved. Furthermore, inhibition of intrinsic factor activity was found in pernicious anemia patients who had been treated only with parenteral vitamin BIZ or in untreated patients (159, 160). In a study of the nature of the serum factor inhibitory to intrinsic factor ( l e l ) , it was shown that when radioactive labeled BIZ was complexed with intrinsic factor it migrated with the (%globulinon electrophoresis, but when immune serum from rabbits injected with intrinsic factor was added to labeled Blzintrinsic factor complex, there was retardation of migration and radioactivity was detected in the y-globulin region. This technique was used in studying the reaction between pernicious anemia serum and B12-human factor complex, and it was shown that in vitro binding of Blzintrinsic factor by pernicious anemia serum correlated with inhibitory activity in the in vivo studies (162). The active fraction of serum was present in y-globulin and appeared to be an antibody. The intrinsic factor antibody was found in 20-30 % of

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patients with pernicious anemia and in one patient with atrophic gastritis without pernicious anemia. In close similarity with the studies of thyroid autoantibodies in Hashimoto’s disease, further studies revealed additional antibodies in pernicious anemia, and certain pernicious anemia sera have been shown to fm complement in the presence of saline extracts of human gastric mucosa (163, 164). The reacting antigen in the saline extracts was not intrinsic factor. The incidence of antibody in the complement fixation test was considerably higher than that in the other studies, and an incidence of 75 % positive complement fixation tests in a group of treated and untreated pernicious anemia patients has been reported ( 164).An extensive study of pernicious anemia patients confirmed that two distinct antibodies were present (165). The tests employing inhibition of intrinsic factor activity and retardation of electrophoretic mobility detected antibody to intrinsic factor, whereas complement fixation and immunofluorescence detected antibody to cytoplasm of gastric parietal cells. The immunofluorescent test appeared to be more sensitive for detection of cytoplasmic antibody, and the authors found an incidence of 86 % as against 62 % by complement fixation. The results of fluorescent antibody studies were confirmed, and it has been shown that cytoplasmic antibodies reacted with autologous gastric parietal cells (166). The incidence of gastric antibodies in other disease states has been investigated by a number of authors. Anti-intrinsic factor antibody was found in atrophic gastritis (162), and antibody to parietal cell cytoplasm has been reported to be higher than controls in iron-deficiency anemia (la),Hashimoto’s disease (166, 167), spontaneous hypothyroidism (166), and diabetes mellitus (168). The presence of gastric antibodies does not show a positive correlation with either achlorhydria or pernicious anemia in these other disease states. The significance of circulating gastric antibodies in pernicious anemia is at present unknown. There is no evidence for any biological activity of antibody to gastric parietal cell cytoplasm. Somewhat more information is available regarding antibody to intrinsic factor. This antibody has been shown to inhibit intrinsic factor activity in in vim B12 absorption studies. However, it was apparent in many of the earlier studies that there was no consistent correlation between refractoriness to intrinsic factor and the presence of circulating antibody. This question was examined again recently (169). Two pernicious anemia patients were immunized parenterally with hog intrinsic factor until both developed circulating antibody as evidenced by immediate-type skin reaction and precipitating antibody in the serum to intrinsic factor. However, both these patients were not

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refractory to orally administered hog intrinsic factor. Inhibition of Blz absorption could, however, be demonstrated when hog intrinsic factor was given orally together with postimmunization serum in one of the two subjects. The results of this and earlier studies suggested that antibody to intrinsic factor had to be present in the lumen of the gut to prevent B12 absorption (162). The presence of antibody in the lumen of the gut could conceivably account for the disease in the small percentage of patients with pernicious anemia refractory to oral intrinsic factor but would not explain the disease in the larger proportion of patients not refractory to orally administered intrinsic factor. It is obvious that with the information available at the present time the role of autoantibodies in pernicious anemia is not apparent, but further studies of these clearly demonstrated antibodies seem warranted,

E. MYASTHENIA GRAVIS Considerable interest in immunological mechanisms as a possible etiological factor in myasthenia gravis has arisen recently. In 1960, a hypothesis was advanced that myasthenia gravis could be due to antibody to neuromuscular end-plate which acted as a competitive blocking substance at this region (170). About the same time that this hypothesis was advanced, other authors (171) reported their results on a series of studies in myasthenia gravis. In searching for a circulating neuromuscular blocking agent, they noted that serum samples from some myasthenic patients caused lysis of frog muscle fibers. It was postulated that if such cytolytic activity occurred in uiuo, complement might be taken up in the process. Complement activity in the sera of myasthenic patients was found to fluctuate over a wide range, but it was reported that there was some evidence that complement activity was low in exacerbations of the disease and was normal or above normal in periods of remission (172). These results could be consistent with fixation of complement in an in uiuo antigen-antibody reaction, and, with other ancillary data, these authors also advanced the hypothesis that myasthenia gravis might be a disease involving a destructive autoimmune mechanism. Thereafter, a study was soon reported in which a circulating serum factor was found in myasthenia gravis which had a5nity for muscle fiber (173). The fluorescent antibody technique was employed, and pooled serum globulin from ten myasthenics, conjugated with fluorescein isothiocyanate, was used for direct staining of a variety of muscles obtained at biopsy. The authors reported that the cross striations of skeletal muscle, but not of cardiac or smooth muscle, were stained by the fluorescein-conjugated, pooled, myasthenic serum. Conjugated, normal

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serum globulin did not show this staining. The reaction between skeletal muscle fiber and myasthenic serum globulin was also shown to bind guinea pig complement by an immunofluorescent method. The morphological character of bound guinea pig complement, however, was somewhat different in that it did not stain cross striations evenly but appeared to be a partial or particulate stain of the cross striations. It was postulated that the latter phenomena might have been due to cytolytic activity of complement fixed by the interaction between myasthenic globulin and muscle. When inhibition tests were performed in the direct staining reaction, it was demonstrated that all myasthenic sera tested showed partial inhibition of staining by the pooled serum conjugate. Using both the direct and indirect methods of immunofluorescence, two myasthenic sera with high titer activity were studied (174). The cross-striation staining of muscle fibers reported in the earlier study was confirmed, and on the basis of different staining properties of these sera it was suggested that there were two types of muscle antibodies in myasthenia gravis: “S” antibody directed against skeletal muscle and “ S H antibody directed against skeletal and heart muscle. In inhibition studies one of these sera failed to block staining of cross striations by the conjugate of the other serum. In a recent study (175) it was found that 42 % of a large series of myasthenic patients showed serum factors that reacted with muscle. Three patterns of staining were reported, the cross-striation staining described previously, staining of alternating muscle fibers, and sarcolemmal staining. The third type was also present in a small percentage of normal sera. With an antiglobulin consumption test (176) it was shown that certain myasthenic sera contained antibodies directed against thymus. The separate identity of muscle and thymus antibodies was established by cross absorptions of myasthenic sera with muscle or thymus powder. In addition to these apparently organ-specific antibodies, antinuclear antibodies have also been described by the majority of investigators (174, 177-180). There have been several recent reports of the association of myasthenia gravis with systemic lupus (181-183). The frequent association of thymic pathology with myasthenia gravis is a well-documented observation (184), and the recent recognition of the role of the thymus in autoimmune disease in animals (185,186) lends further support to the hypothesis that myasthenia gravis might be a disease of autoimmunity. It is also known that some infants born of myasthenic mothers have a transient myasthenic syndrome lasting for a few weeks (187), and this could conceivably be due to the placental transfer of a serum factor, possibly antibody. However, the data at hand, demon-

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strating circulating antibodies with affinity for cross striations of muscle fibers, is difEcult to reconcile pathogenetically with the known pharmacological and physiological data pointing to the neuromuscular junction as the primary site affected in myasthenia gravis. In this disease, the anatomical site of a physiological defect in an organ system has been generally accepted as established, and the demonstration of antibody related to pathogenetic mechanism could conceivably be antibody having specificity for that anatomic site or be shown to affect physiological function at that site. It would seem that an opportunity has been provided in this disease to determine whether the circulating factors that have been demonstrated play a physiological role, or whether they arise as a secondary phenomenon resulting from primary muscle injury and thus fall into the large class of relatively insignificant autoantibodies. The answers to these questions will prove of considerable interest.

F. SCLERODERMA AND DEWATOMYOSITIS There is a large body of literature on the clinical and pathological aspects of scleroderma and dermatomyositis which has pointed to a possible autoimmune disorder as the underlying pathogenetic mechanism in these diseases (188-193). This has been particularly true of the “intermediate” or “transitional” forms of these diseases, which clinically and pathologically may be indistinguishable from systemic lupus erythematosus. It is not surprising that because of this similarity many investigators have looked for antinuclear antibodies. The fluorescent antibody technique has been predominantly used in these studies. In scleroderma, the presence of antinuclear antibodies has been regularly reported by a number of investigators (194-200). In a recent study an incidence of 78 % antinuclear antibodies was reported in a group of thirty-two scleroderma patients ( 198 ) . Different morphological patterns of nuclear staining have been reported and have included nuclear membrane with chromatin staining, nucleolar staining, and homogeneous and speckled nuclear staining (6, 196, 197). A correlation was not noticed between the pattern of nuclear staining and any particular clinical aspect of the disease. Precipitating antibodies against organ extracts have also been reported in a small percentage of scleroderma patients and some of these appeared to be identical to precipitating autoantibodies in Sjogren’s disease ( 198). There have been few studies on antinuclear antibodies in dermatomyositis, and it is impossible to assess its incidence in this disease since most reported series have been concerned with extremely small numbers of cases. With the fluorescent antibody technique, antinuclear antibodies

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have been reported in one or two cases by a few groups of investigators (6,194, 195, 199). It is not possible at this time to arrive at any conclusion regarding these findings. An interesting clinical observation in dermatomyositis has been its association with malignancy. Reports of the incidence of malignant tumors in dermatomyositis have varied from 7 to 34 %, but all authors agreed that the incidence was higher than in control groups (201-204). In a critical analysis of cases associated with malignancy (192) it was stated that malignancy appeared to have preceded the onset of dermatomyositis in all cases, and there have been many published reports of improvement of dermatomyositis following treatment of the malignancy (192, 203, 204). There has been no good explanation for this close association, but some recent studies raise the possibility of an immune reaction to the tumor or tumor products. A patient with dennatomyositis and breast cancer was skin-tested with extract of autologous cancer tissue and autologous normal tissue (205). There was an immediate-type skin reaction with wheal and erythema formation only to the tumor extract. Further evidence that humoral factors were concerned was obtained by passive transfer of the patient’s serum to the skin of a normal volunteer. Subsequent challenge of the transfer site with tumor extract produced an immediate skin reaction. The intimate association of the malignancy with dermatomyositis was also manifested in the clinical events. There was improvement in the patient’s disease when the breast tumor underwent partial regression after oophorectomy, and exacerbation of skin lesions followed renewed tumor growth. Similar techniques employed in the study of a patient with dennatomyositis and a metastatic lung tumor also demonstrated immediate skin reactions to autologous tumor extract ( 206). The type of reaction manifested as skin sensitivity to tumor extract has also been reported in other situations. In patients with tumors showing evidence of local inflammatory reactions, it was found that five patients with breast cancer, three with Hodgkin’s disease, and one with lymphosarcoma had positive skin reactions to autologous tumor extracts (207). Only one of these patients had dermatomyositis, and it was suggested that dermatomyositis might represent a relatively rare, generalized manifestation of a more common sensitivity reaction in cancer. If this sensitivity reaction was, indeed, due to circulating antibodies as the positive Prausnitz-Kustner reaction would suggest, it might be postulated that the antibodies were raised against tissue components in cancer that showed antigenic cross reaction with certain normal tissue components in skin and muscle, Further studies are necessary to demonstrate conclusively cross-reacting antibodies of this type.

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G. PANCREATIC DISEASE Although the evidence for antibodies in pancreatic disease is still meager, the available information with regard to certain similarities in the human disease and experimental animal studies is of interest. It was reported in 1959 that precipitin reactions were demonstrated between saline extract of pooled human pancreas and the sera of patients with chronic pancreatitis and pancreatic carcinoma (208). In these initial studies precipitating factors in serum were shown to be organ- and species-specific. One serum with high titer precipitins against pooled pancreas extract did not react with extract of autologous pancreas, whereas other pancreatitis sera regularly showed precipitins with this latter extract. This observation was extended, and, in four patients with chronic pancreatitis who underwent surgery and had high-titer circulating precipitins to pooled pancreas extract, three failed to react with their own pancreas. The fourth serum gave a positive reaction with autologous pancreas (209). In a survey of positive reactions with pancreas extracts in a group of 228 unselected hospital patients, the authors found an incidence of approximately 5 %, whereas the incidence in chronic pancreatitis, pancreatic carcinoma, and cystic fibrosis was generally very high (210). They concluded from this series of studies that antibodies were demonstrated in certain pancreatic diseases which were organspecific for the pancreas. These antibodies were primarily isoantibodies and were probably formed as a result of injury to the pancreas. Some attempts were made to characterize the pancreatic antigen concerned, and it was reported that the antigen was probably not related to pancreatic enzymes but to be present in the endoplasmic reticulum of the acinar cell (211). Other authors have confirmed the presence of precipitating antibodies to pancreas extracts in patients with chronic pancreatitis and pancreatic carcinoma and also demonstrated that in some of these reacting systems, as many as four precipitin lines were present in Ouchterlony plates (212, 213). In animal studies (214), rabbits immunized with pancreas extracts have shown serological reactions which are relevant to some of the observations in human pancreatic disease. When saline extracts of hog, dog, beef, and human pancreas were injected with Freund's adjuvant into rabbits, antibodies were produced which were organ-specific or could be made organ-specific by absorption of antisera with nonpancreatic organ extracts of the particular species. These pancreas-specific antisera showed very little cross reaction with pancreas extracts of other species tested and could be considered both organ- and species-specific. This finding

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was in contrast to experimentally produced and human thyroid antibodies which were organ-specific but showed a broad cross reaction with thyroid extracts of other species. A further difference from experimental thyroiditis was observed when rabbits were immunized with pooled homologous pancreas extracts (215). The resulting antisera did not react with the rabbit’s own pancreas extract, but each antiserum reacted selectively with a panel of other pancreas extracts. The pancreas antibodies, therefore, showed primarily isospecificity, and, within the stock of rabbits used by the authors, at least four pancreas-specific antigens were detected. The antibodies against these pancreas antigens were not absorbed out by serum, erythrocytes, or other homologous organ extracts. As a sequel to the animal studies, the authors examined the sera of several patients with chronic pancreatitis but did not find pancreas-specific iso- or autoantibodies. The reason for the discrepancy between this study and those of other authors in human pancreatitis is not clear, since it would appear that the careful serological studies in rabbits provided confirmation of the positive findings in human disease. H. ADDISON’SDISEASE The presence of antibodies to adrenal tissue was first established in the sera of two cases of Addison’s disease in which complement-fixing antibodies could be demonstrated against both adrenal and thyroid tissue (216). This initial observation has been confirmed by a number of subsequent investigators. In a 13-year-old girl with Hashimoto’s thyroiditis, which was diagnosed a few months before the onset of symptoms of adrenal insufficiency, complement-fixing antibodies to adrenal and thyroid extracts were present in the serum, and by cross absorptions with the respective antigens, the antibodies were shown to be separate factors (217). With the fluorescent antibody method, adrenal antibodies have been shown to react with cytoplasm of cells in all three zones of the adrenal cortex, but occasionally only cells of the fasciculata fluoresced (218). There was no reaction with cells in the medulla. Sixteen of thirty patients with idiopathic Addison’s disease showed antibodies by this method. No adrenal antibodies were present in patients with adrenal hyperplasia or in twenty-two patients with Hashimoto’s thyroiditis who had thyroid antibodies but no evidence of adrenal insufficiency. In sixteen patients with Addison’s disease and adrenal antibodies, seven had thyroid antibodies by the fluorescent antibody technique. It is interesting that pathological studies in patients with Addison’s disease had long indicated the presence of lymphoid infiltration in the thyroid gland, much resembling Hashimoto’s disease (219,220). At present, the relation-

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ship between these two diseases is not clear. A recent study (221) has shown that patients with Addison’s disease have antibodies to adrenal, thyroid, and stomach, and it was postulated that Addison’s disease might be associated with a fundamental immunological disturbance and circulating antibodies might not simply be the result of tissue damage. This perhaps receives some support from studies which showed that adrenal antibodies were found in some patients with idiopathic hypoparathyroidism who had no evidence of adrenal insufficiency (218). A possible relationship of Addison’s disease with hypoparathyroidism has also been reported by others (222). Experimentally, adrenalitis has been produced in guinea pigs (223225) and rabbits (225) by immunization with autologous or homologous adrenal in Freund’s adjuvant, There has been some question whether the adrenal lesion produced was similar to that in Addison’s disease (224). Lesions were also present in animals injected with Freund’s adjuvant alone (225). Circulating antibodies in animals have been demonstrated by precipitin, complement fixation, and tanned cell agglutination techniques (225). Antibodies were specific for the adrenal and have been shown to be autoantibodies. In many respects the results in experimental adrenalitis appear to be similar to those in experimental thyroiditis, but the antigens concerned in both Addison’s disease and experimental adrenalitis have not yet been characterized.

I. ULCERATIVE COLITIS The possibility of an immunological mechanism as the basis for the intestinal lesions of ulcerative colitis has been raised for a number of years (226, 227). Initial studies postulated a local sensitivity reaction in the colon as the result of a dietary or bacterial antigen. Some support for such a concept was gained by the production of local inflammatory lesions in sensitized animals by the local injection of antigen or by the localization of antigen and antibody in formalin-inflamed areas of the intestine (228). More recently, a number of workers have reported autoantibodies to colon tissue in the sera of patients with ulcerative colitis (229,230). These have been demonstrated with various extracts of colon by a variety of immunological procedures including precipitin, complement fixation, hemagglutination, and fluorescent antibody techniques (231). Other workers have failed to demonstrate specific antibodies of this type (232, 29). The positive results have been most striking in children with ulcerative colitis. Some of the studies, particularly those with the fluorescent antibody technique, are complicated by the localiza-

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tion of blood group antibodies to similar sites as the presumed colon antigen. Further studies are required to evaluate this important factor. Evidence has also been obtained for a specific damaging effect of white blood cells from ulcerative colitis patients on fetal colon cells in ~ i t r o(233). This reaction required complement and was not obtained with normal white cells pretreated with ulcerative colitis serum. The damaging effect was detected through the release of labeled phosphate or labeled amino acids from the colon cells into the medium. These observations are of considerable interest and will certainly stimulate further work in this area. bERENCES

1. Coombs, R. R. A., Coombs, A. M., and Ingram, D. G. (1981). “The Serology of Conglutination and its Relation to Disease” Thomas, Springfield, Illinois. 2. Miescher, P., and Strassle, R. ( 1958). In “Immunopathology, 1st International Symposium” (P. Grabar, and P. Miescher, eds.) 1959, p. 455. Schwabe, Basel, Switzerland. 3. Seligmann, M. (1958). Reo. Franc. Etudes Clin. Btol. 3, 558. 4. Holman, H., Deicher, H., and Kunkel, H. G. (1959). BUZZ. N.Y. Acad. Med. 35, 409. 5. Friou, G. (1983). Arthritis Rheumat. 5, 407. 8. Beck, J. S . (1981). Lancet I, 1203. 7. Lachmann, P. J., and Kunkel, H. G. (1981). Lancet 11, 438. 8. Holman, H., and Deicher, H. (1959). J. Clin. Inuest. 38, 2059. 9. Deicher, H., Holman, H., and Kunkel, H.G. (1959). J. Exptl. Med. 109, 97. 10. Vorlaender, K. O., and Ross, J. (1981). Klln. Wochschr. 39, 605. 11. Atchley, W. A. (1961). Arthritis Rheumat. 4, 471, 12. Anderson, J. R.,Gray, K. G., Beck, J. S., Buchanan, W. W., and McElhinney, A. J. (1982). Ann. Rheumatic Diseases 21, 380. 13. Ross, J., Tan, E. M., and Kunkel, H. G. (1983). Arthritis Rheumut. 8, 790 (Abstr.) 14. Lachmann, P. ( 1981 ). Doctoral thesis, Trinity College, Cambridge, England. 15. Ceppellini, R., Polli, E., and Celada, F. (1957). PTOC.SOC. Exptl. Blol. Med. 96, 575. 18. Holman, H., and Kunkel, H. G. (1957). Science 128, 182. 17. Robbins, W. C., Holman, H. R., Deicher, H. R., and Kunkel, H. G. (1957). PTOC.SOC.Exptl. Biol. Med. 96, 575. 18. Miescher, P., and Stdssle, R. ( 1957). Vox Sanguinis 2, 283. 19. Miescher, P. ( 1955). Vox Sanguinis 5, 121. 20. Kayhoe, D. E., Nasou, J. P., and Bozicevich, J. (1980). New Engl. J . Med. 263, 5. 21. Stollar, D., Levine, L., and Marmur, M. (1982). Biochim. Biophys. Acta 81, 7 . 22. Stollar, D., Lehrer, H. I., and Van Vunakis, H. (1982). PTOC.Natl. Acad. Sci. US.48, 874. 23. Christian, C. L., DeSimone, A. R., and Abruzzo, J. L. (1983). Arthritis Rheumat. 6, 788.

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Effect of Bacteria and Bacterial Products on Antibody Response

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J MUNOZ Nationol lnstitutes of Health. Notional Institute of Allergy and Infedious Diseases. Rocky Mountain Laboratory. Hamilton. Montana

I . Introduction ............................................... A Scope ................................................. B. Historical Notes ........................................ C Possible Action of Adjuvants ............................. I1. Acid-Fast Bacteria .......................................... A Original Observations .................................... B Effect of Mycobacteria in Oil-in-Water Emulsions ........... C Characterization of Active Material' ........................ D. Mode of Action of Mycobacteria .......................... I11 Effect of Endotoxins from Gram-Negative Bacteria ............... A. Nature and General Biological Characteristics of Endotoxins . B Original Observations ................................... C. Effect of Various Endotoxin Preparations ................... D. Mode of Action of Endotoxins ............................ IV Effect of Bordetella pertussts ................................. A General Remarks ....................................... B. General Biological Effects ................................ C. Characterization of Active Material ........................ D. Possible Modes of Action of Bordetella pertussis ............. V . Miscellaneous Bacteria and Bacterial Products . . . . . . . . . . . . . . . . . . VI Possible Mechanisms of Bacterial Adjuvants .................... VII Summary .................................................. References .................................................

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A. SCOPE The factors that affect production of antibodies have been under investigation since the beginning of immunology Great strides have been made in the last half.century. especially during the last decade Very early in the history of immunology it was discovered that stimulation of the antibody-forming mechanism by an antigen could be modi6ed by many substances and conditions including certain bacterial infections. whole. killed bacteria. and some metabolic products of various microorganisms. This review will cover present knowledge of the stimulatory effect of acid-fast bacteria. endotoxins of gram-negative bacteria. and Bordetella pertussis on the antibody response. No attempt will be made

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to include all the literature in these fields, and when discussing related subjects only key references or review articles will be given. Of necessity, certain aspects of delayed hypersensitivity and isoallergic disorders will be discussed, but these fields will not be extensively covered. Fortunately many recent reviews and published symposia are available (Shaffer et al., 1959; Waksman, 1958, 1959; Paterson, 1959; Kies and Alvord, 1959; Grabar and Miescher, 1959, 1962; Lawrence, 1959; Holub and Jaro6kovl, 1960). The adjuvant effect of many chemicals including water-in-oil emulsions will not be reviewed to any extent. These topics have also been adequately covered in the past (Holt, 1950; Haas and Thomssen, 1961; Freund, 1947, 1951, 1956; Jacotot, 1962). The word “adjuvant” is herein defined as a substance that enhances antibody response to antigens injected simultaneously with it or within a period of time closely spaced to the injection of antigen. This meaning is extended to substances that enhance hypersensitivity reactions directly related to antibodies or suspected to be associated with the antibody response. B. HISTORICAL NOTES Some 30 years after the discovery of circulating antibodies (see Bullock, 1938), it was observed that certain agents could, when given with the antigen, enhance production of antibodies. Thus, Ramon in 1925 and Glenny et al. in 1926 found that antigens absorbed on particulate matter stimulated production of antibodies more efficiently than the same amount of unabsorbed antigen. These observations were followed by a number of investigations confirming and expanding these findings. Absorbents such as alum (Glenny et al., 1926) and aluminum hydroxide (Hektoen and Welker, 1933), lanolin in oil, cholesterol, and salts of magnesium and calcium (Ramon et al., 1935), particulate substances such as tapioca (Ramon, 1937; Schmidt and Steenberg, 1936), and bacterial products such as typhoid vaccine (Ramon and Zoller, 1926, 1927; Ramon, 1931, 1936, 1937), staphylococcus toxin (Burky, 1934; Schultz and Swift, 1934; Swift and Schultz, 1936a,b), tubercle bacilli (Lewis and Loomis, 1924, 1926), and certain killed gram-positive cocci ( Clark et d.,1922); Pseudornonas aeruginosa (Khanolkar, 1924), Brucellu abortus (Ramon et al., 1950), and various other bacteria and microbial products (Stanley, 1950; Thompson, 1922) were found to have an adjuvant effect. As will be discussed later, B. pertussis and most gram-negative bacteria enhance antibody production. Other substances found to have an adjuvant effect include phosphorylated hesperidin (Moss et al., 1956), calcium alginate ( Amies, 1959), polyvinylpyrrolidone ( Amies,

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1962), various lipids (Dresser, 1961), silica particles (Vigliani and Pernis, 1959; Antweiler, 1959; Pernis and Paronetto, 1962) , carbon tetrachlorideinduced cell destruction (Heuer et al., 1962), deoxyribonuclease digests of BruceZZu cell extracts (Braun, 1961), and even introduction of diffusion chambers into the peritoneal cavity ( Adler and Fishman, 1962). It has also been found that the serum of animals containing heterophile antibody (Kalinin et al., 1935) stimulates the production of antibodies in certain animals and that tuberculin given together with an antigen to tuberculin-sensitive animals also acts as an adjuvant (Good et al., 1957; Humphrey and Turk, 1963). Antigen-antibody complexes made in the region of antigen excess have been found more effective than the antigen alone (Terres and Wolins, 1961; Terres and Stoner, 1962). Certain dosages of X-irradiation given at the proper time during antigenic stimulation increase antibody production (Graham et al., 1956; Taliaferro and Taliaferro, 1951, 1956; Dixon and McConahey, 1963), and some cytotoxic agents have a similar effect (Berenbaum, 1960). Abscess formation increased antitoxin formation in horses (Ramon, 1926), and damage to the liver by virus hepatitis or partial removal of the organ (Havens, 1959) as well as removal of adrenal glands (Murphy and Sturm, 1947; Rose, 1959; Char and Kelley, 1962) have been shown to increase the titers of antibody produced. The scope of this review, however, does not permit discussion of many of these aspects of adjuvant action.

c.

POSSIBLE ACTIONOF ADJUVANTS From what has been said, it is clear that many substances and conditions can stimulate antibody formation. It is also apparent that more than one mechanism must be responsible for adjuvant action. Explanations of some of the most obvious mechanisms have long been made. Ramon and Falchetti (1935) believed that the adjuvant effect of oil emulsion and tapioca was mainly owing to the inflammatory reaction and to the slow release of antigen into the body. This has also been shown to be important in the adjuvant effect of alum (Holford et al., 1943; White et al., 1955a) and water-in-oil emulsions (White et al., 1 9 s ; Freund, 1956). Another possible effect of an adjuvant is the stimulation of the antibody-forming cells, which has also been proposed as a mechanism of action of Freund's adjuvant (White d al., 1955b) and of alum (White et al., 1955a). It is also possible that an adjuvant may act by a general stimulation of y-globulin formation irrespective of its serological specificity ( Humphrey, 1963). From the response of antigens given alone (Wilson and Miles, 1957),

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it can also be safely stated that an adjuvant can make an antigen more effective by protecting it from destruction or excretion from the body, by improving its distribution throughout the body before it is eliminated, and by prolonging the exposure of the animal to the antigen (Freund, 1958). Other less obvious possibilities have also been suggested, such as supplying possible necessary substances needed for the process of antibody formation at the proper time during antigenic stimulation (Jaroslow and Taliaferro, 1956; Taliaferro and Jaroslow, 1960; Jaroslow, 1960; Braun, 1961) These possibilities indicate that the exact mode of action of any one adjuvant may be completely different from that of others, and that in many cases the answer is not a simple one. The adjuvant effect of substances can be further affected by the type and amount of antigen used (Weigle d al., 1960; Shaw et al., 1962), the route of administration (Lipton and Freund, 1953; Paterson, 1959), the number of injections (Miles and Pirie, 1939; Farthing and Holt, 1962), the animal species (Rice, 1947), the age (Miles and Pirie, 1939), the nutritional status (Axelrod and Pruzansky, 1955), the particular strain of animal (Lewis and Loomis, 1925; Schneider, 1959), and even the particular individual animal used, as well as the environmental conditions under which the animals are kept. After these brief introductory remarks, the rest of this review will be devoted to the adjuvant effect of acid-fast bacteria, endotoxins from gram-negative bacteria, Bordetella pertussis, and other bacterial cells or their products.

.

