Advances in Immunology Volume 63

ADVANCES IN Immunology EDITED BY FRANK J. DIXON Research lnstitute of Scripps Clinic La Jolla, California ASSOCIATE EDI...

Author: Frank J. Dixon

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ADVANCES IN

Immunology EDITED BY FRANK J. DIXON Research lnstitute of Scripps Clinic La Jolla, California ASSOCIATE EDITORS

Frederick Alt K. Frank Austen Tadamitsu Kishimoto Fritz Melchers Jonathan W. Uhr

VOLUME 63

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

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Copyright 0 1996 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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International Standard Serial Number: 0065-2776 International Standard Book Number: 0-12-022463-1 PRINTED IN THE UNITED STATES OF AMERICA 96 97 9 8 9 9 00 01 EB 9 8 7 6 5

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CONTRIBUTORS

Numben in parentheses indicate the pages on which the authom’ contributions begin

Paula M. Chilton (269), Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, Kentucky 40292 Lisa B. Clark (43), Biochemistry Graduate Program, Dartmouth Medical School, Lebanon, New Hampshire 03756 Rafael Fernandez-Botran (269), Division of Experimental Immunology and Immunopathology, Department of Pathology, School of Medicine, University of Louisville, Louisville, Kentucky 40292 Teresa M. Foy (43), Corixa Corporation, Seattle, Washington 98109 Javier G6mez (127), Department of Immunology and Oncology, Centro Nacional de Biotecnologia, Universidad Autdnoma de Madrid, 28049 Madrid, Spain Robert Hooghe (377),Department of Pharmacology, Medical School, Free University of Brussels, B-1090 Brussels, Belgium Elisabeth L. Hooghe-Peters (377), Department of Pharmacology, Medical School, Free University of Brussels, B-1090 Brussels, Belgium Lionel B. Ivashkiv (337), Department of Medicine, Cornell University Graduate School of Medical Sciences, Hospital for Special Surgery, New York, New York 10021 Hajime Karasuyama (l),Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan Ron Kooijman (377), Department of Pharmacology, Medical School, Free University of Brussels, B-1090 Brussels, Belgium Yuhe Ma (269), Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, Kentucky 40292 Carlos Martinez-A. (127), Department of Immunology and Oncology, Centro Nacional de Biotecnologia, Universidad Autdnoma de Madrid, 28049 Madrid, Spain Fritz Melchers (l),Basel Institute for Immunology, Basel, Switzerland ix

Donald E. Mosier (79), ISep~-tnientof InimlInolopy. The Scripps search Institute, La Jolla, C ‘1. l’f i oriiia 9203’7 Randolph J. Noelle (43),Department of Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756 Davina Opstelten (197),Department of Biochemistry,University of Hong Kong, Hong Kong, China Angelita Rebollo ( 127), Department of Iinrnunology and Oncology, Centro Nacional de Biotecnologia, Universidad Authoma de Madrid, 28049 Madrid, Spain Antonius Rolink (l),Basel Institute for Immunology, Basel, Switzerland

ADVANCES IN IMMUNOLOGY. V O L 63

Surrogate tight Chain in B Cell Development HAJIME KARASWAMA,' ANTONIUS ROUNKt, AND FRITZ MELCHERSt 'Chtparhmnt of lmmunobgy,

lha Tokyo M.kopdilon Inslihrk d Mdicol S c h , Tokyo 1 13, hpan; and Insfor Immu-, e~sa/,swi-nd

1. Introduction

The antigen specificity of individual lymphocytes is determined by the distinctive structure of antigen receptors expressed on their cell surface. The antigen binding site of the receptors is composed of two different polypeptides in both B and T cells: heavy and light chains of immunoglobulin (Ig) on B cells, and a and j3 chains (or y and 6 chains) of T cell receptor (TCR) on T cells. The diversity of antigen specificity among B and T cells is generated through the rearrangement of gene segments encoding parts of the variable region of the antigen receptor during the differentiation of lymphocytes in bone marrow and thymus, respectively (Tonegawa, 1983; Yanagi et al., 1984; Hedrick et al., 1984). In precursors of B and T cells, the rearrangements take place in an ordered fashion. In B-lineage cells, initially DH segments are joined to JH segments, then VH segments are joined to DH-JH within the heavy chain locus (Alt et al., 1984; Reth et al., 1985; Alt et al., 1987). This is followed by rearrangements in the light chain loci. Consequently, p heavy chains are produced prior to light chains ( K or A chains) during the B cell development. Such an ordered production of the two chains of the antigen receptor is also observed in T cell development. TCRP chains are produced prior to a chains (Raulet et al., 1985; Snodgrass et al., 1985; Haars et al., 1986). The reasons for the ordered rearrangement of Ig- and TCR-gene loci are not immediately obvious since other species such as chicken appear to rearrange Ig heavy and light chain loci simultaneously to produce surface Ig' B cells (Weill and Reynaud, 1995). It will become clear in this review that B and T cells in mouse and human use the stepwise rearrangement procedure during their differentiation to select productively VDJ-rearranged precursor cells over those with nonproductively rearranged loci. The productively rearranged, Ig heavy chain-, and TCRP chain-expressing cells shut off rearrangements at the Ig heavy chain and TCRP chain loci to secure allelic exclusion of the second allele, before they start rearrangements at the Ig light chain and TCRa chain loci, respectively. p heavy chains alone cannot be transported to the cell surface (McCubrey et al., 1985). Consistent with this, earlier studies failed to detect p heavy chain on the surface of precursor B cells in which p heavy chains but not 1 Coppght ED 19% by Academic Press. Inc All nghts of rrpnxliirtinn 111 m y form rpsrwrd

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HAJIME KARASUYAMA E T A L .

light chains have been produced (Levitt and Cooper, 1980; Siden et al., 1981), although some reports were at variance with these observations (Melchers, 1977; Rosenberg and Parish, 1977). Hence, it was believed that p heavy chains were retained in an intracellular compartment in precursor B cells. On the other hand, the analysis of Ig transgenic mice indicated that the expression of membrane-bound but not secreted p heavy chain is essential for allelic exclusion, a mechanism to inhibit further rearrangements in the second heavy chain locus at the preB cell stage (Nussenzweig et al., 1987, 1988; Manz et al., 1988). In fact, in mice heterozygous for a deleterious mutation in the membrane exon of p heavy chain gene, allelic exclusion was abolished for the transmembrane-deleted allele of p heavy chain (Kitamura et al., 1991; Kitamura and Rajewsky, 1992). Furthermore, transfection experiments using in vitro preB cell lines suggested that the membrane-bound but not the secreted form of p heavy chain was capable of developing rearrangements in the light chain loci (Reth et al., 1987; Iglesias et al., 1991, 1993; Tsubata et al., 1992). Thus, it was puzzling that at the preB cell stage prior to light chain production p heavy chains had to be membrane-bound to function although they appeared not to be on the cell surface. The discovery of two genes, A5 and VpreO,has cast a new light upon the expression and functions of p heavy chains before light chain gene rearrangements (Sakaguchi et al., 1986; Sakaguchi and Melchers, 1986; Kudo et al., 1987b; Kudo and Melchers, 1987;Jongstra et al., 1988;Bauer et al., 1988~;Hollis et al., 1989; Schiff et al., 1990). The genes are transcribed specifically at early stages of B cell development, and their expression is turned off as precursor cells differentiate to mature B cells. The homologies of their sequences with Ig domains provided clues for their possible funcgene has weak but significant homoltions in B cell development. The VpreO ogies with Ig and TCR V regions. The amino-terminal region of the As gene, again, has weak homology to the V regions while the carboyterminal region has strong homology to the J- and C-encoded segments of A light and As genes are not rearranged during chains (Melchers et al., 1983). VpreO B cell development. It has been proposed that VpW0and As proteins could associate with each other to form a light chain-like structure (pseudo-light chain), now termed surrogate light chain (Kudo et al., 1989).This possible structural similarity with conventional light chain, together with the selective expression pattern in preB cells, made the surrogate light chain a good candidate for a partner molecule associated with p heavy chain in preB cells before conventional light chains are produced. Indeed, it was then reported that some mouse and human preB cell lines expressed on their surface p heavy chain together with polypeptides distinct from light chains (Pillai and Baltimore, 1987, 1988; Hollis et al., 1989; Kerr et d.,1989). Fi-

