ADVANCES IN
Immunology V O L U M E 34
CONTRIBUTORS TO THIS VOLUME
MARILYN L. BALTZ ROBERT M. FRIEDMAN A. GONWA THOM...
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ADVANCES IN
Immunology V O L U M E 34
CONTRIBUTORS TO THIS VOLUME
MARILYN L. BALTZ ROBERT M. FRIEDMAN A. GONWA THOMAS TED H. HANSEN F. L. OWEN KEIKOOZATO M. B. PEPYS B. MATIJA PETERLIN DAVIDH. SACHS NATHANSHARON JOHN D. STOBO STEFANIEN. VOGEL
ADVANCES IN
Immunology EDlTED
BY
F R A N K J. DIXON
HENRY G. K U N K E L
Scripps Clinic and Research Foundation La Jollo, California
The Rockefeller University N e w York, New York
V O L U M E 34
1983
ACADEMIC PRESS A Subridictry of Horcovrt Bvace Jovanovich, Publishen
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COPYRIGHT @ 1983, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC O R MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
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LIBRARY OF CONGRESS CATALOG CARD NUMBER: 6 1 1 7057
I S B N 0- 12-022434-8 PRINTED IN THE UNITED STATES OF AMERICA
83 84 85 86
9 8 76 5 43 2 1
CONTENTS CONTRIBUTORS ............................................................. PREFACE...................................................................
T Cell
Alloantigens Encoded
by
vii ix
the IgT-C Region of Chromosome 12
in the Mouse
F. L. OWEN I. Introduction ............................. ............. 11. Preparation of Conventional Anti-Tsu Serum ............................ 111. Preparation of Monoclonal Antibodies Specific for Tpre, Tthy, Tind, and Tsu .............................................................. IV. Genetic Characterization of the IgT-C Linkage Group ................... V. Products of the IgT-C Region Define a Unique T Cell Differentiation Pathway ............................................... VI. Evidence That Tpre, Tthy, Tind, and Tsu Are Excluded from Developing B Cells and B Cell Products .... ...................... VII. Cross-Reactive Determinants Shared by T Cell Alloantigens in This Linkage Croup and Soluble T Cell Factors ............................. VIII. In Vitro Functional Role of Cells Expressing Tpre, Tthy, Tind, and Tsu . . IX. I n Viuo Studies on the Function of Tsu and Tind Bearing Cells .......... X. Preliminary Immunochemical Characterization of Tsu and Ti XI. Concluding Remarks ..................................... References . . . . . . . . .........................................
1
3 4
9 14 22 24 27 32
35
Heterogeneity of H-2D Region Associated Genes and Gene Products
TED H. HANSEN,KEIKO
OZATO, AND
DAVIDH. SACHS
I. Introduction .......................................................... 11. Antigenic Heterogeneity of Gene Products Encoded in the Dd Region .... 111. Chemical Heterogeneity of Gene Products Encoded in the Dd Region . . , . IV. Quantitative Comparisons of Gene Products Encoded in the Dd Region ... V. Functional Studies of H-2L" Gene Products ............................. VI. Searches for Allelic Products of H-2Ld in Other Haplotypes .............. VII. Studies Using Genomic Clones of H-2D Region Loci .................... VIII. Evolutionary Models and Future Approaches ........................... References ...........................................................
39 41 46
50 52 54
58 64 67
Human Ir Genes: Structure and Function
THOMAS A. GONWA,B. MATIJA PETERLIN,AND JOHND. STOBO I. Introduction .......................................................... 11. Structure of Ir Gene Products in Mice and Humans ..................... V
71 71
vi
CONTENTS
111. Ir Gene Function in Humans .......................................... IV. Conclusions .......................................................... References ...........................................................
80 92 92
Interferons with Special Emphasis on the Immune System
ROBERTM . FRIEDMAN AND STEFANIE N . VOGEL I . Introduction .......................................................... I1 . Interferon Production ................................................. I11. Actions of Interferons ................................................. IV Interferons and Defense against Viral Infections ......................... V. Interferons and Other Mechanisms Related to Immunity and Inflammation ..................................................... VI Antitumor Effects of Interferons in Animal Systems ..................... VII . Clinical Studies with Human Interferons ............................... References ...........................................................
.
.
97 99 101 128 129 130 132 133
Acute Phase Proteins with Special Reference to C-Reactive Protein a n d Related Proteins (Pentaxins) a n d Serum Amyloid A Protein
.
M . B PEPYS AND MARILYNL. BALTZ I . Introduction .......................................................... I1. Induction and Control of Synthesis of Acute Phase Proteins .............. I11. C-Reactive Protein. Serum Amyloid P Component (SAP). and Related Proteins (Pentaxins): Definition and Nomenclature ...................... IV. C-Reactive Protein .................................................... V. Serum Amyloid P Component ......................................... VI . Serum Amyloid A Protein ............................................. VII Summary ............................................................. References ........................................................... Note Added in Proof ..................................................
.
141 145 151 156 183 190 198 199 211
Lectin Receptors as Lymphocyte Surface Markers
NATHANSHARON I . Introduction
..........................................................
IV. Human Lymphocyte Subpopulations ................................... V. Lymphocytes of Other Animals ........................................ VI Concluding Remarks .................................................. References ...........................................................
213 223 230 265 281 287 291
INDEX..................................................................... CONTENTS OF PREVIOUS VOLUMES ..........................................
299 303
I1. Methodology ......................................................... I11. Murine Lymphocyte Subpopulations ...................................
.
CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
MARILYNL. BALTZ (141),Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, England ROBERT M. FRIEDMAN (97), Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
THOMAS A. GONWA (71), The Howard Hughes Medical lnstitute and the Department of Medicine, University of California, Sun Francisco, California 94143 TEDH. HANSEN(39),Department of Genetics, Washington University School of Medicine, S t . Louis, Missouri 63130
F. L. OWEN (l),Department of Pathology and Cancer Research Center, Tufts Medical School, Boston, Massachusetts 02111 KEIKOOZATO(39),Laboratory of Developmental and Molecular lmmunity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205 M. B . PEWS (141), Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, England B. MATIJAPETERLIN(71),The Howard Hughes Medical lnstitute and the Department of Medicine, University of California, Sun Francisco, California 94143 DAVIDH. SACHS(39), Transplantation Biology Section, Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 NATHAN SHARON (213), Department of Biophysics, The Weizmann lnstitute of Science, Rehovoth, lsrael vii
viii
CONTRIBUTORS
D. STOBO(71), The Howard Hughes Medical Znstitute and the Department of Medicine, University of California, Sun Francisco, California 94143
JOHN
STEFANIE N. VOGEL (97), Departments of Pathology and Microbiology, Unij'ormed Services University of the Health Sciences, Bethesda, Maryland 20814
PREFACE
The selection of subjects presented in this volume reflects the broad scope of immunologic interest. Most of the progress in our field depends upon elucidation of the genetic basis underlying the immune system’s structure and function, and three important genetic areas are represented. They include presentation of a new group of T cell alloantigens with many similarities to the immunoglobulin isotype markers for B cells, discussion of the recently recognized heterogeneity of Class I MHC antigens, and a review of the structure and function of human Ir genes. Additional areas of expanding interest are indicated by three reviews that derive in part from neighboring fields of science but deal with matters of considerable immunologic importance. These are a discussion of interferon, particularly as it relates to and influences immunologic events, a review of the acute phase response to injury which has many paraIlels to and interfaces with the immune response, and, finally, a description of the lectin receptor markers of immunocytes and the imaginative lectin technology that has contributed significantly to the identification of the various functionally heterogeneous lymphocyte populations. A new group of T cell alloantigens encoded by a cluster of tightly linked genes on murine chromosome 12 is described in the first article by Dr. F. L. Owen. Drawing heavily on his and his associates’ work, he defines the gene cluster, designated IgT-C because of its proximity to the immunoglobulin genes, and its four recognized structural genes Tpre, Tthy, Tend, Tsu. The products of these genes appear on T cells at characteristic points during their maturational pathway in the order just listed. Although these antigens are distinct from the Lyt series of markers, their presence is related to T cell regulatory function. Cells bearing three (thy, end, and su) of these markers appear to have distinct suppressing and/or delaying effects on immunologic responses in uitro, and the pre-marker appears to be associated with a nonregulatory, perhaps precursor cell. The maturational pathway defined by these markers is presented in detail with its functional and anatomical correlates and its relationship to other T cell markers. The apparent ,function of these gene products is discussed with special emphasis on the possibility that they represent constant regions of T cell antigen receptors distributed differentially on various T cell subsets in the same way that immunoglobulin isotypes serve as differentiation markers for B cells. ix
X
PREFACE
In the second article, Drs. Hansen, Ozato, and Sachs present recent research that is revealing a newly appreciated serologic, molecular, genetic, and functional heterogeneity of Class I H-2 antigens. The focus of this review is on the H-2D region associated genes and their products, a subject to which the authors have been major contributors. Exactly how many genes exist in each region is not yet certain; however, it is clear that the past dogma citing only one gene product for each H-2K or H-2D region is incorrect, at least for some haplotypes. The emerging picture is one of Class I genes as multigene families in which certain members undergo continuous evolutionary expansion and contraction. Finally, the contribution of the concept of Class I multigene families to our understanding of the evolution of these genes and to the roles played by recombination, duplication, and gene conversion in the process is presented and clearly related to appropriate experimental data. A timely view of the structure and possible function of human Ir genes appears in Article 3 by Drs. Gonwa, Peterlin, and Stobo. The genetic basis and chemical characterization of human Ia molecules, HLA-DR, and related HLA-DC and HLA-SB are described and compared to those of their less complex murine counterparts. The possible mechanisms by which Ir gene products might regulate immunologic responsiveness are reviewed along with examples of such apparent regulation. Particularly pertinent is the authors’ work on the immune response of humans to collagen indicating the HLA-DR4 relationship, the genetic characteristics, and the cellular events involved. Current knowledge about the several varieties of interferons such as their cells of origin, modes of induction, control of synthesis, and numerous actions, particularly those related to the immune system, is presented by Drs. Friedman and Vogel in the fourth article. Although most of this information on the actions of interferon comes from studies employing naturally derived and therefore limited amounts of interferon, it provides an essential background for intelligent exploitation of the large amounts of interferon now being made available by recombinant DNA technology. Apparently, all the interferons, a, p, and y , can either modulate immunologic mechanisms directly and/or retard the growth of pathogens-the targets of immune responses. Gamma interferon, the product of stimulated T cells, is quite properly considered an immunoregulatory lymphokine which can enhance macrophage function, suppress responding B cells, and inhibit T suppressor activity. Another striking immunologic effect of interferon is its stimulation of natural killer (NK) cells presumably via the accelerated differentiation of pre-NK cells to fully cytolytic forms. One of the least
PREFACE
xi
well understood yet most challenging aspects of interferon is its apparent antitumor activity. This complex area is thoroughly discussed, and the several mechanisms of antitumor action, immunologic and nonimmunologic, elicited by interferon are analyzed and evaluated. The acute phase response is the name given to a characteristic increase in concentrations of numerous serum proteins following a wide variety of infections, inflammations, or other tissue injuries and constitutes a significant component of the overall systemic reaction to injury. Although this paraimmunologic event has been well recognized since the identification of C-reactive protein, one of its major constituents, some 50 years ago, its precise role in host defense is poorly understood. However, the fact that many components of the acute phase response have enjoyed evolutionary conservation throughout the vertebrate kingdom would suggest that they subserve a beneficial function. In the fifth article, Drs. Pepys and Baltz review this subject covering the factors initiating and controlling the response, the chemistry of its more prominent components, their biologic properties and functions, and, finally, their role in the diagnosis and monitoring of human disease. From initiation of the acute phase response via injury-induced activation of macrophages and interleukin-1 formation, which then stimulates synthesis of most of the acute phase reactants by hepatocytes, to the interaction of these reactants with microbial or endogenous molecules that may result in complement activation and modulation of inflammation, the parallelism between the acute phase and immune responses is evident. The former is a relatively nonspecific, extremely rapid defense in contrast to the latter specific but delayed reaction. With recognition of the great functional heterogeneity and extensive cooperative interactions that mark cells of the immune system comes the need for means to identify and isolate the separate and distinct cellular entities. Two major tools to achieve this end have been developed: antibodies reactive with lymphocyte surface antigens and lectins reactive with surface saccharides. I n the final article, Dr. Sharon discusses lectin receptors as lymphocyte surface markers and draws on his extensive experience in detailing the use of lectins in the recognition and purification of lymphocyte subpopulations. Cell surface lectin receptors are carbohydrates that reside in the oligosaccharide sequences of membrane glycoproteins or glycolipids as secondary gene products, just as ABO blood group determinants do. Lectins, which are largely of plant origin, are oligomeric proteins with several sugarbinding sites per molecule, and these sites interact with their target noncovalently primarily via hydrophobic and hydrogen bonds. Al-
xii
PREFACE
though the functions of lectin receptors on lymphocyte and other cell surfaces are not known, a large number of such markers have been identified and correlated with cell surface antigens as well as with maturational and functional characteristics of cells. Techniques capable of recognizing lectin receptors on cells in situ and of separating and purifying specific cellular populations have been developed by using a variety of lectins. The use of this technology in diverse experimental situations as well as its potential clinical application in the preparation of non-graft-versus-host reactive bone marrow transplants are also presented.