II. Acid-Fast Bacteria

A. ORIGINAL OBSERVATIONS That bacteria enhanced the immune response was noted by various workers early in the development of immunology. Lewis and Loomis (1924) were among the first to note this phenomenon when they observed that the same amount of sheep red blood cells produced a higher titer of hemolysins in tuberculous than in normal guinea pigs. This observation was confirmed by Dienes (1927c, 1928, 1929, 1936), who not only found an increased production of antibodies to egg white, horse serum, and other antigens inoculated into the tuberculous lesion, but also made the interesting observation that two types of hypersensitivities were produced in these animals: one, an immediate or anaphylactic type, and the other, a necrotic, delayed type. Delayed sensitivity was produced more easily when the infection was active. Antigens injected into tubercles produced by killed tubercle bacilli were not as effective in produc-

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ing this necrotic type of sensitivity (Freund, 1951). Dienes (1936) later found that in most cases the delayed reaction preceded the appearance of the immediate type (see also Salvin, 1958). As early as 1916, LeMoignic and Pinoy (1916a,b) reported that typhoid and paratyphoid vaccines mixed with petroleum jelly plus lanolin to form an emulsion produced better antibody response than vaccines given alone. These “lipovaccines” were also used by Ramon and coworkers (Ramon and Zoeller, 1927; Ramon and Falchetti, 1935; Ramon et d.,1937) to increase antitoxin production in animals. Coulaud (1935) found that an intense and persistent tuberculin sensitivity could be produced in rabbits with killed tubercle bacilli incorporated in para5n. This observation was confirmed by Saenz (1935, 1937) who employed vaseline (petroleum) oil instead of paraffin. With these observations at hand, Freund started his classic experimentation on the production not only of delayed sensitivity to tuberculin with killed tubercle bacilli but also of antibodies to antigens given in water-in-oil emulsions containing killed mycobacteria (Freund et al., 1937, 1948; Freund and McDermott, 1942; Freund and Bonanto, 1946). Freund’s observations have been reviewed by him in various articles (1947, 1951, 1956). The powerful antibody-stimulating effect of Freund’s adjuvant (light mineral oil, an emulsifier such as Aquaphor or Arlacel A, killed mycobacteria, and aqueous solution of antigen) has not been equalled by any other known adjuvant. This mixture has made it possible to produce antibodies even in animals which were considered to be rather poor antibody producers, such as the mouse and the rat (Havas and Andre, 1955). Two or more milligrams of antibody nitrogen per milliliter of blood have been obtained regularly in mice receiving egg albumin in complete Freund’s adjuvant ( Anacker and Munoz, 1961; Munoz, 1963a). Moreover, with this adjuvant it has been possible to stimulate the production of organ-specific antibodies and an actual isoallergic disease in animals (Kopeloff and Kopeloff, 1947; Morgan, 1947; Freund et al., 1947, 1953, 1954; Freund and Lipton, 1955; Waksman, 1958, 1959; Paterson, 1959). Thus, it has been possible to produce experimental allergic encephalomyelitis ( EAE ) , neuritis, thyroiditis, aspermatogenesis, adrenal damage, nephritis, uveitis, etc. ( Paterson, 1959; Witebsky, 1959). As pointed out by the early investigations of Dienes (1936) in tuberculous pigs and by Coulaud (1935), Saenz ( 1937), and later by Freund ( 1956) employing oil emulsions, mycobacteria not only increase the antibody and sensitization responses but, in some way, also change the characteristics of sensitization from the so-called immediate-type to the delayedtype reaction. It should be noted that in most cases a strong, immediate

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reaction is obtained which is then followed by a necrotic, delayed reaction (Dienes, 1927a,b,c, 1928, 1929), although Dienes ( 1936), and recently Salvin (1958) and others, have observed that a delayed sensitivity precedes the development of immediate or anaphylactic-type reaction in guinea pigs. Contact hypersensitivity to picryl chloride and other simple chemical compounds has also been easily produced by giving these substances in complete Freund's adjuvant ( Landsteiner and Chase, 1941, 1942; Chase, 1943, 1954). At least two main actions of complete Freund's adjuvant should be distinguished. The first is the striking enhancement of antibody production, and the second is the marked modification of the type of sensitivity produced (one could perhaps add the marked ability of Freund's complete adjuvant to produce isosensitization, but probably this property is closely related to the other two effects). Although these two actions seem to be independent (Raffel et d.,1949), it is usually difficult to dissociate one entirely from the other. This report will concentrate mainly on the increase of antibody formation.

B. EFFECTOF MYCOBACTERIA IN OIL-IN-WATER EMULSIONS Since the original observations of Lewis and Loomis (1924) and Dienes (1927a) most work involving the adjuvant effects of mycobacteria has been done with water-in-oil emulsions, From the investigations of Freund and co-workers (Freund, 1947, 1951, 1958), it is clear that the antibody response to bacterial cells such as those in typhoid vaccine (Freund and Bonanto, 1946) can be greatly stimulated by the use of water-in-oil emulsions. Without mycobacteria ( Freund's incomplete adjuvant) the effect is pronounced not only in intensity but also in duration. This important effect of the oil and emulsifier mixture will not be fully discussed here. Addition of killed Mycobacterium tuberculosis, Mycobacterium butyricum, or Mycobacterium phlei to the water-in-oil emulsions further augmented the antibody formation to typhoid vaccines, although this effect was not striking (Freund, 1947). In similar experiments employing egg albumin as antigen and measuring antibody quantitatively, it was found that addition of killed tubercle bacilli to the water-in-oil emulsion produced a substantial increase in antibody production. This increase in antibody correlated with formation of granuloma at the site of injection and hyperplasia in regional lymph nodes and spleen (E. E. Fischel et al., 1952). Tissue response to adjuvant without mycobacteria was minimal. This marked stimulation by mycobacteria on antibody response to soluble protein antigens and viruses has been observed by various workers (Weigle et aZ., 1980; Coe and Salvin, 1964;

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Shepel and Klugerman, 1963). Whole mycobacteria are not needed for this effect since it has been found that lipids extracted from mycobacteria are also effective (Freund et al., 1948; Freund and Stone, 1959; White et al., 1955b; White, 1959). Most acid-fast organisms, including M . butyricum, M . tubmculosis (human, bovine, and avian types), M . phlei, and Mycobacterium smegmatis (Freund, 1947; White, 1959), have been found active in stimulating the antibody response. Freund (1947) found that M . butyricum was slightly better and M . phlei slightly inferior to M. tuberculosis. This was also reported by Friedewald (1944) employing influenza virus as the antigen and by Shepel and Klugerman (1963) employing Venezuelan equine encephalomyelitis virus. The acid-fast Nocardia asteroides has also been found active (Freund and Lipton, 1948), as well as Nocardia brasiliensis and Nocardia rhotochrous (White, 1959). Freund's adjuvant with or without mycobacteria has also been effective in producing anaphylactic sensitization in animals thought to be rather resistant to anaphylaxis, such as the rat and mouse. Thus, local anaphylaxis in the lip of rats and mice has been produced as well as Arthus-type reactions in mice (Benedict and Tips, 1951; Lipton et d., 1956; Freund and Stone, 1956). Fatal anaphylactic sensitization of mice can be induced easily (Wheeler et al., 1950; Morgan et al., 1957, 1959; Munoz, 1963) when either complete or incomplete Freund's adjuvant is used with the sensitizing antigen. Recently, Crowle (1959a,b) claimed the production of delayed-type hypersensitivity in mice employing various antigens in complete or incomplete Freund's adjuvant. When we used egg albumin, one of the antigens employed by Crowle, the reactions observed were of the Arthus type, which could be duplicated by passive transfer of preformed antibodies ( Munoz, 1963b). The Arthus reactions are more severe, and necrosis increased when complete Freund's adjuvant was used. It is, however, extremely difficult to differentiate a delayed-type component after a severe Arthus reaction (Uhr et al., 1957; Letterer, 1959; Boughton and Spector, 1963). Arthus reactions are produced in mice that have high titers of circulating antibodies to the antigen used (Munoz, 1963b). Most likely, mice also develop a sensitivity similar to the delayed type found in the rabbit, guinea pig, and man, but this has not been conclusively demonstrated by skin test ( Pappenheimer and Freund, 1959; Salvin, 1963b). In this connection, it is interesting that Salvin et al. (1962) could not demonstrate delayed hypersensitivity in the skin of sensitized newborn guinea pigs, although they could transfer delayed sensitivity with the cells of such newborn pigs to adult animals. It is possible that in mice, as in newborn guinea

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pigs, a lack of some factor in the skin might account for the failure to develop typical delayed reactions. The use of Freund’s adjuvant has led to intensive investigations on experimental isoallergic diseases and to the development of a new branch in immunology known as “immunopathology” ( see Grabar and Miescher, 1959, 1962). Acid-fast bacilli are thought to be important for the production of isoallergic disorders, such as EAE, aspermatogenesis, uveitis, and thyroiditis ( Paterson, 1959; Constantinesco et al., 1959), even though Rivers et d. (1933) had been able to produce EAE in monkeys after giving a great number of injections of brain and spinal cord suspensions, and man, receiving rabies vaccines prepared from an infected rabbit’s spinal cord, on rare occasions develops encephalomyelitis. Acid-fast bacteria are not essential for the production of EAE in rats (Bell and Paterson, 1960; Steigman and Lipton, 1960;Levine and Wenk, 1961; Paterson and Bell, 1962) or in guinea pigs (Shaw et al., 1964). Shaw et al. (1964) were able to produce EAE by substituting a variety of gram-negative bacteria ( BordeteUa pertussis, Salmonella typhsa, Salmonella typhimurium), purified endotoxins, and some gram-positive bacteria ( Corynebucteriun d r u m , Coccidiodes immitis, and Streptococcus species ) for mycobacteria. Pseudomonas uerugitwsa has also been successfully used (Lipton and Steigman, 1963). It is evident, however, that acid-fast bacteria as a rule are the most potent.

C. CHARACTERIZATION OF AWE MATERIAL As was indicated in the previous section, mycobacteria, whether pathogenic or saprophytic, and various other acid-fast microorganisms enhance the production of antibodies and hypersensitivity reactions when incorporated in water-in-oil emulsions. Since other bacteria also are effective, it seems possible that more than one substance is capable of producing this adjuvant effect. According to Raffel and co-workers (Raffel, 1948; Raffel and Forney, 1948; Raffel et al., 1949) the “wax D” fraction obtained by the method of Anderson is the responsible component of the tubercle bacillus that directs the type of hypersensitivity produced. With wax D, either alone or in water-in-oil emulsion, it was possible to produce delayed hypersensitivity to egg albumin (Raffel et al., 1949) and to produce constant sensitivity to picryl chloride in guinea pigs (Raffel and Forney, 1948). However, wax D alone did not increase antibody formation to these antigens. Raffel et al. (1949) concluded that the glycolipid fraction of tubercle bacilli was responsible for directing the immune response toward the delayed-type sensitization. It is signifi-

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cant that Freund et al. (1948) had found that relatively large amounts of lipid were effective in stimulating antibody response but did not induce tuberculin sensitization in guinea pigs (Pound, 1958). Employing other immunological criteria, such as the production of EAE, various workers have reported successful experiments by substituting wax fractions, especially wax D of Anderson, for whole killed tubercle bacilli (Waksman and Adams, 1953; White and Marshall, 1958; Kies et al., 1958; Colover, 1958). Freund and Stone (1959), however, in a carefully controlled study, questioned the role of wax D in the production of isoallergic diseases. They found that only 0.015mg. of whole tubercle bacilli was needed to produce aspermatogenesis, whereas 0.1 mg. of wax D was required; to produce fatal E M only 0.04mg. of tubercle bacilli was needed, whereas 0.4 mg. of wax D was essential. Finally, they found that 0.004 mg. of whole Mycobacterium tuberculosis was capable of producing tuberculin sensitivity in guinea pigs, whereas 0.4mg. of wax D was needed. The latter observation indicated that wax D was probably contaminated with tuberculoproteins. They concluded that the wax, if it indeed is the active compound in tubercle bacilli, is far less active when purified than when present in the whole cell. Freund and Stone (1959) also stated that tuberculolipid fractions have been found contaminated with intact bacilli, but that the active material is not necessarily whole mycobacteria or even fragments of mycobacterial cells. From the amount of wax D needed to sensitize animals to tuberculin it was calculated that wax D could be contaminated with approximately 0.2 % of whole cells (assuming that whole cells were the only active material). Thus, the amount of mycobacteria in 0.2mg. of wax D would not be capable of inducing EAE or aspermatogenesis, and wax D preparations actually must have an active substance which is not whole cells. Furthermore, chromatographically purified wax D in l-mg. amounts was able to induce EAE (Colover, 1958). Wax D causes a cellular reaction that cannot be distinguished from that caused by whole tubercle bacillus (White et al., 1955b; Freund, 1958), and even though it is not a well-defined compound, there seems to be little doubt that the wax is active in stimulating antibody production, in directing the type of hypersensitivity produced in an animal, and in producing isoallergic diseases (White and Marshall, 1958; Kies and Alvord, 1959). Since wax is much less effective in the production of EAE or aspermatogenesis than whole mycobacteria (Freund and Stone, 1959), it is possible that it deteriorates during the purification procedure leaving only some of it fully active. This problem, however, still remains to be solved.

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In the original investigations only wax D preparations made from human strains of tubercle bacilli were effective as adjuvants in the production of EAE or of antibodies (Lederer, 1959). Recently, however, White et al. (1964)found that certain fractions obtained by high-speed centrifugation of wax D preparations from Mycobacterium phlei, M . tuberculosis, and several bovine strains of M . tuberculosis were active. All active fractions regardless of origin contained a peptide composed of D- and L-alanine, D-glutamic acid, and meso-u,a’-diaminopimelic acid (White et al., 1964). The presence of this peptide in all active preparations is of great interest. It is intriguing that White et al. (1964)found that the bovine and saprophytic strains of mycobacteria studied had relatively little of the active wax D fraction, whereas the strains of human origin contained a large percentage of the active peptide containing glycolipid (peptidoglycolipid). When whole cells are used, however, it has been found that Mycobacterium butyricum is superior to M . tuberculosis horninis and that M . phlei is only slightly inferior to the human mycobacteria ( Friedewald, 1944; Freund, 1947; Shepel and Klugerman, 1963). It may be that the peptidoglycolipid from bovine, avian, or saprophytic mycobacteria is more unstable than that from human strains. The structure of the active peptidoglycolipid corresponds closely in amino acid, amino sugar, and hexose composition to the mucopeptide of bacterial cell walls (White et al., 1964). ,Completely delipified tubercle bacilli were found still active in the production of EAE (White and Marshall, 1958). These were essentially cell walls and contained the three amino acids found in active wax D preparations from human strains of M . tuberculosis. In this connection it should be pointed out that Larson et al. (1963)found that cell walls of M . tuberculosis had an adjuvant effect in the production of EAE. Active fractions of essentially similar composition as wax D have been reported also by Colover and co-workers (Colover, 1954; Colover and Consden, 1956). It is noteworthy that Lederer (1959)has stated that typical delayed hypersensitivity can be induced by a variety of mycolic acid esters of carbohydrates (cord factor, wax D, etc. ) and that even a simple mycolate of a hexose (glucose, galactose, manose) or a pentose (arabinose) is sufficient to induce delayed hypersensitivity. Not all of these, however, are effective in stimulating antibody formation or production of EAE. We know of no confirmatory reports of these very interesting observations. Recently it has been shown that the type of emulsion formed by water-in-oil adjuvants is extremely important in the production of EAE and that an optimal ratio exists between the quantity of tubercle bacilli

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and the amount of the encephalitogenic material (Shaw et al., 1962; Lee and Schneider, 1962). It has also been found that lipids of the tubercle bacilli facilitate the formation of a stable emulsion (Shaw et al., 1963). These observations may explain many discrepancies in the literature. The exact role of the various fractions of acid-fast bacteria is still far from settled. The studies in this field have been complicated by (1) the complexity of Freund's adjuvant, (2) the variety of assay methods used, and (3) the lack of standard reagents with which to prepare Freund's adjuvant. The effect of the type of emulsifier as well as of the particular mineral oil employed usually has been ignored. It is, however, known that there are marked differences in mineral oils employed (Salk et al., 1952; Salk and Laurent, 1952; Salk, 1953). Mineral oils of low viscosity, such as Bayol F, do not produce as frequent local reactions in human subjects as have oils of higher viscosity (Henle and Henle, 1945). Recently, differences in the emulsifier have also been noted (Finger, 1962; Berlin, 1963). Very few investigations have been done dealing with the critical evaluation of various types of oils or emulsifiers ( Berlin, 1960, 1963; Haas and Thomssen, 1961) . Although various types of acid-fast bacilli differ with respect to their adjuvant effect ( Friedewald, 1944; Freund, 1947; Shepel and Klugerman, 1963), the possible variations of different strains of the same bacterium or various culture media on the potency of the cells have not been established.

D. MODEOF ACTIONOF MYCOBACXERIA The enhancement of antibody response by mycobacteria and other acid-fast microorganisms has been so intimately associated with water-inoil emulsions that it is difficult to avoid some discussion on the effect of water-in-oil emulsions when discussing the mechanism of action of acid-fast bacteria. As previously mentioned, three different types of immunological responses have been studied in evaluating the adjuvant effect of waterin-oil emulsions either containing or lacking mycobacteria. These are ( 1 ) the stimulation of the antibody response, (2 ) the production of delayed-type hypersensitivity, and ( 3 ) the production of isoallergic diseases. It is also difficult to separate these three effects of complete Freund's adjuvant. It is, in fact, still not clearly known whether the three effects are different or are produced by a similar mechanism. Certain phenomena which were attributed to delayed-type hypersensitivity, such as the tissue homograft rejection, can be elicited by means of factors which appear to be antibody (Najarian and Feldman, 1962, 1963; Kret-

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schmer and Perez-Tamayo, 1962). It is also suspected that antibody might be involved in EAE and aspermatogenesis (Katsh, 1959; Paterson and Harwin, 1963). As pointed out before, acid-fast bacteria are not essential in the production of EAE or aspermatogenesis (Katsh, 1959; Shaw et al., 1964). Various hypotheses have been advanced to explain the enhancement of antibody production and the induction of isoallergic diseases by Freund’s adjuvant: ( 1 ) Both oil and mycobacteria produce a specific cellular reaction at the site of injection, the regional and distant lymph nodes, and the spleen. In these sites, cells such as lymphocytes, macrophages, epithelioid cells, and plasma cells, which are thought to be involved in antibody formation, proliferate (White et al., 1955b; Rupp & al., 1960). (2) Water-in-oil emulsions have the ability of retaining antigen at the site of inoculation and protecting it from rapid cellular destruction, thus providing a slow and prolonged antigenic stimulus to antibody-forming cells (Halbert et al., 1946; Freund, 1956; Holub, 1957; McKinney and Davenport, 1961). ( 3 ) The oil-trapped antigen is distributed throughout the body in oil globules, thus providing a more extensive stimulation of the antibody-forming cells ( Freund, 1956; Holub, 1957). ( 4 ) The adjuvant produces permeability changes or cellular damage which make the antigen more accessible to the specific antibodyforming cells (Jones and Roitt, 1961). ( 5 ) The adjuvant, especially the mycobacteria, or oil-soluble lipopolysaccharide extracted from the bacteria, may act as a “schlepper” to increase the molecular weight and antigenicity of the antigen (Lipton, 1959; Katsh, 1959). ( 6 ) The adjuvant impairs the homeostatic mechanism controlling immune tolerance, thus allowing forbidden clones of immunologically competent cells to appear and produce antibody directed against the host’s own tissue antigens ( Burnet, 1959). Any one of these postulates does not explain completely the mode of action of complete Freund’s adjuvant, and it is perhaps a combination of at least some of these effects that may explain the mode of action of water-in-oil emulsions containing mycobacteria. It is also possible that additional unsuspected effects of these substances might be involved. It can hardly be questioned that Freund’s complete adjuvant provides a depot of antigen which remains there for a considerable length of time (Talmage and Dixon, 1953; White et aZ., 1955b; Freund, 1956) and that it stimulates the proliferation of cells associated with antibody formation not only in local lymph nodes but also in lymph nodes far removed from the site of inoculation, as well as in the spleen (E. E. Fischel et al., 1952; White et al., 1955b; Askonas and White, 1956; Steiner

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et al., 1960). The retention of the antigen is mainly due to the oil emulsion, but the cellular response is greatly increased by the addition of mycobacteria (Fischel et al., 1952). Granulomas develop not only at the site of inoculation, but elsewhere (Fischel et d.,1952; White et d., 1955b; Freund, 1956). Oil droplets containing antigen can be found in cervical lymph nodes 3 months after injection of incomplete Freund’s adjuvant into the rabbit’s footpad (Herdegen et d.,1947; Freund, 1951; Talmage and Dixon, 1953; White et al., 1955b). Higher antibody titers have been recovered in fluid obtained from sites injected with toxoid in water-in-oil than in the blood or other areas of inflammation (Freund et al., 1952), and granulomatous tissue in the rabbit produced, according to Askonas and Humphrey (1955), a significant amount of the antibody. Removal of the granuloma at the site of injection showed, however, that the effect is not confined to the local granuloma (Freund, 1951; White, et al., 1955b), and in the guinea pig, lymph nodes far removed from the injected footpads produced significant amounts of antibody (Askonas and White, 1956). In addition to these effects, paraffin oil attracts about the antigen cells such as large monocytes (some like epithelioid cells), lymphocytes, and plasma cells that may take part in antibody formation. All these effects can be seen without the tubercle bacilli, but the tubercle bacillus greatly increases the cellular response in the animal. Whether these same factors are involved in the production of isoallergic diseases by Freund’s adjuvant is still not understood. One may postulate that this effect is, indeed, similar to that of the antibodyenhancing effect of Freund’s adjuvant, Other mechanisms, such as increased permeability and cell destruction associated with Freunds adjuvant, may also play a distinct role in the production of isoallergic diseases (Jones and Roitt, 1961). Another possibility is that a substance in acid-fast bacteria as well as other substances of bacterial origin can combine with the organ-specific antigen to produce a complex with specificity still similar to the organ antigen, but different enough so as to make it “foreign” to the antibody-forming cells (Freund, 1956; Lipton, 1959). To explain the mode of action of mycobacteria in producing delayed hypersensitivity to an antigen which normally produces an immediatetype response, it may be well to analyze some of the experimental observations. ( I ) In Dienes’ original work, it was necessary to inject antigen into tubercles during active infection in order to produce the delayed, necrotic type of hypersensitivity. Since tubercles produced by dead tubercle bacilli were usually not effective in this respect, it appears

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that living tubercle bacilli favor the production of delayed hypersensitivity (Freund, 1956). Delayed hypersensitivity has, however, been produced even without using Freund's adjuvant ( Dienes, 1936; Salvin, 1958). (2) The injection of antigen emulsified in complete Freund's adjuvant produced delayed hypersensitivity. If, however, the antigen is given at a site removed from that at which the Freund's adjuvant is given no such effect is shown (Freund, 1956). (3) Water-in-oil emulsions are effective but oil-in-water emulsions are not active (Freund, 1956). The antigen has to be in the water phase surrounded by the oil in order to obtain an adjuvant effect. ( 4 ) Delayed hypersensitivity, in the absence of immediate type, can be produced by minute quantities of protein antigen such as egg albumin or diphtheria toxoid, but large amounts of these antigens also give immediate-type sensitivity (Salvin, 1958). ( 5 ) Pure polysaccharide antigens as well as haptens attached to a protein do not produce delayed-type sensitivity, but do stimulate antibodies and Arthus-type sensitivity (Gell and Benacerraf, 1959; Benacerraf and Gell, 1959; Gell and Benacerraf, 1961; Salvin 1963a,b; Salvin and Smith, 1960). ( 6 ) Delayed hypersensitivity can be produced. with antigen-antibody complexes made in antibody excess but not with complexes made in antigen excess (Uhr et d.,1957). ( 7 ) Tuberculin or tuberculin-type antigens when used as skin testing materials give delayed-type reactions in hypersensitive animals, but do not, although fully antigenic, induce a delayed, necrotic hypersensitivity in animals (Freund, 1956; Gell and Benacerraf, 1961). ( 8 ) Denatured proteins can be used to produce delayed hypersensitivity to undenatured proteins ( Gell and Benacerraf, 1958, 1959). All these observations seem to indicate that one of the most important factors in the production of delayed hypersensitivity resides in ( I ) the antigen used in performing the skin test and ( 2 ) the form in which the antigen is used for sensitization. It is not presently known what function the acid-fast bacteria or other bacteria play in the production of immunopathological disorders ( Paterson, 1959). As pointed out before, the organ extracts have to be given together with the adjuvant for best results, and the emulsion and amount of acid-fast bacteria have to be controlled carefully (Shaw et al., 1982; Lee and Schneider, 1962). The antibody-stimulating property of incomplete adjuvant is not per se capable of producing the disease (Constantinesco et d.,1959). If TB or other bacteria or their products are essential to produce these diseases, it may be possible that a substance from these bacteria can combine with a substance from the organ to produce a modified antigen which can then stimulate antibodies to organ-specific groupings ( Freund, 1956; Grabar, 1959; Lipton, 1959;

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Lipton and Steigman, 1963). It is attractive to think that the organspecific substance has to be altered before producing antibodies in the same species of animal. All previously successful experiments in production of isoallergic disorders may have sufficiently altered the native antigen to accomplish this change. If indeed this happens, the powerful antibody-stimulating action of Freund’s mixture could explain its effectiveness in producing these disorders. It is noteworthy that when EAE has been produced without the aid of bacteria, heterologous brain material was more effective (Bell and Paterson, 1960). This also indicates that “foreignness” of the antigen may be an important factor in producing EAE. 111. Effect of Endotoxins from Gram-Negative Bacteria

A. NATURE AND GENERAL BIOLOGICAL CHARACTERISTICS OF ENDOTOXINS The term endotoxin is perhaps an unfortunate one because it implies that these toxic substances are found inside the bacterial cell. Actually, the material associated with toxicity characteristic of endotoxins is found in the cell walls (Ribi et al., 1959). Another term that has been widely accepted for these substances is that of lipopolysaccharides, although the exact chemical nature is not well known. Until recently the high lipid content of endotoxin preparations was considered one of the most important chemical properties of endotoxins ( Westphal, 1957; Kabat, 1961 ) . Present evidence, however, indicates that material with typical endotoxin activity may have little lipid and no protein or polypeptide components ( R i b et al., 1961, 1964). In this review, however, the terms endotoxin and lipopolysaccharides will be used interchangeably. Endotoxins are large molecular weight substances which exhibit typical toxicity, pyrogenicity, and antigenicity (Ribi et al., 1962). They are easily extracted by well-known procedures such as the trichloroacetic acid extraction (Boivin et d.,1933), the phenol-water procedure (Westphal et al., 1952), and others (Ribi et al., 1964). The material extracted by these methods consists mainly of polysaccharides with various amounts of lipid, phosphorus, and nitrogen. The chemical composition of these extracts varies considerably with the type of organism employed and with the medium in which the organism is grown (Fukushi et al., 1963; Ribi et al., 1964). Although typical endotoxins with similar biological activities ( toxicity, pyrogenicity, tumor damage activity, production of Shwartzman reaction, and the epinephrine-enhancing reaction) are extracted mainly from gram-negative bacteria (Thomas, 1954), substances with at least some

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similar biological activities have been extracted from gram-positive bacteria by some workers (Stetson, 1956; Cremer and Watson, 1960; Isenberg et al., 1963). They have also been obtained from various other sources (Antopol, 1937a,b; Antopol and Click, 1938; Landy and Shear, 1957). Although the composition of endotoxins extracted from gram-negative bacteria varies according to the method of extraction, the strain of bacteria used, and the medium in which they are grown (Fukushi et al., 1963), the qualitative biological activity, nevertheless, seems to be somewhat constant. Endotoxins are antigenic and can be differentiated serologically (Wilson and Miles, 1957). Antibodies to these substances can be detected in sera of normal animals even when they have been reared in a sterile environment (Michael et al., 1961, 1962; Landy et al., 1962). These antibodies are specific and can be absorbed by the appropriate endotoxin (Michael et al., 1962). Antibodies in normal animals probably results from the inescapable contact with endotoxins from the innumerable bacteria of the intestinal tract or, perhaps, from the ingestion of these substances. Endotoxins are quite resistant to heat and would not be destroyed during the sterilization procedures. Conceivably, germfree animals could be also exposed by ingesting antigenically competent endotoxins (Wagner, 1959; Sterzl et al., 1960, 1961, 1982; Michael et al., 1962). Some workers have considered that most animals are hypersensitive to endotoxins and that some activities of endotoxin might be due to the hypersensitivity reaction (Weil and Spink, 1957; Stetson, 1959; Sell and Braude, 1961). Some of the most prominent effects of endotoxins seem to be on the reticuloendothelial system (Biozzi et al., 1955; Zweifach et al., 1957; Howard, 1959; Thomas, 1958, 1959), and since endotoxins seem to fix to these tissues it is probable that a direct action might be responsible for some of their effects (Rubenstein et al., 1962). Endotoxins enhance the reactivity of the skin to epinephrine, produce local and generalized Shwartzman reactions, and damage tumor cells (Thomas, 1958, 1959), increase the so-called properdin (Landy and Pillemer, 1956) and lysozyme levels in the blood (Hook et al., 1960), and decrease complement (Gilbert and Braude, 1962; Kostka and Sterzl, 1962). Endotoxins have also been reported to immobilize sperm cells (Dennis, 1962), to increase the permeability of the blood-brain barrier (Eckman et al., 1958), to affect the adrenal and pituitary glands (Wexler et al., 1957; Egdahl, 1959; Egdahl et al., 1959; Nadel et al., 1961), to increase the phagocytic efficiency of macrophages ( Jenkin and Palmer, 1960), etc. Many of these phenomena have been produced with endotoxins

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which have been practically freed of protein and contain only small amounts of lipid (Ribi et al., 1962, 1964). These polysaccharide-lipid complexes are polymerized and have a high molecular weight. They can be easily depolymerized by acid hydrolysis which results in fragments of about 10,000 molecular weight with chemical composition similar to endotoxin. These fragments can precipitate with antiserum but lack most of the typical biological activities of native endotoxin (Ribi et al., 1962).