SURROGATE LIGHT CHAIN IN B CELL DEVELOPMENT

3

nally, gene transfection experiments proved the noncovalent association of VPEBand h5 proteins and the formation of a disulfide-bonded complex of p heavy chain and V,,,B/& surrogate light chain as shown in Fig. 1 (Karasuyama et al., 1990, 1993; Tsubata and Reth, 1990). The critical role of surrogate light chain in B cell development was highlighted by creating mice deficient for As (Kitamura et al., 1992; Rolink et d., 1993). In these mice B cell differentiation was severely impaired at

FIG.1. Structure of preB cell receptor composed of p heavy chain, As,and VprpB proteins. VpreR and A5 proteins associate noncovalentlywith each other to form a surrogate light chain; p heavy chain is disulfide-linked to the surrogate light chain to form an Ig-like complex. The As protein is drawn with constant and J regions resembling those of conventional light chains, and with avariable region-like subdomain at amino terminal end, based on a computer analysis (Melchers et al., 1993). Note that this structure of A5 protein has not been confirmed by X-ray crystallograhic structural analyses.

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an early stage. The number of later B cell precursors and immature B cells in bone marrow was drastically reduced, resulting in poor generation of B cells in the peripheral lymphoid compartments. This review focuses on recent progress in our understanding of the functions of the surrogate light chain in B cell development. II. Surrogate Light Chain Genes and Their Regulation

In mice two highly homologous genes of VprrR,Vprc.BI, and VpreBP and one As gene have been identified (Sakaguchi et al., 1986; Sakaguchi and Melchers, 1986, 1987; Kudo et al., 1987b; Kudo and Melchers, 1987; Jongstra et nl., 1988). All these genes are located on chromosome 16 which also harbors the A light chain genes (Kudo and Melchers, 1987; Kudo et ul., 1987a). The VpreBgenes are composed of two exons while the A5 gene is composed of three exons. Restriction fragment length polymorphisin analysis revealed that the VpreRlgene has been maintained within 10 kb of the A5 gene in the Mus genera (D’Hoostelaere et al., 1988).The two genes have changed little in the 9-12 million years since the various Mus genera diverged (Bauer et al., 1988a). The VpERgene encodes a polypeptide of 142 amino acids including a signal peptide of 19 amino acids while the As gene codes for a polypeptide of 209 amino acids including a signal peptide of 30 amino acids. Nucleotide sequence of VpreBB is 97% identical to that of VpreB1. The nucleotide sequence differences signif) changes in four amino acids which are different between the two Vpn,Bproteins (Kudo and Melchers, 1987). In human, only one VpreB gene has been found whereas several &-like genes have been identified, which are called 14.1, 16.1, FA1 (t,!118.1), and GA1 (Chang et al., 1986; Bauer et al., 1988b; Hollis et al., 1989; Schiff et ul., 1989, 1990; Bossy et al., 1991; Evans and Hollis, 1991). They are all located on chromosome 22 which also carries the A light chain genes (Bauer et al., 198813; Evans and Hollis, 1991; Mattei d al., 1991). Among the &-like genes only 14.1 was demonstrated to encode a protein (Hollis et al., 1989). The human VpreB gene encodes a polypeptide of 139 amino acids including a signal peptide of 19 amino acids while the 14.1 human A5 gene codes for a polypeptide of 213 amino acids including a signal peptide (Bauer et al., 1988b; Hollis et al., 1989; Schiff et al., 1990). The human VpreBshows 76% amino acid sequence homology with the mouse VpreB, whereas the human A5 has 55% homology with the mouse As (Bauer et al., 1988~;Schiff et al., 1990). It is remarkable that longer parts of framework 2 and 3 nucleotide sequences within VpwBshow sequence identity between mouse and man, even in the third positions of the codons.


5

The lineage- and stage-specific expression of the VpreB and hs genes is controlled by several cis-regulatory elements upstream of the genes, and by transacting proteins bound to them. The promoters of the VpreBand h5 genes lack a TATA box, and transcription is initiated at multiple sites (Kudo et al., 1989). Sequence comparison of the promoter region of the VprpBz,and human VpreBgenes showed that the region up to mouse VpreBlr -200 bp from the most 5’ of the multiple starting sites is strongly conserved. This region is required for the preB cell promoter activity (Okabe et al., 1992a). Two separate regions were identified in the promoter of the As gene, referred to as AA5and BA5.Region AA5functions as a basal promoter in all cell types whereas region BA5acts as a suppressing region in nonpreB cells and therefore confers the preB cell specificity on the expression of the h5 gene (Martensson and Melchers, 1994; Yang et al., 1995). A preB cell- and B cell-specific DNA-binding protein, EBB1, was identified which bound to the promoter of the VpreBand h5 genes (Okabe et al., 199213). Targeted disruption of the genes encoding the transcription factors Pax5, EBF, and E2A results in arrest of B cell development at the ProB cell stage (Zhuang et al., 1994; Bain et al., 1994; Urbanek et al., 1994; Lin and Grosschedl, 1995).It remains to be determined whether these transcription factors also regulate the expression of the VpreBand h5 genes. 111. Expression of Surrogate tight Chain

A. SURROGATE LIGHTCHAININ MICE

The expression of surrogate light chain has been examined extensively in in vitro transformed B-lineage cell lines as well as in normal precursor B cells in bone marrow of mice. The pattern of the expression observed in transformed cell lines “representing” various stages of B cell development was turned out to be different from that in normal counterparts. 1 . Expression of Surrogate Light Chain in Transformed Mouse Cell Lines The expression of RNA transcripts of VpreB and h5 genes has been studied in a series of transformed cell lines (Sakaguchi et al., 1986; Kudo and Melchers, 1987; Jongstra et al., 1988; Kudo et al., 1992). All p- proB and p+ preB cell lines so far analyzed express the two genes. Both VpreBl and VpreBzgenes are coexpressed in all the cell lines but one. Some immature surface IgM (s1gM)-positive cell lines such as 38C13 and B3-P8-16-1-p were found to express these genes together with a K light chain gene. However, no transcript ofVpreB and h5genes was detected in more differentiated B lineage cells, i.e., in mature B cells and Ig-secreting plasmacytomas. Most notably, the genes have never been found to be expressed in trans-

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H A J I M E KAHASUYAMA E T A L .

formed cells of any other lineages except the B lineage, Thus, the expression of surrogate light chain genes is restricted to transformed cells representing early stages of B cell development. The expression of A5 and Vpreegenes as proteins in preB cells was first seen as two proteins, w and L, which could be coprecipitated with p heavy chains from preB cell lines (Pillai and Baltimore, 1987, 1988). The w chain of 18 kDa was disulfide-linked to p heavy chain while the L chain of 14 kDa was associated noncovalently. Subsequently, the transfection of fibroblast or myeloma cells with As and VpreB genes together with a p heavy chain gene demonstrated that the products of the three genes could form a trimolecular complex immunoprecipitable by p heavy chain-specific antibodies, in which a 22-kDa protein (probably the As protein) was associated covalently with the p heavy chain whereas a 16-kDa protein (probably the Vpn+ protein) was associated to them noncovalently as shown in Fig. 1 (Karasuyamaet al., 1990; Tsubata and Reth, 1990). Finally, the generation of polyclonal as well as monoclonal antibodies specific for As and VprvB made it possible to detect the proteins directly in preB cells and revealed that o (22 kDa) was indeed the As gene product while L (16 kDa) was the VpwBgene product (Misener et al., 1990; Cherayil and Pillai, 1991; Misener et al., 1991; Karasuyama d al., 1993, Shinjo et al., 1994). proteins analyzed in transformed H The expression pattern of As and VpreB lineage cell lines followed essentially that of the RNA transcripts described above (Fig. 2). Traditionally preB cells have been defined as cells with cytoplasmic but no surface p heavy chains. In contrast to this definition,

FK:. 2. Expression of surrogate light chain in in oitro transformed B-lineage cell lines of mice. The production and surface expression of surrogate light chain (SL). p, and 6 heavy chains and conventional K and A light (L) chains in in oitro transfornied B-lineagr cell lines representing different stages of B cell development were analy~etlby iinmunoprecipitation and flow cytometry with specific antibodies. SH, surrogate heavy chain (see text).