FRANKJ. DIXON HENRYG. KUNKEL
ADVANCES IN
Immunology V O L U M E 34
This Page Intentionally Left Blank
ADVANCES IN IMMUKOLOGY, VOL. 34
T Cell Alloantigens Encoded by the IgT-C Region of Chromosome 12 in the Mouse F. L. OWEN Department of Pathology and Cancer Research Center, Tufis Medical School, Boston, Massachusetts
I. Introduction ............................... ............. 11. Preparation of Conventional Anti-Tsu Serum ............................ 111. Preparation of Monoclonal Antibodies Specific for Tpre, Tthy, Tind, and Tsu . . . . . . . . ..................................... IV. Genetic Characterizatio C Linkage Group ......... V. Products of the IgT-C Region Define a Unique T Cell Differentiation Pathway ............................................... VI. Evidence That Tpre, Tthy, Tind, and Tsu Are Excluded from Developing B Cells and B Cell Products ............................... VII. Cross-Reactive Determinants Shared by T Cell Alloantigens in This Linkage Group and Soluble T Cell Factors ........................ VIII. I n Vitro Functional Role of Cells Expressing Tpre, Tthy, Tind, and Tsu .............................. ...................... IX. I n Vivo Studies on the Function of Tsu and Ti Bearing Cells ......................................................... X. Preliminary Immunochemical Characterization of Tsu and Tind ....... ........................................... XI. Concluding Remarks ..................................... .......
.............................
1 3 4 14 22 24 27
32 33 34 35
I . Introduction
A new group of T cell alloantigens is encoded by a cluster of tightly linked genes on the murine chromosome 12. This gene cluster, designated IgT-C because of it8 close proximity to the immunoglobulin genes (diagrammed in Fig. l),includes at least four structural genes which code for Tpre, Tthy, Tind, and Tsu. Classical in vivo animal genetic studies predict the gene cluster lies no more than 3 map units distal to alpha, and is therefore spatially closer to the immunoglobulin constant region genes than are the most loosely linked variable region genes (V,nase, 8 map units). The close physical location of these two gene clusters, one specific for B cells (immunoglobulins) and a second apparently specific for T cells (IgT-C),raises questions about a possible evolutionary relationship between the two groups of genes. It has been proposed that these genes may code for constant regions on T cell antigen receptors and may, in fact, have arisen evolutionarily from duplication of a primordial gene coding for both T and B cell antigen 1 Copyright 0 1983 by Academic Press, Inc.
All rights of reproduction in any form reserved. ISBN 0-12-023434-8
2
F. L. OWEN
I
Igh-V
Ly 18,19
I
1
1
Iqh-C
I 1
IpT-C
i
b
2 5 H Rscombinolion
2% Recombinofion 5% Recombinalion
FIG. 1. Map of genes linked to the immunoglobulin locus on chromosome 12 in the mouse. The region proposed to be named IgT-C is distal to alpha and includes four gene products named Tpre, Tthy, Tind, and Tsu. The subscript m (Tpre,) indicates monoclonal antibodies, presumably epitope specific, were used to map these genes. Although Trpe, is more distal than Tsu, the order with respect to the centromere of Tthy,, Tind,, and Tsu, have not been determined.
.
receptors. Some support for this hypothesis is found in the serological cross-reactivity of a monoclonal antibody recognizing Tind and an antigen-specific T augmenting factor, TaF (Section VII). Earlier studies with antiserum had shown stearic blockade of T cell antigen binding. These suggestive data are encouraging. However, the conclusion that products of IgT-C are constant region genes for T cells is critically dependent upon the cloning of a segment of DNA which maps to this region. Use of that genetic material to produce a synthetic polypeptide which shows biological properties of antigen binding and T cell functional replacement and also expresses determinants recognized by one of our monoclonal antibodies would be acceptable evidence that one of these gene products is indeed the elusive C, (Owen and Spurll, 1981; Kronenberg et al., 1980; Schrader, 1979). Similar information for each of the four gene products in this region is required to assume that all four represent isotypes of C.rl-4.Although this is our working hypothesis, it is possible that several different types of structural products may be encoded in this segment of the chromosome, possessing unrelated functional roles. This work was initiated based on the assumption that T cell receptors are unique antigen binding structures and that fine specificity for antigen is a finite property of T cells endowed solely through surface receptor recognition of nominal or self antigens. Difficulties in demonstrating direct antigen binding by helper T cells in vitro or on clones of antigen-specific proliferating cells have led to speculation that T cells may play a physiological, non-antigen binding role in regulating immunoglobulin synthesis. The serological approach outlined below describes an attempt to produce antiserum specific for “constant determinants” on T cell antigen binding structures. A cluster of immunoglobulin genes on chromosome 12 (Honjo and Katavka,
T CELL ALLOANTIGENS
3
1978) specify variable region and constant region genes; despite the fact that extensive descriptions of serological cross-reactivity between anti-VHreagents and T cells have been published (Eichmann, 1978, review; Lonai et aZ., 1978), attempts to demonstrate immunoglobulin constant region genes on T cells have led to negative evidence (Krawinkel et aZ., 1978). If one assumes the serological cross-reactivity of T cell receptors and Igh-V implies at least an evolutionary relationship, if not an identity between the genes encoding immunoglobulin and T cell antigen recognizing structures, then it is biologically most conservative to assume a T cell gene cluster will lie close to the Igh-V genes. If immunoglobulin constant region genes do not encode T cell receptors, then it is assumed that a unique bank of Igh-1 linked T cell genes must exist. Antiidiotype recognizing T cells have been described in mice immunized with rabbit antiidiotypic antiserum and KLH * Ars (Owen et aZ., 1977b) as well as other systems (Bona and Paul, 1979). Surprisingly, the T cells of A/J animals (Igh-le) and C.AL-20 (Igh-Id on a BALB/c congenic background) exhibit the same T cell antiidiotypic specificity in contrast to BALBlc T cells (Owen et aE., 1977a). This finding prompted the choice of strains and serological approach to produce antiallotypic reagents specific for constant region gene products (Owen et al., 1979; Tokuhisa and Taniguchi, 1982a; Aihara et al., 1983). The experiments summarized in the following sections describe attempts to identify target tissues expressing the T cell antigens encoded by the IgT-C region, to identify functional role for cells expressing these antigens, and to look for serological relationships with antigen binding factors. The genetic work at present is confined to identification of surface antigens in recombinant inbred lines of mice. Genetic analysis at a more molecular level must await amino acid sequence data on these antigens and/or good biochemical data with peptide map analysis of possible polymorphisms only suggested by the serological data. II. Preparation of Conventional Anti-Tru Serum
The strains selected for production of polyclonal anti-Tsu were based on the fact that the recombination events in and around the immunoglobulin gene complex in C.AL-20 animals, congenic with BALB/c, is well documented. BALB/c animals accept first set C.AL-20 grafts (Riblet and Congelton, 1977; Owen et al., 1979). Therefore, the antigenic determinants [H(I,)] presumably encoded by the minor histocompatibility locus on chromosome 12, between alpha and preal-
4
F. L. OWEN
bumin, present a favorable unidirectional graft barrier in this strain combination. In addition, the accessibility of a T cell-specific antigen binding system in the C.AL-20 animal (Owen et al., 1976) made this an attractive strain for production for T cell receptor probes. The reagents produced in this effort consequently react with antigens linked to either the Igh-ld or Igh-le immunoglobulin alleles, which are found in strains less often used by cellular immunologists. Briefly, BALB/c AnN animals were injected with C.AL-20 spleen cells grown for 24 hours in 10% FCS containing media with 5 pg/ml concanavalin A. Intraperitoneal injections (5x at 1 week intervals) of cells fractionated on discontinuous BSA gradients (Steinman et al., 1978) and selected for approximately 20% of surviving blast cells resulted in production of an antiserum reacting preferentially with C.AL-20 and not BALB/c cells. Positive antiserum was selected from individual BALB/c mice by testing in visual surface immunofluorescence assays on C.AL-20 spleen cells eluted from nylon wool (Julius et al., 1973). Indirect staining with FITC-goat anti-IgG was used as a screening assay. Serum samples were not adsorbed before testing. Serum titers were typically 1-10 or 1-20 and not greater than 20% of immunized mice in any group were sero-positive. Antiserum from pooled positive samples was aliquoted and frozen at -70°C. Freezethawing resulted in loss of activity. A typical tissue distribution of the antigen(s) detected by surface fluorescence is shown in Table I. Because 10 donor C.AL-20 mice were required for every 1 BALB/c immune animal and only 1 mouse in 5 produced antiserum, this procedure was costly and inefficient. Practical considerations encouraged attempts to produce monoclonal antibodies specific for Tsu. The identification of other gene products encoded in this region was fortuitous and a by-product of our first attempts to produce monoclonal anti-Tsu. Two independent attempts to produce an antiserum specific for T cells by immunization with concanavalin A activated T cell blasts have been reported. Both utilize BALB/c anti-CB.20 serum. Antiserum was screened by antibody and complement-mediated 51Cr release from Con A activated cells. Similar results were obtained, suggesting that the polymorphism of T cell “allotypes” may involve at least three alleles: BALB/ca, CB.20b, and C.AL-20d (Tokuhisa and Taniguchi, 1982b; Aihira et al., 1983). Ill. Preparation of Monoclonal Antibodies Specific for Tpre, Tthy, Tind, and Tsu
Efforts to produce anti-Tsu resulted in the incidental production of monoclonal IgGIK anti-Tind. Continued screening, using the same ap-
5
T CELL ALLOANTIGENS
TABLE I DISTRIBUTION OF REACTIVITYO F ANTI-TS~ ANTISERUM O N CAL.20 LYMPHOID TISSUE Tsd bearing cells Tissue
(%)
Spleen Splenic T cells" Splenic B cellsb Ly 2+ spleen cells Ly 1+ spleen cells Thymocytes (unfractionated) Mature thymocytesC Lymph node Bone marrow Con A blastsd LPS blasts
2-6 5- 12 90% of human, adherent peripheral blood macrophages display HLA-DR molecules, approximately only 50% also display HLA-DS (DC). Most importantly, the HLA-DS+, HLA-DR+, but not the HLA-DS-, HLA-DR+ population is effective in reconstituting antigen induced proliferation among T en-
HUMAN
Ir
83
GENES
TABLE I RELATIVE ABILITYOF HLA-DR+, HLA-DS+ vs HLA-DR+, HLA-DS- MACROPHAGES TO SUPPORT ANTIGEN-INDUCED T CELL PROLIFERATION" M 4 added
70 M 4 added
HLA-DR+, HLA-DS+
HLA-DR+, HLA-DS-
0 0.1 1.0 10.0
210 4016 6002 9934
210 652 755 8 16
a The indicated final % M 4 was added to autologous T enriched M$J depleted cells and reactivity to three concentrations of C. alhicans tested. Results are presented as maximal Acpm.