B. ORIGINAL OBSERVATIONS For the purpose of this review, the most pertinent action of endotoxin is the stimulation of antibody response. As pointed out elsewhere, various investigators observed many years ago that killed whole cells of Pseudomonas aeruginusa (Khanolkar, 1924), Salmonella typhosa (Ramon and Zoeller, 1926, 1927), Brucella abortus (Ramon et al., 1950), Bordetella pertussis (Greenberg and Fleming, 1947, 1948; Fleming et al., 1948), and other bacteria were capable of enhancing the antibody response to various antigens. The substance responsible for the effect may not be identical in all these cases, but the active substance is probably endotoxin. With S . typhosa and other members of the Salmonella group, as well as Escherichia coli, Proteus vulgaris, Serratia marcescens, Pseudomoms aeruginma, and Brucelh melitemis, the active substance has been identified as lipopolysaccharide complex extracted by various methods (Johnson et al., 1956). Similar extracts made from other organisms have also proven active in stimulating antibody production to antigens administered with them (Condie et al., 1955; Farthing, 1961; Malmgren and Ribi, 1962). These preparations have the various effects of typical endotoxins and, perhaps, other effects (Condie et al., 1962; Staab et al., 1962). All the work on the adjuvant effect of endotoxins known to the author has been performed with lipopolysaccharides which chemically are still rather crude preparations. C. EFFEC~ OF VARIOUS ENDOTOXIN PREPARATIONS

The most extensive work on the adjuvant effect of purified endotoxins has been done by Johnson and co-workers (1956). They found that purified endotoxins from S . typhosa, P . vulgaris, Pseudomonas aeruginosa, S . marcescens, B. pertussis, and Brucella melitensis stimulated antibody response to egg albumin (EA). Employing endotoxin from S . typhma they found that three intravenous injections of as little as 1 pg. mixed with 2 mg. of egg albumin given 3 days apart produced a perceptible increase in antibody response to EA. As much as 20 times more antibody was produced in rabbits receiving endotoxin plus EA as compared to

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controls receiving EA alone. A single intravenous injection of 1 pg. of endotoxin plus 10 mg. EA also gave detectable antibody enhancement. Endotoxin was as effective whether it was given mixed with antigen or injected separately in opposite ear veins. The adjuvant effect was observed with all protein antigens tested (EA, bovine serum albumin, diphtheria toxoid, plague capsular protein). Strangely enough, no antibody-stimulating effect was observed when Vi antigen, pneumococcus Type 3 polysaccharide, and Pasteurella tularensis vaccine were used. It is not surprising that the adjuvant effect was not observed when whole cells of a gram-negative bacterium were used as antigen, since these cells already possess endotoxin. In the case of pneumococcal polysaccharides and Vi antigen, however, it is more difficult to explain the failure. It is possible that these polysaccharides, as has been shown for pneumococcus polysaccharides (Felton, 1949), remain in the animal body for quite some time, thus providing a good antigenic stimulus which is difficult to improve. Minute amounts of these polysaccharides are extremely good antigens (Heidelberger et al., 1950). According to Johnson et al. (1956), endotoxins exert their adjuvant effect only in animals susceptible to their toxic action. In guinea pigs, an animal which is rather resistant to endotoxin (LD60 = 0.5-1 mg.), no enhancement of antibody was observed; neither was this effect shown in rabbits made tolerant to endotoxin. Farthing and Holt ( 1962), however, have shown that lipopolysaccharides from B. pertussis and E . coli act as an adjuvant in the guinea pig and that lipid A obtained from these lipopolysaccharides was also effective without producing the hyperemia associated with endotoxins. This suggests that the symptoms of stress produced by lipopolysaccharides are not directly related to their ability to enhance antibody formation. It has also been found that in chickens endotoxins have a pronounced adjuvant effect at levels which are not obviously toxic (Luecke and Sibal, 1962). Adjuvant effect of endotoxin has also been observed in mice, which are rather resistant to endotoxin (Merritt and Johnson, 1962; Malkiel and Hargis, 1959). Endotoxin stimulates antibody response in rabbits only when given less than 6 hours before and not more than 4 days after the antigen (Johnson et al., 1956). On the other hand, endotoxin was effective in mice when given 7 days before or 6 days after the antigen (Merritt and Johnson, 1962). It should be mentioned here that B. pertussis vaccine increased capillary permeability in mice for a period of at least 8 days, and since endotoxin also produces this effect (Munoz, 1961), it is possible that this increase in permeability might be responsible for the adjuvant effect of endotoxin.

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The quantities of meningococcal endotoxin which increase the antibody response are similar to those needed to prepare the skin of rabbits for the Shwartzman reaction (Condie et al., 1955). An optimal amount of this endotoxin was found which, if exceeded, showed little or no adjuvant effect (Condie et al., 1955). Luecke and Sibal (1962) have studied the effect of endotoxin from Salmonella abortus equi on the antibody response in chickens. This endotoxin increased the primary antibody response of chickens in three different ways: the serum antibody was detected at an earlier time (4 or 5 days after antigen injection); severalfold increase in antibody titers were obtained; and antibody persisted for a longer period of time in the circulation. A dose of 5 pg. of endotoxin was effective but 10 pg. was better. The effect was demonstrated best when antigen was injected simultaneously with endotoxin. It was also shown when endotoxin was given 24 hours after antigen, but not when endotoxin was given 24 hours before antigen. The doses of endotoxin caused no untoward reactions in chickens, even though a marked adjuvant effect was observed (Luecke and Sibal, 1962). In addition to these observations, Sibal (1961) and Sibal and Olson (1958) found that spleen cells from previously immunized chickens implanted on the chorioallantoic membrane of developing hens’ eggs could produce antibodies. The antigen had to be given to the donor chicken some 48 hours before the spleens were removed in order to obtain antibody production in the chorioallantoic membrane transplants. Transplants made 1 hour after the antigen was given did not produce antibody and those made 24 hours after the antigen produced antibody only in a few cases. Ten micrograms of endotoxin given simultaneously with 32 mg. of bovine serum albumin to the donor chicken induced a change in the donor so that it was then possible to obtain antibodies by spleen cells transplanted 1 hour after the antigen-endotoxin injection. In addition, spleens taken 48 hours after antigen-endotoxin injection were not as satisfactory as those obtained 24 hours after or even those taken 1 hour after. No increase in antibody due to endotoxin was observed in these experiments. McKenna and Stevens (1957) reported enhancement of antibody response to bovine y-globulin by rabbit spleen cells in tissue culture. Titers in the culture fluid were higher only when endotoxin was given 24 hours before the administration of the antigen. Purified protein antigens added in vitro to splenic fragments of rabbits which had received endotoxin 24 hours previously produced antibodies within 1 hour following incubation of the cells in the tissue culture (Stevens

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and McKenna, 1958; McKenna and Stevens, 1960). Unfortunately, these extremely interesting observations (production of antibody entirely in tissue culture) have not been confirmed (Sterzl and Rychlikovh, 1958; P. Kind and Johnson, 1959; Stavitsky, 1961; Fishman, 1961). Endotoxins also increase anaphylactic sensitivity of animals to antigens administered with them. Animals that are poor antibody producers and that have been considered to be highly resistant to anaphylaxis can be made susceptible by administering the antigen with endotoxins (Malkiel and Hargis, 1959; Munoz, 1963a). This increase in anaphylactic sensitivity may well be due to the adjuvant action of these lipopolysaccharides. Pretreatment of previously sensitized mice with small amounts of endotoxin up to 3 hours prior to challenge with antigen also enhanced the incidence of fatal anaphylaxis (Einbinder et d.,1962). Obviously, this effect is not due to production of antibodies and must be the result of some still unknown action of endotoxin. In contrast, if endotoxin is given from 4 to 24 hours prior to challenge an actual protective effect was produced (Einbinder et d.,1962). The protective effect of endotoxin given 24 hours before challenge has also been observed in this laboratory when nearly lethal doses are given (Munoz, 1963b). This must be caused by an action of endotoxin which is unrelated to the immune response. It is well known that endotoxins from gram-negative organisms modify the response of experimental animals to various forms of injury such as thermal burns (Lasker and Fox, 1959), hemorrhage (Smiddy and Fine, 1957), tourniquet shock ( Oldstone, 1959), whole body irradiation (Zweifach et aZ., 1959), anaphylaxis (Einbinder et d.,1962), and massive bacterial (E. W. Hook and Wagner, 1959a) or viral infections (E. W. Hook and Wagner, 1959b). Thus, depending on circumstances endotoxin may either increase mortality or confer some protection, These effects of endotoxin are not associated with antibody production.

D. MODEOF ACTIONOF ENDOTOXINS It can be stated at the outset that the exact mode of action of endotoxins in stimulating the antibody response is not known. The febrile response and the increase in granulocytes observed after injection of endotoxin do not seem to be responsible for the adjuvant effect, since amounts of endotoxin which can produce these effects are not active as an adjuvant (Condie et d.,1955). Amounts that can prepare the rabbit for generalized Shwartzman reaction, however, have been effective (Condie et d.,1955). It has been suggested by some

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investigators that the toxic properties of endotoxins might not be involved in their adjuvant effect (Luecke and Sibal, 1962; Farthing and Holt, 1962), although toxicity has been suspected by others to play an important role (Johnson et al., 1956). An adjuvant effect has been demonstrated in chickens, guinea pigs, and mice which are relatively resistant to the lethal effect of endotoxin (Malkiel and Hargis, 1959; Luecke and Sibal, 1962; Farthing and Holt, 1962; Munoz, 1963a). It is not possible to say with certainty that amounts of endotoxin which produce adjuvant effects are completely free of toxicity to these animals. It is noteworthy that the effect of endotoxin is mainly observed in the primary response (Johnson et al., 1956; Prochazka, 1961), and that no adjuvant effect was demonstrable in rabbits made tolerant to endotoxin (Johnson et al., 1956). The adjuvant effect of Bordetella pertussis vaccine can also be reduced by a previous injection of this vaccine given without antigen (L. S. Kind and Roffler, 1961; Farthing and Holt, 1962). Prochazka (1961) found that the adjuvant effect of endotoxin from Salmonella typhdmurium could be demonstrated by repeated intravenous injections of the endotoxin with diphtheria toxoid. The increase in antibody formation produced by endotoxin was noticed even when the toxoid was alum-precipitated. It is well known that marked changes occur in the reticuloendothelial cells (Biozzi et al., 1955; Thomas, 1959) and in the spleens of rabbits after receiving endotoxin (P. Kind and Johnson, 1959; Ward et al., 1959, 1961; Johnson et aZ., 1962; Langevoort et al., 1963). Other changes which are known to occur in animals receiving endotoxin may somehow affect the antibody response. Those giving rise to the Shwartzman reaction, for example, might have some effect on the antibody-forming mechanism as well as the increased sensitivity of tissues to epinephrine. The known hemodynamic alterations produced by endotoxin may somehow influence the distribution and retention of antigen ( Condie et al., 1955). The adjuvant effect of endotoxins in rabbits can be shown when they are given simultaneously, 6, 12, 24, and 48 hours after but not 24, 12, or 6 hours before or 4 days after antigen injection (P. Kind and Johnson, 1959). This indicates that there is a critical temporary change produced by endotoxins during which time protein antigens become more effective. Taliaferro and co-workers ( Taliaferro and Talmage, 1955; Taliaferro and Taliaferro, 1957) have shown that there is a critical early step in antibody formation that occurs prior to the actual incorporation of the amino acids into the antibody. P. Kind and Johnson (1959) postulated that this is the phase of antibody formation influenced by endotoxin and that the effect may be a direct or indirect one on the primitive reticular

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cell described by Wissler and co-workers ( 1957). These primitive cells are assumed to require antigenic stimulation to proliferate and differentiate into the large pyroninophilic cell associated with actual antibody production. According to P. Kind and Johnson (1959), endotoxin could not affect these latter cells because it is active for a limited time during the early phase of antibody formation. Endotoxin restores antibody formation in X-irradiated rabbits (P. Kind and Johnson, 1959) and rabbits treated with cortisone (Ward and Johnson, 1959). Rabbits receiving a single injection of protein antigen show marked histologic changes in the follicular areas of the spleen. Only minimal changes are seen in the pulp, Following injection of endotoxin plus antigen, no new cellular types appear, but a profound augmentation of the cellular response is observed. The augmentation appears to account for the higher levels of antibody initiated by this adjuvant (Ward et al., 1959, 1961; Langevoort et al., 1963). It is interesting that Ward et al. (1959) observed no increase in plasma cells but rather in what they called modified reticular cells. Endotoxins potentiated all phases of antibody production-the induction period was shortened by approximately 4 days, antibody titers were higher, and the antibody response prolonged for several days. It is noteworthy that Ward et al. ( 1959) did not observe this sequence of readily discernible events in rabbits receiving only endotoxin. Endotoxins are antigenic and should initiate changes similar to those produced by other antigens. It may be that the amount of endotoxin given was too small to make these changes noticeable. No splenic follicular response was observed in cortisone-treated rabbits receiving antigen alone. When endotoxin was also administered, however, there were marked follicular activity and antibody production to the antigen injected (Ward et al., 1961). Johnson and co-workers concluded from these studies that endotoxins stimulate the follicles of the spleen to accelerate reticular cell diff erentiation. Since increased antibody production was not accompanied by an augmented plasma cell or mature lymphocyte response, these workers concluded that the reticular cell of the white pulp must be the important cell in the primary production of antibody. However, when they attempted to demonstrate antibody in the various cells of the spleen by direct fluorescent staining they found no antibody in the hyperplastic germinal centers of the white pulp. Instead, as has been found by others, they found antibody in the plasma cells, modified reticular cells, plasmoblasts and in Russell body cells (Coons, 1958; Vazquez. 1961; Johnson et al., 1962).

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Weissman and Thomas (1962) and Martini (1959) have demonstrated that intravenous injection of endotoxin in rabbits gives rise to significant increase in the release of two glycosomal enzymes from the large granular fractions of liver homogenates prepared in sucrose. Both cathepsin and 0-glucuronidase activities were increased by endotoxin treatment of rabbits. Fractions prepared from animals made tolerant to endotoxins or from animals pretreated with glucocorticoids no longer responded to incubation and irradiation by an augmented release of these two hydrolases (Weissman and Thomas, 1962). These very interesting observations may explain some of the modes of action of endotoxins, and further studies along these lines should prove fruitful in elucidation of endotoxin action. From all these observations it seems clear that endotoxins do not enhance antibody production by creating a depot of antigens or by producing local proliferation of cells as might be the case for Freund's adjuvant and alum, Endotoxins do have a marked effect on the reticuloendothelial system (Biozzi et al., 1955; Thomas, 1959), have maked effects on the hemodynamic flow (Thomas, 1959), produce marked changes in permeability (Eckman et d.,1958; Thomas, 1959; Munoz, 1961), have a marked effect on the adrenal glands (Egdahl, 1959; Egdahl et al., 1959; Nadel et al., 196l),are toxic to various cells (Dennis, 1962), and produce, among other phenomena, hypersensitive-like reactions in animals (Stetson, 1959). All these effects have been suspected by various workers to increase antibody production. Thus, proliferation of cells has been given as the main mode of action of Freund's adjuvant ( Freund, 1956). Alteration in the distribution of antigen based on changes in circulatory patterns in key organs has been suspected to increase the efficiency of an antigen ( Condie et al., 1955). Changes in permeability and adrenal insufficiency have been suspected to increase antibody response (Rose, 1959; Munoz, 1961; Char and Kelley, 1962). Substances released from tissues or damage to certain tissues have been observed to be beneficial to antibody production (Taliaferro and Jaroslow, 1959, 1960; Braun, 1961; Heuer et al., 1962; Dixon and McConahey, 1963). Liver damage has been shown to increase antibody production (Havens, 1959), and, finally, antigen-antibody reactions in the animal at the time of antigenic stimulation have been shown to increase antibody formation (Good et al., 1957; Terres and Wolins, 1961; Terres and Stoner, 1962). The action of endotoxins on antibody formation may then not be a single action, but an additive effect of a multitude of actions.

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IV. Effect of Bordetella perfussis

A. GENERALREMAW BordeteZla pertussis, as do most other gram-negative organisms, also contains endotoxin which has the ability to enhance the antibody response. There are some reasons, however, for considering this organism separately. First, injection of B. pertussis vaccine into mice produces various hypersensitive conditions which are not directly ascribable to endotoxin activity, and second, fractionation of these cells by methods that usually do not extract endotoxins has yielded materials which are highly active in producing hypersensitivity in mice to various substances and stresses, including anaphylaxis.

B. GENERALBIOLOGICAL EFFEC~S 1, Hypersensitivity Reactions In 1942 Eldering isolated a crude lipopolysaccharide fraction from B. pertwsis which rendered mice highly sensitive to intraperitoneal challenge with living cells; in 1944 Ospeck and Roberts found that mice immunized with toxoid prepared from culture filtrates often died of shock following challenge with B. pertussis toxin; and 3 years later Parfentjev et aZ. (1947a,b,c) found that mice inoculated with B . pertussis vaccine could be killed a number of days after with proteins and nucleoproteins from B. pertussis. These observations seemed to show that B. pertussis-inoculated mice were either highly sensitive to toxins or to anaphylaxis induced with antigens of B. pertussis. Indeed, these possibilities were conclusively shown later by Parfentjev and co-workers who found that B. pertussis vaccine increased the sensitivity of mice to endotoxin, bacterial infections, viral infections, and histamine ( a review of the literature on this field is given by L. S. Kind, 1958), and Malkiel and Hargis (1952a) who showed that B. pertussis vaccine given with other antigens makes mice highly sensitive to anaphylactic shock by the antigen administered. Soon after these observations it was found that B. pertussistreated mice also became highly sensitive to serotonin, to cold stress, to X-rays, to anoxia, and to passively induced anaphylaxis (see Pittman, 1957; L. S. Kind, 1958; Schweinberg, 1961). The phenomenon most widely studied has been the sensitization to histamine. Normal mice of all strains tested have been found to be extremely resistant to histamine, although dserences in degree of resistance are found among them (Munoz and Schuchardt, 1953). BordeteZZu pertussis vaccine or active fractions increase markedly the susceptibility of certain strains of mice. Most strains, however, do not

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become as hypersensitive to histamine after injection of B. pertussis (Munoz and Schuchardt, 1953; Malkiel and Hargis, 1960; Munoz 1963b). This might also prove to be the case with serotonin, but not enough work has been reported to establish this point. The lack of relationship between the increased susceptibility to histamine or serotonin and increased anaphylactic susceptibility of B. pertussis-treated mice has been amply demonstrated. This phenomenon of sensitization of mice to histamine, serotonin, cold stress, X-rays, anoxia, viral and bacterial infections, etc., is not within the scope of this review and will not be discussed fully. Those interested will find good coverage of the subject in reviews by Pittman (1957), L. S. Kind (1958), Schweinberg (1961), and Munoz (1964). 2. Antibody Response and Anaphylactic Sensitivity Greenberg and Flemming (1947, 1948) and Flemming et d. (1948) showed that B. pertussis given with diphtheria toxoid enhances antitoxin production in guinea pigs as well as in children. Similar observations were reported by Ordman and Grasset (1948) who also noted that agglutinins to B. p e r t w d were not increased when the mixed vaccine was used. This adjuvant effect of B. pertussis on diphtheria and tetanus toxoids was later confirmed by many others (Ferago and Pusztai, 1949; Ungar, 1952; Barnes and Holt, 1955; Barr et al., 1957; Bousfield and Holt, 1957). Bordetella pertussis vaccine also enhances the production of antibodies in mice to particulate antigens such as chicken red blood cells (L. S. Kind, 1957) and to purified, soluble protein such as egg albumin and bovine serum albumin (Malkiel and Hargis, 1959; Munoz, 1963a). The adjuvant effect of B. pertussis in mice is striking, but not nearly as pronounced as that produced by Freund's complete adjuvant. In degree it compares to the effect given by endotoxin preparations. Farthing (1961) and Farthing and Holt (1962) found that endotoxin prepared from B. pelzzcssls had an activity similar to whole B. pertussis cells. Furthermore, they found that lipid A prepared from these endotoxins was also active. In the case of lipid A, the activity was greatly reduced as compared to the intact lipopolysaccharide. Except for Farthing's work, most of the studies on the adjuvant effect of B. pertussis have been done with whole cells. I t is obvious from the work on endotoxin, that whole cells are not needed. We have made extracts from B. pertussis (Munoz and Hestekin, 1962, 1963) which are also active in stimulating the antibody response to protein antigens. Even highly purified fractions possessed the adjuvant effect (Munoz, 1963b). Whether these fractions contain endotoxin is still not known. However, the methods used in

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preparing them usually extract little or no endotoxin from other gramnegative bacteria. Although, the preparations of endotoxin used by Farthing and co-workers (Farthing, 1961; Farthing and Holt, 1962) were most probably not highly purified, there seems to be little doubt from their work that endotoxin from B. pertussis is an active adjuvant as is endotoxin from other gram-negative bacteria. As stated above, B. pertussis cells have repeatedly been shown to increase anaphylactic sensitivity of mice. This is true not only when the animals are sensitized by active means, but also when they are sensitized passively (Pittman, 1957; L. S. Kind, 1958; Schweinberg, 1961; Munoz, 1964). Mouse strain differences have been noticed with respect to their ability to be sensitized to anaphylaxis (Tokuda et al., 1963). Certain antigens, such as bovine serum albumin, which are highly anaphylactogenic in other animals seem to be poor in the mouse (Munoz, 1963b). Tetanus toxoid failed to produce anaphylactic sensitization in the mouse ( Malkiel and Hargis, 1952b). Bordetella pertussis cells do not noticeably affect anaphylactic reactions which are carried out in uitro. The Schultz-Dale reaction performed with uteri of mice passively sensitized with graded doses of antibody showed that identical concentrations of antibody were effective in sensitizing the uterine muscle of normal or B. pertussis-treated mice (Munoz and Maung, 1961). It has also been shown that passive cutaneous anaphylaxis (PCA) sensitivity of the skin of mice was not increased by previous administration of B. pertussis. In fact, an actual decrease in sensitivity of PCA in mice that had received B. pertussis 4 days before was noticed (Munoz and Anacker, 1959). This was perhaps due to a faster disappearance of antibody from the skin site of B. pertussistreated mice than from similar sites in the skin of normal mice. When the anaphylactic sensitivity of intestinal strips or uteri from mice receiving antigen alone or antigen with B. pertussis was tested, it was found that an earlier sensitivity appeared in the muscles from mice which had received B. pertussis (Munoz and Maung, 1961). This finding is in agreement with the observations that antibody can be detected earlier and higher titers produced in B. pertussis-treated mice. Borddella pertussis vaccine enhances the production of EAE in mice receiving brain or spinal cord injections as shown by Lee and Olitsky ( 1955), and it is effective in promoting EAE in guinea pigs (Shaw et d., 1984). It should also be noted that adrenalectomy also increases susceptibility to develop EAE (Levine et al., 1962). Recently, Mota (1958, 1963a,b, 1964) reported that B. pertussis

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induces the production of a special antibody in rats which he called the "mast cell sensitizing" antibody. It can be demonstrated by treating mast cells with serum from rats that had received B. pertussis and an antigen and subsequently exposing the sensitized mast cells to the specific antigen. The mast cells degranulate and release histamine. The antibody is found early in the period of immunization and disappears by the thirtieth day even though high titer of circulating antibodies are demonstrable by the hemagglutination technique. The function of this antibody in the production of general hypersensitivity reactions in rats, mice, or other animals is not clear at present. The mast cell sensitizing antibody is heat labile at 56" C. for hour and attaches firmly to the skin. For these reasons it has been compared to the reaginic antibody found in certain allergic patients (Mota, 1964). C. CHARACTEIUZATION OF A c n v ~ MATERIAL Little work has been done on the purification of the adjuvant and sensitizing substances of B. pertussis. The so-called histamine-sensitizing factor (HSF) has been shown to be a surface component of the cell and found mainly in the cell wall (Yoshida et al., 1955; Munoz et al., 1959; Billaudelle et al., 1960). It is found in supernatant of lysed cultures end can be extracted from fresh whole cells by various methods (Maitland and Guerault, 1958; Niwa, 1962; Munoz and Hestekin, 1962). The exact nature of the HSF is not known, but Niwa (1962) reported that his best preparation contained 5.76 % N, 2.80 % P, 2.14 % sugar, and 0.4 % glucosamine. We have reported on the purification of HSF (Munoz and Hestekin, l962), but no chemical analysis was done on the most highly purified preparations. The activity of HSF is destroyed by heating at 75" C. for hour, and certainly its activity is not due to endotoxin as extracted by the various methods commonly used (trichloroacetic acid, phenol-water, etc.). The HSF seems to be closely associated, if not identical, to the protective antigen of B. pertmsis (Munoz and Hestekin, 1963), although the point is still questioned by some (Dolby, 1958; Sutherland, 1963). Endotoxin from this organism does not protect against intracranial challenge, or sensitize mice to histamine (Munoz and Schuchardt, 1955; Malmgren and Ribi, 1962; Sutherland, 1963). Crude, cell-free saline extracts from B. pertussis have the ability to increase the sensitivity of mice to actively and passively induced anaphylaxis as well as to increase the permeability of capillaries to Evans blue (Munoz, 196313). As indicated above, endotoxin prepared by the phenol-water method of Westphal et al. (1952) was active in stimulating antibody response in

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the guinea pig in amounts of 0 . 2 5 pg. (Farthing, 1961), and lipid A prepared by acid hydrolysis of the lipopolysaccharide was also active, but to a lesser extent (even 150 pg. did not produce as good a response as 0.5 pg. of the lipopolysaccharide) (Farthing, 1961). The results of Farthing (1981) clearly show that endotoxin preparations from B. pertussis act as adjuvants to antibody production. Whether there are other factors in B. pertussis which may also act as an adjuvant is not at all certain and further quantitative work with purified fractions is needed.

D. POSSIBLE MODFSOF ACTIONOF Bmdetella pertussis The mechanism or mechanisms by which B. pertussis increases sensitivity of mice to histamine, serotonin, endotoxin, and pmsiuely induced anaphylaxis seem to be similar, at least in time relationship. The increased susceptibility to these agents follows a similar course (Munoz, 1957; L. S. Kind, 1959), except that susceptibility to passively induced anaphylaxis decreases somewhat sooner (Munoz et al., 1958). Since adrenalectomy of certain strains of mice mimics the B. pertussis effect closely, one of the most attractive hypotheses to explain its mode of action is that B. pertussis interferes with adrenal function (L. S. Kind, 1958; Munoz et al., 1958). This view, however, has not been directly proved. In fact, most evidence has shown no direct effect on adrenal glands. However, no extensive work along these lines has been done (L. S. Kind, 1958; Schweinberg, 1961).Evidence that adrenal function might somehow be involved in the B. pertwsis effect was obtained by Schayer and Canley (1961) who showed that B. pertussis cells do not stimulate the adrenal glands, whereas substances such as endotoxin and Freund's adjuvant do stimulate them to produce more steroids. Since B. pertussis cells have endotoxin, it seems that they may also have substances that prevent the stimulation of the adrenal glands by endotoxin. Schayer and Ganley (1961) also showed that B. pertwsis cells, endotoxin, and Freund's adjuvant increased the histidine decarboxylase of various tissues and postulate that an increase in tissue permeability was produced through the action of histamine released by this enzyme. In the absence of an adrenal compensatory effect in B. pertussis-treated mice, the increase in permeability of tissues leads to hypersensitivity to histamine, other toxic materials, and stresses of various kinds (Schayer and Ganley, 1961). Independently, we found (Munoz, 1961) that the permeability of capillaries and perhaps tissues in general are increased in B. pertussls-treated mice and that this increase in permeability correlates with the hypersensitivity to histamine. Endotoxin also increases permeability of capillaries but not sensitivity to histamine. According to

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Schayer and Ganley (1961), however, endotoxin increases the adrenal output and this effect may account for the observed lack of sensitivity to histamine. The increased permeability of tissues and cells uncompensated by adrenal steroid, then, could explain the increased susceptibility of mice to histamine, serotonin, endotoxin, to passively induced anaphylaxis, etc. Interestingly, the flavonoid, quercetin, which is known to have vitamin P activity (it increases capillary ,tone and decreases permeability) was found to be active in protecting B. pertussis-treated mice against histamine (Thiele and Schuchardt, 1952). We have repeatedly confirmed this observation; however, other flavonoids with vitamin P activity were not effective (Munoz and Schuchardt, 1955). C. W. Fishel et al. (1962) have recently noticed that dichloroisoproteranol (OCI), a (3-adrenergic blocking agent, when given to mice makes them more susceptible to histamine whereas an a-adrenergic blocking agent (dibenzyline) did not produce this effect, and actually protected mice from histamine. Fishel et al. (1962) hypothesize that the B. pertussis effect might be due to a functional imbalance between the two types of adrenergic receptors. Bordetelh pertussis either has a substance that directly blocks the receptors or stimulates the mouse to produce such blocking substance. In connection with this, it should be pointed out that parabiosis between a normal and a B. pertussis-treated mouse renders both mice resistant to histamine rather than both sensitive (Chedid and Boyer, 1955). This observation suggests that there is no significant amount of @-adrenergicsubstance produced by a B. pertussis-treated mouse. It should also be emphasized that the a-adrenergic blocking agent that Fishel and co-workers employed (dibenzyline) has a marked antihistamine effect which should protect mice from histamine shock. It is indeed difficult with the evidence at hand to determine how B. pertussis acts in inducing sensitivity to histamine, serotonin, endotoxin, passively induced anaphylaxis, and to various stresses and infections. Since mice are normally rather resistant to histamine and serotonin, as well as to anaphylaxis, it was suspected that B . pertussis increased the anaphylactic sensitivity of mice by increasing their susceptibility to histamine and serotonin. This was an attractive hypothesis because of the role that these two amines seem to have in anaphylaxis of other animals. No direct experimental evidence has been obtained to support this view, however. On the contrary, it has been shown that susceptibility to anaphylaxis of B. pertussis-treated mice does not follow the development of increased susceptibility to histamine or serotonin (Munoz, 1957;

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L. S.Kind, 1958; Munoz et al., 1958), and antihistamine and antiserotonin drugs, in concentrations that protect against the respective amines, fail to protect mice from anaphylaxis (L. S . Kind, 1958). Since B. pertussis cells also increase antibody response to antigens given simultaneously with them (L. S. Kind, 1958; Munoz, 1963a), it is probable that this adjuvant effect is mainly responsible for the increased sensitivity to actively induced anaphylaxis. Some workers have expressed some doubt regarding the significance of the adjuvant effect on increased anaphylactic susceptibility of B. pertussis-treated mice (L. S . Kind, 1957; Malkiel and Hargis, 1959). In the author's hands, however, the adjuvant effect of B. pertussis seems to be the most important factor in increasing susceptibility of mice to actiwely induced anaphylaxis ( Munoz, 1963a). Obviously the adjuvant effect is not involved in passively induced anaphylaxis, and in this case a different mechanism must be involved as has been indicated above. Freund's adjuvant is also effective in producing fatal sensitization of mice to protein antigens such as egg albumin. The titer of antibodies in these mice is much higher than in B. pertussis-treated mice receiving the same amount of antigen. Other adjuvants used with various antigens have also been shown to produce anaphylactic sensitivity in mice. Thus, alum (Solotorowsky and Winsten, 1954) endotoxin, zymosan, and other known adjuvants (Malkiel and Hargis, 1959),as well as repeated injections of antigen (Fox et al., 1958), have been successfully used to produce active anaphylaxis in mice. From these results there seems to be little doubt that when the antibody response is stimulated adequately, mice become sensitive to fatal anaphylactic shock, There is an indication that when B. pertussis cells are given, an earlier and more uniform sensitivity to fatal anaphylactic shock develops. This might result from the early changes known to be produced in the mouse by B. pertussis. It is during this period that the mice have been shown to be highly susceptible to histamine, serotonin, cold stress, X-rays, anoxia, and various viral and bacterial infections in addition to passively induced anaphylaxis. During this period of high sensitivity to various agents, it would be expected that actively induced anaphylactic shock should also produce higher mortality rates. Freund's adjuvant, in our hands, was even more effective in producing sensitization of mice to anaphylactic shock when judged by the amount of antigen required to produce fatal anaphylactic sensitivity, i.e., less antigen was required to produce anaphylactic sensitivity, as well as antibodies, than when B. pertussis was used (Munoz, 1963a). If the amount of antigen (egg albumin) administered in the sensitizing dose exceeded 0.2 mg., and the challenge was performed within the first 3 weeks, there was no significant difference in the efficiency with which