7

p heavy chains were detected in association with VPreBlAS surrogate light chains on the cell surface of all the p heavy chain-positive preB cell lines

so far tested (Cherayil and Pillai, 1991; Misener et al., 1991; Karasuyama et al., 1993). Flow cytometry analyses have estimated that the number of p heavy chaidsurrogate light chain complexes expressed on the surface of such preB cell lines is 10- to 100-fold lower than that of IgM on the surface of mature B cells. This could be one of the reasons why p heavy chains have previously not been detected on the surface with less sensitive methods. It is presently uncertain what makes a difference in transportation to and deposition on the cell surface between the complex and IgM. The association of p heavy chain either with surrogate light chain or with conventional light chain may have different effects in masking retention signals. A different set of chaperons bound to them may also determine their destination (Melnick and Argon, 1995). One particular preB cell line, 300-19, has been shown to produce a truncated form of p heavy chain, called a D, protein. It is transcribed and translated from the heavy chain locus DH-JHrearranged in reading frame I1 (Reth and Alt, 1984; Ichihara et al., 1989). In this cell line D, protein is associated with VpreB/h,5 surrogate light chain and transported to the cell surface (Tsubata et al., 1991). However, when an expression vector coding for the D, protein was introduced into a proB cell line that produced surrogate light chain, D, protein could not be detected on the surface of most of the cells. This suggested that the Dplsurrogate light chain complex might need another component to be transported to the cell surface. It remains to be determined whether a putative VH protein encoded by VH segments specifically expressed in preB cells is a missing component (Yancopoulos and Alt, 1985; Tsubata et al., 1991). Two cell lines, 38C13 and B3-P8-16-1-p, were found to coexpress p heavy chaidsurrogate light chain complex and IgM on their surface (Karasuyama et al., 1993). A mixed complex composed of p heavy chain, surrogate light chain and conventional light chain was also detected in these cell lines. Other properties of these transformed cells suggested that they represented cells at the immature B cell stage (Bergman et al., 1977). A similar observation was made with the NFS-5 cell line where variants expressing either no conventional light chain, K light chain, or K and A light chains all coexpressed surrogate light chain (Kudo et al., 1992). These results indicate that in transformed cell lines the expression of conventional light chains does not necessarily switch off the expression of surrogate light chain genes. h5 and VpreB proteins were found to be synthesized in all proB cell lines which could not yet synthesize p heavy chains, i.e., in which heavy chain gene loci were either in a g e r m h e configuration, DHJH-rearranged, or out

8

H A J I M E KAHASUYAMA E T A L .

of frame VHDHJH-rearranged (Misener et d., 1990, 1991; Karasuyama et al., 1993). The As and VprrBproteins were shown to associate noncovalently

with each other to form a surrogate light chain even in the absence of p heavy chain. This capacity to associate with each other was also seen in experiments in which Ig-nonproducing myeloma cells transfected with the A5 and VprrBgenes formed surrogate light chain (Karasuyama et d., 1993). Surprisingly, VprrRIASsurrogate light chain could be detected on the cell surface of those proB cell lines in the absence of p heavy chain (Misener et nl., 1991; Karasuyama et nl., 1993). Since neither the As nor the VpreB proteins have a transmembrane portion, this suggested that the surrogate light chain might be associated with (a) membrane-bound polypeptide(s) distinct from p heavy chain. A N-linked glycoprotein with apparent molecular weight of 130 kDa was found to be coprecipitated with surrogate light chain in immunoprecipitations using surrogate light chain-specific monoclonal antibodies (Karasuyama et nl., 1993; Shinjo et nl., 1994). In contrast to p heavy chain, this protein was not disulfide-linked to the As protein. The biochemical characterization showed that the protein was distinct from CD43 and BP-l/6C3 antigen which both have approximate molecular weight of 130 kDa and both are expressed on early precursor B cells. Besides the 130-kDa protein, other glycoproteins (200, 105, and 65-35 kDa) were coprecipitated with surrogate light chain (Karasuyama et nl., 1993). In summary, the surrogate light chain can be brought to the cell surface in association with different partners in different transformed cells representing different stages along B cell development (Fig. 2). It is first associated with a 130-kDa glycoprotein, tentatively termed “surrogate heavy chain,” in proB cells, and then with D, protein or p heavy chain in preB cells. It can be associated with p heavy chain together with K or A conventional light chains to form a mixed complex in some immature B cells. Finally, conventional light chains take over the place of surrogate light chains to form IgM molecules with p heavy chains in immature and mature B cells. This pattern of expression is derived from analyses of surrogate light chain in transformed cell lines, and differs from that observed in normal B-lineage cells of mouse fetal liver and bone marrow as discussed below.

2. Expression of Surrogate Light Chain in N o m l Mouse B-Lineage Cells The generation of monoclonal antibodies specific for either the A5 or the VprrBproteins has made it possible to analyze the expression of surrogate light chain in normal B-lineage cells from mouse fetal liver and bone marrow (Karasuyama et al., 1993, 1994). These analyses revealed that the

SUHHOGATE LIGHT CHAIN IN B CELL DEVELOPMENT

9

production of surrogate light chain is confined to two successive stages of early B cell development in bone marrow, namely to proB/preB-I cells and a part of large preB-I1 cells, as shown in Fig. 3 (Karasuyama et al., 1994; for the nomenclature of B-lineage subpopulations, refer to Melchers et al., 1993).In agreement with the observation of transformed cell lines, surrogate light chains were detectable on the surface of the proB/preB-I and large preB-I1 cells in bone marrow, albeit in lower levels (Karasuyama et al., 1994; Winkler et al., 1995). However, in contrast to the observation in transformed cell lines, no surrogate light chain was detectable in small preB-I1 cells, the major population of preB cells, or immature B cells in bone marrow. The differential expression of surrogate light chain, together with c-kit, CD25 ( IL-2Ra chain, Tac), and p heavy chains, has allowed a separation of CD45R (B220)' B-lineage bone marrow cells into five subpopulations. These are c-kit+CD25-surrogate light chain+pheavy chain- proB/preB-I cells, c-ki-CD25+surrogate light chain+pheavy chain+large, cycling preBI1 cells, c-kit-CD25+surrogate light chain-p heavy chain+ large, cycling preB-I1 cells, c-ki-CD25+surrogate light chain-p heavy chain+ small, rest-

FIG.3. B cell development in murine bone marrow. The expression of surrogate light chain is confined to two successive stages of early B cell development, i.e., the proB/preBI cell stage and the early phase of the large preB-I1 cell stage. At the transition from proB/ preB-I cells to large preB-I1 cells, when the g heavy chain/surrogate light chain complex (preB cell receptor) is formed, the expression of TdT, c-kit, CD43, and RAG is downregulated while that of CD25 and CD2 is up-regulated, and cells are driven into active cell cycle. Concerning the nomenclature of various stages of B cell development used by different laboratories, refer to other reviews by Rolink et al. (1994b) and Lijffert et al. (1994).