riched, macrophage depleted cells (Table I). The HLA-DS-, HLADR+ population of macrophages does not suppress antigen induced proliferation and cannot reconstitute reactivity even when exogenous interleukin I is added. This suggests that the difference in the two populations to support antigen-induced T cell proliferation represents differences in their ability to suitably present antigen. This in turn suggests that HLA-DS is more important than HLA-DR in restricting T cell-macrophage interactions in this model of reactivity to C. albicans. However, it is possible that the display of HLA-DS simply correlates with some other property or metabolic function of macrophages required for antigen presentation by HLA-DR. A second observation which implicates HLA-DS molecules in antigen presentation is the observation that antigen induced T cell proliferation can be blocked by anti-HLA-DS reagents (Table 11). The fact that the anti-DS reagent did not completely block reactivity is consistent with the possibility that with a complex antigen such as C . albicans, some determinants would be restricted by HLA-DS and others by HLA-DR. These blocking studies, however, are subject to the same alternative explanations as those outlined for blocking with antiHLA-DR reagents. The results of the blocking studies implicate both HLA-DR and HLA-DS in the T dependent proliferation to complex antigens. The relative role that each plays in this function remains to be established. Another approach to establishing HLA-D linked genes in controlling immune reactivity has been to link specific immune reactivity in families or the general population with given HLA-D linked gene products. Scher et al. (1975) immunized 61 normal volunteers with the
84
THOMAS A. GONWA ET AL.
TABLE I1 ABILITY OF ANTI-HLA-DSTO BLOCK ANTIGEN-INDUCEDT CELL PROLIFERATION" Dilution of serum used
Expt. 1 NRS Anti-HLA-DS
1/200
1/400
1/800
8,982 481
13,093 3,002
10,544 5,160
25,045 9,268
26,479 10,549
29,018 13,355
Expt. 2
NRS Anti-HLA-DS
"Normal rabbit serum (NRS) or anti-HLA-DS was added to peripheral blood mononuclear cells to achieve a dilution of sera as indicated; 1 x los cells were tested for responsiveness to C . albicans. Results are expressed as maximal Acpm. Acpm in cultures with no antisera added were 9,018 and 36,749 in Expt. 1 and 2, respectively.
synthetic terpolymer, L-glutamic acid L-lysine L-tyrosine (GLT). Thirty-four of these individuals demonstrated a positive delayed hypersensitivity skin test in response to challenge with GLT. This responsiveness showed no association with known HLA-A or HLA-B determinants. HLA-DR typing was not performed. Three different groups of investigators have demonstrated that a substantial portion of normal, nonimmunized individuals acquire immune reactivity to several synthetic polypeptides. Young and Engleman (1980) demonstrated that 57, 85, and 71% of normal volunteers demonstrate reactivity to the synthetic polypeptides glutamic acid : tyrosine (GT), glutamic acid : alanine : tyrosine (GAT), and poly(L-tyrosine, L-glutamic acid)-poly(DL-alanine) poly(L-lysine) (T,G)-AL, respectively. Responsiveness, as measured by in vitro proliferation, occurred despite the fact that none of the individuals was purposely immunized with the antigens and responsiveness was not noted using cord blood lymphocytes. Hsu et a2. (1981) similarly demonstrated a naturally occurring T proliferative response to poly(Lhistidine, L-glutamic acid)-poly(DL-alanine)-poly(L-lysine), (H,G)-AL and (T,G)-AL in 64 and 54% of normal volunteers, respectively. Katz and colleagues (1981) demonstrated that T cells from 50% of normal donors could be induced to secrete an antigen-specific, T cell replacing factor upon challenge with (T,G)-AL. The precise nature of the naturally occurring immunogen which leads to this reactivity is unknown. Nonetheless, the presence of reactivity to simple, defined anti-
HUMAN
Ir
GENES
85
gens provides an opportunity to determine if it is controlled by HLA-D linked genes. In a study of nine families, Young and Engleman (1980) could find no association between a specific haplotype and responsiveness or unresponsiveness to GT, GAT, or (T,G)-AL. For example, offspring of nonresponders were unpredictably responders and vice versa. HLAidentical siblings were discordant for responder status. Based on the results of these studies, the authors concluded that a single dominant immune response gene did not control reactivity to the three synthetic antigens tested. Hsu et al. (1981) arrived at a slightly different conclusion in investigating the inhertance of immune reactivity to (H,G)-AL and (T,G)AL. These investigators showed concordant inheritance of HLA and responsiveness in families although there was no association between a specific HLA-A, Byor D allele in the general population and immune reactivity to either peptide. Based on the observation in two families that the mating between two nonresponders could produce responder offspring, the investigators proposed that gene complementation was involved in determining responder status. This hypothesis is consistent with the demonstration of a requirement for gene complementation in determining responder status to some synthetic polypeptides in mice. One very interesting family in this study (family 4000) demonstrated a recombination between HLA-B and HLA-D. The response pattern to both (T,G)-AL and (H,G)-AL indicated that Ir genes controlling these reactivities was telomeric and not centromeric to HLA-D (i.e., between HLA-D and B, but not between HLA-D and HLA-SB). This is of particular interest in view of the fact that HLA-DS (DC) may also be telomeric and not centromeric to HLA-D (see Fig. 1). While the studies of Hsu et al. provide convincing data to implicate HLA linked genes in controlling immune reactivity, they indicate that such control is polymorphic and not simply mediated by a single HLA-D gene. Moreover, not all inheritance patterns of responsiveness could be explained by a requirement for gene complementation. Three possibilities could explain a failure to show a significant association between a single HLA-D gene product in the general population and reactivity to synthetic polypeptides. First, it is possible that genes separate from, but closely linked to HLA-D actually represent the human Ir genes for reactivity to (T,G)-ALand (H,G)-AL. Likely candidates could be HLA-DS (DC) genes. According to our analysis, responsiveness to (T,G)-AL or (H,G)-AL in the study by Hsu et al. did
86
THOMAS A. GONWA ET AL.