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B. pertussis cells or Freund’s adjuvant induced fatal anaphylactic sensitivity. In studies of this type, however, one should keep in mind that there are marked differences among certain strains of mice with respect to their ability to develop active anaphylaxis (Tokuda et d., 1963). As stated above, some workers have questioned that the B. pertussis adjuvant effect is wholly responsible for the sensitization of mice to actively induced anaphylaxis (L. S. Kind, 1957; Malk‘lel and Hargis, 1959). Mota’s discovery that B. pertussis increases the formation of “mast cell sensitizing antibody” (Mota, 1963b, 1963c) could be interpreted as meaning that B. pertussis changes the antibody response in rats and possibly mice, not only quantitatively but also qualitatively. The function of the mast cell sensitizing antibody in anaphylaxis is still questionable. Guinea pigs sensitized either actively or passively show mast cell damage and histamine release upon injection of antigen (Mota and Vugman, 1956; Humphrey and Mota, 1959; Mota, 1959). Mota (1961, 1962, 1983a), however, found that passively induced anaphylaxis in the rat or mouse is not accompanied by mast cell damage or histamine release. In addition, it was found that B. pertussis which enhanced the mast cell damage and histamine release in actively sensitized rats and mice (Mota, 1963a) did not enhance this phenomenon in animals challenged with antigen-antibody complexes ( Mota, 1962,1963a). Austen and Humphrey (1961) stated that only rats receiving horse serum and B. pertussis showed mast cell damage and release of histamine after challenge with specific antigen. No such damage or release of histamine has been observed by these workers in animals that received human serum albumin, bovine serum albumin, human y-globulin, or hernocyanin, either absorbed on alum or mixed with Freund’s adjuvant, despite the presence of abundant antibody to the respective antigen in the animals tested. It should be pointed out, however, that these tests were made several weeks after the first injection of adjuvant, and that any antibodies made early would have disappeared. For these reasons it is premature to assign great significance to the mast cell sensitizing antibody in the mechanism of anaphylactic-enhancing effect of B. pertwsis. The mechanism by which B. pertwsis cells or fractions thereof increase the antibody response is still not understood. It has been shown that guinea pigs that received B. pertussis vaccine with diphtheria toxoid produce a much earlier response of antitoxin than those guinea pigs that received the toxoid alone (Farthing and Holt, 1962) and that the effect was mainly on the primary response of the antibody-forming mechanism; little effect on the secondary response was noticed. It is

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also known that a previous injection of B. pertussis cells interferes with

the adjuvant effect of a second injection of the same cells when given with the antigen (L. S. Kind and Roffler, 1961), and that B. pertussis produces a marked increase in the size of the spleen as well as a leucocytosis (Parfentjev and Manuelidis, 1956). Fichtelius and Hasler ( 1958) also found that repeated injection of B. pertussis vaccine in adrenalectomized rats produces a more rapid formation of lymphocytes in the thymus and lymph nodes in these animals. Since all these experiments have been conducted with whole B. pertus& cells, one cannot exclude the role of endotoxin in all these phenomena, and, from the results of Farthing (1961) and Farthing and Holt (1962), it seems that endotoxin extracted from the B. pertussis cell has the same activities as the endotoxins extracted from other gramnegative organisms including the effect on reticuloendothelial cells and spleen. Bmdetella pertussis cells, just as the endotoxins from gram-negative organisms, probably do not act by retaining the antigen in a depot form as was believed by Ferago and Pusztai (1949), or by combining with the antigen. Most likely, B. pertassis acts by producing a stimulation of the antibody-forming cells and also by facilitating antigen distribution by increasing the permeability of tissues and capillaries. The other abovementioned possibilities on the adjuvant action of endotoxins could also be involved in the action of whole B. pertussis cells, V. Miscellaneous Bacteria and Bacterial Products

As pointed out in the Introduction, other bacteria have also been effective in stimulating antibody response *toantigens. As early as 1922, Clark et al. found that killed gram-positive cocci had a stimulatory effect on the production of agglutinins to typhoid vaccines. Schroeder (1923) also showed that higher titers of hemolysins were produced in rabbits which had developed abscesses at the site of immunization, and that pneumonococcal infections had similar effects. Soon after these observations Khanolkar (1924) showed an increase in the production of agglutinins to Salmonella paratyphi or Salmonella enteritidis by a previous treatment of the animal with killed P s e u d o m m aeruginosu, Escherichia coli, and S . paratyphi B, or Shigella dysenterae. Ramon and co-workers then showed that TAB vaccine (typhoid, paratyphoid A, paratyphoid B) increased the production of antitoxins to tetanus or diphtheria toxoids both in animals and man (Ramon and Zoeller, 1927). Schultz and Swift (1934) found that the reactivity of rabbits to horse serum was greatly increased by previous sensitization of the animal with repeated small

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intracutaneous inoculations of nonhemolytic streptococci or large intravenous injections of either nonhemolytic or hemolytic streptococci. The skin reactions to horse serum were more severe and sensitization appeared earlier. These latter workers also observed that a higher precipitin titer to horse serum was obtained in animals receiving streptococci. Swift and Schultz ( 1936a) observed that staphylotoxin enhances the response of animals to beef lens protein. It is significant that no adjuvant effect was obtained among animals previously immunized to the toxin. The adjuvant effect of staphylotoxin must have been due to the action of active toxin on antibody-forming cells. Rice in 1947 found that Clostridium botulinum type A toxoid was an adjuvant for C. botulinum type B toxoid. The effect of C. botulinum type A toxoid could be demonstrated easily in mice but had only minimal effect in guinea pigs. This adjuvant effect could not be shown when diphtheria or tetanus toxoids were used as antigens. Ramon and coworkers showed that Brucella abortus has a stimulatory effect on the production of diphtheria and tetanus antitoxins (Ramon d d.,1950). Various organisms have been effective as substitutes for the tubercle bacilli in complete Freund's adjuvant for the production of EAE. Thus it has been reported that Pseudomonas pseudomallei (Steigman and Lipton, 1960), Bordeteh pertussis (Wiener et al., 1959; Lumsden, 1962), Salmonella typhosa (Kabat, 1957), Salmonella typhimurium (Katsh, 1959), Coccidioides immitis (Kabat, 1957), Streptococcus ( Campbell, 1962), Nocardia asteroides, Mycobacterium butyricum, Mycobacterium phlei, Mycobacterium smegmatis, E . coli, and Pseudomonas aeruginosu (Shaw et al., 1964) enhance the production of EAE. Some gram-positive organisms were also active, such as Cmynebacterium rubrum which was found to be as effective as Mycobacterium tuberculosis in the production of EAE. StaphylococMIs aureus and Coynebactem'um diphtheriae were only weakly active (Shaw et al., 1964). Although other reports on the adjuvant effect of bacteria have probably appeared in the literature, no extensive work on any one bacterium has come to our attention. The variety of bacteria or their products which can stimulate the antibody response is noteworthy suggesting that the substances involved are probably varied. VI. Possible Mechanisms of Bacterial Adjuvants

A few remarks regarding the factors that affect antigenicity of various substances seem appropriate. It is well-known that antigens vary considerably in their ability to stimulate antibody formation. Large molecular

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weight substances are usually better antigens than substances with very low molecular weight; as a rule, larger doses of antigen are better than smaller doses; multiple injections of antigen are better than a single injection even when the total dose is comparable in both cases; a secondary injection of an antigen gives a "booster" effect in the animal; and easily digested or excreted substances are less antigenic than substances ,that remain in the body for a long period of time (Boyd, 1956; Wilson and Miles, 1957; John and Gergl, 1960). It is also well accepted that the lymphoid tissue is mainly associated with antibody formation and that the plasma cell is at least one of the cells involved in antibody formation (Coons, 1958; Harris and Harris, 1980; Vazquez, 1961; Nossal, 1962). From these observations it is safe to assume that the stimulation of the lymphoid ,tissue may lead to the formation of higher antibody titers. The participation of other cells, however, in antibody formation has long been suspected and the stimulation of the reticuloendothelial system may also be important (Wilson and Miles, 1957; Fishman, 1961). From the facts just mentioned it becomes apparent that a substance can increase the formation of antibodies by various mechanisms: (1) by prolonging the antigenic stimulation of the substance given; (2) by protecting against rapid excretion or destruction of the antigen; (3) by increasing the number of cells concerned in antibody formation; ( 4 ) by combining with an antigen so as to make it more resistant to digestion or excretion; ( 5 ) by supplying intermediate factors needed for antibody formation; and (6) by allowing a better distribution of the antigen, either by physical means (water-in-oil emulsions) or through changes produced in the animal, such as increasing permeability of the tissues. These seem to be the most obvious ways in which the antibody response to an antigen can be improved, and an effective adjuvant might well act by having more than one of these characteristics. Freund's adjuvant seems to act by stimulating the antibody-forming cells, by protecting the antigen, by providing a continuous antigen release, by distributing the antigen throughout the body, etc. The mode of action of most adjuvants, as is the case with Freund's adjuvant, must be complex. Many bacterial cells, especially the acid-fast and gram-negative organisms, may act by increasing the cellular response and by improving the distribution of the antigen throughout the body through changes in tissue permeability or by preventing its destruction through interference with certain cells or enzyme systems. Some believed that substances from the bacterial cells, especially acid-fast cells, actually combine with the antigenic substance to make it effective ( Lipton, 1959). It is also possible that some bacterial substances may release, through

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cellular damage, materials essential to antibody-forming cells ( Jaroslow, 1960; Braun, 1981). It is clear that the exact modes of action of bacterial adjuvants, or adjuvants in general, are not definitely known. The phenomenon is complex and cannot be expected to have a simple answer. There are many factors which affect the antibody response and many ways in which the antibody response can be modified. It can be safely said that adjuvants of different kinds act differently and that any one adjuvant may have more than one mechanism by which it increases the antibody formation. VII. Summary

1. Many bacteria stimulate antibody formation in animals. The most effective are acid-fast and gram-negative bacteria, but a few grampositive cocci and certain fungi are also active. The materials in these bacteria responsible for the adjuvant effect seem to be different in different organisms. Highly active waxes have been isolated from acidfast bacteria. The lipopolysaccharides in the gram-negative bacteria, and perhaps also to a certain extent in the acid-fast bacteria, associated with endotoxin activity act as adjuvants. Not enough work has been done with other bacteria to know which compounds are most Iikely responsible for the stimulation of the antibody response. 2. There are many conditions involving proliferation of bacteria in the animal body and toxic fractions of bacterial cells which also stimulate antibody-forming cells. Little work has been done along these lines. 3. The adjuvant effect of bacteria is probably due to many different mechanisms. For most bacteria and their products, the most important mechanisms are possibly the stimulation of antibody-forming cells and the better distribution of antigen throughout the body because of increased permeability of the tissues and capillaries. 4. Although acid-fast microorganisms have been widely used as adjuvants in the production of isoallergic diseases, especially experimental allergic encephalomyelitis, other microorganisms and endotoxins have also been effective. 5. Considerably more work is needed to elucidate completely the mechanisms by which substances of bacterial origin stimulate the antibody response. REFERENCES Adler, F.L.,and Fishman, M. (1962).Proc. Soc. Exptl. B b l . Med. 111, 691-695. Amies, C.R. (1959).I. Puthol. Bucteriol. 77, 435-442. Amies, C.R. (1962).J . Hyg. 80, 483-493.

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Tokuda, S.,Weiser, R. S., Munoz, J., and Laxson, C. (1963). 1. Infectious Dkreasea 112, 77-83. Uhr, J. W., Salvin, S. B., and Pappenheimer, A. M., Jr. (1957). 1. Exptl. Med. 105, 11-24. Ungar, J. (1952). Proc. Roy. SOC. Med. 45, 674-676. Vazquez, J. J. (1961). Lab. Inuest. 10, 1110-1125. Vigliani, E. C., and Pernis, B. (1959). J . Occupational Med. 1, 319; cited from Pernis and Paronetto (1962). Wagner, M. (1959). Ann. N.Y. Acad. Scl. 78, 261-271. Waksman, B. H. ( 1958). Progr. AZZergy 5, 349-458. Waksman, B. H. (1959). In “Mechanisms of Hypersensitivity,” Henry Ford Hospital Symp., 1958 (J. H. Shaffer et al., eds.), pp, 679-691. Little, Brown, Boston, Massachusetts. Waksman, B. H., and Adams, R. D. (1953). J. Infectious Diseases 93, 21-27. Ward, P. A., and Johnson, A. G. (1959). J . Immunol. 82, 428-434. Ward, P. A., Johnson, A. G., and Abell, M. R. (1959). 1. Exptl. Med. 109, 463-474. Ward, P. A., Abell, M. R., and Johnson, A. G. (1961). Am. 1. Pathol. 38, 189-205. Weigle, W., Dixon, F. J., and Deichmffler, M. P. (1960). Proc. SOC. Exptl. Biol. Med. 105, 535-541. We& M. H., and Spink, W. W. (1957). 1. Lab. Cltn. Med. 50, 501-515. Weissmann, G., and Thomas, L. (1962). 1. Exptl. Med. 118, 433-450. Westphal, 0. (1957). In “Polysaccharides in Biology,” 2nd Josiah Macy, Jr., Conf., 1958 (G. F. Springer, ed.), pp. 115-220. Madison Printing Co., Madison, New Jersey. Westphal, O., Liideritz, O., and Bister, F. (1952). 2. Naturforsch. 7b, 148-155. Wexler, B. C., Dolgin, A. E., and Tryczynski, E. W. (1957). Endocrinology 61, 300-308. Wheeler, A. H., Brandon, E. M., and Petrenco, H. (1950). 1. Immunol. 85, 687-700. White, R. G. ( 1959). In “Mechanisms of Hypersensitivity,” Henry Ford Hospital Symp., 1958 (J. H. Shaffer et al., eds.), pp. 637-645. Little, Brown, Boston, Massachusetts. White, R. G., and Marshall, A. H. E. (1958). ImmunoZogy 1, 111-122. White, R. G., Coons, A. H., and Connolly, J. M. (1955a). J . Eleptl. Med. 102, 73-82. White, R. G., Coons, A. H., and Connolly, J. M. (1955b). 1. Ex@. Med. 102, 83-104. White, R. G., Jolles, P., Samour, D., and Lederer, E. (1964). Immunology 7, 158-171. Wiener, S. L., Tinker, M., and Bradford, W. L. (1959). A.M.A. Arch. Pathol. 67, 694-699. Wilson, G. S., and Miles, A. A. (1957). “Topley and Wilson’s Principles of Bacteriology and Immunity,” 4th ed. Williams & Wilkins, Baltimore, Maryland. Wissler, R. W., Fitch, F. W., LaVia, M. F., and Gunderson, C. H. (1957). J. Cellular C ~ m pPhystol. . 50, 285-301. Witebsky, E. (1959). In “Allergic Encephalomyelitis” (M. W. Kies and E. C. Alvord, Jr., eds. ), pp. 321-334. Thomas, Springfield, Illinois. Yoshida, N., Tanaka, S., Takaishi, K., Fukuya, I,, Nishino, K., Kakatani, I., Inci, S., Fukui, K., Kono, A., and Hashimoto, T. (1955). Tokushfma 1. Exptl. Med. 2, 11-19. Zweifach, B. W., Benacerraf, B., and Thomas, L. (1957). J . Exptl. Med. 106, 403414. Zweifach, B. W., Kivy-Rosenberg, E., and Nagler, A. L. (1959). Am. 1. Physiol. 197, 1364-1370.

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 Abd-el-Malek, Y., 276, 282 Abe, S., 145,167 Abell, M. R., 417, 418, 440 Aberg, B., 206, 238 Ablaza, I., 388(228), 395 Abrams, R., 136, 171 Abruzzo, J. L., 357(23), 359(38), 365 (38), 368(38), 389, 390 Ackerman, G. A., 26,27,28,29,96 Ada, G.L., 126, 171 Adams, M.H., 261, 262,283 Adams, R. D., 232,247,405,440 Adamson, C. A., 12, 96 Adelsberger, L.,37, 96 Adler, F. L., 166, 169, 192,241, 399, 431 Adler, R. H., 59, 97, 383(174), 394 Adner, M. M., 32, 57, 112 Afzelius, B. A., 195, 238 Ahlinder, S., 339,342 Aho, K.,364(64),390 Akiyama, H.J., 144, 148, 155,167, 169 Akiyama, T., 128, 146, 158, 159, 160,

Alvin, A., 25, 105 Alvord, E. C., Jr., 398, 400, 404, 405, 407, 408,410,422, 429, 435,439 Amano, S., 176, 177,237, 240 Amano, T., 135, 167 Amid, F. E., 39, 96 Amies, C. R., 398, 431 Amiraian, K.,336, 337, 342 Amos, D. B., 162, 165, 167, 235, 242 Anacker, R., 401, 432 Anacker, R. L., 411, 412, 413, 422, 434,

437, 438 Anderson, D., 47, 96 Anderson, H.C., 262, 282 Anderson, J. R., 353( 12), 355(12), 377 (131), 378(138), 379( 149), 384 (197, 198), 387(216), 389, 392, 393, 394 Anderson, S. G., 11, 96 Anderson, T.F., 196, 197, 237, 242, 244 Ando, S., 28, 96 Andr6, A., 47, 96 Andre, J., 401,434 Andres, G. A., 203, 209, 211, 219, 222, 172, 173 223, 225, 237, 240 Alarcon-Segovia, D., 216,243 Andres, G., 218, 241 Albert, F.,47, 96 AngeU, F. L., 39, 105 AIberty, R. A., 5, 6, 100 Antopol, C.,412, 432 Alden, M. W., 39, 96 Antweiler, H.,399, 432 Alepa, F. P., 360(43), 390 Alexander, W. R. M., 384(199), 385 Appel, S. H., 232, 238 Arai, S., 190,237 (199),394 Arakawa, T.,216, 220, 237 Algire, G. H., 162, 167, 173 Aratake, H., 208, 237 Alksne, J. T., 202, 237 Araujo, P.,259, 282 Allan, W. S. A., 379(152),393 Arbesman, C. E., 288, 336, 349 Allen, B. M., 90, 96 Archer, G. T., 188,205,237 Allen, F. W., 73, 96 Allen, J. C.,275, 282, 361(48, 49), 367 Archer, 0.K., 31, 32,33, 38, 39, 49, 51, 52, 56, 57, 96, 102, 113, 114 (49),390 Archibald, A. R., 272, 277,281,282, 283 Allen, R. D., 121,167 Ardenne, M., 198,237 Allerhand, J,, 412, 435 Arhelger, R. B.,222,237 Allgower, M.,142, 167 Armstrong, J. J., 270, 272, 277, 282, 283 Allison, M.J., 151,167 Armstrong, S. H., 217,243 Almeida, J., 197, 199, 237, 290, 342 Amason, B. G., 32, 57, 96, 105, 114 Altchek, A,, 221,237

441

442

AUTHOR INDEX

Arnaud, L. E., 402, 404, 437 Arnold, J. D., 216, 218, 237, 246 Aronson, M., 160, 167 Artman, M., 143,167 Arts, G., 205, 247 Arundell, F. D., 385(204), 395 Aschoff, L., 91, 96 Asherson, G. L., 357( 24), 388(227), 390, 395 Ashworth, C. T., 228, 242 Askonas, B. A., 20, 105, 183, 237, 291, 321, 342, 408, 409, 432 Asofsky, R. M., 417, 418, 435 Aspinall, R. L., 27, 30, 50, 56, 96, 108 Aster, R. H., 5, 112, 230, 246 Atchley, W. A,, 353( l l ) , 389 Attardi, G., 340, 342 Attleberger, M . H., 27, 106 Atwater, E. C., 371(97), 391 Aub, J. C., 191, 246 Auerbach, R., 26, 28, 29, 30, 96, 97 Augustin, R., 12, 96, 335, 342 Aust, J. B., 48, 97, 107 Austen, K. F., 427, 432 Avery, 0. T., 261,282, 285, 286 Axelrod, A. E., 400, 432 Aycock, W. L., 11, 97

B Bachrach, H. L., 196, 237 Baddiley, J., 267, 269, 270, 271, 272, 275, 276, 277, 278, 279, 280, 281, 282, 282, 283, 284, 285, 286 Badertscher, J. A., 30, 97 Baehr, G., 384( 188). 394 h e r , B. S., 135, 171 Baer, J. G., 62, 97 Baggenstoss, A. H., 371( 94), 391 Baglioni, C., 341, 342 Bailey, M. L., 407, 438 Baker, R. F., 140, 169, 188, 190, 241 Baldridge, C. W., 130, 167 Baldwin, F. M., 90, 97 Bale, W. F., 6, 108 Balfour, B. M., 377( 123), 392 Ball, W. D., 26, 28, 30, 97 Ballabio, C., 365(70), 391 Balls, M., 94, 97 Baney, R. N., 61,97, 177,237

Banfield, W. G., 205, 246 Bang, F. B., 67, 97 Bangham, D. R., 7, 9, 97, 333, 342 Barandun, S., 330, 342 Barbieri, G., 378( 144), 393 Bardawil, W. A., 8, 97, 191, 220, 238, 246, 384( 194), 385( 194), 394 Bardier, A., 218, 246 Baril, E. F., 82, 97 Barker, S. A., 265, 266, 284 Barkulis, S. S., 252, 258, 283, 284 Barland, P., 233, 238 Barnes, D. W . H., 53, 103 Barnes, H. A., 32, 97 Barnes, J. M., 421, 432 Bamett, E. V., 371( 97), 391 Bamett, J. A., 44, 97 Barr, M., 339, 346, 421, 432 Bartel, A. H., 82, 97 Barth, W. F., 338, 339, 349 Bass, B. H., 378( 136), 393 Battaglia, S., 212, 238 Battisto, J. R.,48, 49, 97 Baughman, W. H., 19, 99 Baumann, J. B., 12, 13, 114 Baumgartner, L., 19,23, 97 Baxandall, J., 195, 238 Baxter, H., 4, 102 Bayles, T. B., 373( log), 384( 194), 385 ( 194), 392, 394 Beall, G. H., 358( 31), 390 Beard, J., 29, 90, 97 Beck, J. S., 353(6, 12), 355(12), 384 (6,197, l98), 385(6), 389,394 Becker, E. L., 337, 348 Becker, H., 130, 167 Beech, M., 379( 147), 393 Behring, 2, 97 Beierwaltes, W . H., 379( 150), 393 Beiler, J. M., 398, 436 Beith, E. M., 413, 421, 433 Bekierkunst, A., 143, 154, 167, 172 Bekman, J. I., 412, 435 Bell, E. T., 26, 29, 97 Bell, J., 404, 411, 432, 437 Bell, J. F., 406, 435 Bell, S. D., 335, 342 Belton, F. C., 265, 286 Belyavin, G., 377( 122), 392

AUTHOR INDEX

Benacerraf, B., 33, 91, 113, 124, 125, 128, 134, 151, 152, 154, 167, 169, 171, 172, 173, 234, 241, 243, 288, 298, 303, 309, 311, 316, 321, 325, 336, 342, 343, 346, 347, 348, 410, 412, 417, 419, 432, 434,440 Bender, M. A., 177, 181, 245 Benditt, E. P., 219, 247 Benedict, A. A., 403, 432 Benenson, A. S., 11, 105 Bennett, B., 126, 165, 167, 171 Berenbaum, M. C., 399,432 Berenson, G. S., 262, 263, 284 Beres, J., 218, 220, 242 Berg, O., 232, 238 Berggard, I., 331, 332, 342, 345 Bergstrand, A., 213, 218, 220, 238 Berk, R. S., 160, 171 Berkovici, B., 9, 10, 111 Berlin, B. S., 407, 432 Berlin, R. B., 11, 115 Bernard, J., 186, 238 Bernard, L., 39, 97 Bernhard, W., 177, 238 Bemheimer, A. W., 64, 65, 91, 97, 132, 139, 168, 169, 173 Bems, A. W., 220, 238 Berrill, N. J., 75, 97 Berry, G. P., 404,438 Berson, S. A., 325, 342 Berthillier, Y., 123, 171 Berthrong, M., 142, 145, 167 Bessis, M., 177, 186, 190, 199, 238, 245 Beutner, E. H., 59, 97, 377(133), 378 (133), 383( 174), 393, 394 Bevans, M., 222, 242 Beveridge, W. I . B., 18, 97 Bezer, A. E., 402, 408, 409, 433 Biemond, A., 383( 177), 394 Biering-SBrensen, K., 39, 114 Bierring, F., 50, 97 Billaudelle, H., 423, 432 Billingham, R. E., 5, 34, 47, 48, 87, 96, 97, 98, 162, 167, 171 Binet, J. L., 235, 238 Bingley, M. S., 121, 167 Biozzi, G., 91, 98, 123, 124, 125, 151, 152, 154, 167, 168, 169, 171, 412. 417, 419, 432

443

Birke, G., 339, 342 Bir6, Z.,58, 104 Birzu, N., 404, 410, 433 Bishop, D. W., 21, 98 Bisset, K. A., 3, 62, 63, 72, 73, 98 Bister, F., 411, 423, 440 Bjorklund, B., 265, 266, 284 Bj@rneboe,M., 3,98 Blackmon, J. R., 132, 169 Blaese, M., 48, 52, 107 Bland, J. H., 369(85), 391 Blaw, M. E., 59, 110 Blaylock, H. C., 385(201), 395 Blizzard, R. M., 387( 218), 388(218), 395 Bloch, E. H., 124, 170, 196, 230, 238 Bloch, H., 129, 142, 143, 156, 167, 168, 172 Bloch, K. J., 288, 336, 342, 347, 371 (98), 372(101), 391, 392 Block, M., 22, 32, 57, 98, 106 Bloom, B., 312, 313, 343, 345 Bloom, G., 206, 238 Blozis, G. C., 228, 238 Blumenthal, H. T., 220, 238 Boder, E., 59, 98 Bohle, A., 213, 221, 222, 238, 244 Boivin, A,, 411, 432 Bonanto, M. V., 401, 402, 433 Bonomo, L., 378( 144), 393 Boon, M. C., 50, 106 Borg-Petersen, C., 21, 101 Borin, P., 176, 245 Bomstein, M. B., 232, 238 Bosch, H., 58, 104 Boughton, B., 403, 432 Boughton, J., 161, 170 Bouissou, H., 218, 219, 238, 245, 246 Bourrillon, R., 331, 343 busfield, G., 421, 432 Bouthillier, Y., 125, 154, 168 Boutke, E., 134, 169 Bowden, D. H., 59,98 Bowen, T. J., 263,283 Boyd, W. C., 430,432 Boyden, E. A., 27, 30,98 Boyden, S. V., 120, 121, 122, 126, 161, 164,168,173,358(32),390 Boyer, F., 425, 432

444

AUTHOR INDEX

Boyer, S. H., 325, 328, 342,348 Boyles, T.B., 220,238 BOyse, E. A., 126, 165, 167, 171 Bozicevich, J., 356( 20), 389 Bradford, W.L.,23, 98,429,440 Bradley, S. G.,30, 45, 51,109 Brambell, F. W. R., 7, 8, 98, 333, 334, 342, 345 Brandon, E. M., 403,440 Brandsen, J., 268, 284 Brandt, P. W.,190, 238 Brandtzaeg, B., 328, 342 Braude, A. I., 412,434, 438 Braun, D.,129, 142, 146, 168 Braun, W.,153, 156, 161, 170,171, 172, 399, 400, 419, 431, 432, 434 Braunsteiner, H., 176, 179,230,231, 238, 239 Braverman, N., 8, 100 Brecher, G.,185,239 Breed, R. S., 276, 283 Breese, S. S., 196,237 Brenner, J. M., 384(199), 385(199), 394 Brent, L., 34, 47, 48, 97, 98, 162, 167, 168 Breton-Gorius, J.. 199, 245 Bricka, M.,199,238, 245 Bridges, R. A., 8, 14, 15, 20, 24, 25, 32, 33, 47, 48, 49, 98, 112, 176, 241 Brieger E. M.,188,239 Briggs, J. D.,65, 91, 98 Briggs, M.,268,283 Brini, A,, 186,241 Brittin, G. M.,185, 239 Broberger, O.,388(227, 229, 231), 389 (233),395 Broman, B., 11, 98 Brown, A. L.,216,243 Brown, D.M.,289, 348 Brown, J. B., 162, 168 Brown, P. C.,3'77(1321,392 Brown, R. A., 197, 242 Brown, R. C.,377(124), 392 Browne, J. T.,216, 239 B ~ d t tW., , 135,168 Brunson, J. G.,216, 218, 222, 237, 247, 412, 419, 433 Brunsting, L. A,, 385(202), 395

Brunton, C., 380( 161),393 Bnosko, W.J., 379( 155),393 Bubis, J. J., 232, 239 Buchanan, J. G.,270,271,277,282,283, 285, 286 Buchanan, W. W.,353( 12), 355( 12), 378( 142) , 389, 393 Bucht, H., 213,218,220,238 Buckley, C. E.,291,342 Buddingh, G.J., 19,110 Butler, C.,360(42),390 Buettner-Janusch, V.,291,303, 348 Bullock, W.,398, 432 Bunim, J. J., 372( 101),392 Burke, F. G.,13,109 Burkholder, P. M., 59, 113, 215, 222, 239, 382( 173),394 Burky, E. L., 398, 432 Burnet, F. M.,2, 3, 18, 19, 30, 47, 56, 59, 94, 97, 98, 114, 358(29, 30), 383(185), 388(29), 390, 394, 408, 432 Burnett, J. P., 291, 344 Burnham, T. K., 375(116), 392 Burrows, B. A., 59, 106 Burtin, P.,6,98,319, 320, 330, 342, 344 Burton, S. W., 220, 242 Butler, V. P.,364(63),390 Buxton, C.L., 13,47,98, 108

C Caesar, R., 187,212,239 Cdcagno, P. L.,222,227,239 Calhoun, M.L.,27, 30, 98 Calkins, E.,212, 213, 239 Calmette, A., 38,98 Cameron, G. R., 63, 64, 66, 67, 68, 69, 91, 98 Cammack, K. A., 289, 342 Campana, L.,28,98 Campbell, B., 48, 61, 99, 106, 429, 432 Campbell, D. H.,3, 12, 69, 72, 77, 92, 98,99,104,192,239 Campbell, P. N., 376( 120), 377( 126), 392 Cann, J. R., 65, 113 Cannon, J. A,, 35, 98 Cantacuzhe, J., 62,64,77,99 Carbonara, A. O.,304, 342

AUTHOR INDEX

445

Cinader, B., 47, 99, 197, 199, 237, 290, 328, 342, 343 Circo, R., 142, 169 Clark, A. H., 28, 99 Clark, L. G., 369(85), 391 Clark, P. F., 398,428, 432 Clark, S. L., 8, 99, 191,239 Clarke, D.A., 134, 152, 171, 173 Clarke, F. H., 288, 349 Clarke, P.H.,270,273,283 Clem, L. W., 77, 79,99, 112 Coca, A. F., 19,99 Cochrane, C. G., 41, 99, 121, 168, 202, 205, 208, 239 342,343,345,348 Cock, A. G., 48, 99 Cecchi, E., 230, 239 Coe, J. E., 402, 432 Cecil, R., 302, 343 Cohen, A. S., 212,213,239, 245 Cedergren, B., 188, 239 Cohen, E., 77,99,278,284 Celada, F., 355, 389 Cohen, J. O., 128,168 Ceppellini, R., 355, 389 Cohen, P., 11, 99 Chadboum, W. A., 179, 239 Cohen, S., 292, 298, 299, 301, 303, 304, Chan, P. C. Y.,335, 343 305, 310, 318, 317, 318, 322, 323, Chandler, R. W., 387(218), 388(218), 324, 325, 330, 332, 338, 339, 343, 395 346 Chang, T. S., 27, 30, 50, 99,102 Chapeau, M. L., 209, 211, 222, 237, 246 Cohen, S. G., 422,436 Chaplin, H.,292, 305, 343 Cohn, M.,340, 342 Chapman, G. B., 130, 168 Cohn, Z.A., 127,130,131,132,133,134, 135, 137, 138, 139, 140, 141, 168, Chapman-Andresen, C., 191, 239 169, 188, 205, 239, 242 Char, D.F. B., 399, 419, 432 Colberg, J. E., 340, 343 Charache, P.,295, 345 Chase, M.W., 2,34,47,49,99,398, 402, Colbert, E. H.,75, 78,99 432, 435, 438 Cole, F.J., 90, 99 Chase, P. H.,374( 113), 392 Cole, L. J., 135, 169 Chedid, L.,425, 432 Cole, R. M.,252, 253, 284 Cheny, W.B., 128, 168 Colle, E.,219, 247 Chimori, M.,144,170 Collet, A,, 178, 188, 205, 245 Chin, D., 148, 155, 157, 158, 159, 160, Colombot, 72, 111 186, 169 Colover, J., 388(223),395,405,408, 432 Chiquoine, A. D., 190, 244 Colwell, C. A., 144, 168 Chodirker, W.B., 338,343, 365(89),391 Condemi, J. J., 371(97), 391 Choi, M. H.,90, 99 Condie, R. M.,8, 14, 15, 20, 21, 24, 25, 32, 33, 48, 49, 71, 72, 73, 74, 77, Chorine, V., 64, 99 79, 82, 83, 85, 87, 88, 89, 98, 99, Christensen, H.E., 212, 242 101, 106, 107, 109, 176, 232, 239, Christian, C. L., 357(23), 359(38), 385 241, 399, 413, 415, 416, 417, 419, (38),388(38), 374(113),389, 390, Carey, W. F., 412, 435 Carlisle, J. W., 218, 245 Carlson, A. S., 132, 168 Caroli, J., 212,247 Carr, 0.B., Jr., 222,237 Carss, B., 270, 283 Casals, J., 401,433 Casals, S. P., 374(112),392 Caspari, E.,64, 91, 97 Castleman, B., 59, 99, 383( 1841, 394 Caulfield, J. B., 191, 246 Cave-Browne-Cave, J. E., 268, 283 Cebra, J. J., 298,297, 301, 312, 313, 337,

432, 433,434, 439

392

Christianson, H. B., 385(202), 395 Chure. 1.. 221. 222., 239. 243 EI..I.