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HAJIME KARASUYAMA ET A L .

ing preB-I1 cells, and sIgMt surrogate light chain- immature and mature B cells (Fig. 3). The analysis of the status of Ig gene rearrangements in single cells by PCR in these different subpopulations has demonstrated that B cell development follows the order given above (ten Boekel et al., 1995).The majority of c-kit'CD25- proB/preB-I cells are DHJH-rearranged whereas more than 90% of c-kit-CD25' large preB-I1 cells have at least one heavy chain locus VHDHJH rearranged. Rearrangements of at least one allele of the K light chain loci become detectable in 65% of c-ki-CD25' small preB-I1 cells. Similarly, an ordering of the Hardy's fractions A, B, C, C', D, E, and F based on the expression of CD43, HSA, and BP-1 (Hardy et al., 1991) has been achieved by PCR analysis of the status of the Ig gene loci (Ehlich et al., 1993, 1994). Although the latter analysis has not included surrogate light chain expression, those two analyses allow a comparison of the ordering of B-lineage subpopulations in mouse bone marrow defined by these different sets of markers (Lijffert et al., 1994; Rolink et al., 199413). The analyses of surrogate light chain expression in different subpopulations of mouse bone marrow B-lineage cells (Karasuyamaet al., 1994) agree well with RT-PCR analyses of surrogate light chain-specific transcripts in bone marrow precursor B cells (Li et al., 1993). The expression of both As and Vpreegenes was found largely restricted to stages prior to light chain gene rearrangements, and was hardly detectable in sIgMt immature B cells. In line with these observations, long-term proliferating, IL-7/stromal cell-dependent proB and preB-I cell lines, which express on their surface surrogate light chains together with surrogate heavy chains, are induced to differentiate to sIgM+ cells by the removal of IL-7, with a concomitant loss of the expression of surrogate light chain (Grawunder et al., 1993). In contrast to these results are findings by Cherayil and Pillai (1991) using a h5 specific antiserum, which showed that As-positive cells in bone marrow coexpressed p heavy chains and K light chains on their surface, while no h5surface-positive cells without p heavy- or K light-chain expression were detectable. These &-positive cells composed of 5-10% of all Blineage cells in mouse bone marrow, and appeared to be at the transitional stage from preB to immature B cells. We have obtained this antiserum from one of the authors and have tried to reconcile the differences between their and our experiments. We found that the antiserum stained p heavy chain- proB cells from bone marrow of RAGS-deficient mice, not unlike our h5-specific monoclonal antibodies. Our analyses indicate that normal preB cells isolated ex uiuo from bone marrow elicit a pattern of surrogate light chain expression which differs from that of transformed preB cell lines (Karasuyama et al., 1993, 1994). Surrogate light chain expression in normal B-lineage cells is turned off


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after the stage of a p heavy chaidsurrogate light chain complex-expressing large preB cell, whereas it remains expressed in transformed cells even when they express sIgM. In normal cells surrogate light chain expression appears to be turned off as soon as the production of p heavy chains allows the formation of the complex with surrogate light chain. This suggests the possibility that the p heavy chaidsurrogate light chain complex, in fact, signals the down-regulation of its surrogate light chain. In line with these observations are experiments in which a transgenic p heavy chain was introduced into RAG2-deficient mice (Karasuyama et al., 1994). In the bone marrow of these mice the precursor B cell compartment is expanded to the stage of small preB-I1 cells. These preB cells no longer express surrogate light chain. Since this small, resting preB-I1 cell compartment is the largest part of all B-lineage cells in bone marrow before sIgM expression, the traditional definition of preB cells as surface p heavy chain-, cytoplasmic p heavy chain+ cells appears to hold true for most preB cells (Fig. 3). How can the differences in expression of p heavy chains, conventional light chains, and surrogate light chain on transformed preB cell lines and normal preB cells be explained? First of all, they differ at the small preBI1 cell stage. Small preB-I1 cells compose approximately 70% of all preB cells in normal bone marrow, whereas all p heavy chain+ transformed preB cell lines so far tested coexpress surrogate light chains and display the p heavy chaidsurrogate light chain complex on the cell surface. The absence of the small preB-I1 phenotype in transformed cell lines might be explained by the poor susceptibility of noncycling small preB-I1 cells to virus transformation, particularly if the transformation requires a cycling cell. Alternatively, but not mutually exclusively, transformed proB/preB-I cells might continue the rearrangements in heavy chain loci and in some cases even in light chain loci to produce p heavy and light chains in vitro, while they stay at a cellular stage of differentiation that retains surrogate Iight chain expression. Indeed, transformed preB cell lines have been found to continue rearrangements of the second heavy chain gene allele, even when a productive rearrangement has been made on the first allele (Schlissel et al., 1991).This might suggest that, in contrast to normal preB cells, transformed preB cells are frozen in some properties such as surrogate Iight chain expression, while they continue their molecular program of differentiation in others such as immunoglobulin-gene rearrangements. Another difference between normal and transformed preB cells is in the level of the surface expression of the p heavy chaidsurrogate light chain complex. In contrast with the transformed cell lines, the complex was hardly detectable on the surface of bone marrow preB cells (ReichmanFried et al., 1990, 1993; Karasuyama et al., 1994; Shinjo et al., 1994).The

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HAJIME KARASUYAMA ETAL.

possibility existed that the complex could be displayed on the surface of normal preB cells, but at such low levels it is not detectable by flow cytometric analysis. Virus transformation may up-regulate the surface expression of the complex in transformed cells. We assumed that, as soon as the complex is transported to the cell surface, it is rapidly internalized by cross-linking through binding to its putative ligand(s) in the bone marrow environment, and that the complex could be induced to reappear on the surface of preB cells upon culture in vitro. Therefore, we reanalyzed bone marrow B-lineage cells isolated ex uivo and cultured in vitro for the expression of the p heavy chairdsurrogate light chain complex by using a novel monoclonal antibody SL156 (Winkler et al., 1995). This antibody does not recognize free surrogate light chain or its components, nor its complex with so-called “surrogate heavy chain” on proB/preB-I cells. However, it does bind to a conformational epitope on the p heavy chain/ surrogate light chain complex on p heavy chain+ preB cell lines. Thus, SL156 can distinguish cells expressing the p heavy chairdsurrogate light chain complex from those expressing the surrogate heavy chairdsurrogate light chain complex. On bone marrow precursor B cells prepared ex vivo on ice, the expression of the p heavy chainhrrogate light chain complex was very low and almost undetectable by SL156, in accordance with the previous results (Karasuyama et al., 1994). Incubation of the precursor cells for 1 hr at 37°C was found to up-regulate the surface expression of the complex. These results appear to reconcile some of the apparently discrepant results on surface expression of the complex with bone marrow precursor B cells, and show that the p heavy chairdsurrogate light chain complex can be expressed on the surface of mouse preB-I1 cells. It remains to be clarified whether the appearance of the complex on cell surface is essential for its function. In summary, the surrogate light chain is already produced and expressed on the cell surface at the proB/preB-I cell stage. Once p heavy chain is produced, a p heavy chaidsurrogate light chain complex is formed at an early phase of the large preB-I1 cell stage. Soon after, cells terminate the production of surrogate light chain and differentiate to small preB-I1 cells where the rearrangements in light chain loci take place. At the transition from the proB/preB-I to the large preB-I1 stage, the expression of c-kit and terminal deoxynucleotidyl transferase (TdT) is down-regulated while that of CD25 and CD2 is up-regulated (Chen et al., 1994; Rolink et al., 1994a; Young et al., 1994) (Fig. 3). CD43 appears to be down-regulated somewhat later in this transition (Karasuyama et al., 1994; Rolink et al., 1994a). The functional roles of CD2, CD25, and CD43 in B cell development remain to be determined. The temporal order in the expression of surrogate light chain, p heavy chain, and then conventional light chain

SURROCATE LIGHT CHAIN IN B CELL DEVELOPMENT

13

was also observed along B cell development in fetal liver (Lennon and Perry, 1990).