not correlate with the expression of a defined DS or DC molecule (i.e., DC-1, MT-3). A second possibility is that the response to (H,G)-ALand (T,G)-AL is encoded for by HLA genes which are not closely linked to HLA-DR or HLA-DS (DC) (e.g., HLA-SB). This possibility cannot be excluded. A third possibility is that the response to these two synthetic polypeptides is dependent on epitopes shared by distinct families of HLA-DR (or HLA-DS) molecules. In mice, it can be demonstrated that the immune response to a single synthetic antigen is controlled by genes present in several distinct H-2 haplotypes. For example, Ir genes associated with the H-2 haplotypes a, b, d, f, r, u, and v each determine responsiveness to GAT (Dorf, 1981).Although HLA-DR molecules are serologically distinct as defined by certain typing reagents, they may share epitopes which are important in determining immune reactivity to a specific antigen. Stated in another way, HLA-DR1, 4,and 7, for example, might share a common epitope necessary for presenting (T,G)-AL to reactive T cells. This possibility is consistent with the observation that the frequency of immune reactivity in the general population to any of the synthetic antigens tested (e.g., approximately 50%) is greater than the frequency of an individual HLA-DR determinant. It is also consistent with the observation that some serologically defined supratypic determinants actually reside within HLA-DR molecules defined as distinct by conventional typing reagents. Although HLA-DR molecules have been used as an example here, a similar consideration applies to HLA-DS molecules. Structurally distinct HLA-DS molecules can be demonstrated to share common, serologically defined epitopes (Shackelford et aZ., 1981). Several groups of investigators have studied the association between HLA phenotypes and the immune response to either environmental antigens or antigens used for routine immunizations. Marsh and colleagues (1981, 1982a,b) investigated the association between IgE and IgG antibodies with specificity for a highly purified component of ragweed pollen (Ra5) and HLA-D or HLA-DR types in 447 Caucasians who were naturally exposed to ragweed. Seventy-nine to 85% of individuals with IgG antibodies to Ra5 were HLA-D2+ while 93 to lo@% of individuals with IgE antibodies to Ra5 were HLA-D2+. The frequency of HLA-D2 in individuals Iacking anti-Ra5 antibodies was approximately 20%. It was noted that the association between antibodies to Ra5 and HLA-D2 was stronger than that seen with the serologically defined HLA-DR2. Specific MB or MT types were not associated with antibody positive, HLA-DR2-, D2-, or antibody positive HLA-DR2+, D2- phenotypes. Whether this finding indicates that
HUMAN
Ir
GENES
a7
genes closely linked to, but distinct from, those coding for HLA-DR actually constitute Ir genes for antibody responses to Ra5 is not known. It is interesting to note that Marsh and his group were not able to demonstrate a convincing linkage between IgE antibodies until they utilized a very pure Ra5 preparation (>99.% pure). This underscores the necessity for using simple, defined molecules with a single or limited number of antigenic determinants when analyzing the influence of Ir genes. Sasazuki and collaborators (l978,1980a,b) examined the influence of Ir genes on determining reactivity in the Japanese population to three antigens, tetanus toxoid, schistosomal worm antigen, and streptococcal cell wall antigens. Each of these complex antigens contains several different antigenic determinants and thus the immune response to the whole molecule would not be controlled by a single Ir gene. In order to circumvent this and examine the T proliferative response to only a single immunodominant determinant, the authors examined reactivity occurring in response to in vitro stimulation with low concentrations of each antigen (0.2-1 kg). For both tetanus toxoid and schistosomal worm antigen, a low response in the general Japanese population was associated with HLA-Dwl2. Seventy and 58% of low responders to schistosomal worm antigen and tetanus respectively were HLADw212. The respective frequency of HLA-Dw12 in high responders to each antigen was 13 and 11%. Low responsiveness appeared to be controlled by a dominant gene in that all of the low responders were heterozygous. HLA-Dw 12 individuals were not unresponsive to all antigens. They demonstrated normal reactivity to diphtheria toxin and C . albicans. Although the same group of investigators could demonstrate, in families, HLA-linked control of unresponsiveness to immunodominant determinants in streptococcal cell walls, they were not able to show an association between a specific HLA-D gene product in the general population and responsiveness to the same antigen. Sasazuki et at. interpreted these studies as indicating the presence of a dominant immunosuppressor gene which, during natural exposure to three antigens tested, caused preferential activation of suppressor T cells. This conclusion was supported by three findings (Hays et al., 1982). First, T cells from low responders to streptococcal cell wall did not respond to the antigen even when mixed with antigen presenting cells from haplotype-identical high responders. Second, purified T cells from low responders inhibited streptococcal cell wall reactivity when added to haplotype-identical, high responder cells. Finally, removal of suppressor effector cells from low responders by panning
88
THOMAS A. GONWA ET AL.
with the monoclonal antibody Leu 2a resulted in the appearance of streptococcal wall reactivity among the remaining cells. (The Leu 2a determinant is displayed by the T cell population containing suppressor effector cells.) Whether or not similar T dependent suppressive influences are also involved in determining low reactivity to tetanus toxoid and schistosomal worm antigens associated with HLA-Dw 12 linked genes is not known. Another system in which HLA-D linked genes appear to influence the expression of immune reactivity by dictating the relative activity of helper vs suppressive influences is the Ir gene control of collagen reactivity (Solinger et al., 1982; Solinger and Stobo, 1982). These studies were initiated to determine the frequency of T dependent reactivity to collagen in patients with rheumatoid arthritis. The results of these studies indicated that while collagen induced production of the lymphokine, leukocyte inhibition factor (LIF), could be detected in 90% of patients with rheumatoid arthritis, it could also b e detected in approximately 30% of normal controls without any evidence of synovitis. Collagen induced production of LIF was demonstrated to require interactions between T cells and M4, was not specific for any single type of collagen, and could be elicited by the collagen-like synthetic polypeptide (Gly-Pro), which manifests no tertiary helical structure. Since rheumatoid arthritis is significantly associated with the HLA-DR phenotype, HLA-DR4, the association between collagen responsiveness and HLA-DR4 was investigated in both patients and normals (Table 111). All HLA-DR4+ individuals, either patients or COLLAGEN
TABLE I11 RESPONSIVENESSAND HLA-DR4 POSITIVITP Responders
HLA-DR phenotype
Rheumatoid arthritis
Nonresponders Rheumatoid arthritis
Others
~~
HLA-DR4+ HLA-DR4Totals
11 5
12 3
0 2
31
Others
Totals
0
23 38
~
28
30 ~
This is a 2 x 2 analysis of the relationship between collagen reactivity and the presence of the HLA-DR4 phenotype in patients with rheumatoid arthritis as well as in individuals with other forms of arthropathies and normals (these latter two groups are referred to as “others”). Only individuals in whom it was possible to assay for both collagen-induced LIF production and HLA-DR phenotypes are included. The xa for the relationship between collagen responsiveness and HLA-DR4 is 33.7, with a p value of less than 0.0001.
HUMAN
Ir
GENES
89
normals, were collagen responders. Of the 31 collagen responders, 23 were HLA-DR4+. This association is highly significant, p < 0,001,and suggests that genes linked to those coding for HLA-DR4 determine the expression of reactivity to collagen-like determinants inherent in the linear polypeptide Gly-Pro. One interpretation of these studies is that the HLA-DR4 molecule, or another Ia molecule encoded by closely linked genes, is the only human Ia molecule capable of restricting immune reactivity to collagen. HLA-DR4- individuals would be nonresponders by virtue of the fact that their accessory cells lacked the appropriate Ia determinant. Alternatively, HLA-DR4- individuals might contain T cells potentially reactive to collagen which are inhibited by specific suppressive influences. In this situation, HLA-DR4- individuals would be nonresponders by virtue of a predominance of suppressor, and not an absence of reactive, T cells. In order to distinguish between these two possibilities, advantage was taken of the fact that in some systems suppressive influences are radiosensitive while LIF production is radioresistant. Peripheral blood mononuclear cells (PBMC) from 20 HLA-DR4- collagen nonresponders were irradiated with 1000 rads and then assayed for LIF production in response to challenge with collagen. In each case the irradiated PBMC manifested collagen reactivity (Solinger and Stobo, 1982). Two points concerning the appearance of this reactivity among irradiated PBMC should be emphasized. First, it was specific for collagen in that there was no increase in the mean response of the irradiated PBMC to another antigen, C . albicans. In addition, irradiation did not result in the appearance of reactivity to purified protein derivative among the PBMC of two individuals who were unresponsive to this antigen. Second, the cellular requirements for and specificity of reactivity among the irradiated PBMC were identical to those noted among nonirradiated HLA-DR4+ individuals. Collagen-induced LIF production required both T cells and M$J and could be elicited by the collagen-like polypeptide (Gly-Pro),. If the appearance of reactivity to collagen among irradiated PBMC represents the elimination of suppressive influences, then it should be possible to inhibit reactivity among the irradiated cells by the addition of unresponsive, autologous nonirradiated PBMC. Indeed, this proved to be the case (Solinger and Stobo, 1982). Moreover, suppression requires the presence of T cells. Two other findings indicate that the absence of apparent collagen reactivity among HLA-DR4- individuals is due to the presence of suppressive influences. First, fractionation of T cells from' HLA-DR4-
90
THOMAS A. CONWA ET AL.
collagen nonresponders on a 5-step discontinuous bovine serum albumin density gradient results in collagen reactivity among the high density population (Solinger and Stobo, 1982). Addition of low density T cells to this high density population suppressed this collagen reactivity. Second, treatment of PBMC from collagen nonresponders with the monoclonal antibody, OKT8, and complement (a procedure which depletes suppressor effector cells) resulted in the appearance of collagen reactivity among the remaining cells (Solinger and Stobo, 1982). In this system, therefore, the HLA-D linked expression of collagen reactivity represents the influence of these genes on determining the relative activity of collagen specific suppressive influences. Collagen reactive suppressor cells could be detected in association with each HLA-DR phenotype tested except HLA-DR4 (i.e., HLA-DR 1 , 2 , 3 , 5 , 7 , and 8). All HLA-DR4+ collagen responders except for one individual were heterozygous at the HLA-D locus. Their MHC, therefore, contains one of the alleles expressed in HLA-DR4- nonresponders. Irradiation (1000 R) of PBMC from HLA-DR4+ individuals did not result in any increase in reactivity to collagen suggesting that collagen reactivity in these individuals reflects an absolute absence of suppressive influences and not simply a reactive predominance of reactive over suppressor cells. These findings suggest the following two models to link HLA-DR4 positivity with collagen reactivity. First, generation of collagen specific suppressive influences might require complementation between two genes in the trans position. One of these genes would be linked to those coding for each HLA-DR type except HLA-DR4. Therefore, only one of the two required genes would be present in HLA-DR4 heterozygotes. Second, HLA-DR4 linked genes could code for events which prevent either the generation or activation of collagen reactive suppressive cells. This would be similar to the recently described contrasuppression circuit in mice in which it can be demonstrated that Ir genes can modulate immune reactivity by determining activity among cells capable of “suppressing” the activity of suppressor T cells (Gershon et al., 1981). Each of these possibilities is consistent with the observation that collagen reactivity and thus the failure to express collagen reactive suppressor cells is inherited as a dominant trait. In these studies, an association between HLA-D gene products (i-e., HLA-DR4) and expression of collagen reactivity was demonstrated. However, there were several HLA-DR4- collagen responders. Therefore, it is possible that the true collagen Ir genes occupy a locus which is distinct from, but closely linked to HLA-DR. The finding that rheumatoid arthritis is more closely associated with the HLA-DS mol-
HUMAN
Ir
GENES
91
ecule, MT-3, than with HLA-DR4 supports this (Collier and Stobo, unpublished observations). Over the past decade it has been possible to demonstrate a striking association between specific HLA-D gene products and clinical disorders in which a given immune response contributes to the expression of disease (Schwartz and Shreffler, 1980). The association between HLA-DR2, Goodpasture’s syndrome, and the antibody response to basement membrane antigens in lung and kidney is but one example (Rees et al., 1978). In these associations, it has been hypothesized that Ir genes linked to the HLA-D locus determine responder status for the immune reactivity involved in the expression of the disease. However, for most associations, this has not been proven. For example, it has not been demonstrated that HLA-DR2 linked genes confer responder status for immune reactivity to basement membrane antigens. Only in the case of rheumatoid arthritis have HLA-D linked genes been implicated in an immune reactivity (i.e., T cell reactivity against collagen) which may contribute to the clinical manifestations of the disease. Several different autoimmune diseases each characterized by immune reactivity to a different auto-antigen are associated with the HLA-DR phenotype, HLA-DR3. These include systemic lupus erythematosus, Sjogren’s syndrome, dermatitis herpetiformis, myasthenia gravis, Graves disease, juvenile onset diabetes, Addison’s disease, and chronic active hepatitis (Schwartz and Shreffler, 1980). In this association, it has been hypothesized that genes linked to those coding for HLA-DR3 cause a generalized increase in immune reactivity which then provides the basis for a specific autoantibody response. This concept receives support from the demonstration that normal HLA-DR3 individuals exhibit immune hyperresponsiveness as manifest by an increase in spontaneous immunoglobulin production among peripheral blood mononuclear cells (Lawley et al., 1981; Ambinder et al., 1982). Two hypotheses have been provided to explain this immunologic hyperresponsiveness. The first is that the HLA-DR3 phenotype is associated with a generalized decrease in suppressive influences which normally serve to dampen immune reactivity. Ambinder et al. (1982) demonstrated diminished suppressor activity in normal HLA-DR3+ when compared to normal HLA-DR3- individuals. Four of 11 HLA-DR3+ individuals lacked any detectable suppressive influences. The assay system measured the ability of Con A activated T cells to inhibit immunoglobulin secretion by autologous peripheral blood mononuclear cells. A second hypothesis invoked to explain enhanced immune reactivity in HLA-DR3+ subjects is that there is decreased degradation of
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THOMAS A. GONWA ET AL.