I

I

Conestabile, E., 230, 239 Connell. C. E.. . 341.. 3 4

446

AUTHOR INDEX

Connolly, J. M., 3, 61,99, 106, 176, 183, Cushing, J. E., Jr., 3, 69, 70, 72, 73, 76, 193, 240, 399, 403, 405, 408, 409, 77, 99 440 Czerkawski, J. W.,282,283 Consden, R., 406, 432 D Constantinesco, N.,404,410,433 Daems, W. T., 205, 240 Convit, J., 188, 242 Dalling, T.,7, 107 Cooke, J. V., 37, 09 Cooke, R. A., 12, 112, 335, 348 Dalmasso, A. P., 32, 46, 48, 51, 52, 53, Coombs, A. M., 351(1), 389 54, 55, 99, 100, 102, 107 Coombs, R. R. A., 351(1), 389 Dalton, A. J., 177, 179,240 Coons, A. H., 3, 49, 61, 99, 106, 111, Dameshek, W.,3, 32, 49, 57, 100, 111, 112 176, 183, 191, 193, 239, 240, 246, 399, 403, 405, 408, 409, 412, 418, Dammaco, F., 378( 144),393 430, 433, 438, 440 Dancis, J., 4,5, 8, 12, 17,23, 24, 25, 26, 38, 40, 100, 109, 114 Cooper, B. A., 380(lei), 393 Cooper, N. S., 179,248 Danforth, C. H., 35, 100 Corcos, J., 370(88), 391 Dannenberg, A. M., Jr., 131, 168 Cordoba, F.,295, 345 Danon, A,, 191,240 Cornillot, P.,331, 343 Dao, T. L., 385(205), 395 Costea, N.,32, 57, 112 Darden, E. B., Jr., 177, 181,245 Dasinger, B. L., 142, 168 Cote, W.P., 27, 30,56,108 Cottier, H., 51, 58, 104, 113, 330, 342 Davenport, F. M., 408,436 Couchman, K. G., 377( 123), 381(165), Davies, S. H., 381( 164), 393 302, 393 Davis, B. D., 6, 100 Coulaud, E., 401, 433 Davis, D. J., 33, 100 Cowan, S. T., 275, 286 Davis, H.P., 48, 47,100 Cowart, G. S., 128, 168 Davis, J. E., 200,230,240 Cowen, D., 34, 109 Davis, S. F., 39, 100 Davison, A. L., 270, 275, 277, 281, 283 Cowen, D. M., 188, 193, 241 Cowles, R. B., 74, 101 Dawson, I. M., 250, 283 Craig, J. M., 49, 102, 179, 240, 372 Day, E.,23, 98 Day, E. D., 291,345 (102),392 Crampton, C.F., 136, 168 Debray, C., 386(213), 395 Creighton, A. S., 369(84),391 Debrt5, R.,39,97 Cremer, N.,412, 433 Decker, B., 369(81), 391 CritcNey, P.,281, 283 Decker, J. L., 372(100), 391 Cromartie, W . J., 48, 105 de Duve, C., 134, 137, 168 Crowle, A. J., 403,433 Defendi, V., 32, 57, 100, 111 Deibel, R. H., 276, 284 Cruchaud, A., 179, 240 Crumpton, M.J., 291, 292,298,303, 343 Deicher, H.,352(4), 353(4, 8, 9), 355 (8,9, 17), 358(9),357(9,25), 358 Cuenot, L.,89, 99 (25),372(25), 389, 390 Culling, C. F. A., 163, 173 Cummins, C. S.,251, 254, 255, 260, 263, Deichmiller, M. P., 20, 100, 400, 402, 440 204, 283 Curtin, C. C., 179, 240 Delamore, I. W., 381( 184), 393 Delanney, L. E., 34,35,100 Curtis, A. C., 385(201, 206), 395 Curtis, R. M., 13,106 Delaunay, A., 120, 168 Cushing, J. E., 65, 87, 70, 78, 88, 102, Delous. A., 218,246 DeMarsh, Q. B., 178, 242 113

AUTHOR INDEX

de Montera, E., 218,235, 241 Denney, D., 383( 181), 394 Dennis, S. M., 412, 419, 433 Den, F. A., 33, 100 de Petris, S., 183, 184,240 DeSimone, A. R., 357(23), 389 Deutsch, H.F., 5, 6, 71, 100, 323, 343 Deutsch, K., 200, 240 Deutsch, L., 3, 100 de Vries, A., 191, 240 De Vries, T., 417, 418, 435 Dienes, L.,400, 401, 402, 409, 410, 433 Dierheimeier-Vaur, C.,10, 111 Dietel, V., 16, 100 DiLapi, M. M.,414,434 Dilks, E., 18, 19, 115 DiLuzio, N. R., 166,173 Dineen, J. K., 216, 247 di Sant’Agnese, P. A,, 23, 100 Dische, Z.,307, 343 Dixon, F. J., 5, 7, 20, 41, 42, 43, 45,47, 48, 61, 97, 99, 100, 107, 108, 110, 121, 168, 176, 177, 181, 205, 206,

209, 213, 215, 219, 222, 223, 227, 229, 230, 237, 239, 240, 241, 244, 245, 247, 373( lM), 392, 399, 400, 402, 408, 409, 419, 433, 439, 440 Dixon, G. H., 341,348 Dohi, S., 176, 240 Dolby, J. M., 423, 433 Dole, V. P., 254, 285 Dolgin, A. E., 412, 440 Dong, L.,222, 245 Doniach, D., 376, 377(123, 126, 130, 132, 134, 135), 378(137, 140, 141, 142, 146),379( 130, 135, 151, 153), 381( 165, 167), 392, 393, 394 Dorfman, A., 136, 171 Dorward, B.,17,25, 101 Doty, P., 290, 291, 349 Douglas, G. W., 4, 5, 100 Dowling, G. B., 384(192), 385(192), 394 Downs, C. M., 85,100,142,170 Drabkin, D. L., 3, 100 Dray, S., 309, 320, 326, 327, 328, 340, 343 Dresner, E., 366(71),391

447

Dresser, D. W., 47, 48, 49, 100, 399, 433 Dreyer, N. B., 85,100 Duane, R. T., 5, 111 Dubert, J. M., 47, 99 Dubiska, A.,326,343 Dubiski, S.,326, 328, 343 Dubos, R. J., 133, 142, 154, 168, 170 Dukes, C . D., 402, 404, 437 Dukiski, S., 367(78), 391 Dunham, E. C., 12, 100 Dupont, H.C., 219, 238 Durall, G. L., 65,87,70, 88, 113 Duran-Reynals, F.,358(28), 390 Dutcher, T.F., 179,240 Duthie, J. J. R., 384(199), 385(199), 394 Dvorak, H. F., 164, 169

E Eakin, R. M., 36, 100 Earle, D. P., 219, 240, 242 Easley, C . W., 295, 296, 347 East, J., 51, 54,110 Eastlick, H.L., 35, 100 Easton, J. M., 194, 195,240 Easty, G.C., 197,240 Ebbs, J. H., 13, 109 Ebert, J. D., 34,35, 100 Ecker, E. E., 123,168 Eckman, P. L., 412, 419, 433 Edebo, L., 423, 432 Edelhoch, H.,290,291,343, 348 Edelman, G . M., 294,295,297,298,301, 303, 309, 311, 313, 318, 321, 325, 330, 332, 342, 343, 344, 347, 348 Edgerton, M. T., 48, 100 Edney, M.,18,47, 98 Egan, R. W., 376(121), 392 Egdahl, R. H., 35,42,100,164,170,412, 419, 433 Egner, W.,386(208, 209), 395 Ehrich, W.,33, 100 Ehrich, W.E., 3, 50, 100 Ehrlich, P.,2, 100, 332,344 Eichenwald, H.F., 24,26, 100 Einbinder, J. M., 416, 426, 433 Eisen, H. N., 176, 182, 187, 240, 242 Eisenbeis, C. H., 369(84), 391

448

AUTHOR INDEX

Elberg, S. S., 135, 146, 148, 149, 153, Favour, C.B., 69,72, 86,101, 373(log), 155, 157, 158, 159, 160, 166, 168, 392 169, 171 Fawcett, D.W..134,173 Elchlepp, J. G., 388(228),395 Feinstein, A., 288, 293, 308, 309, 330, Eldering, G.,420,433 331, 344 Elek, S. D., 82, 100, 290, 344 Feldherr, C. M., 191,240 Eliasson, S., 59, 60, 104 Feldman, H.A,, 24, 111 ElKhadem, H. S., 268, 283 Feldman, J. D., 8, 101, 158, 169, 203, Elliott, S. D., 260. 261, 279,283, 286 207, 209, 219, 222, 223, 234, 240, Ellwood, D. C., 271, 276, 280, 283, 284 244, 373( 106). 392, 407, 437 Enders, J. F., 6, 100. 123,173 Felix-Davies, F., 221,241 Englberger, F. M., 292, 293, 303, 346 Fellinger, K., 176, 179, 231, 239 Engle, R. L., Jr., 70,77,81,82,101, 115, Feltkamp, T.E. W., 383(175, 177),394 325, 348 Felton, L. D., 47, 49, 101, 414,433 Engleman, E. P., 371 (93),391 Feltynowski, A., 199,246 Epstein, W.V., 365(67), 370(90), 371 Fennell, R. H., Jr., 384(196), 394 (93),391 Fenner, F., 47,98 Erickson, J. O., 192, 240 Fennestad, K. L., 21, 101 Erickson, R. P., 87, 101 Ferago, F., 421, 428,433 Ericsson, J. L. E., 219, 240 Ferguson, K. A,, 35, 111 Eriksson, Z.,335, 342 Femandes, M. V., 163, 164, 170 Essner, E., 130, 139, 168, 190,240 Fernando, N. V. P., 177, 202, 203, 205, Estrada-Parra, S., 258, 283 208, 241, 244 Evans, C.A., 93, 105 Ferringan, L. W., 5, 98 Evans, E. E., 27, 74, 82,101, 106 Fessel, W.J., 321,344 Evans, E. P., 53, 103 Fiaschi, E., 218,221,241 Evans, W.H., 133, 168 Fichtelius. K. E., 50,51,101, 428,433 Field, E. J., 377( 125), 392 F Fields, M.,192,240 Fagraeus, A., 3, 61, 101, 176, 177, 240 Fife, E. H., 357(27), 390 Fahey, J. L., 6, 61, 81, 101, 176, 179. Finby, N.,371(95), 391 182, 240, 245, 246, 316, 320, 321, Finch, H.,196,245 323, 329, 332, 338, 339, 342, 344, Fine, J., 412,416, 438, 439 348, 349 Finger, H., 407, 433 Fahlberg, W. J., 400,404, 407,408, 410, Fink, C.W., 17,25, 101 422, 429, 439 Finkelstein, M. S., 17,25,26. 82,85, 114 Falchetti, E., 399, 401, 438 Finland, M.,332, 346, 347 Fant, W.M., 222,237 Finley, S. C., 39, 100 Farber, M.B., 21, 44, 45, 103, 104 Finotti, A., 27, 111 Fariss, B., 135, 143, 171 Finstad, J., 72, 76, 78, 79, 83, 89, 101, Famham, A. F., 130, 150,172 102, 109 Farquhar, M.G., 216,218,220,221, 240, Fireman, P., 6, 12, 101, 288,335, 344 242, 247 Fischel, E. E., 402,408,409, 433 Farthing, C. P., 291,342 Fischer, H.,130, 167 Farthing, J. R., 400, 413, 414, 417, 421, Fishel, C. W., 425, 433 422,424,427,428,433 Fisher, E. R., 50, 60, 101, 219, 240 F a d , A., 365(70), 301 Fisher, T. N., 132, 169 Faunce, K., Jr., 149, 168 Fishman, M.,91,101, 135,161, 166,169, Fauvert, R., 6, 98, 320, 342, 344 192, 211, 388, 416, 430, 431, 433

449

AUTHOR INDEX

Fitch, F. W., 418, 440 Fitzgerald, P. H., 234, 245 Flack, A., 24, 108 Fleischer, S., 291, 344 Fleischman, J. B., 297, 298, 301, 307, 310, 313, 314, 329, 344 Fleming, D. S., 413, 421, 433, 434 Florey, H . W., 202, 241, 243 Foerster, A., 32, 101 Folli, G., 218, 241 Fong, J., 144, 148, 148, 153, 155, 157, 158, 159, 180, 186, 167, 168, 169 Fonkalsrud, E. W., 386(212), 395 Forbes, I. J., 377(135), 379(135, 147), 393 Forbes, W. A., 27, 29, 101 Ford, C. E., 53, 103 Ford, H., 388(228), 395 Forman, C., 3, 100 Fomey, J. E., 404, 437 Forsen, N. R., 48, 49, 101 Foster, F.,35, 100 Fowler, R., Jr., 38, 47, 101 Fox, C. L., Jr., 418, 428, 433, 435 Fox, J. P., 18, 101 Francis, T., Jr., 149, 173, 262, 286 Frangk, F., 13, 20, 101, 297, 313, 344 Frankenstein, C., 23, 101 Franklin, E. C., 6, 11, 17, 25, 28, 82, 85, 98. 101, 114, 179, 248, 288, 295, 307, 319, 320, 321, 328, 329, 330, 331, 332, 333, 334, 338, 337, 342, 343, 344, 346, 348, 349, 359(33), 382( 56), 383(58), 364( 331, 385 (56, 65), 370(91), 390 Freda, V. J., 11, 101 Frhderic, J., 205, 245 Freedman, P., 215,218, 241 Freeman, B. A., 130, 142, 169, 171 Freeman, E. B., 132, 168 Freeman, T., 338, 343 Freimer, E. H., 253, 283 French, J. E., 139, 173 Frensdorff, A., 212, 239 Freund, J., 19, 37, 102, 398, 399, 400, 401, 402, 403, 405, 408, 407, 408, 409, 410, 419, 433, 434, 436, 437 Freyberg, R. H., 364(61), 390 Friedberger, E., 19, 37, 73, 85, 102

Friedewald, W. F., 357( 28), 390, 403, 408, 407, 434 Friedman, D. I., 30, 51, 109 Friedman, H., 47, 102 Friedrich-Freksa, H., 198,237 Friou, G., 352(5), 353(5), 374( 112), 384( 195), 385( 195), 389, 392, 394 F d i n g , L., 177, 179, 185, 188, 212, 241 Fry, E. S. J., 286, 283 Fudenberg, H., 96, 101, 102, 328, 329, 330, 334, 342, 344, 347, 349, 380 (46), 382( 52), 365( 52), 388( 731, 367( 52, 79), 388( 79), 371( 95), 373(104), 390, 391, 392 Fuji, H., 178, 241 Fujikawa, K., 135, 167 Fujino, K., 76, 102 Fujisaki, S., 218, 241 Fukuki, K., 423, 440 Fukushi, K., 411, 412, 413, 434, 438 Fukuya, I., 423, 440 Fulthorpe, A. J., 421, 432 Funkhouser, J. W., 80;102 Furth, J., 50, 106

G Gabrielsen, A. E., 58, 80,72, 89, 102, 109 Gaby, W. L., 47, 102 Gafni, J., 59, 102 Gaines, S., 413, 414, 417, 435 Gaisford, W., 12, 110 Galbraith, R. F., 383(183), 394 Gale, J. C., 216, 241 Galins, N., 384( 194), 385( 194), 394 Galle, P., 218, 235, 241 Gally, J. A,, 294, 313, 330, 343,344 Ganley, 0. H., 424, 425, 438 Garbutt, E. W., 38, 110 Garrett, A. J., 273, 285 Gartha, S., 380( lei), 393 Garvey, J. S., 92, 98, 192, 239 Gaschen, H., 84, 108 Gedigk, P., 134, 169 Gee, L. L., 72, 102 Geer, J. C., 213, 241 Geitner, M. B., 409, 434 Cell, P. G. H., 292, 308, 325, 326, 330, 331, 342, 344, 410, 432, 434

450

AUTHOR INDEX

Gelzer, J., 129, 140, 169 Gerald, L., 309, 328, 327, 343 Gerard, R. W., 130, 167 Gerbeux, C., 398, 413, 429, 438 German, J. L., 373( 104), 392 Gerlil, T., 430, 435 Ghuysen, J. M., 287, 272, 283, 286 Giacomelli, F., 218, 241 Gibier, P., 72, 102 Gibson, T., 278, 282 Giedion, A., 330, 344 Gilbert, V. E., 412, 434 Giles, B. D., 328, 348 Gilman, P. A., 191, 246 Gilmour, J. R.,28,102 Giltaire-Rolyte, L., 178, 245 Ginsberg, H. S., 132,169 Gisinger, E., 230, 239 Gitlin, D., 49, 58, 102, 111, 179, 240, 294, 295, 330, 332, 333, 338, 344, 345, 348, 347, 372( 102), 392 Givol, D., 294, 298, 297, 314, 343, 344, 348 Glaser, K., 33, 102 Glastonbury, J., 268, 283 Glauert, A. M., 188, 239 Glazer, A. N., 314, 344 Gleich, G. J., 384(63), 390 Glenny, A. T., 398, 434 Click, B., 27, 30, 49, 50, 102 Click, D., 412, 432 Glover, P.,273, 283 Glynn, A. A., 135, 168 Glynn, L. E., 388(223), 395 Goebel, W. F., 281, 282, 283, 286 Gokcen, M., 59, 115, 373( 105), 392 Goldberg, A. F., 186, 241 Goldberg, B., 194, 195, 200, 234, 240, 241 Goldgraber, M. B., 388(228), 395 Goldsmith, M., 152, 171 Goldstein, M., 4, 102 Golins, N., 220, 238 Gonzales, E., 5, 109 Good, R. A., 2, 3, 8, 9, 10, 13, 14, 15, 20, 24, 25, 30, 31, 32, 33, 38, 39, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 58, 57, 58, 59, 00, 61, 72, 73, 74, 70, 77, 78, 79, 82, 83,

85, 87, 88, 89, 92, 96, 97, 98, 99, 100, 101, 102, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 170, 203, 218, 218, 219, 222, 232, 237, 239, 240, 241, 245, 247, 389 (82), 372( 103), 373( 105), 391, 392, 399, 413, 415, 418, 417, 419, 432, 433, 434,439 Goode, G., 411, 413, 438 Goodline, M. A., 420, 437 Goodlow, R. J., 153, 171 Goodman, H. C., 8, 12, 101, 288, 335, 344 Goodman, J,, 222, 245, 387(79), 368 (79), 391 Goodman, J. R., 130, 140, 169, 188, 190, 241 Goodman, J. W., 297, 323, 344 Goodman, M., 76,102 Goodner, K., 85, 102 Goodpasture, E. W., 19, 110 Gordon, A. H., 339, 343 Gordon, H. A., 18, 113 Gordon, M., 88, 105 Gordon, R. S., 59, 107 Gordon, R. S., Jr,, 8, 103 Gordon, W . S., 7, 107 Gormsen, H., 3, 98 Gorrie, J., 53, 109 Gonynski, E. A., 278, 284 Goss, R. J,, 87, 103 Costing, L. J., 5, 8, 100 Got, R., 331, 343 Gotschlich, E. C., 281, 283, 285 Goudie, R. B., 377( 131), 378( 138), 379 (149), 387(218), 392, 393, 395 Gourvitch, A. E., 294, 313, 345 Gowans, J. L., 92, 103, 106, 188, 193, 241 Cowing, N. F., 82, 100 Gowland, G., 47, 48, 98, 103 Grabar, P., 5, 8, 98, 103, 287, 319, 320, 342, 344, 398, 410, 434 Grace, J. B., 285, 286 Grace, J. T., 385(205, 207), 395 Grady, R. C., 39, 103 Graham, J. B., 399, 434 Graham, R. M., 399, 434 Granboulan, N., 177, 238, 241

AUTHOR INDEX

451

Haber, M.H., 222,223,242 Habib, R., 218, 241 Habich, H.,325, 345 Hackel, D.B., 222,227, 228,242 (131), 378( 138), 379( 149), 384 Hackett, E., 379( 147), 393 (198), 387(216), 389, 392, 393, Hadzi, J., 62,103 394, 395 Hassig, A., 325, 330, 342, 345 Green, H., 194, 195, 200, 240, 241 Hahn, J. J., 253, 284 Green, H. N.,200,230,240 Halbert, S. P.,408,409,434 Greenberg, G. R.,270, 283 Hale, J. H.,11, 103 Greenberg, L.,413,421,433,434 Hall, C.E.,197,241,290,345 Hall, R., 378(145), 393 Greene, H. S. N., 4, 103 Gregg, M.B.,39,111,403,438 Halliday, R., 7, 103 Halpern, B. N., 91, 98, 124, 151, 152, Grbgoire, C.L.,33, 103 154, 167, 169, 334, 346, 412, 417, Gregory, K. O.,13,109 419,432 Gresham, E.,291, 344 Hamburger, J,, 218,241 Grey, H.,44, 82, 84, 97, 103 Hamilton, J., 11, 96 Grice, D.S., 372( 102), 392 Hamilton, M.,142, 145, 167 Grigson, J. P., 59, 60, 104 Hammar, J. A., 26, 27, 28, 30, 50, 57, Grisham, J. W.,202,248 90, 103 Grishman, E.,221,222, 239, 243 Hammarsten, C., 423,432 Grobstein, C.,29, 103 Hammer, D.,222, 223, 227, 240, 241 Grogg, E.,134,169 Hammerman, D.,233, 238 Gronwall, J. A., 222,237 Hammond, W.S., 219,247 Gross, D.,297, 323, 344 Hampton, J. C.,190,242 Grossberg, A. L.,291,293,345 Hampton, S. F.,12, 112, 335, 348 Groto, L.,154,172 Grubb, R., 328, 345, 359(36, 40), 362 Han, I., 417, 418, 435 Han, S., 417,418, 435 (53),390 Hanan, R., 47, 103 Gruber, M.,294, 348 Hanaoka, M., 176, 177, 183, 237, 240, Gubemieva, L. M.,294, 345 242 Guelin, A,, 18, 114 Hanks, J. H., 130, 168 Gueradt, A., 423,436 Hansen, A. E.,59, 109 GuCrin, C.,38,98 Guex-Holzer, S., 266, 267, 284, 286 Hansen, R. G., 7, 103 Hanson, E. D.,62, 75, 103 Gugler, E.,10, 13, 16,26, 103, 114 Hanson, L. A., 10, 103, 296, 332, 338, Gulland, G. L.,28, 103 345 Gump, D.,21, 98 Happ, W. M., 23, 103 Gunderson, C.H., 418, 440 Harboe, M., 96,103, 323, 328, 329, 330, Gundobin, N.,32, 103 340, 345, 347, 361(50), 362(50, Gustafsson, B. E.,18,103 55), 363(55), 368(55, 80), 390, Guttmann, R. D., 48, 97 391 Gyllensten, L.,33, 50, 103 Hardin, C. A., 48, 103 H Hardin, R. L.,291,344 Haas, R., 36, 86, 87, 92, 93, 104, 398, Hardy, F.E.,271,283 Hare, W.K., 39, 100 407, 434 Habel, K., 93,103 Hargis, B. J., 414, 416, 417, 420, 421, 422,428,427,436 Haber, E.,325, 346 Grant, L. H., 202, 241 Grasset, E., 18, 19,103,109,421,437 Gray, J. G., 388(232),395 Gray, K. G., 353(12), 355(12), 377

452

AUTHOR INDEX

Harkin, J. C.,219,232, 242, 247 Harrada, N., 188,248 Harrell, E. R., Jr,, 385(201 ), 395 Harris, A,, 6, 100 Harris, H., 251, 255, 260, 263, 283 Harris, J. E., 53, 103 Harris, M.,38, 100 Hams, R. J. C.,47, 103 Harris, S., 3, 21, 38, 44, 45, 104, 430, 434

Harris, T. N., 3, 21, 38, 44, 45, 50, 104, 430, 434 Hart, D A . P., 143, 169 Hartley, M,W., 220,242 Hartley, P., 333, 345 Hartmann, A. F., 274, 284 Hartmann, L., 8, 98, 320, 342, 344 Harwin, S . M., 408, 437 Harwood, S., 222, 227, 228, 242 Hdek, M.,34, 35, 47, 48, 104, 105 Haserick, 3. R., 385(204),395 Hashimoto, S., 144, 170 Hashimoto, T.,423, 440 Haskins, W.T., 411, 412, 413, 434, 438 Hdkovh, V., 4, 35, 104 Hasler, O.,428, 433 Hasson, M. W., 222, 242 Hatfield, W.B., 374( 113),392 Haukenes, G.,271,276, 280, 284 Haurowitz, F., 2, 104, 191, 242, 291, 344

Havas, H. F., 401, 434 Havens, W.P., Jr., 399, 419, 434 Hay, J. B., 272,278, 282, 284 Hayashi, H., 188, 242 Hays, R. C., 9, 109 Heckaman, J. H., 385(206), 395 Heden, C. G., 254,286,423,432 Heefner, W.A., 212,213,214,242 Heidelberger, M., 258, 281, 285, 288, 282, 283, 284, 285, 288, 345, 414, 434

Heim, F., 37, 102 Heimer, R., 384(81),386(74),370(86), 371(99),390, 391 Heimlich, E. M., 12, 104 Hektoen, L.,398, 434 Heller, G.,370(90), 391 Heller, H., 59, 102

Hellman, T. J., 28, 32, 104 Hellstrom, K. E., 93, 106 Helmreich, E,,178, 182, 187,240,242 Hemmings, W.A,, 333,334,342, 345 Henderson, D. W., 153, 169 Henle, G.,407, 434 Henle, W.,407, 434 Hensley, T. J., 192, 240 Herbert, V., 381( 189), 394 Herdegen, M.,409, 434 Heremans, J., 179, 244 Heremans, J. F., 8, 9, 98, 104, 287, 288, 295, 304, 307, 319, 321, 330, 335, 338, 338,342,343,344,345,349 Heremans, M. T., 8, 9, 104, 287, 295, 321, 343, 345 Herrman, C., 11, 104 Hershgold, E. J., 295, 345 Hertig, A. T., 8, 97 Hertzenberg, L. A., 328, 349 Hess, E. V.. 51, 59, 80, 104, 228, 242, 389(83), 391 Hem, M.,360(42), 390 Hestekin, B. M., 421, 423, 437 Heuer, A,, 399, 419, 434 Heymann, H., 258, 284 Heymann, W., 222, 227, 228, 242 Higham, W.,290, 344 Hijmans, W.,378(137), 393 Hildemann, W. H., 36, 72, 88, 87, 92, 93, 97, 104 Hilgar, A., 412, 419, 437 Hilgard, H. R., 55, 104, 115 Hill, B. H., 90, 104 Hill, 0.W., 379(148), 393 Hiller, M. C., 5, 112, 230,246 Hines, W.D., 130, 171 Hinglais, N.,218, 241 Hirata, Y., 220, 238 Hirsch, J. G., 119, 123, 133, 134, 135, 138, 137, 138, 139, 168, 169, 188, 205, 237, 239, 242 Hitzig, W. H., 5, 8, 11, 18, 58, 104, 333, 345