B. SURROGATE LIGHT CHAIN I N HUMAN B CELLDEVELOPMENT 1. Expression of Surrogate Light Chain in Transformed Human Cell Lines The analysis of transformed human cell lines by Northern blotting revealed that RNA transcripts specific for A, and Vprt,B were selectively expressed in cells representing both the proB and preB cell stages (Bauer et al., 1988c; 1991; Hollis et al.; Schiff et al., 1991). Polyclonal antibodies raised against synthetic peptides of human A5 and V detected proteins of 22 and 18 kDa, respectively, in preB cell lines (HOE: et al., 1989; Kudo et al., 1989).The 22-kDa As protein was found to be disulfide-linked to p heavy chain whereas the 18-kDa VpreBprotein is noncovalently associated to them (Kerr et al., 1989; Bossy et al., 1993; Lassoued et al., 1993). In some human preB cell lines, a 16-kDa protein was also detected in a p heavy chain-associated complex together with the A, and VpreBproteins (Kerr et al., 1989; Schiff et al., 1991; Lassoued et al., 1993). This protein is disulfide-linked to p heavy chain like the A, protein and can be recognized by apolyclonal anti-A antibody crossreactive to the As protein. The structure of the 16-kDa protein remains to be identified. It may be an isoform of the A5 gene product, or a product of one of the other &-related genes, or the recently discovered JC, protein (Frances et al., 1994), or a human (Shirasawa et al., 1993). analog of VpreB3 The analysis of a panel of human leukemic proB and preB cell lines with human surrogate light chain-specific monoclonal or polyclonal antibodies revealed that all the cell lines including proB cell lines synthesized surrogate light chain (Hollis et al., 1989; Kerr et al., 1989; Bossyet al., 1993; Lassoued et al., 1993). All the p heavy chain-producing preB cell lines tested expressed surrogate light chain on the surface in association with p heavy chain, some of which coexpressed K or A light chain as well (Bossy et al., 1993; Lassoued et al., 1993). This agrees with the expression of surrogate light chain observed in transformed mouse preB cell lines (Misener et al., 1990, 1991; Karasuyama et al., 1993). In contrast, none of the p heavy chain-negative human proB cell lines examined displayed surrogate light chain on the cell surface (Lassoued et al., 1993). However, it is difficult at present to judge whether the spectra of transformed mouse and human cell lines correspond to each other for early stages of B cell development. 2. Expression of Surrogate Light Chain in Normal Human B-Lineage Cells The surface expression of the p heavy chaidsurrogate light chain complex on preB cells in human bone marrow was first indicated by the

14


presence of a subpopulation of CD19’ cells which expressed p heavy chain but not conventional light chain ( K or A) on the surface (Nishimoto et ul., 1991). Proteins of 22 and 18 kDa were found associated with p heavy chain in these cells. The generation of monoclonal antibodies specific to human A5 or V,,re,reH has made it possible to analyze directly the expression of surrogate light chain (Lassoued et al., 1993; Guelpa-Fonlupt et al., 1994). However, two laboratories obtained partially contrasting results on the expression pattern of surrogate light chain. Lassoued et al.’s study of human bone marrow cells with their A5-specific antibodies demonstrated that around 10% of the sIgM- CD 19’ cells expressed surrogate light chain on their cell surface (Lassouedet al., 1993). None of sIgM’ cells were found to express surrogate light chain: Only one-fifth of the cytoplasmic p heavy chain+ preB cells expressed surrogate light chain. These results are consistent with the findings in mouse bone marrow (Karasuyamaet al., 1994). However, in contrast to mouse bone marrow cells, all the surface surrogate light chain+ cells coexpressed p heavy chain on the surface, that is, no p heavy chain-surrogate light chain+ proB/preB-I cells were detected. Since the p heavy chain’surrogate light chain+ cells were CD34-, TdT-, and CD20+ and expressed intracellular p heavy chain in relatively high levels, a model has been proposed in which the surrogate light chain is displayed on the surface only at the late stage of preB cells, immediately prior to the immature B cell stage. This is in contrast to the distribution pattern observed in mice where the expression of surrogate light chain is confined to the proB/preBI cell and early preB-I1 cell stages (Karasuyama et al., 1994). On the other hand, Guelpa-Fonlupt et al.’s study with another set of antibodies specific to human Vprreshowed a different spectrum of surrogate light chain expression on the surface of human bone marrow cells (GuelpaFonlupt et al., 1994). In contrast to the studies of Lassoued et nl., the antibodies detected surrogate light chain in the absence of p heavy chain on proB/preB-I cells. This result is similar to those found in normal mouse B-lineage cells. In disagreement with both Lassoued et aZ.’s results with human and Karasuyama et d ’ s results with mouse B-lineage cells, the VpreHspecific antibodies stained -20% of sIgMf cells in human bone marrow. It was estimated that p heavy chain-surrogate light chain’ preB cells (PreBl), p heavy chain+surrogate light chain+ preB cells (ProB2), and sIgM+surrogate light chain+ cells (PreB3) composed 23, 4,and 73%, respectively, of all surrogate light chain+ cells. The discrepancies observed in the two studies of human surrogate light chain remain puzzling. They could be explained, at least in part, by differences in specificities of the antibodies. For mouse surrogate light chain, two types of monoclonal antibodies were reported: one (such as LM34)


15

recognized surrogate light chain either in free form or in association with

p heavy chain or surrogate heavy chain while the other (such as SL156) detected surrogate light chain only when associated with p heavy chain

(Winkler et al., 1995). In order to reconcile all the results obtained with normal mouse and human B-lineage cells (Karasuyama et al., 1994; Lassoued et al., 1993; Guelpa-Fonlupt et al., 1994), one would have to postulate that GuelpaFonlupt et aZ.’s antibodies have the same recognition capacity for surrogate light chain as LM34 has. However, the antibodies, in contrast to LM34, could crossreact with V-region of Ig molecules, hence detect sIgM+ cells. Lassoued et d ’ s antibodies, on the other hand, could be similar to SL156, not capable of detecting surrogate light chain in association with surrogate heavy chain. However, in contrast to SL156, those antibodies have been found to precipitate free surrogate light chain. This might suggest that Lassoued et d ’ s antibodies, in contrast to SL156, recognize a determinant on free surrogate light chain which is covered by surrogate heavy chain, but not by p heavy chain. It is clear that additional comparative studies have to be undertaken with those antibodies to resolve the apparent discrepancies in the detection of surrogate light chain in precursor B cells in mouse and human. The analysis of RNA transcripts specific for surrogate light chain in each subpopulation of human B lineage cells and an ordering of human B lineage subpopulations in bone marrow by an analysis of rearrangement status of Ig gene loci may also help to define the stage(s) where surrogate light chain is produced.

N. Functional Roles of Surrogate Light Chain in B Cell Development In order to elucidate the physiological functions of surrogate light chain, mutant mice were created in which the h5 gene had been inactivated by targeted gene disruption in embryonic stem cells (Kitamura et aZ., 1992). The analysis of bone marrow cells by flow cytometry revealed that the number of CD43- small preB cells and of sIgM’ immature and mature B cells was drastically reduced whereas that of CD43+ earlier precursor B cells was normal, if not increased 2- to 3-fold compared with control littermates (Kitamura et al., 1992). An analysis using c-kit, CD25, and surrogate light chain as distinguishing markers showed that c-kit+CD25-surrogate light chain+proB/preB I cells were produced in normal numbers, whereas c-kit-CD25+surrogate light chain+ large preB-I1 cells and c-kit-CD25’surrogate light chain- large and small preB-I1 cells, as well as immature B cells, were at least 40-fold reduced (Rolink et al., 1993). These results indicate that in &deficient mice B cell differentiation is impaired at the transition from the proB/preB-I to the preB-I1 cell stage.