antigen thus resulting in a persistent antigenic challenge. Legrand and colleagues (1982) studied the catabolism of sheep red blood cells by the peripheral blood macrophages of 100 normal individuals and demonstrated that 50% of individuals with diminished antigen degradation were HLA-DR3+. I n contrast, only )3% of individuals with rapid antigen degradation were HLA-DR3+. IV. Conclusions
The structural studies clearly indicate the existence of human l a molecules which are similar to those delineated in mice. However, it appears that there may be more human (4-6) than murine (2-3) Ia molecuIes. The functional studies indicate that HLA-DR and HLA-DS molecules can function as restriction elements which determine reactivity to simple antigens as well as immunodominant determinants present in complex antigens. The mechanisms by which human l a molecules control the expression of immune reactivity have only begun to b e explored. The available studies implicate these molecules in determining the relative activity of helper vs suppressive influences in controlling specific immune reactivity as well as generalized, i.e., polyclonal antibody formation. Future studies will generate genetic and molecular maps characterizing the whole range of human Ir genes and Ia molecules and indicate how they function to modulate immune reactivity in humans.
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Scher, I., Beming, A. K., Strong, D. M., and Green, I. (1975).J. fmmunol. 115,36. Schwartz, B., and Shreffler, D. (1980).In “Clinical Immunology” (C. Parker, ed.), p. 49. Saunders, Philadelphia, Pennsylvania. Schwartz, R. H. (1978). Scand. J . Immunol. 7 , 3 . Shackelford, D. A., and Strominger, J. L. (1979).J.E x p . Med. 151, 144. Shackelford, D. A., Mann, D. L., vanRood, J. J., Ferrara, G. B., and Strominger, J. L. (1981). Proc. Natl. Acad. Sci. U.S.A. 78,4566. Shaw, S., Kavathas, P., Pollach, M. S., Charmot, D., and Marvas, C. (1981). Nature (London) 293,745. Shaw, S., DeMars, R., Schlossman, S. F., Smith, P. L., Lampson, L. A., and Nadler, L. M. (1982).J.E x p . Med. 156, 731. Silver, J. (1981). CRC Crit. Reu. Immunol. 2,222. Silver, J., and Ferrone, S. (1979). Nature (London) 279, 436. Silver, J., and Ferrone, S. (1980).lmmunogenetics 10, 295. Silver, J., and Russell, W. A. (1979).Nature (London) 279,437. Silver, J., Walker, L. E., Reisfeld, R. A., Pellegrino, M. A., and Ferrone, S. (1979). MoZ. Immunol. 16, 37. Snary, D., Barnstable, C., Bodmer, W. F., Goodfellow, P., and Crumpton, M. J. (1976). Cold Spring Harbor Symp. Quant. B i o l . 41, 379. Solinger, A., and Stobo, J. D. (1982).f. Immunol. 129, 1916. Solinger, A., Bhatnagar, R. S., and Stobo, J. D. (1982). Proc. Natl. Acad. S c i . U.S.A. 78, 3877. Springer, T. A., Kaufman, J. F., Siddoway, L. A., Mann, D. L., and Strominger, J. L. (1977a).J . B i o l . Chem. 252,6201. Springer, T. A., Kaufman, J. F., Terhorst, C., and Strominger, J. L. (197%). Nature (London) 268,213. Sredni, B., Volkman, D., Schwartz, R. H., and Fauci, A. S. (1981).Proc. Natl. Acad. Sci. U.S.A. 78, 1858. Steinmetz, M., Minuid, K., Horvath, S., McNicholas, J., Frelinger, J., Wake, C., Long, E., Mach, B., and Hood, L. (1982). Nature (London) 300,35. Stetler, D., Das, H., Numberg, J. H., Saiki, R., Shen-Ding, R., Mullis, K. B., Weissman, S. M., and Erlich, H. A. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 5966. Suciu-Foca, N., Weiner, J., Rohowsky, C., McKiernan, P., Susinno, E., and Rubinstein, P. (1978). Transplant. Proc. 10, 799. Suciu-Foca, N., Godfrey, M., Rohowsky, C., McKiernan, P., Susinno, E., and Broell, J. (1979).Transplant. Proc. 11, 1781. Tanigaki, N., Tosi, R.,Kogama, K., and Pressman, D. (1980). Immunology 39, 615. Termitjelen, A., Boeltcher, B., Burdleg, B. A,, D’Amaro, J., van Leeuwen, A., and van Rood, J. (1980). Tissue Antigens 16, 140. Tosi, R., Tanigaki, N., Centis, D., Ferrara, G. B., and Pressman, D. (1978)J. Exp. Med. 148, 1592. Tsuji, K., Nose, Y., Komori, K., Tajika, R., Nakagawa, M., Inouye, H., and Yamamura, M. (1979). Transplant. Proc. 11, 1792. Uhr, J. W., Capra, J. D., Vitetta, E. S., and Cook, R. G. (1979). Science 206, 292. van Rood, J. J., van Leeuwen, A., Termijtelen, A., and Keuning, J. J. (1976). Transplant. Reu. 30, 122. Wake, C. T., Long, E. O., and Mach, B. (1982a).Nature (London) 300,372. Wake, C. T., Long, E. O., Strubin, M., Gross, N., Accola, R., Carrel, S., and Mach, B. (1982b). Proc. Natl. Acad. Sci. U.S.A. 79,6979.
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Walford, R. L., Grosset, T., Smith, G. S., Zeller, E., and Wilkinson, J. (1975). Tissue Antigens 5, 196. Wiman, K., Larhammar, D., Claesson, L., Gustafsson, K., Schenning, L., Bill, P., Bohme, J., Denaro, M., Dobberstein, B., Hammerling, U., Kust, S., Serrenius, B., Sundelin, J,, Peterson, P. A., and Rask, L. (1982).Proc. Natl. Acad. Sci. U.S.A.79, 1703. Winchester, R. J., Fu, S. M., Wemet, P., Kunkel, H. G., Dupont, B., and Jersild, C. (1975). J . E r p . Med. 141,924. Young, E., and Engleman, E. G. (1980).J . Zmmunol. 125, 352.
ADVANCES IN IMMUNOLOGY, VOL. 04
Interferons with Special Emphasis on the Immune System1 ROBERT M. FRIEDMAN AND STEFANIE N . VOGEL Departments of Pothology and Microbiology, Uniformed Services University of the Healfh Sciences, Betherda, Morylond
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Interferon Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Actions of Interferons .................................. IV. Interferons and Defense against Viral Infections . . ......... V. Interferons and Other Mechanisms Related to Immunity and Inflammation .................................... VI. Antitumor Effects of Interferons in Animal Systems ...................... VII. Clinical Studies with Human Interferons ................................ References . . . . . . . . . . . . . ........................... ........
97 99 10 1 128
129 130 132 133
I. Introduction
Studies on interferons have become a special area of research. While in previous years a reasonably short review could give a complete summary of research on interferons, this is no longer possible; therefore, we shall attempt to highlight the areas of research on interferons that we feel are of special interest to immunologists. Interferons are induced, animal proteins. A variety of stimulating substances can act as interferon inducers and interferons inhibit a wide range of viruses by inducing an intracellular antiviral state; however, many interferons are species-specific in their antiviral activity (Isaacs and Lindenmann, 1957). Interferons were first described in 1957, but there is as yet no complete explanation of their induction, biological role, or biological activities. One reason for this is the impressive potency of interferons; the specific antiviral activity of human interferon, for instance, is more than lo9 international units per mg of protein (Rubinstein et al., 1978). With a molecular weight of approximately 20,000 this means that biological activity resides in 0.4 pg or lo7 molecules or 2 x M . This suggests that a few thousand molecules of interferon may induce an antiviral state; therefore, interferons are among the most active biological substances. As a consequence of this, The opinions or assertions contained herein are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or Department of Defense. There is no objection to its presentation and/or publication.