Hjort, G. H., 212, 242 Hobbs, K. R., 9, 97 Hoglund, S., 290, 345 Hoerlein, A. B., 13, 104 Hoff, E. E., 199, 245

AUTHOR INDEX

Hoffman, H. A., 328, 343 Holborow, E. J., 371(96), 377( 132), 378(137, 142), 391, 392, 393 Holdsworth, E. S., 263, 284 Holford, F. E.,399, 434 Holland, J. J., 93, 105, 129, 142, 146, 169 Holland, N. H., 16, 115 Hollander, J. L.,370(88), 391 Holman, H. R., 6, 201, 216,244, 352(4), 353(4, 8, Q), 355(8, 9, 16, 17), 356(9), 357(9, 25), 358(25), 365(65), 372(25), 373( 107), 374(114), 389, 390, 391, 392 Holmes, M. C., 59, 98 Holt, L. B., 398, 400,414,417,421, 422, 427, 428, 432, 433, 434 Holtzer, H., 191, 196, 242, 245 Holtzer, S., 191, 242 Holub, M.,20, 38, 43, 105, 112, 408, 412, 434, 439 Holzmann, H., 220, 243 Honet, T., 404, 410, 433 Hong, R., 16, 115 Honjin, R., 232, 242 Hook, E. W.,416, 434, 435 Hook, W.A., 412,435 Hopper, J., 220, 221, 240, 242 Horibata, K., 340,342 Home, R. W.,250, 285 Homung, M.O.,262, 263,284 Horowitz, J., 291, 3 4 Horton, S. L., 82, 101 Horvat, J., 20, I05 Hoskins, D. W., 380( 162), 381( 162), 382( 162) , 393 Hoskins, M. M.,90, 105 Hosmer, E. P., 401, 433 Hotchin, J., 47, 105 Hough, H. B.,401,403,405,434 Howard, J. G., 2, 91, 98, 108, 123, 125, 134, 154,169, 173, 412,435 Howatson, A., 197,199,237,290,342 Hraba, T.,34,35,47,48,104,105 Hrubesova, M.,20, 42, 105, 112 Hsu, K. C.,59, 113, 180, 183, 203, 209, 211, 222, 223, 225, 237, 245, 246, 247, 382(173), 394 Huang, J. S., 402,404,437

453

Hubbs, C. L., 75, 105 Hudson, R. V., 376(120)) 377( 134), 392, 393 Huff, C. G., 3,62,63,64,68,70,105 Hughes, R. D.,328, 349 Hughes, W.L., 47, 113 Huhn, D.,218,222,242,244 Hulliger, L.,134,145, 150,173 Hummeler, K., 24, 106, 196, 197, 237, 242 Humphrey, J. H., 20, 105,291, 342, 399, 409, 427, 432, 435 Hung, W.,387(218), 388(218, 222), 395 Hunt, A. D.,Jr., 24,106 Hunter, J. L. P., 222,227,228,242 Huser, H.J., 58, 104 Hutt, M. P.,216, 239 Huxley, J., 94,105 Hyman, L. H., 62, 105

I Ikawa, M., 277, 284 Imaeda, T.,188, 242, 248 Imura, M.,232, 242 Inai, S., 135, 167 Inci, S., 423, 440 Ingram, D. G., 351( l), 389 Ingram, P. L.,7, 97 Ingram, V. M.,75,84,105,294,345 Inman, 0.L.,63, 105 Inoue, Y., 144, 170 Inui, S., 144, 170 Irvine, W.J., 381( 164, 166), 388(221), 393, 394, 395 Irwin, M. R., 76, 105 Isakovic, K.,20, 105 Isenberg, H. D., 412, 435 Ishimoto, Y., 203, 242 Ishizaka, K., 336, 337, 345 Ishizaka, T.,336, 337, 345 Isliker, H., 308, 345 Isliker, H. C.,65, 105 Isola, J. B., 188, 247 Ito, Y., 93, 105 Iviinovics, G.,264,265,266,284,285 Ivtinyi, D.,35, 36, 105 Ivdnyi, P.,35, 36, 105 Izumi, M.,222, 242

454

AUTHOR INDEX

J Jaap, R. G., 27, 30,50,102 Jacobson, A. S., 370(90), 391 Jacobson, E. B., 417, 418, 435 Jacot-Guillarmod, H., 308, 345 Jacotot, H., 398, 435 Jacox, R. F., 371(97), 391 Janczura, E., 277, 284 Janeway, C. A,, 58, 111, 179, 240, 330, 338, 344, 347, 372( 102), 392 Janicki, B., 151, 169 Jankovih, B. D., 20, 32, 57, 96, 105, 114, 164, 169 Jaquet, H., 312, 313, 343, 345 Jaroslow, B. N., 400, 419, 431,435,439 Jebb, W. H. H., 264,284 Jeffries, G. H., 380(162), 381(162), 382 ( 162) , 393 Jenkin, C. R., 125, 128, 129, 154, 169, 170, 172, 412, 435 Jenkin, J., 128, 134, 154,169 Jenkins, G. C., 288, 349 Jennings, R. B., 219, 222,223,240,242 Jensen, D., 75, 105 Jensen, D. R., 399,4153, 434 Jensen, E., 48, 105 Jensen, K., 128, 170 Jeme, N. K., 2, 105 Jerushalmy, A., 191, 240 Jervis, G. A., 24, 106 Jirgensons, B., 290, 345 Johansson, B. G., 296, 345 John, C., 430, 435 Johnson, A., 291, 345 Johnson, A. G., 48, 105, 166, 170, 413, 414,417,418,435,436,440 Johnson, A. J., 416,417,418, 435 Johnson, G. D., 371(96), 391 Johnson, S. A., 231, 244 Jolles, P., 406, 440 Jolly, J., 27, 30, 90, 105 Jones, D., 276, 284 Jones, H. E. H., 379(151), 393, 408, 409, 435 Jones, M. E., 252, 283 Jones, R. E., 334, 345 Jordan, H. E., 68,88, 105 Journey, L. J., 235, 242 Juergens, W. G., 273,274,284,285

Julia, J. F., 12, 23,24, 109 Julianelle, L. A., 270,274, 275, 284, 286

K Kabat, E. A., 5, 6, 65, 100, 105, 113, 275, 282, 288, 323, 345, 348, 402, 408,409,411,429,433,435 Kaeberle, M. L., 13, 14, 20, 111 Kallen, B., 232, 238 Kafig, E., 196, 230, 238 Kahn, M. F., 371(99), 391 Kaiser, A. D., 64, 91, 97 Kajikawa, K., 216, 242 Kakatani, I., 423, 440 Kalinin, W. S., 399, 435 Kallings, L. O., 25, 105 Kallman, K. D., 86, 105 Kalmutz, S. E., 22, 32, 56,105 Kammerer, W. H., 370( go), 391 Kandutsch, A. A., 162, 172 Kantor, F. S., 234,241,252,284 Kaplan, M. E., 381(169), 394 Kaplan, M. H., 191, 217, 227, 239, 242, 254, 284 Kapral, F. A., 127, 131, 168, 170, 172 Kark, R. M., 216, 218, 219, 241, 243, 245, 384( 200), 395 Karlsbad, G., 183, 184, 240 Karnowsky, M. L., 124, 130, 131, 133, 140, 143, 150,168,170,172 Karte, H., 8, 105 Karush, F., 2, 105, 291, 303, 310, 312, 334, 345, 347, 349 Kashiba, S., 135, 167 Katchalski, E., 296, 343 Katsh, S., 408, 429, 435 Kauffmann, G., 47, 49, 101 Kautz, J., 176, 242 Kawakami, M., 149, 171 Kayhoe, D. E., 356(20), 389 Kehn, J. E., 289,299,300,348 Keil, H., 384(190), 394 Keiser, H., 139, 173 Kekwick, R. A., 288, 345 Kelemen, M. V., 270, 276, 281, 283, 284 Kelley, V. C., 399,419,432 Kellner, A., 132, 168 Kelly, J. T., 48, 99

AUTHOR INDEX

Kelly, L. S., 50, 111 Kelly, W. D., 15, 31, 32, 39, 57, 96, 102, 105, 114 Kelman, H.,48, 107 Kelus, A., 292, 308, 325, 326, 328, 330, 331, 343, 344, 345, 346 Kempe, C. H., 11, 105 Kemper, J. W., 371(94), 391 Kempf, J., 177,185,212, 241 Kendall, F. E.,261, 284 Kendig, E.L.,Jr., 39,105 Kent, S. P.,27, 106 Keppie, J., 265, 286 Kerby, G. P., 151, 170 Kern, M.,176,182,187,240,242 Kerr, W.R., 47, 106 Kersey, J., 51, 52, 107 Kessel, R. W.,161, 170 Kevy, S. V., 330, 347 Khanolkar, V. R.,398,413,428,435 Kidd, J. G.,357(26), 390 Kies, M. W.,398, 400, 404, 405, 407, 408,410,422,429,435,439 Kifune, S., 219, 243 Kim, 0.J., 218,220,242 Kim, Y. B., 14,21,106 Kimmelstiel, P.,218, 220, 242 Kind, L. S., 417, 420, 421, 422, 424, 428, 427, 428, 435 Kind, P.,416,417,418,435 King, A. L.,5, 111 King, J. W., 85, 100 King, W.M., 412,419,433 Kingley-Smith, B. V., 290, 344 Kingsbury, B. F., 30, 106 Kirkpatrick, C. M., 30, 106 Kirsner, J. B., 388(226,228), 395 Kitasato, 2, 97 Kitaura, T.,144, 170 Kite, J. H.,377(124), 392 Kivy-Rosenberg, E.,416, 440 Kleczkowski, A., 197,242,290,346 Klein, E.,93,106 Klein, G.,93, 106 Klein, M., 136, 171 Klemperer, P.,384(188, 189), 394 Kling, C.A., 26,28,106 Klinman, N. R., 303, 345 Klugeman, M. R., 403,406,407,439

455

Knisley, M. H., 124, 170 Knoll, W.,27, 57, 106 Knouff, R.A., 26,27,28,29,96 Knox, K. W.,268,269,283,284 Kobayashi, O.,218,219,242,243 Kobayashi, R., 149, 170 Kobrin, S., 188, 247 Koch, F., 16, 106 Kochan, I., 152, 170 Kochen, H.G.,213,215,222,247 Koenig, V. L.,71, 100 Kollmann, M., 68, 106 Kolouch, F.,3, 61, 106 Kondo, T.,385(207), 395 Koniecma-Marczynska, B., 48, 112 Kono, A., 423, 440 Kopeloff, L. M.,401, 435 Kopeloff, N., 401, 435 Koprowski, H.,24, 32, 57, 100,106, 163, 164, 170 Korngold, L., 179, 182, 243, 244, 319, 320, 325, 346, 370(86), 391 Korting, G. W., 220, 243 Koshland, M.E.,292,293,303,346 Kostka, J., 20,36,112,412,435,439 Kotani, S., 144, 170 Kotsevalov, O.,24, 100 Kourilsky, F. M., 336, 342 Krafka, J., Jr., 70, 106 Kramhr, E.,264, 284 Kramer, K. L., 21, 112 Kramer, S. D.,11, 97 Krause, A. K., 160, 170 Krause, R. M., 252, 253, 256, 259, 260, 282, 283, 284 Krecke, H.J., 221, 238 Kretschmer, R. R., 408, 435 Kringelbach, J., 39, 114 Kritzman, J., 179,243,365(68),391 Kross, D.J., 142, 169 Kruse, H.,191, 243 Kiihn, K., 220, 243 Kuge, H.,176, 243 Kulberg, A. Y., 297, 346 Kunkel, H.G.,6, 11, 12, 96, 101, 103, 106, 111, 179, 213, 215, 216, 243, 275, 282, 288, 289, 295, 307, 319, 320, 321, 323, 325, 329, 330, 333, 334, 340, 343, 344, 345, 346, 347,

456

AUTHOR INDEX

348, 352(4), 353(4, 7, 9, 13), 355 (9, 13, 16, 17), 356(9), 357(9,25), 358(25), 359(33, 34, 39), 361(48, 49), 362(52, 54, 55), 363(55, 57), 364( 33, 39, 62), 365( 39, 52, 65, 66, 68), 366(72, 73), 367(52, 75), 368(55), 369(39), 370(91), 372 (25), 373(104, 108, 110), 375 (117), 376(17), 389, 390, 391, 392 Kushner, D. S., 217, 243 Kutsakis, A., 200, 243 Kuzovleva, 0. B., 313, 345 Kyle, M. A., 387(218), 388(218), 395

L Lachmann, P. J., 213, 215, 216, 243, 353(7), 355( 14), 373( 108), 389, 392 Lacomme, 23, 110 Laemmert, H. W., Jr., 18, 101 Lafferty, K. J., 197,243,290, 346 Lagercrantz, R., 11, 114 Lahti, A., 274, 286 Lambie, A. T., 59, 106, 219, 243 Lamm, M. E., 289,299, 300,348 Lampkin, G. H., 47, 96, 97 Lamprecht, S., 212, 239 Law, A., 412, 439 Lancaster, M. C., 153, 169 Lancefield, R. C., 253, 254, 255, 256, 258, 284, 285, 286 Landing, B. H., 338, 344 Landsteiner, K., 402, 435 Landy, M., 120, 124, 170, 171, 411, 412, 413,414, 417, 434,435,436,438 Lange, K., 222, 243 Langer, B., 388(224), 395,408,439 Langevoort, H. L., 417, 418, 435 Lannigan, R., 219, 243 Lanset, S., 328, 348 Lapinska, E., 129, 172 LaPlante, E. S., 48, 49, 106 Largier, J. F., 289, 346 Larkin, L., 358(30), 378( 139), 390, 393 Larson, C. L., 406, 423, 435, 437 Larsson, B., 206, 238 Lasker, S. E., 416, 435

Latta, H., 199, 200, 218, 243 Latta, J. S., 28, 106 Laurel], A. B., 328, 345, 359( 40), 390 Laurel], A. H. F., 289, 346 Laurel], C. B., 18, 103 Laurel], G., 51, 101 Laurent, A. M., 407, 438 La Via, M. F., 22, 32, 106, 418, 440 Lavin, G. I., 261, 262, 283 Law, L. W., 55, 106 Lawler, S. D., 303, 329, 330, 344, 346 Lawrence, H. S., 2, 47, 106, 159, 166, 170 Lawrence, M. E., 338, 344 Laxson, C., 422, 427, 440 Leake, E. S., 135, 143, 152, 171, 188, 244 Leblond, C. P., 53, 111 Lederberg, J., 2, 106 Lederer, E., 406, 436, 440 Leduc, E. H., 3, 61, 99, 106, 176, 183, 191, 193, 239, 240 Lee, J. M., 407, 410, 422, 436 Lee, L., 124,170, 220, 243 Lee, L. H., 11, 103 Lee, R. E., 5, 106, 193, 194, 216, 243, 245 Lehrer, H. I., 356(22), 389 Leikhim, E. J., 336, 337, 342 Leitner, A., 230, 246 Lejeune, G., 47, 106 Lejeune-Ledant, G., 47, 96 Lelihvre, A., 30, 110 Lemetayer, E., 398, 401, 438 Le Minor, L., 124, 125, 154, 167, 168 Lemli, L., 59, 106 Le Moignic, E., 401, 436 LengerovA, A., 5, 34, 47, 48, 104, 106 Lenhart, N. A., 255, 275, 285, 286 Lennox, B., 47, 115 Lennox, E. S., 326, 340, 342,343 Lenoir, J.. 328, 347, 360(44), 390 Leonard, M. R., 125, 171 Leplatre, J., 398, 413, 429, 438 Lepper, M. H., 222,227,229,246 Lerner, A. M., 332, 347 Letterer, E., 403, 436 Levaditi, C., 8, 106 Levey, R. H., 55, 106

457

AUTHOR INDEX

Levin, F. M., 366(74),371(99),391 Levine, L., 356, 389 Levine, S., 404, 422, 436 Lewis, C. W.,188,246 Lewis, E. W.,17,25,26,114 Lewis, F. B., 32, 57, 112 Lewis, M. R.,234,245 Lewis, P.A.,398,400,402,436 Lewis, T.,222, 243 Li, I. W., 255,275,285 Liacopoulos, M., 334, 346 Liacopoulos, P.,334, 346 Libby, R. L., 192,240 Lieberman, R., 328, 343 Liljedahl, S. O.,339, 342 Lillie, F. R., 46, 106 Lffly, M. D., 270,283 Limani, L. R., 33, 102 Lhd, J., 8, 100 Lindsley, D. L., 177,181,245 Lipari, R., 319, 320,346 Lipman, L. N.,86,109,296,346 Lippoldt, R. E., 290, 343 Lips, G., 16, 114 Lipton, M. M., 400, 401, 403, 404, 408, 409, 410, 411, 429, 430, 433, 434, 436, 439 Lisowski, J., 123, 172 List, R. H.,406,435 Little, R. B., 7,112 Liu, T.Y.,261,263,285 Llewellyn-Jones, M., 421, 432 Lochhead, M. S., 94, 111 Locke, R. F., 13, 111 Lods, J. C., 386(213), 395 Loeb, L., 69,86,106 Loebel, J., 154, 172 Lofgren, S., 12, 96 LoGrippo, G. A., 398, 438 Lohmann, D., 16, 100 Loiseau, 23, 110 Longmire, W.P.,35,98,386(212),395 Longsworth, L. G.,13, 106 Loomis, D., 398,400,402,436 Lopez-Castro, G., 123, 168 Losnegard, N.,276, 285 LoSpalluto, J., 17,25, 101, 364(60),390 Lowenstein, L., 380(lsl), 393 Ludden, G. B., 399,434

Luecke, D. H., 414,415,417,436 Liideritz, O.,411,423,440 Lukes, R.J.,21,34,112 Lumsden, C. E., 429, 436 Lundevall, J., 328, 345 Lundgrexr, H.P.,289, 347 Lupton, C. H.,220, 242 Lurie, M., 145,149,151,167,170 Luse, S. A., 232,243 Lycette, R. R., 234,245 Lynn, L. T.,296,347

M Maal@e,O., 123, 170 McCarthy, E. F., 7, 106 McCarthy, J., 179, 243 McCartney, C. P.,221, 246 McCarty, D. J., Jr., 370(87),391 McCarty, M., 149, 173, 253, 256, 257, 258, 259, 261, 262, 270, 278, 279, 280, 282, 283, 284, 285, 365(68), 391 McCluskey, J. W., 234, 243 McCluskey, R. T.,124, 170,234, 243 McConahey, P. J., 399, 419, 433 McCullough, N. B., 59, 107 McCutcheon, M., 120, 121, 122, 170 McDaniel, F. C., 73, 96 McDermott, K., 401, 433 MacDonald, M. K., 219,220, 243, 246 McDougal, D. B., Jr., 232, 243 McDougall, E. I., 7, 106 McDuffie, F. C., 363(58), 390 McElhinney, A. J., 353(12), 355(12), 384(197), 389, 394 McEndy, D. P.,50, 106 McFadden, M. L., 291, 303, 348 McCill, H.C., 213, 241 McGregor, D. D., 92,106, 188, 193, 218, 222, 241, 242, 244 McGregor, I. A,, 339, 346 McGuiness, M. B., 409, 434 Mackaness, G. B., 127, 129, 133, 138, 143, 145, 147, 152, 170, 171 MacKay, I. R., 216, 247, 358(29, 30), 378( 139), 388(29), 390, 393 McKenna, J. M., 415,416,436,439 MacKenzie, M. R., 323, 343 McKinney, R. W.,408, 436

458

AUTHOR INDEX

McLaren, L. C., 93, 105 MacLean, L. D., 50, 106 MacLeod, C. M., 262, 285 McMaster, P. D., 191, 243 McPherson, S. E., 222, 243 Makela, O., 3, 61, 106, 109, 118, 161, 171 MHrtensson, L., 329, 346, 360(46), 390 Maier, P., 288, 336, 349 Main, J. M., 49,93,94,106,110 Main, O., 47, 114 Maitland, H. B., 423, 436 Majno, G., 202, 203, 243 Makinodan, T., 3, 34, 45, 46, 61, 106, 114, 177, 247

Maldonado, J. E., 216, 243 Malkiel, S., 414, 416, 417, 420, 421, 422, 426, 427, 436 Malmgren, B., 413, 423, 432, 436 Malmgren, R. A., 179, 182, 246 Malmnas, C., 12, 96 Mandel, L., 20, 36, 112, 412, 439 Mandell, A., 412, 419, 437 Mandelstam, M. H., 272, 285 Mandema, E., 384(200), 395 Mandy, W. J., 96, 102, 293, 295, 302, 346, 347

Mannick, J. A,, 164, 170 Manniello, J. M., 258, 284 Mannik, M., 320, 321, 323, 325, 329, 330, 340, 345, 346 Manuelidis, E. E., 428, 437 Manwell, C., 75, 84, 107 Marchesi, V. T., 139, 173, 202, 243 Marcus, E., 62, 107 Mariani, T., 2, 48, 49, 107 Mark, R., 44, 107 Markowitz, A. S., 215, 217, 241, 243 Marks, R., 303, 345 Markson, J. L., 381(163), 393 Markus, G., 96, 109 Marler, E., 289, 300, 301, 302, 346 Mannur, M., 356(21), 389 Marrack, J. R., 331, 346 Marshall, A. H. E., 54, 59, 108, 115, 383(178), 394, 405, 406, 440 Martin, C. M., 59, 107 Martin, F., 23, 98 Martin, G., 205, 247 Martin, G. S., 398, 436

Martin, H. S., 232, 245 Martin, N. H., 14, 107 Martin, R. O., 271, 283 Martin, S. P., 142, 170 Martin du Pan, R., 8, 13,16,108, 111 Martinez, C., 2, 32, 46, 48, 49, 51, 52, 53, 54, 55, 58, 60, 97, 99, 100, 102, 104, 107, 112, 115

Martini, E., 419, 436 M a n , Z., 3, 110 Mason, J. H., 7, 107 Mathk, G., 235, 238 Mathias, A. P., 270, 273, 283 Matoltsy, M., 38, 114, 234, 247 Matthews, C. M. E., 339, 343 Maung, M., 422, 437 Maurer, F., 89, 90, 107 Maurer, P. H., 47, 48, 100 Mauro, J., 9, 109 Mautner, W., 221, 222, 239, 243 Maximow, A., 26, 30,90,107 Maxted, W. R., 254, 285 Mayer, M. M., 323, 345 Mazzella, H., 199, 243 Mead, R. K., 387(217), 395 Medawar, P. B., 2, 34, 36, 47, 48, 96, 97,98,107, 162,167,168,170 Meeker, W. R., 87, 107 Meier, R., 120, 170 Melby, J. C., 412, 419, 433 Mele, R. H., 5, 112 Mellander, O., 13, 108 Mellors, R. C., 179, 182, 216, 222, 243, 244, 365(68), 370(86, 92), 373 (107), 379(155), 391, 392, 393 Melly, M. A., 127, 128, 170, 171 Meltzer, M., 96, 101, 329, 330, 344 Mercer, E. H., 197, 240 Merchant, D. J., 132, 170 Merler, E., 120, 170, 294, 330, 332, 344, 346, 347

Merriott, J., 142, 170 Merritt, K., 414, 436 Merryman, C., 316, 346 Merwin, R. M., 177, 179, 240 Meryman, H. T., 196, 230, 238 Mesnil, A., 72, 108 Mesrobeanu, I., 411, 432 Mesrobeanu, L., 411, 432

AUTHOR INDEX

Mester, L., 266, 285 Metalnikov, S. I., 62,64,67,108 Metcalf, D.,50, 108 Metschnikoff, E.,2, 60, 66, 72, 73, 108, 133, 170 Metzgar, R. S., 387(215), 395 Metzger, H.,222, 247, 314, 315, 346 Meyer, R. K.,27, 30,50,51,56,96,108 Miasoedova, K. N., 294, 345 Michael, J. G.,124, 171, 333, 344, 412, 435, 436 Michaeli, D., 59, 102 Michalska, E.,123, 172 Michel, M., 323, 347 Michelson, A. M., 279, 285 Michie, D.,2, 108 Michielsen, P.,218, 241 Michon, J., 331, 343 Middlebrook, G.,142, 170 Miescher, P.,352(2), 356( 18, 19), 389, 398, 434 Miescher, P. A,, 125,172,336,349 Migeon, C. J., 388(222), 395 Migita, S., 295, 321, 346,347 Migliavocca, A., 221, 241 Mika, L. A., 153, 171 Miler, I., 412, 439 Miles, A. A., 399, 400, 412, 430, 436, 440 Milgrom, F., 359(37), 364(37), 367(78, 79), 368(79), 388(225), 390, 391, 395 Miller, B. F., 5,109,136,171 Miller, C.,21, 99 Miller, F., 191, 213, 217, 221, 222, 227, 238, 244 Miller, J., 48, 49, 97 Miller, J. F. A. P., 32, 51, 52, 54, 55, 108, 109, 383(186), 394 Miller, L. L., 6, 108 Miller, W.E.,Jr., 17,25,101 Mills, J. A., 325, 346 Milner, K.C.,411,412,413,434,438 Mita, S., 85, 102 Mitchell, P., 269, 285 Mitchison, N.A,, 45,47,108,162,171 Mitsuhashi, S., 128, 148, 149, 150, 153, 158, 159, 171, 172, 173 Modern, F., 222, 245

459

Mohr, J., 328, 342 Moll, L., 19, 108 Monckeberg, F.,49, 102 Mongan, E. S., 371(97), 391 Monsen, G.,21, 99 Moog, F.,7, 108 Moon, H. D.,163, 171, 220, 221, 240, 242 Moor-Jankowski, J. K., 325,328,346 Moore, D.H.,6,13,100,108,190,244 Moore, J. M., 381(163, 168),393, 394 Moore, R., 222, 245 Moore, R. D.,191,246,408,438 Moore, R. E.,130, 140, 169, 188, 190, 241 Moore, S., 136, 168 Mora, P. T., 65, 108 Mordarska, H.,129, 172 More, R. H.,207, 245 Morgan, C., 180, 183, 203, 222, 223, 225, 237, 245 Morgan, H. R., 132, 170 Morgan, I. M., 401, 436 Morgan, P.,403, 436 Morgan, R. F.,46,47,109 Morioka, T.,135, 167 Moriuchi, M., 219, 243 Morrel, S. C.,48,100 Morris, I. G.,333, 346 Morrison, L. R., 39, 114 Morse, J. H.,373(110), 392 Morse, S. I., 127, 130, 131, 132, 134, 137,168,171,274,275,282,285 Morton, J. A., 380(156, 157),393 Moss, J. N., 398, 436 Mota, I., 422,423,427,435,436,437 Moureau, P., 47, 96 Mouton, D.,91, 98, 123, 124, 125, 152, 154, 167, 168, 169, 171 Movat, H. Z., 177, 202, 203, 205, 206, 213,218,222,241,242,244,246 Moyle, J., 269, 285 Mudd, S., 2, 108, 196, 244, 255, 275, 285, 286, 408, 409, 434 Muehrcke, R. C.,218, 245 Mueller, A. P., 18, 19, 27, 30, 47, 51. 56, 108, 113, 115 Miiller, W.,90, 108

460

AUTHOR INDEX

Miiller-Eberhard, H. J., 8, 101. 213, 215, 218, 243, 307, 346, 359(33), 384 (33), 365(85, 8 8 ) , 387(75, 78), 373(108, 110), 390, 391, 392 Mukheji, P. K., 222,227,239 Muldoon, T. N., 422, 436 Mumow, V. R., 191, 246 Munder, G.,130, 167 Munoz, J., 401, 403, 414, 418, 417, 419, 420, 421, 422, 423, 424, 425, 428, 427, 432, 437, 440 Munroe, J. F., 131, 171 Murphy, J. B., 34,108,399,437 Murray, J., 383( 183), 394 Murray, M. J., 388(208,209,210),395 Murray, M. R., 232, 238 Murray, U.,12, 23, 114 Muschel, L. H.,357(27),390,412,435 Muslow, I., 213, 241 Myers, L., 374(112), 392 Myers, W.L., 13, 111 Myrvik, Q. N., 135, 143, 151, 152, 171, 188, 244

Neil, A,, 178,244 Neilson, J. McE., 381( 188), 394 Nelson, C.A., 289,300,301, 302,346 Nelson, C. T.,416, 428, 433 Nelson, E. L., 180, 171 Neri, L.,399, 434 Nesbitt, R. E.Lo, Jr., 9,109 Neter, E., 278, 284 Neuhaus, F. C.,270, 283 Neumann, C.G., 38,40,114 Neurath, H.,290, 346 Newton, W.L.,412, 435 Nezlin, R. S.. 313, 344 Nichamin, S. J., 23, 111 Nilsson, S., 378(143), 393 Nishimura, S., 135, 167 Nishino, K., 423, 440 Nishiura, M., 188, 248 Nisonoff, A,, 96,102, 109, 197,241, 290, 291, 293, 295, 298, 302, 327, 340, 343, 345, 346, 347 Niven, C.F., Jr., 278,284 Niwa, M.,423, 437 Njoku-Obi, A. N., 147,148,171 N Nolan, C.,307, 346 Naccaroto, R., 218, 221, 241 NoU, H.,143, 171 Nace, G.W.,4, 108 Nordbring, F., 11, 23, 114 Nachman, R. L., 325, 348 Norris, E.A., 59,99,383(184),394 Nadel, E.M., 412,419,437 North, R. J., 138, 152,171 Nadel, H., 388(214), 395 Norton, T.W.,24, 106 Nagai, M.,149, 171 Norton, W.L.,234, 244 Nagler, A. L.,418,440 Nossal, G . J. V., 3, 42, 45, 53, 81, 106, Nagoya, C.,32, 108 109, 118, 128, 181, 171, 340, 347, Najarian, J. S., 5, 43, 108, 159. 169, 430, 437 234,240,244,407,437 Noufflard, H., 188, 245 Nakamura, T., 232, 242 Novelli, G. D.,183,248 Nakano, M., 148,158,159,180,172 Novikoff, A. B., 233, 238 Naki6, B., 48, 108, 109 Nowoslawski, A., 179, 244 Naki6, Z., 48, 109 Nuttall, G., 2, 109 Nance, W.E., 341, 346 Nybelin, O.,73, 109 Nasou, J. P., 356(20), 389 Nyka, W.,153, 171 Nastuk, W. L., 59, 113, 382(171, 172, 0 173), 394 Nathan, P.,5, 109 Oakley, C. L., 47, 103, 333,342 Nathenson, S. G.,271,273,285,286 Oberman, J. W.,13, 109 Nattan-Larrier, L.,19, 109 Obst, B., 123, 172 Naumann, P., 177, 247 O’Dea, J. F., 179,240 Neblett, T.R., 231,244,375(116),392 Odor, L., 190, 244 Neess, J., 18,19,47,115 Oeding, P.,271, 276,280,284,285