16

H A J I M E KARASUYAMA ET AI,

Peripheral B1 and conventional B cells accumulate more slowly as compared with littermate controls. While the small compartment of B1 cells is filled 1 to 2 weeks later after birth, it takes more than half a year to fill even half of the large compartment of conventional B cells. The retarded accumulation of mature B cells in the periphery can be explained by the severely reduced pool size of immature B cells from which these mature cells are formed. In contrast, the number of T cells appears to be unchanged in &-deficient mice. Thus, surrogate light chain plays an important role in B cell development. How does surrogate light chain govern B cell differentiation? Possible functional roles ascribed to surrogate light chain will be discussed below. A. FUNCTIONS OF p HEAVY CIIAIN/SURROGATELIGHTCHAIN COMPLEXPREBCELLRECEPTOR

1 . Selective Expansion of Cells Which Have Succeeded in a Functional Rearrangement of the p Heavy Chain Gene

VHDHJH rearrangements can occur in three reading frames with equal probability, two of them out of frame, one in frame at both heavy chain alleles. However, the analysis of VHDHJH joints in precursor €3 cells in bone marrow demonstrated the strong shift toward an overrepresentation of productive joints over nonproductive joints (Laffert et aZ., 1994; Ehlich et al., 1994). Indeed, over 95% of the large and small CD43-c-kit-CD25' preB-I1 cells in bone marrow were found to express p heavy chain in the cytoplasm (Karasuyama et al., 1994; Rolink et al., 1994a). These results indicated that a cellular selection with preference for p+ cells over pcells takes place during the differentiation from the proB/preB-I to the preB-I1 cell stage. It suggested that the expression of p heavy chain might facilitate cell survival or cell proliferation. A cell cycle analysis revealed that p heavy chainhrrogate light chain+ large preB-I1 cells were the most actively cycling B lineage cells in mouse bone marrow (Karasuyama et al., 1994; Rolink et al., 1994a) (Fig. 3). This actively cycling population was not detectable in RAG2-deficient or in Asdeficient mice, indicating that both p heavy chain and surrogate light chain are necessary to create this population. Furthermore, heavy chain must be membrane-bound, since large, cycling preB-I1 cells are also lacking in mice which cannot deposit p heavy chains in membrane (Kitamura et aZ., 1991). Based on these observations a model of selective amplification of p heavy chain+ preB cells has been proposed (Fig. 4). Once p heavy chains are produced through functional VHDHJH-rearrangement in precursor B cells, a p heavy chaidsurrogate light chain complex, i.e., a preB cell

SURROGATE LIGHT CHAIN I N B CELL DEVELOPMENT

17

FIG.4. Selective expansion of p. heavy chain+precursor B cells through a signal delivered

by a preB cell receptor. SL, surrogate light chain; VDJ’, a productive rearrangement of a

p. heavy chain gene; VDJ-, a nonproductive rearrangement of a p. heavy chain gene. See

text for the details.

receptor, is formed. This preB cell receptor, in turn, delivers a signal for cell proliferation. This amplification step selectively expands cells carrying p heavy chains and creates a late preB-I1 cell pool of sufficient size, in which VJ rearrangements in light chain loci will then be carried out. The lack of this amplification step at the transition from proB/preB-I to preBI1 cells is seen as a consequence of the absence of surrogate light chain, p heavy chain, or both, i.e., of the preB cell receptor. This results in the massive reduction of large and small preB cells and, consequently, of immature and mature sIgM+ B cells as seen in &,-deficient mice (Kitamura et al., 1992; Rolink et al., 1993) (Fig. 4, lower panel). In these mice only 60% of sIgM-CD43- preB cells were found to produce p heavy chains, whereas more than 90% of those cells in normal mice produced p heavy chains (Fig. 5). This result is consistent with the prediction based on the proposed model, i.e., the lack of the selective expansion of p heavy chain+ preB cells in &-deficient mice. Two additional sets of experiments illuminate the proliferation-inducing role of the preB cell receptor. In one, the transgenic expression of p heavy chains in RAG-deficient mice fills up the bone marrow with normal number of large and small preB-I1 cells (Karasuyama et al., 1994; Rolink et al.,

18

H A J I M E KARASUYAMA ET A L .

/BALBlcl Cell surface

Cell surface

CD43 (57)

CD43 (57)

s cu

?i!

%

B0

CvtoDlasm , .

104

Cytoplasm

0.9

I

103

R1 31°2 101

100 1

104

4

190.2

I

3.21 '04j60.0

I

4

FIG.5. The lack of the selective expansion of g heavy chain+ precursor B cells in Asdeficient mice. Bone marrow cells from BALB/c mice (left) or &-deficient mice (right) were depleted of sIgMt cells and stained with APC-anti-CD45R and PE-anti-CD43 on the surface, followed by cytoplasmic staining with FITC-anti-p heavy chain and biotin-anti-& (revealed by RED613-streptavidin). CD45Rt precursor B cell populations were divided into fractions R1 (CD43+)and R2 (CD43-). R2 composes -90% of CD45R' precursor B cells in BALB/c mice whereas that comprises only -10% in &-deficient mice. The hvocolor analyses of cytoplasmic p heavy chain versus h5 in R 1 and R2 fractions are shown in the middle and lower panels, respectively. Note that in &-deficient mice only 60% of R2 cells were found to produce p heavy chains, whereas more than 90% of R2 cells in normal mice produced p heavy chains.

1994a; Spanopoulou et al., 1994; Young et al., 1994). This result is in accord with the proposed model. In the other, the precocious expression of a conventional A2 or K light chain as transgenes in A5-deficient mice restores the large and small preB-I1 cell as well as the sIgM+ immature B cell compartments and generates mature peripheral B cells at normal rates (Rolink et al., 1996). This restoration can be observed only when these light chain transgenes are expressed precociously, i.e., under the


19

control of E, enhancer, but not when expressed normally, i.e., under the control of their own enhancers. This reflects that the surrogate light chain can be replaced by conventional light chains if the light chains are expressed abnormally early. An alternative, but not necessarily mutually exclusive, model was proposed to account for the incomplete block of B cell development or “leaky” B cell generation in As-deficient mice (Ehlich et al., 1993; Lijffert et al., 1994). The model is based on findings that the rearrangement of K light chain gene can occasionally occur even in the absence of the preceding rearrangement of p heavy chain gene. In this model, As-deficient mice generate B cells via a distinct differentiation pathway that does not require the preB cell receptor. In this pathway (pathway 1 or pathway ah)productive light chain rearrangement precedes or coincides with productive p heavy chain rearrangement in a given cell at the CD43+ proB/preB-I stage. Such a cell can develop directly into sIgM+ B cells without passing through the CD43- small preB-I1 cell stage. The rearrangements of both light chain and heavy chain genes are initiated in proB/preB-I cells in which the incidence of rearrangement is low for the light chain loci and high for the heavy chain loci. Therefore, the model predicts that in normal mice pathway 1 is minor and the majority (-95%) of B cells are generated through a preB cell receptor-dependent pathway (pathway 2 or pathway c). In pathway 2, p heavy chains are produced prior to light chains so that p heavy chain+ precursor B cells express a preB cell receptor and progress to the small preB-I1 cell stage before they produce light chains. According to the model, pathway 2 is selectively abrogated in &-deficient mice, thereby resulting in a limited B cell genesis. The model predicts that there is no intrinsic order of opening and closing of first heavy chain then light chain gene loci in As-deficient preB cells. It remains to be determined by + versus the single-cell PCR analysis how frequently one can find p - ~ cells p + ~cells in the CD45R+CD43+population of As-deficient mice. 2. Induction of Allelic Exclusion in the Heavy Chain Locus When one of the two p heavy chain alleles has achieved a productive VHDHJH rearrangement and produces p heavy chain, the VHto DHJH rearrangement at the other allele is inhibited to avoid the generation of B cells with double specificity (reviewed by Storb, 1995). The study with transgenic mice suggested that this feedback inhibition, referred to as allelic exclusion, was mediated by the membrane-bound form of p heavy chain (Nussennveig et al., 1987, 1988; Manz et al., 1988). Indeed, the allelic exclusion was found violated in heterozygous mice carrying a targeted disruption of the membrane exon of the p heavy chain in one locus and an unmutated gene in the other locus (Kitamura and Rajewsky, 1992). B