97 Copyright 0 1983 b y Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-022434-8
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interferon preparations with great antiviral activity contain very little interferon. Interferon assays were, until very recently, biological. They are based on the ability of a preparation to inhibit the production of a virus or a viral product (Buckler, 1977). Although sensitive, they are time consuming and relatively imprecise. Because of the inherent inaccuracies of biological assays, a two- or threefold inhibition of a viral function is considered barely significant to define a unit; therefore, a level of uncertainty is present which is usually intolerable in a biochemical or biophysical system. Recent progress in the purification of interferons and the availability of monoclonal antibodies to interferons will soon lead to their immunochemical assay. One other problem has been that there are many species of interferon (Burke, 1977; Youngner, 1977). There are three general types of human interferon, designated alpha, beta, and gamma. When stimulated with virus, leukocytes in cultures produce predominantly the species called alpha interferon. There are at least 14 distinct genes for human alpha interferon (Nagata et al., 1980). Most alpha interferons contain little or no carbohydrate. Human fibroblast cultures, when stimulated with viruses or a chemical inducer of interferon such as the double-stranded RNA polymer, polyriboinosinic acid :polyribocytidylic acid [poly(I : C)], produce an interferon that is immunologically distinct from alpha interferons, and designated as beta (fibroblast) interferon. It is important to note, however, that leukocytes can produce beta interferon under some conditions, and that fibroblasts can be stimulated to produce some alpha .interferon. Beta interferon, a glycoprotein, is more hydrophobic than alpha interferon, so that beta interferon adheres to hydrophobic ligands such as hydrocarbons, that do not interact with alpha interferons. Beta interferon, but not alpha interferons, bind to lectins such as concanavalin A. One other striking difference between alpha and beta interferons is in their species specificity of antiviral activity. While human beta interferon is usually species-specific and for the most part induces antiviral activity only in human cells, human alpha interferons induce activity in human as well as some animal cell cultures. The third type of interferon, gamma (immune, type 11, or T interferon), like beta interferon, is a glycoprotein that differs from alpha or beta interferons in several fundamental respects (Youngner, 1977). It is antigenically distinct from them, and is more labile to acid. Most alpha and beta interferons are quite stable at pH 2, while the antiviral activity of gamma interferon is significantly reduced by this treatment. Gamma interferon is produced by lymphocytes in response to mito-
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gens or exposure to an antigen to which the cell had already been sensitized. Gamma interferon is, therefore, a lymphokine which appears to play a role in the regulation of immune response, and possibly in the antitumor effects of interferon treatment. Macrophages must be present for the production of gamma interferon by normal T lymphocytes. Also, a T to T cell interaction involving interleukin 2 may be required (Farrar et al., 1981). B lymphocytes produce alpha, rather than gamma interferon, whereas lymphoblastoid cell lines produce mixtures of alpha and beta interferons. Genes of all three types of human interferon have been cloned in microorganisms. So far, single genes have been definitely described only for gamma (Grey et al., 1982) and beta (Taniguchi et al., 1980) interferons, although there are some reports that additional types of beta interferon may be present (Sehgal and Sagar, 1980). There are at least 14 human alpha interferon genes, some of which may be alleles, and others nonexpressed pseudogenes. The various alpha species differ from each other by at most 15 to 30% in amino acid sequence. In contrast, the amino acid sequences of alpha interferons differ by about 85% with that of beta interferon (Nagata et aZ., 1980). It is not understood why there are so many types of alpha interferon, or indeed why there are three major gene types for interferons. II. Interferon Production
Alpha or beta interferons can be induced in animals or in cell cultures by living or killed RNA or DNA viruses (Burke, 1977); in addition, a number of natural and artificial nonviral substances are effective inducers of interferon production. The best studied of these are natural double-stranded RNA forms from fungi or synthetically produced molecules such as poly(1: C) (Vilcek and Kohase, 1977). It is possible that double-stranded RNA molecules are such excellent interferon inducers because they are similar in structure to a natural product that is the actual signal for turning on interferon production by cells. Many, but not all, viruses contain double-stranded RNA as a structural element or produce it during the course of their replication processes. There is a diverse group of additional substances which also induce interferon production in cell cultures or in animals (Merigan, 1973; Grossberg, 1977). These include bacteria that grow intracellularly such as Brucella abortus, Listeria monocytogenes, or Hemophilis influenzae, rickettisiae, mycoplasmae, protozoae, clamydiae; microbial products such as lipopolysaccharides; organic polymers, such as pyran copolymers or polyvinyl sulfate; and a variety of low-molecular-
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weight substances such as cycloheximide, kanamycin, tilorone, and toluidine blue. Some of the above induce interferon production only in animals; others, in vivo and in tissue cultures. This group is so divergent in structure, however, that the inducers may act by causing the production and release of a common intracellular substance such as a cellular double-stranded RNA. There are in animal cells naturally occurring double-stranded RNA forms that are themselves good interferon inducers (Stern and Friedman, 1970). Alternatively, the diverse substances which induce interferons and double-stranded RNA forms may interact with similar receptors on the cell surface. Many types of cells make interferons. Both T, B, and non-T, non-B lymphocytes produce alpha interferons; macrophages and most fibroblast cultures can also be induced to make both alpha and beta interferons. T cells produce predominantly gamma interferon. The inability of some cell lines to produce interferons has not been explained, but one interesting system involves mouse teratocarcinoma cell cultures: when undifferentiated, these cells do not produce interferons; however, after differentiation, production of interferons can be induced (Burke et al., 1978). The control of interferon production is not well understood, but it is likely that it involves a repressor mechanism. Since the cellular content of interferon mRNA can be assayed by several methods, the mRNA forms for alpha and beta interferons have been shown to be poly(A)-rich, distinct 8-12 S molecular forms (Cavalieri and Pestka, 1977). The best evidence for a repressor mechanism for interferon production is the phenomenon of superinduction in human fibroblast cultures (Vilcek and Kohase, 1977). Superinduction is the greater than normal production of beta interferon in fibroblast cultures that are induced to form interferon in the presence of inhibitors of protein synthesis (cycloheximide or puromycin), or of RNA synthesis (actinomycin D), or processing (dichloro-l-D-ribofuranosylbenzimidazole, DRB). Increased production of interferon is observed when the inhibitors of protein synthesis are removed or, if actinomycin D is added, after exposure to the inducer. Superinduction is at least in part due to a prolongation of the half-life of interferon mRNA. This could be related to an inhibition of the production of a repressor of interferon mRNA function; the repressor may normally be made so that interferon synthesis is usually not constitutive. There does appear, however, to be at least one cell line in which interferon synthesis is semiconstitutive; in this case the proposed repressor of interferon production may be defective (Jarvis and Colby, 1978). Gamma interferon production, like that of other lymphokines, is
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generally induced by substances that stimulate a mitogenic response in lymphocytes; these include antigens to which the donor of the lymphocytes had been exposed. The antigens can be viral or nonviral in origin and may be bacterial products such as tetanus and diphtheria toxoids or PPD. Plant-derived mitogens, e.g., phytohemagglutinin A (PHA), pokeweed mitogen (PWM), or concanavalin A (Con A), as well as other mitogenic stimulants, such as staphylococcal enterotoxin A (SEA) or antilymphocyte serum, are also effective in stimulating in vitro gamma interferon production; however, a recent report has demonstrated production of gamma interferon in the absence of cell proliferation (Landolfo et al., 1981). Unfortunately, the purification of gamma interferon has been difficult and has only recently been achieved (Yip et al., 1982). As noted above, lymphocytes produce gamma interferon. T cells produce gamma interferon within 3 days of exposure to mitogens. Interferon production in this system is dependent on the presence of macrophages in the culture so that pure cultures of normal lymphocytes cannot be stimulated to produce gamma interferon; however, lymphocytes depleted of macrophages can still be stimulated by viruses to produce alpha interferons. In addition, certain T cell lines have been shown to be capable of producing gamma interferon in the absence of macrophages. It is not known what factor the macrophages must supply to lymphocytes in order for them to produce gamma interferon, but the type of the interferon is determined by the species of lymphocyte which produce the interferon, not by macrophages in the culture (Youngner, 1977). Ill. Actions of Interferons
1. The Antiviral State
The mechanism of interferon action, for the sake of discussion, may b e divided into two phases (Friedman, 1977).The first relates to how interferon treatment induces an antiviral state or other activities in cells; the second relates to how these induced states are expressed on various cellular activities such as virus growth, cell replication, or the immune response. In order to bring about its effects on cells, interferons must first interact with the plasma membrane (Grollman et al., 1978). The significant reaction is an initial binding of interferons that is not an energy requiring process. The bound interferon can be released from the cell surface without inhibiting the later development of an antiviral state. Interferons seem to bind to a specific cell surface receptor; evidence
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for the specificity of the binding site is the finding that interferon action can be blocked in human cells by an antibody to a product of chromosome 21 (Revel et aE., 1976) or competitively by thyroid stimulating hormone, chorionic gonadotropin, or cholera toxin. Since the last three appear to have a similar or identical binding site, it follows that interferon must bind to the same or very similar site as substances in this general group (Grollman et al., 1978). Current work with purified, radioactive interferons should help to elucidate the nature of the binding reaction and the binding site(s) (Auget, 1980). The location of the putative interferon binding site appears to be on the outer surface of the plasma membrane. This was determined by stimulating human fibroblasts to produce interferon in the presence of antibody to beta interferon. Antiviral activity failed to develop in cells producing interferon; therefore, interferon had to be externalized to induce an intracellular antiviral state (Vengris et al., 1975). The chemical nature of the interferon receptor appears to be a complex of both ganglioside and glycoprotein components (Besancon and Ankel, 1977; Chang et al., 1978). How these interact to bind interferon on the cell surface and transmit information to an intracellular site is not clear, since interferons bind to either gangliosides or glycoprotein components. The glycoprotein component may represent an activation or amplification site for the induction of intracellular antiviral activity (Grollman et al., 1978). Once interferon has interacted with its receptors on the cell surface it is not clear what steps follow immediately. It is uncertain whether interferon is taken up by the cell, although interferons bound to Sepharose beads are active in inducing an antiviral state; however, it is uncertain in such studies how tightly the interferons are bound to the carrier. For the most part the activity of interferons can be accounted for by reactions initiated at the cell surface, but further studies with purified interferons will be necessary to answer definitively the question of whether interferon uptake is required for its biological activities. The great specific activity of interferons suggests that one or fewer molecules per cell can induce an antiviral state.
2 . The Inhibition of Virus Replication The development of antiviral activity following treatment with interferon requires cellular protein and RNA synthesis and in human cells chromosome 21 (Tan et al., 1977). After human interferons are bound to a receptor, biochemical reactions occur. This results in the production of specific mRNA forms which are, in turn, translated to
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give rise to proteins related to the antiviral state. In cultures of interferon-treated, virus-infected cells, viral messenger RNA was not efficiently translated. In some cases where virion-associated transcriptases were present, interferon treatment did not inhibit viral mRNA synthesis. In cell-free protein synthesizing systems derived from mouse cells treated with low concentrations of interferon, there was no inhibition of viral mRNA translation, unless the cells had also been infected with a virus. This suggested that interferon treatment induced a potential antiviral state which was not fully developed until the cells were virus infected (Friedman, 1977). Furthermore, addition of minute quantities of double-stranded RNA to cell-free extracts from interferon-treated cells resulted in the inhibition of virus-directed protein synthesis (Kerr et d.,1974). This might be related to the requirement for viral infection of interferon-treated cells in order to demonstrate an inhibition of translation of viral mRNA, because in many viral infections double-stranded RNA species are produced. Treatment of cell extracts from interferon-treated cells with doublestranded RNA resulted in an increase in the activity of three substances (Fig. 1) that might be related to antiviral activity (Farrell et al., 1978). These are a protein kinase, a series of adenosine polymers having more than two adenosines with a 2'5' linkage, the most important of which in most animal cells is pppA2'p5'A2'p5'AoH (2'5'A for short), and a synthetase capable of producing 2'5'A. The protein kinase, an enzyme activated by double-stranded RNA in interferon-treated cells, phosphorylates the small subunit of the protein synthesis initiation factor eIF-2. This is consistent with several observations strongly suggesting that initiation of viral protein synthesis is inhibited after interferon treatment. The 2'5'A and yet another enzyme, an endoribonuclease, that is usually constitutive, are closely related in the following manner: 2'5' adenylate synthetase, that is induced following interferon treatment, is activated by double-stranded RNA. It uses ATP as a substrate to form 2'5'A polymers that in turn may inhibit virus protein synthesis b y activating the endoribonuclease. The latter may be the active element in inhibition of virus protein synthesis, because it can hydrolyze viral mRNA. Thus, there are at least two ways in which interferon treatment inhibits viral protein synthesis. It is uncertain which if either of these is the more significant in a given virus infection of interferon-treated cells; it is also possible that both contribute to an antiviral state. In fact, rather than being unique antiviral mechanisms, the processes employed in the interferon system may well be adaptations of the normal systems that control cell growth and differentiation. It was not entirely
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ROBERT M. FRIEDMAN AND STEFANIE N. VOGEL ENDONUCLEASE (Inacllwe) ENDONUCLEASE
SYNTHETASE
MET-1RNAf GTP elF-2 40s RIBOSOME COMPLEX
(PHOSPHORYUTED)
405 RIBOSOME
PHOSPHORYIATED (37K)
elF-2 (37K) (PROTEIN SYNTHESIS INITIATION FACTOR)
FIG.1. Double-stranded ribonucleic acid (dsRNA)-related steps in the mechanism of interferon action. In the presence of dsRNA and adenosine triphosphate (ATP), an active protein kinase with a molecular weight of about 67,000 (67K)is induced. The phosphate added to the kinase may be removed by a phosphatase that is inhibited in the presence of dsRNA. The function of the active protein kinase appears to be to add a phosphate group to a subunit with a molecular weight of 37,000 (37K)of protein synthesis initiation factor eIF-2. Ordinarily, eIF-2 acts together with a ribosomal subunit (40 S), initiator transfer RNA (Met-tRNA,), and guanosine triphosphate (GTP) to initiate protein synthesis. In the presence of the phosphosylated 37K subunit of eIF-2, the initiation of protein synthesis is inhibited. In the case of the 2‘,5’-A synthetase, the addition of dsRNA activates the enzyme which forms oligoadenylate polymers (2’,5‘A,,) from ATP. Several “degradases” may inactivate 2‘5‘A,, but, if it is not destroyed, the 2 ’ 5 ’ k interacts with an endonuclease that is present in most cells. The active endonuclease degrades messenger RNA (mRNA). This in turn inhibits protein synthesis by stopping the elongation of proteins. Thus, both of these pathways may converge to inhibit viral protein synthesis.