AUTHOR INDEX

Oertelis, S. J., 197,243,290,346 Oesterberger, R., 13, 108 Ogburn, C.A.,21,44,45,103,104 Oglesby, R. B., 372( 101), 392 Ohno, S., 128, 172 Oishi, M.,219, 242 Old, L. J., 126, 134, 152, 165, 167, 171, 173 Oldstone, M., 416, 437 Olhagen, B., 339, 342 Olins, D.E.,301,313,321,344,347 Olitsky, P. K.,422, 436 Olson, V. H.,42, 112,415,439 Ono, E.,219, 242 Onstad, T.,48, 107 Oort, J., 205, 240 Oosterhuis, H. J. G. K., 383( 175, 176, 177), 394 Ordman, D.,421, 437 Orfanos, C.,206, 244 Orihara, Y., 135, 167 Orlandini, O.,13, 109 Orlova, 0.K.,263, 285 Ortega, L. G.,216, 222, 244, 373(107), 392 Ortiz, J., 124, 172 Osborn, J. J., 12,23,24, 109 Osebold, J. W.,147, 148, 171 Oshima, S., 143, 152,171,188,244 Osler, A. G.,373(lll), 392 Osoba, D.,55, 109 Ospeck, A. G.,420, 437 Osserman, E.F.,180,183,245 Osserman, K. E.,59, 113, 382( 171, 172, 173),383( 180),394 Osterland, C. K., 96,103, 323, 329, 330, 340, 345, 347, 362(54, 55), 363 (55),368(55), 390 Oswald, E., 179, 239 Ottinger, B.,47, 49, 101 Oudin, J., 305, 323, 326, 327, 343, 347, 361, 390 Ovary, Z., 288, 309, 311, 312, 313, 321, 334, 336, 342, 343, 344, 347, 348 Owen, R. D., 46, 47, 87, 109 Owen, S. G.,378(145), 393 Owens, C. T., 220, 238 Oyama, J., 47, 103

401

P Pachter, M. R., 231, 244 Packrnan, L., 153, 169 Page, A. R., 39, 59, 109, 114 Page, E. W.,221, 242 Page, R. H.,24, 113 Pagel, W.,384( 191), 394 Paghs, J., 120, 168 Paige, B. H.,34, 109 Paillot, A., 64,109 Pain, R., 297,301, 313, 314,329,344 Pain, R. H., 289, 298, 347 Paine, J. R., 376(121), 392 Pakesch, F., 176, 179, 230, 231, 238, 239 Palade, G. E., 177, 190, 202, 203, 243, 244 Palmer, D. L., 154,169,412,435 Palmer, J. L.,82,97,295,302,347 Palmstiema, H.,423, 432 Papermaster, B. W.,30, 31, 32, 45, 49, 51,52, 56,57,72, 73, 74,76, 77,78, 79, 82, 83, 85, 88, 89, 96, 101, 102, 109, 219, 247 Pappas, G. D., 190, 201, 221, 238, 244, 245 Pappenheimer, A. M.,289, 347 Pappenheimer, A. M.,Jr., 403, 410, 437, 440 Parakevas, F., 179, 244 Parfentjev, I. A., 420,428, 437 Parker, R. H.,379( 150), 393 Parks, H.F.,190, 244 Paronetto, F., 213, 215, 216, 243, 373 ( 108), 392, 399, 437 Parrott, D.M.V., 51,52,54,109,110 Parrott, R. H.,388(222),395 Parsons, D.F.,177,181,245 Pasteur, L., 2, 110 Paterson, P. Y., 221, 232, 245, 398, 400, 401,404,408,410,411,432,437 Patnode, R. A., 151,169 Patterson, R., 7, 110 Paul, W. E.,213, 245 Paulhg, L.,2, 110 Pavillard, E.R. J., 134,137,171 Peacock, S., 153, 169 Pearmain, G.,234, 245 Pearse, A.G.E., 134,169

462

AUTHOR INDEX

Pearson, G. R., 130, 171 Pearson, H. A., 5, 112 Pease, G . L., 218, 243 Pedersen, K.O., 8,110,288,345 Peer, L. A., 5, 110 Pekin, T. J., 370(89), 391 Peller, H., 191, 242 Pembroke, R. H., Jr., 13,106 Pencea, I., 404,410,433 Pepe, F. A,, 198, 245 Perez-Tamayo, R., 408, 435 Perkins, E. H., 125, 171 Perkins, F. T., 12, 110 Perkins, H. R., 251n., 277, 282, 283, 284, 285 Perlmann, G., 254, 285 Perlmann, P., 195, 238, 388(229, 231), 389 ( 233), 395 Pemis, B., 183, 184, 240, 399, 419, 434, 437, 440 Perramant, M. F., 334, 346 Perrault, A., 120, 170 Perrine, T. D., 411,438 Perry, H. O., 385(202), 395 Person, N. G., 12,23,114 Pert, J, H., 81,101 Peters, J. H., 218, 241 Peterson, R. D. A., 30, 31, 59, 102, 110, 203, 219, 245, 247 Peterson, W. J., 34,45, 48, 106 Petrenco, H., 403, 440 Petroff, S. A., 37, 110 PfeifTer, R., 3, 110 Phillips, J. H., 85, 87, 110 Phillips, P. H., 7, 103 Philipson, L., 9, 110 Philipsson, L., 51, 101 Pickett, M. J., 129, 142, 146, I68 Pickles, M. M., 5, 110 Piel, C., 222, 245 Piernme, T. E., 383(182), 394 Pierce, A. E., 7, 110 Pierce, C. H., 142, 170 Pierce, J. C., 32, 51, 52, 96, 102 Pih, K., 21, 99 Pillemer, L., 412, 435 Pinoy, P. E., 401,436 Pinto, L., 378( 144), 393 Pirani, C., 216,218,241,245

Pirie, N. W., 400,436 Pisani, T. M., 401,403,405, 434 Pittman, M., 420, 421, 422, 437 Pizzarello, D. J., 87, 110 Plagge, E., 89, 110 Plantin, L. O., 339,342 Plescia, 0. J.. 337, 347,382( 172), 394 Pliszka, F., 73, 110 Podliachouk, L., 325, 347 Polcack, J., 388(230), 395 Policard, A., 176, 188, 190,205,245 Polk, A., 19, 110 Pollack, A. O., 384( 188), 394 Pollak, V. E., 218, 218, 219, 241, 243, 245, 384(200), 395 Pomales-Lebrh, A., 129, 142, 145, 158, 168, 171, 172 Poncher, H. G., 33,102 Ponder, E., 199, 245 Pope, C. G., 398,434 Popper, H., 182, 246 Porcile, V., 33, 110 Porte, A., 177, 179,185,188,212,241 Porter, D., 181, 245 Porter, K,A., 35,110 Porter, K. R., 201, 245 Porter, R. R., 85, 98, 110, 289, 295, 298, 297, 298, 300, 301, 304, 307, 308, 310, 313, 314, 318, 317, 318, 325, 329,333,342,343,344,347 Potter, E. V., 123, 171 Potter, M., 178, 177, 179,240,245 Podik, M. D., 297, 298, 309, 318, 325, 343, 347 Pound, A. W., 405,437 Powell, A., 198,230,238 Pratt, G. T., 177, 181,245 Pregennans, S., 205, 245 Prehn, R. T., 49, 93, 94, 106, 110, 182, 167, 173 Prescott, B., 47, 49, 101 Press, E. M., 98, 110, 295, 298, 305, 307, 308, 310, 343, 344, 347 Pressman, D., 216, 222, 243, 288, 291, 293, 313, 338, 345, 346, 347, 349 Prochazka, O., 417, 437 Pruzansky, J., 400, 432 Puck, T. T., 118,171

AUTHOR INDEX

Pullinger, E. J., 153, 171 Pdvertaft, R. J. V., 377(134), 379(153), 393 Pund, E. R.,33,110 Pusztai, S., 421, 428, 433 Putnam, F. W., 294, 295, 296, 316, 321, 346, 347

R Radzimski, G., 291,313,345,347 Raffel, S., 402, 404, 437 RajBhandary, U. L., 271,281,283,285 Rallison, M. L., 216, 245 Ralston, D. J., 135, 171 Ramel, A., 297, 347 Ramon, G., 19, 109, 398, 399, 401, 413, 428, 429, 438 Ramon, P., 12, 110 Ramos, A. J., 59, 110 Ramsdell, S. G., 70, 110 Ramseier, R., 135, 136, 143, 144, 152, 162, 171 Randall, E., 278, 285 Rantz, L. A., 278, 285 Rao, M. A., 50,108 Rapacz, J., 326, 343 Rawles, M. E., 35, 36, 110, 115 Rebers, P. A., 258,282,283,285 Rebuck, J. W., 199,245 Recant, L., 219, 242 Record, B. R., 265,285,288,345 Rees, R. J. W., 38,110, 143,169 Rees, T. A., 82,100 Reger, J. F., 216,239 Regnier, C., 218,219,238,245,246 Reid, R. T. W., 218,222,241,245 Reimer, E., 179, 239 Reisman, R. E., 288, 336, 349 Reiss, A. M., 337, 347 Remington, J. S., 332, 346, 347, 381 (169), 394 Repaske, R., 135, 171 Retterer, E., 30, 110 Reyniers, J. A,, 18,113 Rezaian, J., 384(200), 395 Rheins, M. S., 27,50,99 Rhoads, J., 50, 104 Ribadeau-Dumas, L., 23, 110 Ribble, J. C., 151, 171

463

Ribi, E., 408, 411, 412, 413, 423, 434, 435, 436, 437, 438 Rice, E. C., 13,109,400,429,438 Rich, A. R., 234,245 Richards, C. B., 331, 346 Richou, R., 12, 110, 398, 401, 413, 429, 438 Ricken, D., 59, 97, 383( 174)) 394 Ridley, A., 377(125), 392 Rieder, R. F., 327, 347 m i n d , R. A., 180, 183, 203, 222, 223, 225, 237, 245 Riha, I., 13, 20, 36, 43, 101, 105, 110, 112, 114, 412, 439 Ritter, D. B., 411, 413, 438 Rivat, L., 328, 347 Rivers, T. M., 404, 438 Robbins, W. C., 201,245, 355(17), 389 Roberts, M. E., 420, 437 Roberts, W. K., 282, 285 Robertson, D. M., 207, 245 Robertson, J. D., 75, 110 Robertson, M., 47, 106 Robineaw, J., 205, 245 Robinson, A. C., 339, 344 Roboz, E., 405, 435 Robson, J. S., 219, 220, 243, 246 Rockey, J. H., 6, 12, 111, 288, 346 Rodgers, W. L., 39, 105 Rodnan, G. P., 369(84), 384( 196), 391, 394 Rotstein, J., 15, 59, 102, 111, 372(103), 392 Roffler, S. K., 417, 428, 435 Roger, S., 179, 241 Rogers, D. E., 127, 128,170,171 Rogers, H. J., 251n., 272, 273, 277, 282, 283, 284, 285 Rogers, J. B., 90, 111 Roholt, O., 313, 347 Roitt, I. M., 376, 377(123, 126, 130, 132, 134, 135), 378(137, 140, 141, 142, 146)) 379(130, 135, 151, 153)) 381 (165, 167), 392, 393, 394, 408, 409, 435 Romanowski, W., 199, 246 Romer, A. S., 75, 76, 79, 111 Roosa, R. A., 32,57,100, 111 Ropartz, G., 328, 347, 360(44), 390

464

AUTHOR INDEX

Rose, B., 331,332,349, 399,419,438 Rose, N. R., 376(118, 119. 121), 377 (124, 127, 129), 386(214), 387 (215),392, 305 Rose, R. L.,383(181),394 Rose, S. R., 152, 170 Rosen, F. S., 58,111, 179,240, 330, 333, 344, 347

Rosenau, W., 163, 164, 171 Rosevear, J. W., 307, 348 Ross, J., 353( 10, 13), 355( 13), 389 Ross, M. H.,221, 244 Ross, S., 13, 109 Rossanda, M.,220, 246 Roth, I. L., 188, 246 Roth, N.,8, 17, 26, 111 Rothbard, S., 229, 246 Rothenberg, M. S., 209, 211, 222, 237, 246, 247 Rous, P., 133, 171

Rowe, D.S., 331, 332,348 Rowell, N. R., 384( 197, 198), 394 Rowlands, D.T., Jr., 22, 32, 106 Rowley, D.,119, 125, 128,154,169, 170, 172, 173, 228,238 Roy, J. H. B., 7,97 Rozansky, R., 9, 10, 111 Rubenstein, H.S., 412, 438 Rubin, B. A., 48, 111 Rubin, H.,93, 111 Rubin, M. I., 222,227, 238 Rudd, E.,366(74), 391 Rumpianesi, G.,27, 111 Rupp, J. C., 408, 438 Rusan, S., 404, 410, 433 Ruska, H.,190, 244 Russell, E. F., 19,99 Ruth, R. F., 29, 111 Rychlikovh, M.,42, 112, 418,439 Rydon, H.N., 266, 283 Ryser, H.,191, 248 Rytel, M. W., 123, 124,172 Rywosch, M.,18, 111

S Sabesin, S. M., 205, 206, 246 Sabin, A. S.,24, 111 Sabin, F. R., 26, 28, 111 Sabour, M. S., 220, 246

Sabrazks, 72, 111 Saenz, A., 19, 114, 401,438 Sainte-Marie, G.,53, 111 Saito, K., 128, 146, 150, 153, 158, 159, 160, 171, 172, 173 Sakaguchi, H., 222, 246 Sako, W.,23, 111 Salk, J., 407, 438 Salkind, J., 90,111 Salteri, F., 365(70), 391 Salton, M. R. J., 250, 251 n., 252, 256, 285

Salvin, S. B., 39, 111, 401,402,403,410, 432, 438, 440

Samour, D., 406, 440 Samuels, B. D., 4, 5, 100 Sanderson, A. R., 270,271,273,274,283, 284, 285, 286

Sandstrom, C.J., 34, 111 Sanger, F.,314, 344 Sargent, L. J., 277, 286 Sasena, K. M., 378(145),393 Sass-Koxtsak, A., 13, 109 Sato, I., 128,146,150,153,171, 172, 173 Saukkonen, J. J., 273,274, 286 Saxer, F.,26, 28, 111 Sbarra, A. J., 124,130,131,172,191,246 Scadron, S. J., 11, 99 Scalabrino, R., 220, 246 Schachman, H.K., 297, 347 Schaedler, R. W., 154, 168 Schar, B., 120, 170 Schaffner, F.,182, 246 Scharrer, B., 94, 111 Schatz, D.L., 388(224),395,408,439 Schayer, R. W., 424, 425,438 Schechtman, A. M., 7, 115 Scheidegger, J. J., 6, 8, 16,98, 111, 330, 344

Schereschewskaja, N. I., 399, 435 Schinckel, P. G., 35, 111 Schlaff, S., 291, 343 Schlange, H.,40, 111 Schlossman, S., 124, 167 Schlumberger, H.G., 67, 111 Schmidt, G. A., 34, 114 Schmidt, S., 398, 438 Schmidt, W.C., 256, 286 Schneegans, G.,10, 111

AUTHOR INDEX

Schneider, H. A., 400,407, 410, 436, 438 Schneider, P.,146, 148, 153,168, 169 Schoefl, G. I,, 202, 203, 243 Schoen, E. J., 5, 111 Schoenberg, M. D.,191,246, 408, 438 Schofield, R., 191, 247 Schooley, J. C.,50, 111 Schramm, G.,196, 237 Schroeder, M. L., 428, 438 Schrohenloher, R. E.,296, 348 Schryver, E. M.,409, 434 Schubert, W.K.,38,47, 101 Schuchardt, L. F., 420, 421, 423, 424,

465

Shapland, C., 381( 165),393 Sharpe, M. E.,288, 286 Shaw, C.,275, 286 Shaw, C. M.,400, 404, 407, 408, 410,

422, 429, 439

Shayegani, M. G.,127, 170, 172 Shear, J., 412, 435 Shear, M. J., 120, 170 Shedlovsky, T.,261, 282, 283 Shepel, M.,403, 408,407,439 Sherman, J. D.,32, 57, 112 Sherman, W.B., 12, 112, 335, 348 Sherwood, N. P.,403, 436 Shillam, K.W.G., 7, 97 425, 426, 437, 439 Schultz, M.P.,398,428,429,438,439 Shinefield, H.R., 24, 112 Schultze, H. E.,6, 9, 18, 104, 106, 287, Ship, I. I., 372(101),392 Shirley, W.,191, 246 288, 293, 307, 345, 348 Schumaker, V. N., 191,246 Shoemaker, A., 142, 170 Schur, P. H.,337, 348 Shulman, N. R.,5,112,230, 246 Schwartz, E. R., 384(61), 390 Shulman, S., 377(127, 128), 392 Schwartz, J. H.,295,330,348 Sibal, L. R., 42, 112, 414,415,417, 438, Schwartz, M.,380(158, 160),393 439 Schwartz, R., 32,49,57,111, 112 Sicard, 73, 115 Schweinberg, H.,420,421,422,424,438 Sick, T.,18, 17, 114 Schwick, G.,16,106, 288, 348 Siege], M.,216,222,243,414,434 Sebestyen, M. M.,124, 151, 167 Siekevitz, P.,177, 244 Sedgwick, R. P.,59, 98 Sigel, M. M.,77, 79,99, 112 Seegal, B. C.,59, 113,203,209,211, 217, Silman, H.I., 296,297,343, 348 222, 223, 225, 227, 237, 242, 246, SilobrW, V., 48, 108,109 Silverman, J. J., 11, 115 247, 382(173), 394 Seelig, A., 73, 102 Silverman, M. S., 135, 169 Silvers, W.K.,5,98,182,167 Segal, W.,156, 272 Segre, D.,13, 14, 20, 111 Silverstein, A. M.,21,34, 112 Sehon, A. H.,12,111,331,332,349 Simer, P. H.,222, 246 Simmel, H.,19, 102 Seifter, J., 50, 100 Seijen, H, G.,294, 348 Simms, E. S., 178, 182,240 Seki, Y., 135, 167 Simon, H.J., 366(73), 391 Sela, M.,294,314, 344 Simons, K.,384(84), 390 Seligmann, M.,352(3), 353(3),355,374 Simons, P. J,, 47, 103 Simonsen, M.,48,48,99,105, 112 (31,389 Selikowa, R. E., 399, 435 Simonton, L. A., 357(27), 390 Sell, S., 412, 438 Simpson, J. A,, 382( 170), 383( 179), 394 Sengson, B. L., 179, 244 Simpson, L. O.,48, 115 Sercan, E.,49, 111, 178,246 Singer, S. J., 314, 315, 346 Sever, J. L,,158, 157, 158, 172 Sirotinin, N. N.,3, 72, 112 Shabarova, 2. A., 282, 286 Sitte, H.,221, 222,238,244, 246 Shaffer, J. H.,398,438 Sjodin, K.,46, 52, 53, 54, 55, 100, 104, Shands, J. W.,Jr., 125,172 115 Shapiro, F.,2, 48,51, 107, 112 Sjogren, H. O., 93, 106

488

AUTHOR INDEX

Skane, B., 378(143), 393 Skarnes, R. C.,3,91,112, 136, 172 Skinner, D.,5, 112 Skowron-Cendrzak, A., 48, 112 Skurski, A., 123, 129, 172 Skvdil, F.,298,348 Slater, R. J., 149,173,218,244, 246, 325,

348 Slater, R. S., 8, 112 Slayter, N. S., 197,241, 290, 348 Sleisenger, M. H.,380(162), 381(l62),

382( l62), 393 Slocumb, C.H.,371(94),391 Slopek, S., 123, 172 Sloper, J. C.,387(220), 395 Small, P. A,, 289, 299, 300,348 Smiddy, F. G.,416,439 Smith, E. L.,289,291,303,307,346,348 Smith, H.,285,288, 286 Smith, H.W.,149, 172 Smith, J. M.,2,48,49,107, 112 Smith, N., 129, 170 Smith, P. N.,222, 237 Smith, R. F.,39, 111,403,410,438 Smith, R. T.,17, 21, 25, 26, 40, 47, 48,

49, 112, 114

Sorkin, E., 20, 112, 181, 168, 358(32),

390 Soulier, J. P., 186, 238 Sparck, J. V., 42, 112 Spargo, B., 216, 218, 221, 222, 228, 237,

238, 246, 248 Spector, W. G.,121, 172, 403, 432 Speidel, C. C.,88, 105 Speirs, R. S., 192, 246 Spiegelberg, H.L.,125, 172 Spink, W.W.,412,419,433,440 Spiro, D.,213,218,232,246,247 Sprague, L.,78, 99

Sprick,

M. G., 133, 172

Sprunt, D.M.,404, 438 Staab, E. V., 413,433,439 Stiihelin, H.,130, 140, 143, 150,172 Stambaugh, M. K.,293, 346 Stanley, J. L., 265, 286 Stanley, N. F.,398, 439 Stanley, W.M.,198, 237 Stanworth, D.R., 98,101, 321, 329, 330,

336, 344, 348 Stary, Z.,291, 344 Stader, R., 328, 348 Stavitsky, A. B., 20, 112, 416, 439 Steblay, R. W., 222, 227,229,246, 248 Steenberg, E.,398, 438 Stefanini, M.,5, 112 Steigman, A. J., 404, 411,429,436,439 Stein, S., 325, 348 Stein, W. H.,136, 168 Steinbeg A. G., 328, 348, 360(41, 43,

Smith, S. W., 218, 239 Smith, T.,7, 112 Smith, W., 73, 112 Smith, W. W., 72, 102 Smithies, O.,318, 341, 348 Smolens, J., 408, 434 Snell, E. E.,277, 284 45), 361(51), 390 Snell, G. D.,182, 172 Steiner, J. W.,206, 218, 222, 242, 244, Soderberg, R., 403, 436 246, 388(224), 395, 408, 439 Sokuma, T.,218, 243 Solomon, A., 179, 182, 246, 320, 339, Steiner, R. F.,290, 243, 348 Stella, J. A., 222,243 344, 348 Solomon, I. L.,377( 135),379( 135,147), Stellwagen, E.,297, 347 Stemke, G. W., 308, 327, 348 393 Stem, E. R., 401, 434 Solotorowsky, M.,426, 439 Sterzl, J., 13, 20, 36, 38, 42,43, 45, 101, Sommer, H. E.,401,403,405,434 112, 123,172,412,418,435,439 Sommers, S. C.,59, 108 Stetson, C. A,, 165, 167, 172, 412, 419, Sonkin, L. S., 379(155), 393 439 Soons, J. B. J., 339, 348 Stevens, K. M.,415,416,436, 439 Soothill, J. F.,331, 338, 348 Stevens, W.H.,399, 434 Sorel, R., 218, 246 Sorenson, G. D.,181,212,213, 214, 242, Stevenson, C.T.,320, 331, 332,348 Stevenson, H. N.,34, 113 246

AUTHOR INDEX

Stewart, C. R., 373( lll),392 Stewart, F. W., 37, 110 Stewart, W. A., 254, 286 Stieda, L., 29, 113 Stiffel, C., 91, 98, 123, 124, 125, 152, 154, 167, 168, 169, 171 Stinebring, W. R., 129, 142, 146, 156, 168, 171, 172 Stitt, J. M., 275, 286 Stobbe, H., 177, 247 Stockard, C. R., 90, 113 Stockell, A., 291, 303, 348 Stoeckel, E., 186, 241 Stoeckenius, W., 177, 247 Stoerk, H. C., 402, 408, 409,433 Stokes, J., Jr., 24, 50, 104, 106 Stollar, D., 356(21, 22), 389 Stollerman, G. H., 123, 124,171, 172 Stone, H. W., 398, 428, 432 Stone, J. D., 18, 47, 98 Stone, S. H., 403, 405, 433, 436 Stoner, H . B., 383(187), 394 Stoner, R., 51, 104 Stoner, R. D., 399, 419, 439 Stormont, C., 77, 99 Striissle, R., 352(2), 356( 18), 389 Strange, R. E., 265, 286 Straus, H. W., 40, 113 Strauss, A. J. L., 59, 113, 382(171, 173), 394 Strominger, J. L., 271,272, 273,274, 275, 283,284,285,286 Strong, J. P., 213, 241 Strunk, S. W., 219, 247 Stuart, A. E., 379( 152), 393 Stulberg, C. S., 24, 113 Sturgeon, P., 33, 113 Sturm, E., 399, 437 Stiittgen, G., 206, 244 Sugahara, T., 336, 337, 345 Sugiyama, T., 128, 146, 173 Sulitzeanu, D., 154, 172 Sumita, Y., 216, 242 Summerskill, W. H . J., 383(183), 394 Sundberg, R. D., 92, 113, 177, 247 Surgenor, D. M., 124, 173 Suter, E., 3, 113, 119, 123, 125, 129, 130, 134, 135, 136, 140, 142, 143, 144, 145, 148, 149, 150, 152, 155, 157, 158, 159, 169, 171, 172, 173

467

Sutherland, D. E. R., 31, 33, 56, 96, 113 Sutherland, I . W., 423, 439 Suzuki, Y., 222, 246 Svennerholm, L., 13, 108 Swahn, R., 362(53), 390 Sweet, L. K., 39, 96 Swift, H. F., 254, 286, 398, 428, 429, 438, 439 Syverton, J. T., 93, 105 Szenberg, A., 30, 31, 56, 86, 92, 113, 114 Szentivanyi, A,, 425, 433 Szongott, H., 264, 286

T Taft, L. I., 216, 247 Takaishi, K., 423, 440 Taliaferro, L. G., 399, 417, 439 Taliaferro, W. H., 118, 173, 339, 348, 399, 400, 417, 419, 435, 439 Talmage, D. W., 2, 65, 113, 339, 348, 408, 409, 417, 425, 433, 439 Tan, E. M.,353( 13), 355( 13), 365(67), 375( 117), 378( 17), 389, 391, 392 Tanaka, S., 423, 440 Tanaka, T., 128, 146, 149, 153, 171, 172 173 Tanaka, Y., 185, 239 Tanford, C., 289, 291, 300, 301, 302, 342, 346 Taranta, A., 336, 337, 348 Tarkhanova, I. A., 297, 346 Tarr, H. L. A., 63, 113 Taylor, H. E., 163, 173 Taylor, K. B., 378(146), 380(156, 157, 159), 381(165, 167), 393, 394 Tempelis, C., 18, 19, 47, 113, 115 Tennyson, V. M., 190, 244 Terasaki, P. I., 35, 92, 98, 113 Terplan, K. L., 222, 227, 239, 376( 121), 392 Terres, G., 47, 113, 399, 419, 439 Terry, R. D., 232, 247 Terry, R. J., 7, 9, 97 Thal, A. P., 386(208,209,210,211), 395 Thiele, E. H., 425, 439 Thiery, J. P., 176, 185, 186, 187, 192, 212, 238, 247, 398, 413, 429, 438 Thomas, L., 94, 113, 188, 221, 244, 247, 411, 412, 417, 419, 439, 440

468

AUTHOR INDEX

Thomason, D., 191, 247 Thomison, J. B., 127, 128, 170 Thompson, C. M., 121, 167 Thompson, D., 388(232), 395 Thompson, G., 399, 419, 434 Thompson, G. E., 401, 434 Thompson, H. L., 398, 439 Thompson, K. J., 401, 403, 405, 434 Thomssen, R., 398, 407, 434 Thorbecke, G. J., 18, 33, 84, 91, 113, 124, 134, 173, 178, 247, 321, 348, 417, 418, 435 Thornburg, W., 176, 242 Thorne, C. B., 282, 286 Tillett, W. S., 149, 173, 281, 282, 286 Timbury, G. C., 387(218), 395 Timmerman, W. A., 12, 113 Tinker, M., 429, 440 Tips, R. L., 403, 432 Tiselius, A., 5, 113, 288, 348 Tobler, R., 58, 113 Todd, C. W., 305, 309, 330, 331, 348 Tokuda, A., 188, 242 Tokuda, S., 422, 427, 440 Tomasi, T. B., 338, 343, 348, 365(88, 89),371(95), 391 Tomcsik, J., 264, 265, 288, 287, 282, 284, 286

Tompsett, R., 127, 171 Toone, E. C., 389(81), 391 Topley, W. W. C., 149, 173 Toumanoff, K., 84, 113 Townes, A. S., 373( l l l ) ,392 Townsend, J. I., 328, 349 Townsend, T. E., 24, 112 Toy, B. L., 8, 97, 220, 238, 384(194), 395( 194), 394 Trainin, N., 55, 106 Trapani, R. J., 120, 170, 411, 413, 438 Traub, E., 47, 113 Treip, C. S., 384( 191), 394 Trentin, J. J., 49, 113 Treuting, W. L., 23, 111 Trevorrow, V. E., 18, 113 Triedman, R. S., 222, 247 Triplett, E. L., 65, 67, 70, 88, 93, 113 Tmka, Z.,20, 42, 43, 112, 113, 114 Trombley, P., 386(71), 391 Trotter, W. R., 377( 122), 392

Truant, J. P., 231, 244 Tryczynski, E. W., 412, 440 Tudvad, F., 39, 114 TuiTanelll, D. L., 384( 193), 394 Tullis, J. L., 124, 173 Tumanova, A. E., 313, 345 T’ung, T., 284, 286 Turk, J. L., 399, 435 Tumer, J. E., 291, 344 Tumer, K. J., 125, 173 TveterAs, E., 9, 110 Tymms, P. W., 200, 230, 240