20

IIAJIME KAHASUYAMA ETAI..

cells producing p heavy chains from both alleles were readily detected i n the heterozygous mutant mice, whereas such cells were barely found in wild type mice. The feedback inhibition for allelic exclusion is expected to take place as soon as a productively rearranged p heavy chain gene becomes expressed, that is, at the earliest phase of the large preB-I1 cell stage, in which a membrane-bound form of p heavy chain becomes associated with surrogate light chain (Karasuyama et al., 1994; Winkler et al., 1995). Therefore, the preB cell receptor appears to participate in signaling allelic exclusion. In contrast to this prediction, the allelic exclusion seemed to be maintained in &-deficient mice as long as splenic B cells were examined (Kitamura et al., 1992). However, a recent analysis of bone marrow cells in these mice revealed that the p heavy chain genes were not allelically exluded at the precursor B cell stage (Uffert et al., 1996). Though it remains uncertain how the cells producing two p heavy chains are eliminated along the differentiation from preB to B cells, this result supports the idea that both p heavy chain and surrogate light chain, i.e., the preB cell receptor, are involved in the process of allelic exclusion of the preB cell stage. The gene products of two recombination activating genes, RAG1 and RAG2, are crucial for the rearrangements of Ig genes. In fact, mice deficient for either of the two have all Ig heavy and light chain gene loci in germline configuration (Mombaerts et al., 1992b; Shinkai et al., 1992). Hence, one of the ways to stop the rearrangement at the second allele could be to down-regulate the expression of the RAG genes as soon as a p heavy chain has been expressed. Therefore, the pattern of RAG expression along B cell development was examined on mRNA level by RT-PCR. Earlier work had shown that the expression of both RAG genes was maintained at high levels through the proB and preB cell stages until it dropped at the immature B cell stage (Li et al., 1993). However, a recent study on mRNA level by RT-PCR and on protein level by RAG-specific antibodies together with more detailed fractionation of preB cell populations has illuminated a sharp and transient drop of RAG-1 and -2 expression at the earliest phase of the large preB-I1 cell stage, coincident with the expression of the preB cell receptor (Grawunder et al., 1995) (Fig. 3). As expected, high levels of RAG transcripts and RAG2 proteins were detected in the proB/preB-I and the small preB-I1 stages where the rearrangements of heavy chain and light chain genes took place, respectively. In contrast, the large preB cells between those stages expressed at least 50-fold less RAG transcripts and RAG2 proteins. Among them, especially, the cells expressing preB cell receptors did not express any detectable levels of RAG transcripts or protein. The coincidence of the transient down-regulation of RAG and


21

the expression of the preB cell receptor strongly suggests an involvement of the receptor in allelic exclusion of the heavy chain locus through this down-regulation of the RAG genes. It has been shown that the level of RAG2 protein oscillates in cell cycle and the proteins are rapidly degraded during the S, G2, and M phases (Lin and Desiderio, 1993, 1994, 1995). This suggests that RAG2 proteins produced during the proB/preB-I cell stage might be degraded quickly after cells progress to the actively cycling large preB-I1 cell stage. Taken together, the shut-down of recombination activity at the large preB-I1 cell stage appears to be guaranteed by the down-regulation of RAG expression at both the transcriptional and post-transcriptional levels. Thus, the preB cell receptor might induce allelic exclusion in several different modes; one to shut down the expression of RAG genes, another to drive cells into cell cycle. Once large preB-I1 cells have shut down RAG activity, they are expected to close the second p heavy chain allele in order to prevent further rearrangements while they open the light chain gene loci before the RAG genes are reexpressed. In human, the correlation of the expression of RAG with the preB cell receptor has not yet been studied. If the receptor participates in allelic exclusion also in human, one can assume that it functions at the earliest phase of preB cell stage like in mice. However, it is difficult to reconcile this prediction with the report that the surface expression of the receptor is restricted to the late preB cells immediately prior to the sIgM' immature B cell state in human (Lassoued et al., 1993), unless one assumes that 80%of the cytoplasmic p heavy chain+ cells which are negative for surrogate light chain are, in fact, the cells homologous to the small preB cells in mouse. The study with one strain of y2b transgenic mice demonstrated that the expression of y2b inhibited the rearrangements of endogenous p heavy chain, but was unable to promote B cell development (Roth et al., 1993). Thus, the y2b heavy chain in this particular strain of mice can replace the p heavy chain in inducing allelic exclusion but might not induce preB cell proliferation and, hence, efficient B cell maturation. This suggested that the signals for these two events may be different. However, subsequent studies with other y2b transgenic mice showed that both the feedback inhibition of p heavy chain genes and B cell development could be induced by y2b heavy chain (Roth et al., 1995; Kenny et al., 1995). It remains unclear why the same transgenic y2b heavy chain can show different signaling properties. One possibility might be that in the one transgenic line which is able to separate signals for allelic exclusion and for B cell maturation, the y2b heavy chain transgene is inserted so that (a) endoge-

22

HAJlME KARASUYAMA ET AL

nous gene(s) contributing to the signaling for B cell maturation is inactivated. 3. Arrest of Maturation of PreB Cells Expressing D, Protein There are 15 DH gene segments in the heavy chain locus of mice (Kurosawa and Tonegawa, 1982).They can in principle be used in three reading frames (RFs). However, a strong bias for the expression of one particular RF, RF1, has been noticed in murine antibodies (Kaartinen and Makela, 1985; Ichihara et al., 1989).This overrepresentation of RF1 was detected already at precursor B cell stage, i.e., independent of antigenic selection (Gu et al., 1990, 1991).Although the physiological meaning of this biased usage of RFs remains to be determined, it has been noticed that several anti-DNA autoantibodies use RF2 and RF3 in their DH elements, suggesting that sequences encoded by those RFs may bring some undesirable feature to antigen specificity (Eilat et al., 1988; Marion et al., 1990; Smith and Voss, 1990). Short stretches of sequence homology between the DH and JH segments might promote the preferential usage of RF1 (Gu et al., 1990; Gerstein and Lieber, 1993). Two additional mechanisms appear to counterselect other RFs, RF2 and RF3. The counterselection of RF3 is attributed to the frequent Occurrence of stop codons when DH elements are read in this RF (Ichihara et al., 1989).In contrast, RF2 allows the expression of a truncated p heavy chain consisting of DH, JH and C,, the so-called D, protein, from most DH-JH joints (Reth and Alt, 1984; Gu et al., 1991). Therefore, the counterselection of RF2 usage could be based on the selection against cells expressing D, proteins. Interestingly, the underrepresentation of RF2 was not observed when DHJH joints were analyzed in B cells where the membrane exon of the p chain is disrupted by homologous recombination on one allele (Gu et al., 1991). Since all the DHJH joints in such B cells should be derived from the targeted allele in which membrane-bound forms of p heavy chain and D, protein cannot be produced, the result indicates that the expression of membrane-bound forms of D, protein is responsible for the underrepresentation of B cells of normal mice which carry DH-JH joints in RF2. It has been shown that D, protein can be associated with surrogate light chain to form a complex expressed on the surface (Tsubata et al., 1991; Home et al., 1996).The analysis of DHJH joints in bone marrow precursor B cells from &deficient mice revealed that RF2 was not suppressed in the absence of surrogate light chain (Haasner et al., 1994).These results indicate that the D,/surrogate light chain complex is involved in the counterselection of RF2 usage. In analogy with the p heavy chaidsurrogate light chain preB cell receptor, the D,/surrogate light chain complex might