unexpected, therefore, to find that interferons also have effects on the immune system, and on the growth of uninfected cells (Gordon and Minks, 1981). There are, however, several reports of interferon inhibition of steps in the virus replication cycle other than at the level of virus protein synthesis. This has been especially noted in tumor virus replication. Interferon treatment results in a marked decrease in the production of some oncogenic viruses and in the efficiency of cell transformation by virus, but, while interferons were originally thought to inhibit tumor viruses through the same process as that involved in the inhibition of other viruses, the mechanism of interferon action for at least some of these viruses appears to be more complex. Treatment of cells with
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interferon at various times after infection with SV40 virus yielded different results (Friedman, 1977; Revel, 1979). If interferon was added to cells prior to SV40 infection, virus production and viral T-antigen synthesis were inhibited, effects possibly resulting from blocking the transcription of SV40 DNA molecules by the cell RNA polymerase (Metz et al., 1976). Infection of these cells with SV40 DNA instead of intact virions, however, overcame the antiviral effect of interferon, which suggested that interferon was also inhibiting SV40 uncoating (Yamamoto et uZ., 1975). The latter results, however, are difficult to interpret since the large amount of infectious D N A used in these studies may be analogous to infecting with multiplicities of SV40 high enough to overcome the interferon block (Revel, 1979). Addition of interferon to cells during the early phase of SV40 infection (before viral DNA synthesis), however, failed to inhibit production of SV40 early RNA; but, addition during the late phase of the virus lytic cycle resulted in inhibition of viral protein synthesis at the level of translation with no inhibition of viral mRNA synthesis or of host cell protein synthesis (Revel, 1979). Transcription and translation of the SV40 T-antigen were not sensitive to interferon treatment in SV40transformed cells, although T-antigen production was sensitive during the late phase of the SV40 lytic cycle. Therefore, a single viral gene may be sensitive to interferon in certain phases of virus growth and resistant in others. Similar to SV40-transformed cells, treatment of cells acutely or chronically infected with C-type leukemia or B-type mouse mammary tumor virus (MMTV) viruses resulted in no inhibition of viral RNA or protein synthesis. RNA tumor viruses are, however, sensitive to an interferon mechanism that appears to act at a late stage of virus maturation. In some systems, interferon treatment resulted in marked inhibition of virus release, while in others, virus particle production appeared normal; however, the released virus was deficient in infectivity (Friedman, 1977). The data suggest that interferon may be inhibiting these viruses by altering the membrane through which these viruses are exported out of the cell, or b y altering cellular or viral protein(s) necessary for proper maturation of the virus particle. Studies with cells infected with an adeno-SV40 hybrid virus, that contains a combination of the interferon-sensitive (SV40 virus) and an interferon-insensitive (adenovirus) genome, have yielded interesting findings. I n simultaneous infection of cells with both complete viruses the sensitivity of adenovirus or of SV40 T-antigen production was characteristic of infection with either virus separately. I n contrast, in cells infected with an SV40-adenovirus hybrid, production of both
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T-antigens was as resistant as adenovirus T-antigen production in infection with adenovirus alone (Oxman et aZ., 1967). The SV40 genome is covalently linked to the adenovirus genome in the hybrid or to cellular DNA in SV40-transformed cells. The mRNA produced by the. integrated SV40 genome contained host sequences, and the mRNA of the hybrid had adenovirus and SV40 sequences. The resistance to interferon treatment of SV40 T-antigen production directed by an integrated viral genome may indicate that the primary sequence in the mRNA that specifies the viral protein does not determine sensitivity to interferon, but that other sites on the genome such as those concerned with initiation or control of genetic expression may be the loci of interferon action. This may also explain the lack of interferon-induced inhibition of murine RNA tumor virus protein production in chronically infected cells where the proviral DNA is integrated into host DNA. In experimental animal systems with virally induced tumors, such as hamsters inoculated with pol yoma virus or chickens infected with Rous sarcoma virus, interferon may inhibit development of tumors by inhibiting virus multiplication or an early virus-dependent step involved in cell transformation. It is unlikely, however, that the antiviral activities of interferons are responsible for inhibition of tumors that are apparently not virus-induced, or tumors in which virus replication is not involved in the progression of development. Interferons may inhibit replication of the tumor cell itself or may have effects on the host’s capacity for tumor rejection. For example, L1210 leukemia cells, that were resistant to the cell growth inhibition activity of interferons in uitro, could be inhibited in viuo. This suggested that the antitumor effect of interferons in these mice was not a result of direct inhibition of tumor cell multiplication and might be the result of interferon action on the immune system (Gresser et aZ., 1972). On the other hand, human interferon is effective in inhibiting the growth of human tumor transplants in nude mice. This suggested a direct antitumor effect of interferons (Taylor-Papadimitriou, 1980).
3. Znhibition of CeZZ Proliferation Since purified interferons inhibit both virus replication and cell multiplication (Knight, 1976; Gresser et aZ., 1979), it is now accepted that interferons inhibit the growth of a wide range of cell types. The sensitivity of cells to the growth inhibitory effects of interferons ranges from very sensitive to resistant and the same cell types can show varying sensitivities under varying assay conditions: for instance, growth of colonies in agar is more sensitive than growth on a solid support and
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sparsely seeded cultures appear to be more sensitive than the same cells seeded at high densities (Taylor-Papadimitriou, 1980). Tumor cells may be more sensitive to the growth inhibitory effects of interferons than are normal cells. The multiplication of HeLa cells was inhibited to a greater extent than that of human fibroblasts (Friedman, 1977); similarly, the inhibition of the multiplication of human osteosarcoma cells was greater than that of nontumor cells (Strander and Einhorn, 1977). In contrast, the multiplication of oncornavirus carrier cells (Billiau, 1975), or of X-ray transformed cells (Brouty-Boye et al., 1979) derived from C3H fibroblasts was less inhibited than that of nontransformed cells. Moreover, in the comparison of normal human mammary epithelial cells to breast cancer cells, or of 3T3 cells to SV40-transformed 3T3 cells, the normal cells were at least as sensitive to the growth inhibitory effects as the analogous transformed cells (Balkwill et al., 1978). There are also studies that suggest similar effects in uiuo. Interferons inhibited the multiplication of tumor cells and normal cells in animals (Gresser and Bourali, 1970b), of allogeneic lymphocytes and syngeneic bone marrow cells, when these were transferred into irradiated mice (Cerottini et al., 1973), and of regenerating liver cells in partially hepatectomized mice (Frayssinet et al., 1973). There are many possible sites at which interferons could inhibit the complex process of cell multiplication. Different approaches are being used to examine aspects of control of cell growth and what effects interferons have on these processes. These include (1)examination of interferon’s effects on the cell cycle; (2) study of cellular functions that may be involved in control of cell growth or cellular parameters that are altered in malignant cells; and (3) determination of whether the molecular mechanisms thought to be implicated in interferon’s antiviral activities play any role in its antiproliferative activities.
4 . Znterferons and the Cell Cycle Interferons do not arrest cells that are dividing asynchronously but synchronizes them into one phase of the cell cycle. Interferon treatment reduced the rate of entry into S phase and increased the duration of the G, and S + G2 phases (Balkwill and Taylor-Papadimitriou, 1978), so that the increased length of cell cycle time observed in treated cultures (Collyn d‘Hooghe et al., 1977) is probably due to the extension of these phases. Quiescent cells that can be stimulated to divide synchronously by mitogens provide an excellent system to study how interferon affect the events in G, crucial to the initiation of DNA synthesis. The events that occur after stimulation of cells with mitogens, but precede DNA synthesis, can be divided into “early” and “late.”
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Early events occur within minutes of mitogen stimulation, they are not dependent on cellular protein synthesis, and they include changes in intracellular CAMP levels and increased uptake of ions, nucleotides, and sugars. Late events, that occur hours after mitogen stimulation, are protein synthesis dependent; they include secondary increases in sugar and ion uptake and increases in the activities of certain enzymes. One of these enzymes, ornithine decarboxylase (ODC), catalyzes the first rate-limiting step in the synthesis of polyamines, that are involved in the regulation of various cellular reactions, including transcription and translation. Increases in ODC activity are associated with the proliferative response of cells in culture, in tumors, and also with tumor promotion (Janne et al., 1978). Interferon treatment of quiescent Swiss 3T3 cells at the time of mitogen stimulation had no effect on the early increase in uptake of ions, nucleosides, or sugars; however, it had a differential effect on protein synthesis-dependent events: induction of ODC activity was inhibited, while the second phase of stimulation of 2-deoxyglucose uptake was not affected. These results were observed with addition of serum, of a combination of growth factors, or of a tumor promoter serving as a mitogen (Sreevalsan et al., 1979, 1980). Similar findings were recently reported on the inhibitory effect of interferon on the induction of S-adenosyl-L-methionine decarboxylase, another enzyme involved in polyamine biosynthesis (Lee and Sreevalsan, 1981). There seems then to be a common interferon-sensitive' step involved in the stimulation of DNA synthesis by serum, tumor promoters, or growth factors. Further evidence for an interferon-sensitive step in DNA synthesis comes from the study of two clones of Swiss 3T3 cells with differential sensitivities to both the antiviral and antiproliferative activities of interferons. One clone was more sensitive to interferon in terms of inhibition of cell division, DNA synthesis, and induction of ODC activity, when interferon was added at the time of serum stimulation. Under the same conditions, NIH 3T3 cells sensitive to the antiviral effect of interferon against murine leukemia virus exhibited no inhibition of cell division, DNA synthesis, or ODC induction (Czarniecki et al., 1981). There is, however, evidence that interferoninduced inhibition of DNA synthesis is not dependent on the inhibition of ODC activation also caused by interferon. Concomitant inhibition of DNA synthesis and of activation of the enzyme was observed only when polypeptide hormones were used as stimulants; for instance, cholera toxin-stimulated DNA synthesis was inhibited, while toxin-stimulated ODC activation was not (Lee et al., 1980). These results indicated that a poor correlation exists between the
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activation of ODC and DNA synthesis in quiescent 3T3 cells, that are stimulated to proliferate.