U Udaka, K., 188, 242 Uhr, J. W., 12, 13, 17, 21, 25, 28, 38, 40, 82, 85, 112, 114, 188, 247, 403, 410, 440 Unanue, E., 219, 229, 247 Ungar, G., 188, 247 Ungar, J., 421, 440 Unno, G., 177, 237 Uriuhara, T., 203, 206, 244 Urquhart, A., 222, 247 Urso, P., 3, 81, 114, 177, 247 Ushiba, D., 128, 148, 149, 158, 159, 180, 170, 172, 173

Utsumi, S., 310, 312, 349 Utsunomiya, S., 218, 220, 237

V Vaerman, C., 288, 338, 345, 349 Vaerman, J. P., 8, 12, 104, 288, 335, 338, 345, 349

Vahlquist, B., 11, 12, 23, 114 Vainio, T., 274, 286 Valentine, R. C., 290, 349 Valtis, J., 19, 37, 114 Vandenbroucke, J., 5, 114 Van der Geld, H., 383(175, 176, 177), 394

Van der Scheer, J., 288, 349 van Leuwen, G., 325, 346 van Loghem, J. J., 383(177), 394 Vannier, W. E., 6, 12, 101, 104, 288, 335, 344

Van Vunakis, H., 356(22), 389 Varco, R. L., 15, 39, 50, 87,102, 105,106, 107

AUTHOR INDEX

Vaughan, J. H., 359(35), 364(63), 369 (35,82), 371(97), 373(109), 390, 391, 392 Vaughn, R. B., 126, 173 Vazquez, J. j.,3, 5, 61, 97, 106, 114, 182, 188, 209, 213, 215, 222, 227, 240, 241, 247, 373( 108), 384( 196), 392, 394, 418, 430, 440 Vernier, R. L., 216, 218, 219, 222, 237, 240, 245,247 Verstraete, M., 5, 112 Verwey, W. F., 424, 426,437 Vial, J. D., 232, 247 Vickrey, H. M., 158, 169 Victor, J., 153, 171 Vigliani, E. C., 399, 440 Villa, L., 365(70), 391 Virion, M. E., 420, 437 Vischer, W. A., 148, 173 Vivell, O., 16, 17, 114 Vogt, A., 213,215, 222,247 Vogt, M., 70, 114 Voisin, C., 205,247 Vojtiikovii, M., 34,47, 104 Vokurka, V., 388(230), 395 Volpe, R., 388(224), 395 von Muralt, G., 10, 11, 13, 16, 26, 103, 111, 114 von Piquet, C., 2, 114 Vorlaender, K. O., 353( l o ) , 389 Vugman, I., 427, 437

W Wachstein, M., 222, 243 Wada, H., 218,219, 242, 243 Waddams, A., 377( 122), 392 Waddington, C. H., 34, 114 Waddington, H., 398, 434 Wagner, M., 18, 113, 412, 440 Wagner, R. R., 416, 434, 435 Wake, R. G., 302, 343 Waksman, B., 221, 241 Waksman, B. H., 32, 38, 39,57, 96, 105, 114, 232, 234, 247, 398, 401, 405, 440

Waldenstrom, J., 59, 115, 179, 244 Waldmann, T. A., 338, 339, 348, 349 Walker, B. L., 222, 237 Walker, R. C., 388(232), 395

469

Walker, W. G., 216, 218,248 Wallace, J. H., 130, 168 Wallace, U., 398, 434 Waller, M.,328, 349, 367(77), 369(81), 301 Wallis, R. G., 265, 285 Walter, A. W., 414, 434 Walter, H., 191, 242 Walter, P. C., 131, 168 Wannamaker, L. W., 11, 114 Ward, H. K., 123, 173 Ward, J. R., 370(91), 391 Ward, P. A., 417,418,440 Ward, S. M., 325, 348 Wardlaw, A. C., 123, 125,169, 173 Warner, L., 124, 170, 196, 230, 238 Warner, N. L., 30, 31, 56, 86, 92, 113, 114 Warwick, W. J., 38, 39, 40, 114 Watanabe, K., 218, 243 Watson, D. H., 197,247,248 Watson, D. W., 3, 14, 21, 30, 45, 48, 51, 91, 105, 106, 109, 112, 136, 172, 412, 433 Watson, I., 219, 229, 247 Watson, R. F., 201, 229, 245, 246 Weaver, J. M.,162, 187, 173 Webb, T., 331, 332, 349 Webster, H. deF., 232, 247 Weigand, H., 47, 105 Weigle, W. O., 7, 20, 41, 42, 43, 45, 100, 110, 121, 168, 205, 239, 400, 402, 440 Weil, M. H., 412, 440 Weinberg, M., 18, 114 Weiner, D., 87, 107 Weir, D. M., 371(96), 391 Weir, R., 292, 303, 305, 307, 310, 349 Weiser, R. S., 135, 151, 171, 422, 427, 440 Weiser, W. J., 203, 206, 244 Weiss, D. W., 38, 47, 114 Weiss, L., 212, 239 Weiss, L. P., 134, 173 Weissmann, G., 139, 169, 173, 188, 247, 374(115), 392, 419, 440 Welker, W. H., 398, 434 Wellensiek, H. J., 177, 192, 247 Weller, E. M., 7, 115

470

AUTHOR INDEX

Williams, R. P., 188,246 Williamson, J. R., 202, 248 Willier, B. H., 35, 115 Willkens, R. F., 372(100), 391 Willoughby, D.A., 121, 172 Wilson, A., 383(187), 394 Wilson, A. T., 254,256,280, 286 Wilson, D.,32, 57, 111 Wilson, D.B., 162, 163, 167,173 Wilson, D.R., 177,244 Wilson, G.S., 399,412,430,440 Wilson, J. A,, 328, 348, 361 (51),390 Wilson, J. B., 142, 168 Wilson, S., 222,227, 228,242 Winemiller, R., 221, 222, 246, 248 Winkelmann, R. K., 384(193), 394 W i d e r , M.,290,291, 349 435, 436 Winn, H.J., 182,172, 173 White, R. G., 54, 59, 108, 115, 176,227, Winquist, G.,206, 248 248, 288, 349, 378(136), 379( 154), Winsten, S., 426, 439 383(178), 393, 394, 399, 403, 405, Winter, A. R., 27, 50,99 406,408,409,432,440 Wischnewezkaja, L. J., 27,28, 32,115 Whitney, P. L., 291, 342 Wissig, S. L., 190, 248 Wicht, W. C., 406, 435 Wider, F. C., 96,109. 296,346 Wicken, A. J., 278, 279,284, 286 Wider, R. W., 418, 440 Widal, 73, 115 Witebsky, E.,59, 97, 359(37), 364(37), Wieczorek, Z.,129, 172 376, 377(127, 128, 129, 133), 378 Wiedermann, G.,336, 349 (133), 383(174), 386(214), 387 Wieghard, C.W., 270,275, 284, 286 (215), 388(225), 390, 392, 393, Wieme, R., 6, 98 394, 395, 401, 440 Wieneke, A. A,, 139, 173 Witt, D. B., 23, 111 Wiener, A. S., 11, 115 Wochner, R. D., 338, 339, 349 Wiener, E.,138, 168 Woerdeman, M. W., 4,115 Wiener, S. L., 429, 440 Woedey, D. L., 296, 346 Wigzell, H.,48, 115 Wofsy, L., 314, 346 Wildy, P., 197,247, 248 Wohl, M. J., 372( 101),392 Wiley, G.G., 280, 286 Wolf, A., 34, 109 Wilkinson, J. M., 291,292, 298, 303, 343 Wolf, J., 370(90), 391 I Wilkinson, P. C., 288, 349 Wolf, J. K., 59, 115 Wilkinson, R. D., 385(204), 395 Wolf, J. L., 373(105),392 Williams, A. W., 381( 164), 393 Wolfe, H.R., 18, 19, 27, 30, 47, 51,56, Williams, C.A., 5,103,287,344 108, 113, 115 Williams, E., 378(136), 393 Wolins, W., 399,419, 439 Williams, J. W., 289, 347 Wollheim, F.,59, 115 Williams, L. H., 47,97 Wollman, E.,73, 115 Williams, R. C., 323, 325, 346, 359(39), Wolsky, A., 87, 110 363(57,59),364(39,62),365(39), Wong, S. C., 284, 286 386(72), 368(59), 389(39), 385 Wood, C., 227, 248 (203),3eo, 391, 395 Wood, H.F., 149, 173

Wells, A. Q., 129, 170 Wells, H. G., 387(219), 394’ Wells, P. A., 357(27), 390 Welsh, R. A,, 177, 186,247 Wenckebach, K. F., 29, 115 Wenk, E. J., 404, 422,436 Wennersten, C.,32, 57, 96 Werder, A. A., 48,103,403,436 West, C. D., 16, 38,47, 101, 115 Westenbrink, H. G. K., 339, 348 Westendorp-Boerma, F.,6, 98 Westphal, O., 411, 423, 440 Wetzel, G.,32, 115 Wexler, B. C., 412, 440 Wheeler, A. H.,385(206),395, 403, 440 Whipple, A., 338, 344 Whitby, J. L., 124, 128, 171, 173, 412,

AUTHOR INDEX

Wood, W. B., Jr., 126, 173 Woodin, A. M., 138, 139, 151, 173 Woodruff, M. F. A., 36,47,107,115 Woods, K. R., 70, 77, 81, 82,101,115 Woods, M. W., 412, 435 Wooles, W. R., 166, 173 Work, E., 251n., 286 Worthen, H. G., 218,219, 247 Wostman, B., 18, 113 Wostmann, B. S., 18, 115 Wozniczko, G.,367(78), 391 Wright, K. A., 399, 434 Wunderlich, J., 328, 349 Wunderly, C.,6, 98 Wust, C. J., 183, 248 Wyckoff, R. W. G., 288, 349

Y Yagi, Y.. 288, 336, 349 Yalow, R. S., 325, 342 Yamaguchi, Y., 149, 171 Yamamoto, N., 196, 237 Yamamoto, T., 188, 248 Yamauchi, H., 221, 242 Yamura, T., 188, 247 Yardley, B. J., 65, 110 Yardley, J. H., 216, 218,248 Yendt, E. R., 218, 246 Yetts, R., 12, 110 Yoffey, J. M., 50, 115 Yolac, A., 222, 238 Yoshida, A., 255, 275,285,286 Yoshida, N., 423, 440

471

Yoshikawa, T., 219, 242 Young, B., 412, 419, 437 Young, B. G., 65, 108 Young, G. O., 309,326,327,343 Young, J. Z., 75, 77, 115 Young, W. J., 325, 342 Youngner, J. S., 7, 110 Yunis, E.J., 55, 115 Z Zak, S. J., 8, 13, 14, 15, 20, 24, 25, 32, 33, 50, 98, 102, 106, 115, 413, 415, 416, 417, 419, 432 Zalusky, R., 381(169), 394 Zappasodi, P., 151, 167 Zavate, O., 404, 410, 433 Zellmer, C. E., 398, 428, 432 Zernoff, 64, 115 Ziff, M., 59, 60, 104, 228, 234, 242, 244, 364( 60), 369( 83), 390, 391 Zigelbaum, S., 338, 348 Zimmerman, M., 291, 344 Zinder, N. D., 313, 344 Zinneman, H. H., 179,239 Zoeller, C., 398,401,413,428,438 Zucker-Franklin, D., 138, 173, 179, 181, 248 Zuckerman, A., 278, 285 Zuelzer, W. W., 24, 39, 103,113 Zvaiiler, H. J., 371( 98), 391 Zvaifler, N. J., 370( 89), 391 Zwartouw, H. T., 265,266,286 Zweifach, B. W., 412,416,440

SUBJECT INDEX A Acid-fast bacteria, see Mymbacteria Adaptive immunity phylogeny and, 80-94 Addison's disease, autoantibodies and, 387-388 Adenine, nucleic acid antibodies and, 358 Adjuvants, Bordetella pertussis and, 420-428 definition of, 398 endotoxins of gram-negative bacteria and, 411-419 historical notes, 398-399 *cellaneous bacteria and* 428-429 mycobacterial, mode of action of, 407-411 nature of active material, 404-407 possible action Of2 429-431 Adult, cell transfer to, 45-46 Agammaglobulinemia, fetal, 14-18,24-25 rheumatoid arthritis and, 372-373 t h p u s and, 58-59 Allotypes, immunoglobulins and, 325-331 Amino acid( s), immunoglobulins and, 291-293,305, 306 Amyloidosis. pathology of, 212-215 Anaphylactic sensitivity, Bordetelh pertussia and, 421-423 Anaphylactoid purpura, pathology, ultrastructure and, 219 Anaphylaxis, invertebrates and, 70 pathology, ultrastructure and, 203 Anthrax bacillus, cell wall carbohydrates Of, 264-266,281 Antibodies, see also Immunoglobulins antinuclear, 352-357 characterization of, 81-84 fixation to skin, 334338 formation, ultrastructure and, 176-185 y-globulins and, 6 production, ontogeny of, 18-26 storage or release, ultrastructure and, 185-187 synthesis of, 339-341 transfer to fetus, 11-13,332-334

Antibody-combining site, immunoglobulins and, 309-318 Antibody-forming capacity, evolution in vertebrates, 74-81 Antibody response, Bordetella pertussis and, 421-423 Antigen( s), antibody production and 18-22 capsular, 281-282 diffusible, 281-282 endogenous, ultrastructure and, 193198 exogenous, ultrastructure and, 189-193 nonliving, invertebrates and, 64-68 vertebrates and, 72-74 polysaccharide, cell walls and, 255-269 streptococcal cell wall and, 252-254 Antigen-antibody complexes, circulating, 202-221 Antigen-antibody union,

389-4009

ultrastructure, in LlftrO, 198-201 in vivo, 201 Antigenicity, immunog'obulins and' 319-325 lower vertebrates and, 84-85 Anti-y-globulins, function of, 351-352 Anti-Gm factors, occurrence of, 380-362 Antimicrobial substances, intracellular kill of microorganisms and, 133-137 Antitoxins, immunoglobulins and, 288 Appendix, immunity and, 31, 58, 58 Arthus reaction, pathology of, 203-207 Autoantibodies, Addison's disease and, 387-388 dermatomyositis and, 384-385 diseases associated with, 388-389 lupus erythematosis and, 373-376 myasthenia gravis and, 382-384 pancreatic disease and, 388-387 pernicious anemia and, 380-382 physiogenic, 351 rheumatoid arthritis and, 388-373 scleroderma and, 384-385

472

SUBJECT INDEX

473

thyroiditis and, 376-380 C ulcerative colitis and, 388-389 Capsule, antigens of, 281-282 widely prevalent, 352-368 Carbohydrates, Autoimmunity, anthrax bacillus, 264-288 pathology, ultrastructure of, 221-233 bacilli cell wall and, 263-269 p-Azobenzenearsonic acid, immunoglobuimm~oglobdinsand, 305-308 lin composition and, 292-293 streptococcal cell wall, 256-261 p-Azophenyltrimethylammonium, immu- Cell( s 1, noglobulin composition and, 292-293 sensitized, ultrastructure of, 187-189 Cell transfer, immunologic development B and, 41-46 Bacilli, cell wall carbohydrates of, 263- Cellular ( delayed) sensitivity, 269 pathology, ultrastructure and, 233-235 Bacillus megatedum, cell wall carbo- Cell walls, hydrate of, 286-268,281 gram-positive, Bacillus subtilis, cell wall teichoic acids isolation and composition of, 250-252 of, 277 polysaccharide antigens of, 255-264 protein antigens of, 252-255 Bacteria, teichoic acid of, 269-277 acid-fast, see Mycobacteria Chemotaxis, homeostasis and, 119-122 antinuclear antibodies and, 357 Chloroquin, nucleic acid antibodies and, gram-negative, 356 cell walls of, 249-250 Chondosteans, immunity in, 79-81 endotoxins of, 411-419 gram-positive, cell wall antigens of, Chymotrypsin, immunoglobulins and, 294 Colostrum, antibodies and, 7, 8,13,20-21 250-277 miscellaneous, adjuvant effects of, 428- Complement, autoantibody to, 351 Complement fixation, immunoglobulins 429 and, 336-337 Bence-Tones proteins, immunoglobulins Corticosteroids, immunity and, 73 &d, 294i95, 319 Cortisone, alkaline phosphatase and, 7 Biliary cirrhosis, antibodies in, 354 Corynebactedum diphtheriae, Birds, lymphoid tissue in, 27, 29-31 cell wall, Blood, antigens of, 254-255 formed elements, autoimmunity and, carbohydrates of, 263-264 230-231 Cyclostomes, immunity in, 77-80 Blood cells, invertebrate, 68-69 Cytoplasm, antibodies to, 354,357-359 Body fluid, proteins, invertebrate, 70-71 D Bone and joint, autoimmunity and, 233 Defense mechanisms, nature of, 3-4 BorakteUa pertussis, Delayed allergic response, ontogeny of, as adjuvant, 37-41 characterization of active material, Deoxyribonucleic acid, antibodies to, 423-424 353-357,374 general biological effects, 420-423 Dermatomyositis, autoantibodies and, general remarks, 420 384-385 possible modes of action, 424-428 Detergents, immunoglobulins and, 290 Brucella abortus, infections, antibodies and, 288, 336 Diabetic glomerulosclerosis, pathology, ultrastmcture and, 220 Bursa of Fabricius, immunity and, 49-60

474

SUBJECT INDEX

Disease, unknown etiology, pathology of, 215-

221 Disulfide bonds, immunoglobulin, stability of, 302-303

E Elasmobranchs, immunity in, 79, 80 Embryo, cell transfer to, 41-44 immunoglobulin transfer to, 6-11 Endocarditis, rheumatoid factors in, 366 Endotoxins, adjuvant effects, mode of action, 418-419 original observations, 413 various preparations and, 413-416 nature and biological characteristics of,

composition of, 278-279 serological specificity of, 279-281 Craft-versus-host reaction, thymectomy and, 52-55 Guanidine, immunoglobulins and, 291 Guinea pig, immunoglobulins of, 288

H

a-Helix, immunoglobulins and, 290 Hematogenous organs, evolution of spleen and, 88-89 Heymann’s “autoimmune” renal disease, ultrastructure and, 227-228 Histone, antibodies to, 353 nucleic acid antibodies and, 356-357 Holosteans, immunity in, 79-81 Homografts, immune cells and, 182-185 411-413 Homotransplantation immunity, inverteEnzymes, intracellular kill of microorgan69-70 brates and, isms and, 133-137 Escherichia coli, rheumatoid factors and, Horse, immunoglobulins of, 288, 289, 292, 303, 305 385 Experimental allergic encephalomyelitis, Human, antibody production, development of, pathology of, 231-233 23-28 F immunoglobulins of, 289,292,295, 305 lymphoid tissue in, 27-28 Fetus, Hypersensitivity, antibody transfer to, 11-13, 332-334 delayed-type, cellular immunity and, relationship to mother, 4-5 160-162 Flagella, invertebrates and, 70 bacterial, antibody and, 290 Hypersensitivity reactions, Bordetella 0 pertussis and, 420-421 y -Globulins, I antibodies to, Immune reaction, definition of, 118 buried determinants and, 368 Gm and InV specificity of, 359-363 Immune response, vertebrate, 03-94 nonrheumatoid, 367-388 types of, 2 normal sera and, 386-367 rheumatoid factors and, 383-368 Immunity, antigenic form of, 384-365 cellular, buried determinants, antibodies to, 388 bacterial residence and, 155-158 characterization of, 81-84 delayed hypersensitivity and, 180y2-Globulin, antibodies in, 6 162 Glomerulonephritis, demonstration of, 145-149 pathology, ultrastructure and, 216-219 homografts and, 182-185 Glycerol teichoic acids, invertebrates and, 68-88 intracellular, 277-278 in oitro acquisition of, 149-150

475

mJEmINDEX mononuclear phagocytes and, 150153 nonspecific manifestations of, 153155 transfer of, 158-180 humoral, invertebrates and, 84-88 ontogeny of, 4-80 Immunoconglutinin, function of, 351 Immunocytes, vertebrate, 91-93 Immunoglobulin(s ) , see also Antibodies allotypes of, 325-331 amino acid analysis of, 291-293, 305,

306 antibody-combining site of, 309-318 antigenic properties of, 319-325 biological properties of, 319-341 carbohydrate content of, 305-308 complement fixation and, 338-337 distribution and turnover of, 338-339 disuEde bonds, stability of, 302-303 electron microscope studies of, 290 enzymatic splitting of, 295-297 fragments, urinary excretion of, 331332 heterogeneity of, 318-319 molecular weight of, 289 nature of, 5-6 nomenclature of, 287-288 passive transfer to embryo, 8-11 peptide patterns of, 294-295 reduction of, 297-302 structural relationships of, 303-305 summary, 341-342 synthesis, ontogeny of, 13-18 tertiary structure of, 290-291 three types of peptide in, 308-309 Immunoglobulin A, antibody activity of, 288 Immunologic activity, nature in invertebrates, 71 Immunologic capacity, immunologic tolerance and, 48-49 Immunologic development, cell transfer and, 41-48 Immunologic reactions, pathology of, 202-235 ’ ultrastructure and, 198-235 Immunologic system, vertebrate, 91

Immunologic tolerance, immunologic capacity and, 48-49 Inflammatory reaction, invertebrates and, 88-88

Influenza virus, antibody and, 290 Invertebrates, anaphylaxis and hypersensitivity in, 70 blood cells of, 88-89 body fluid proteins of, 70-71 “cellular immunity” in, 88-88 homotransplant immunity in, 89-70 humoral immunity in, 84-86 immune responses in, 82-71 infection with microorganisms, 82-84 nature of immunologic response in, 71 nonliving antigens and, 64-88 Isoguanine, nucleic acid antibodies and, 358

K Kidney, ultrastructure, autoimmunity and, 222230 1 Lactobacdllus, cell wall carbohydrates of, 288-289 cell wall teichoic acids of, 278-277 intracellular teichoic acids of, 281 Lactoglobulins, immunoglobulin transfer and, 7 Leucocytes, polymorphonuclear, degranulation of, 137-139 Lipid, anticytoplasmic antibodies and, 357 Lipid nephrosis, pathology, ultrastructure and, 218-219 Liver disease, autoimmune complement fixation and, 358 rheumatoid factors in, 388 Lupiis erythematosus, ’ antibodies in, 352, 373-376 pathology, ultrastructure and, 215-218 rheumatoid arthritis and, 371-372 Lymphoid cells, vertebrate, 91-93 Lymphoid tissue, development of, 28-34, 50, 53-54 evolution of, 88-90 immunity and, 2-3, 18, 22

476

SUBJEcr INDEX

M

0

Macromolecular aggregates, circulating, 202, 221 Malignancy, dermatomyositis and, 385 8-Mercaptopurine, nucleic acid antibodies and, 358 Metabolic activity, phagocytosis and, 130-132 Micrococci, cell wall teichoic acids of, 270-278 Microorganisms, consequences of residence in immune cells, 155-158 infections, in invertebrates, 62-84 in vertebrates, 72 intracellular digestion of, 139-141 intracellular kill, degranulation and, 137-139 enzymes and, 133-137 physicochemical conditions and, 133 Microsomes, antibodies to, 357 Milgrom factors, reactions of, 387-388 Mitochondria, antibodies to, 357 Mother, immunoglobulin transfer by, 8-11,332334 relationship to fetus, 4-5 Myasthenia gravis, autoantibodies and, 382-384 Mycobacteria, adjuvant effects, characterization of active material, 404-407 mode of action, 407-411 oil-in-water emulsions and, 402-404 original observations, 400-402 Myelomatosis, immunoglobulins and, 289, 294-295, 317-320

Oil-in-water emulsions, effect of mycobacteria in, 402-404 Oligonucleotides, deoxyribonucleic acid antibodies and, 358 Ontogeny, antibody production and, 18-26 delayed allergic response and, 37-41 immunity and, 4-80 immunoglobulin synthesis and, 13-18 transplantation immunity and, 34-37 Opsonins, phagocytosis and, 123-128,144 Opsonization, virulence and, 126129

P Pancreatic disease, autoantibodies and, 388-387 Papain, immunoglobulins and, 290, 293295, 301, 303, 308-307,321, 323, 330, 331 Parasites, facultative intracehlar, 141144 Parasitic disease, rheumatoid factors in, 388 Pathology, circulating antigen-antibody complexes and, 202-221 Pepsin, immunoglobulins and, 294, 298, 302, 323 Peptide chains, number in immunoglobul i n ~ 308-309 , Pernicious anemia, autoantibodies and, 380-382 Phagocyte( s ) , delayed-type hypersensitivity and, 180182 demonstration of cellular immunity in, 145-149 digestion of bacteria by, 139-141 facultative intracellular parasites of, 141-144 N homografts and, 182-185 immune, properties of, 144-185 Nargase, immunoglobulins and, 294 immunity and, 118 Nephrotoxic serum nephritis, ultrastrucinvertebrates and, 68-88 ture and, 222-227 in w h o acquisition of cellular immunity Nervous system, autoimmunity and, 231by, 149-150 233 Newborn, cell transfer to, 41-44 killing of microorganisms by, 133-139 Nucleoprotein, antibodies to, 352-355, metabolic activity, particle ingestion 357 and, 130-132 Nucleus, antibodies to, 352-357 mononuclear, immunity in, 150-153

SUBJECT INDEX

nonspecific manifestations of immunity in, 153-155 normal, postengulfment period and, 129-144 transfer of cellular immunity and, 158180 Phagocytosis, factors affecting, 122-129 physicochemical conditions and, 133 Phosphatase, alkaline, appearance of, 7 Phosphate extract, antibodies to, 353, 354 Phylogeny, adaptive immunity and, 6094 Placenta, immunologic tolerance and, 4-5 Plasma cells, antibodies and, 18, 20-22, 33-34 Pnewnococei, C and F polysaccharides of, 281-283 capsdar antigens of, 282 Polysaccharides, antigenic, cell wall and, 255-289 pneumococcal, 281-283 Poststreptococcal glomerulonephritis, pathology, ultrastructure and, 219 Proline, immunoglobulins and, 290-293 Protein, antigenic, cell walls and, 252-255 body fluid, invertebrate, 70-71 Protochordates, immunity in, 77 Purine, nucleic acid antibodies and, 358

477

buried determinants and, 388 specificity of, 363, 365 Ribonucleic acid, antibodies and, 20, 188 immunity transfer and, 158-160,164

S Scleroderma, autoantibodies and, 384-385 pathoIogy, ultrastructure and, 219-220 Sensitivity, immediate and delayed, 85-88 Sera, normal, rheumatoid factors in, 388387 Serological reactivity, cell wall teichoic acid and, 273-275 Serum sickness, pathology of, 207-212 Sialic acid, immunoglobulins and, 293 Sjogren’s syndrome, rheumatoid arthritis and, 372 Skin, antibody fixation to, 334-338 Spleen, evolution of, 88-89 Staphylocmcw aureus, cell wall antigens of, 255, 282 cell wall teichoic acid, serological reactivity of, 273-275 Structure Of, 270-273 intracellular teichoic acid of, 281 Staphylococm eptdmidfs, cell wall teichoic acid of, 275-278 Steblay’s “autoimmune” renal disease, pathology of, 229 Streptococci, cell wall, protein antigens of, 252-254 R group A, cell wall carbohydrate of, 258-259 Rabbit, M proteins of, 252-253 imm~oglobulinsOf, 288-292,294,305 other protein antigens of, 253-254 disulfide bond stability of, 302-303 group C, cell wall carbohydrate of, Reagins, 259-280 amounts in plasma, 334-335 group D, cell wall carbohydrate of, immunoglobulins and, 288 260 Reticuloendothelial system,infection and, group G, cell wall carbohydrate of, 165-167 280 Rh antibodies, rheumatoid factors and, 387 T Rheumatoid arthritis, Teichoic acids, anti-y-globulins and, 352, 380, 382 cell wall, 289-277 antinuclear antibodies and, 357 glycerol, autoantibodies and, 368-373 composition of, 278-279 Rheumatoid factors. seroiogical specfacity of, 279-281

478

SUBJEcr INDEX

Teleosts, immunity in, 80, 81 exogenous antigens and, 189-193 Temperature, immune response and, glomerulonephritis and, 216-219 72-74 immunologic reactions and, 196-235 Theobromine, nucleic acid antibodies lipid nephrosis and, 218-219 and, 356 lupus erythematosus and, 215-216 Thymus, nephrotoxic serum nephritis and, 222abnormalities of, 58-60 227 evolution of, 89-90 scleroderma and, 219-220 immunity and, 2, 4, 22, 31-32, 49-60 sensitized cells and, 187-189 lymphopoiesis and, 27-29, 46 serum sickness and, 207-212 Thyroiditis, autoantibodies and, 376-380 toxemia of pregnancy and, 220-221 Tobacco mosaic virus, antibody and, 290 Urea, immunoglobulins and, 290-291 Toxemia of pregnancy, Urine, immunoglobulin fragments in, pathology, ultrastructure and, 220-221 331-332 Transfusions, anti-y-globulins and, 361 V Transplantation immunity, Vertebrates, ontogeny of, 34-37 antibody-forming capacity, evolution vertebrates and, 86-88 of, 74-81 Trypsin, immunoglobulins and, 294-296 evolution of lymphoid tissue in, 88-90 Tumor immunity, vertebrate, 93-94 y-globulins in, 81-84 Tyrosine, immunoglobulins and, 290-291 immediate and delayed sensitivity in, 85-86 U immune responses in, 71-94 Ulcerative colitis, autoantibodies and, tumor immunity and, 93-94 388-389 immunocytes of, 91-93 Ultrastructure, immunologic system of, 91 amyloidosis and, 212-215 infection with microorganisms, 72 anaphylactoid purpura and, 219 killed organisms and, 72-74 anaphylaxis and, 203 lower, antigenicity in, 84-85 antibody formation and, 176-185 transplantation immunity in, 86-88 antibody storage or release and, 185- Virulence, opsonization and, 126-129 187 antigen-antibody union, W in Uitro, 196-201 Wart virus, antibody and, 290 in uko, 201 Wax D, adjuvant effects of, 404-406 Arthus reaction and, 203-207 autoimmunity and, 221-233 X cellular sensitivity and, 233-235 Xanthine, nucleic acid antibodies and, diabetic glomerulosclerosis and, 220 356 disease of unknown etiology and, 215Y 221 Yolk sac, immunoglobulins and, 7-8 endogenous antigens and, 193-196