5. Eflects of Interferons on Other Cellular Parameters Information concerning the many varied effects of interferons on cell structure and function is increasing rapidly (for reviews see Gresser, 1977; Stewart, 1979; Taylor-Papadimitriou, 1980). Interferon treatment of cells resulted in significant alterations of the cell surface including increased expression of certain cell surface antigens or receptors (see below), increased net negative charge on the cell surface (Knight and Korant, 1977), decreased thymidine uptake (Brouty-Boye and Tovey, 1977), and alteration in the density of the plasma membrane (Chang et al., 1978). Interferon treated cells also exhibited changes in the binding of cholera toxin and thyrotropin (Kohn et al., 1976). Alterations in the cell membrane resulting from interferon treatment may play a role in the inhibition of murine leukemia viruses discussed earlier. Additionally, SV40 transformed cells that normally produce and release plasminogen activator (PA) seem to accumulate PA at the plasma membrane after interferon treatment (Schroeder et ul., 1978), so that interferon might alter the cell surface in a manner that prevents C-type virus shedding. There is, however, an increase in secretion of plasminogen activator after exposure of human macrophages to leukocyte interferon (Hovi et al., 1981). Since cell to cell contact plays a role in cell growth regulation, alterations induced in the plasma membrane could well cause alterations in cell DNA synthesis and growth. A direct negative effect on cell growth would, of course, be an ideal mechanism of action for an antitumor agent. In many respects interferons would seem to be growth control factors; but, they are an unusual class of biological substances, since almost all growth factors that have been studied stimulate cell replication. Indeed, many of the “toxic” effects observed in interferon therapy, such as leukopenia, thrombocytopenia, and hair loss, may be extensions of its growth inhibitory properties. Changes in the cytoskeIeton, composed of microtubules, microfilaments (actin and myosin), and the cytoplasmic matrix, have been observed in conjunction with transformation. Such changes may be a cause of or result in transformation. The cytoskeleton may be altered by interferon treatment so that microfilament and fibronectin organization are changed and there is increased rigidity of the plasma membrane lipid bilayer (Pfeffer et al., 1979, 1980a,b). Drugs such as colchicine, that disrupt the cytoskeleton, inhibit the development of the
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antiviral action of interferon, while compounds such as sodium butyrate, which has been reported to promote cytoskeletal organization, appear to enhance interferon action (Taylor-Papadimitriou, 1980). The antiviral activities of interferons were shown to be potentiated by dibutyryl-CAMP (Friedman and Pastan, 1969). Interferon-treated cells contained increased levels of cAMP (Weber and Stewart, 1975; Meldolesi et al., 1977). Since it appeared that increased levels of cAMP might be involved with inhibiting cell growth rates of several systems (Rozengurt, 1979), it was thought that membrane adenylate cyclase activities might play a role in the antiproliferative response to interferons; however, a longer exposure to interferon was necessary for alteration of cAMP levels than for detection of cell growth inhibition (Tovey et al., 1979). Additionally, growth of Schwann cells, human keratinocytes, and human mammary epithelial cells is stimulated by both cholera toxin and cAMP analogs (Taylor-Papadimitriou, 1980); therefore, the relationship between interferons and cAMP appears to be more complex than originally suggested.
6. Antiviral and Antiproliferative Activities of Interferons One important question is whether the proteins induced by interferon treatment are involved in interferon’s cell growth inhibitory activity. In one report, mouse embryonal carcinoma stem cells that were insensitive to the antiviral and antiproliferative effects of interferon did not demonstrate kinase induction after interferon treatment. After differentiation, kinase activity was induced and growth of these cells was inhibited (Wood and Hovanessian, 1979); however, many changes occur within a cell upon differentiation, and it is difficult to assign responsibility for acquired sensitivity to any one of these changes. The role of the synthetase in these antiproliferative effects has been studied by examining the inhibitory effects of 2’51 directly on cell protein synthesis (Williams and Kerr, 1978; Hovenessian et al., 1979). The dephosphorylated trimer, that can apparently pass through the cell membrane, has also been shown to inhibit DNA synthesis in lymphocytes stimulated by lectins (Kimchi et al., 1979). NIH 3T3 cells were sensitive to the antiviral effects of interferon against murine leukemia virus; however interferon treatment resulted in no antiviral activity against a lytic virus such as encephalomyocarditis virus (EMC), and no inhibition of cell division, or DNA synthesis. Both synthetase and kinase activities were induced by interferon but the endonuclease ordinarily activated by 2’5’ oligoadenosine appeared to be absent. The results suggested that kinase activity is not sufficient for cell growth inhibition, and also indicated a possible role of the 2’5’A activated
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pathway in the antiproliferative activities of interferons (Epstein et al.,
1981).
7 . Effects of Interferons on the Immune System. Effects of Interferons on the Humoral Immune System The immunoregulatory functions of interferons were recognized only within the past decade. The effects of interferons on the production of antibodies were among the first examined. Interferons can have both suppressive or enhancing effects on the production of antibodies, depending upon the dose and time of administration of interferon relative to antigenic challenge. Early in vivo studies indicated that when low doses of interferon were administered simultaneously with sheep erythrocytes (SRBC), an augmented antibody response was seen; however, if high doses were administered with the antigen, the antibody response was depressed (Braun and Levy, 1972). Subsequently, it was observed that when interferon was administered to an animal prior to antigenic challenge, both the primary antibody response (Chester et al., 1973; Merigan et al., 1975) as well as the induction of an anamnestic response (Brodeur and Merigan, 1975) were suppressed. The antigens used in these various studies were shown to fall in both T-dependent (i.e., SRBC) and T-independent (i.e., lipopolysaccharide) classes. The results of these in vivo studies were confirmed and extended in vitro. Gisler et al. (1974) and Johnson et al. (1974, 1975) demonstrated interferon-mediated suppression of the antibody response to SRBC in vitro when the interferon was added simultaneously with the antigen, but found an augmentation of the antibody response if added to the cultures 2-4 days after the antigenic stimulus. Pretreatment of purified B cells with interferon and the subsequent co-culture of these cells with T cells, macrophages, and antigen, resulted in significant inhibition of antibody production (Gisler et al., 1974), suggesting a direct suppressive effect on B cells. Interferon was found to reduce dramatically the number of clones that proliferate in response to antigen (Booth et al., 1976a,b).The in vivo findings were further confirmed in vitro using the T-independent antigen, lipopolysaccharide (LPS), as the antigenic stimulus (Johnson et al., 1975), although the T-dependent anti-SRBC response was found to be more readily induced by interferon (Johnson and Baron, 1976; Johnson, 1977). The finding that the T-dependent, B cell antibody response was more sensitive to suppression by interferon than the T-independent response to LPS suggests differences in the mechanisms by which each antigen initiates proliferation. Recent findings by Attallah et al. (1980), that
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interferon fails to suppress pokeweed mitogen-driven, human B cells to produce plaque-forming cells or to secrete antibody, suggest that polyclonal activiation may overcome the antiproliferative effects of interferon on B cells. Gamma (Type I1 or immune) interferon was subsequently tested for its effects on the antibody response. It is important to remember that until very recently, all gamma interferon preparations contained a mixture of a number of lymphokines. Several different groups have demonstrated that gamma interferon also suppresses the in vitro and in vivo immune response to SRBC (Virelizier et al., 1977; Sonnenfeld et al., 1977; Lucero et al., 1980). Two of these reports found that on the basis of antiviral activity, gamma interferon was significantly (20-250 times) more suppressive than preparations of alpha or beta interferons (Virelizier et al., 1977; Sonnenfeld et al., 1977). As observed for alpha and beta interferon preparations, gamma containing preparations also enhanced the production of anti-SRBC forming cells if administered in vivo or in vitro 48 hours after antigen stimulation (Sonnenfeld et al., 1978). The in vitro response to LPS was also inhibited by gamma interferon-containing preparations 24 hours prior to antigen; however, the gamma preparations failed to augment the PFC response when added to LPS-stimulated cultures at 48 hours. These findings (1) strongly support the role of gamma interferon as an immunoregulatory lymphokine, and (2) are consistent with the hypothesis that interferons suppress the antibody response b y exerting antiproliferative effects on those B cells which would normally be proliferative in response to antigen, but augment the immune response to antigen later by inhibiting the proliferation of suppressor T lymphocytes. The suppressive effects of interferons on the production of plaqueforming cells are mimicked by the addition to cultures of oxidized glutathione, and the effects of both are reversed by the addition of sulfhydryl reducing agents, such as 2-mercaptoethanol (2-ME). p-Hydroxymercuribenzene sulfonic acid binds sulfhydryl groups only at the cell surface and its suppressive effects on the antibody response are not inhibited by 2-ME (Johnson, 1980). Johnson (1980) proposed that interferons suppress by binding to sulfhydryl groups on the cell surface, or by functioning as a thiol-oxidizing agent. Since both interferon- and oxidized glutathione-treated cells or cell lysates possess a ribosome-associated protein kinase that may inhibit protein synthesis (Farrell et al., 1978), inhibition of protein synthesis may underlie the inhibition of B cell proliferation, or provide a second mechanism leading to the depression of antibody synthesis. The ability of interferons to modulate the antibody response of
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human cells has only recently been addressed (Parker et al., 1981). When human peripheral blood lymphocytes were stimulated with a preparation of leukocyte interferon, a rather different pattern from that seen in murine cultures was observed. In contrast to murine cultures, where interferon pretreatment suppressed the production of antibody, interferon pretreatment of human cells stimulated the plaque-forming cell response (PFC) to a T-dependent antigen; however, simultaneous addition of interferon and antigen to the cultures led to a suppression of the PFC response. The kinetics of antibody production or the ratio of T to B cells in the human vs murine cultures might underlie the apparent differences following interferon treatment; however, until these studies are verified using highly purified interferon preparations (the preparation used in this study was