23

also deliver a signal for feedback inhibition to prevent further rearrangements on p heavy chain loci (Reth et al., 1985; Gu et al., 1991; Lijffert et al., 1994). This would make it impossible for the D,-expressing preB cell to rearrange VHto this DHJH-rearranged heavy chain locus. This incapability of producing normal p heavy chains will destine cells to die, as is observed in p heavy chain-deficient mice such as RAG-, JH-, and pm-deficient mice (Kitamura et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992b;Ehlich et al., 1993; Chen et al., 1993a). In addition, the D,/surrogate light chain complex may not be able to provide a signal for cell survival or cell proliferation in contrast to the preB cell receptor. Thus, cells expressing D, proteins could be counterselected. In this respect, D, protein must be functionally equivalent to y2b heavy chain expressed in the one y2b transgenic mouse strain quoted above where feedback inhibition of the rearrangement of endogenous p heavy chain genes was observed in the absence of B cell development (Roth et al., 1993).It remains to be demonstrated that early precursor B cells in bone marrow and fetal liver indeed express D, proteins. 4. Promotion of Transition from the ProB to the Small PreB Cell Stage The expression of p heavy chain but not light chain transgenes allows developmentallyarrested proB cells in RAG-deficient mice to progress up to the small preB-I1 cell stage (Karasuyama et al., 1994; Rolink et al., 1994a; Spanopoulou et al., 1994; Young et ul., 1994). Thus, the preB cell receptor appears to be responsible for the transition from the proB/preBI cell to the large and subsequently small preB-I1 cell stages. At this transition the expression patterns of several molecules change. Expression of c-kit, TdT, and RAG1 and 2 is rapidly down-regulated and that of CD43 and surrogate light chain more slowly, whereas the expression of CD25 and CD2 is up-regulated (Chen et al., 1994; Rolink et al., 1994a; Young et al., 1994)(Fig. 3).Although it has not yet been measured, it is predicted that the second p heavy chain allele is closed when it is still in germline or DHJH rearranged configuration, to avoid VH to DHJH rearrangements. It is presently uncertain whether all these changes are directly controlled by signals delivered by the preB cell receptor.

5. Promotion of Light Chain Gene Rearrangements Ample evidence has been accumulated that neither p heavy chain nor surrogate light chain is prerequisite for the rearrangement of light chain loci. Several transformed cell lines were found to have K chain gene rearrangements in the absence of heavy chain expression (Blackwell et al., 1989; Schlissel and Baltimore, 1989; Kubagawa et al., 1989; Felsher et al., 1991). Cell lines with two nonfunctionally rearranged heavy chain alleles, i.e., without the capacity to produce heavy chain, could be induced to

24


differentiate to K light chain gene rearrangements and K light chain production (Grawunder et al., 1993). Furthermore, when the capacity of c-kit' proB/preB-I cells derived from normal and &--deficient mice was compared in uitro to differentiate to sIg' cells, the numbers of sIg' cells and the kinetics of their appearance were found to be indistiguishable between them (Rolink et al., 1993). These observations do not appear to be in in uitro artifacts, since studies with a series of knock-out mice clearly demonstrated that neither p heavy chain nor surrogate light chain was required for the induction of K chain gene rearrangement (Kitamura and Rajewsky, 1992; Kitamura et al., 1992; Ehlich et al., 1993; Chen et al., 1993a). Thus, the preB cell receptor appears to be dispensable in the rearrangement of light chain loci. In apparent conflict with this conclusion are experiments in which the capability of p heavy chain was tested to induce K light chain rearrangements. In these experiments in uitro transformed proB cell lines were transfected with p heavy chain expression vectors (Reth et al., 1987; Iglesias et al., 1991;Tsubataet al., 1992;Iglesias et al., 1993).Membrane-bound but not secreted form of p heavy chain was found functional in the induction of K light chain rearrangements. The transfection of a truncated p heavy chain gene lacking both VHand CH1 domains hence incapable of associatingwith surrogate light chain could not induce light chain gene rearrangements, though truncated p heavy chains were detected on the cell surface in the absence of surrogate light chain (Tsubata et al., 1992). However, crosslinking of the truncated p heavy chains on the surface of the transfected cells by anti-p antibodies induced the rearrangement of K light chain genes. These results suggested that membrane-bound i . ~heavy chains, possibly in association with surrogate light chains, could facilitate the rearrangement of light chain genes. It was observed in RAG-deficient mice that the sterile transcription from germline K chain gene loci was induced when the p heavy chain transgene was expressed (Spanopoulou et d.,1994; Young et al., 1994). From this observation the authors concluded that a signal delivered by the preB cell receptor may facilitate to open the light chain gene loci for the rearrangement (Schlisseland Baltimore, 1989).Alternatively, one could explain these results on a cellular differentiation level. Since the preB cell receptor signals proliferative expansion and differentiation into the large and subsequently small preB-I1 cell compartments, cells can develop in which light chain rearrangements can take place.

6. Other Possible Functions of the PreB Cell Receptor The association of p heavy chain with surrogate light chain may provide an opportunity to check the quality of p heavy chains prior to the association


25

with conventional light chains. Malformed p heavy chains which cannot be assembled with light chains also may not associate with surrogate light chain. As a consequence, preB cells expressing such p heavy chains will not be rescued from cell death by proliferation, because the signal normally delivered by the preB cell receptor cannot be given. It is not yet clear whether p heavy chains with given V regions prefer to associate with surrogate light chain. It also remains to be determined whether preB cells can be clonally selected by self antigens in the primary lymphoid organs, i.e., bone marrow, either positively or negatively, on the basis of the V, regions of p heavy chain which they express. In any case the proliferative expansion of preB cells through the preB cell receptor in bone marrow predicts that the productively VHDHJH-rearranged heavy chain alleles are repeated in the final repertoire of sIg+ B cells with different light chains.

B. FUNCTIONAL ROLEOF SURROGATE LIGHTCHAIN COMPLEX EXPRESSED ON PROBCELLS The surrogate light chain can be detected in the absence of p heavy chain on the surface of proB cells in vivo and in vitro both in mice and in human (Misener et al., 1991; Karasuyama et al., 1993, 1994; Shinjo et al., 1994; Guelpa-Fonlupt et al., 1994). Several molecules distinct from p heavy chain have been identified in association with surrogate light chain (Karasuyamaet al., 1993; Shinjo et al., 1994).The functional role of these surrogate light chain complexes has not yet been clarified. The complexes appear not essential for the generation and maintenance of the c-kittCD43+ proB/preB-I cell population, since that population of cells was present in normal numbers in &,-deficient mice (Kitamura et al., 1992; Rolink et al., 1993, 1994a; Ehlich et al., 1993). The characterization of the so-called “surrogate heavy chain” associated with surrogate light chain should cast light on its functional role. V. tigand and Signal Transdudion of Surrogate tight Chain Complex

A. DOESA LICAND EXISTFOR THE PREBCELLRECEPTOR? From the structural similarity to IgM expressed on mature B cell, it would be reasonable to consider the p heavy chaidsurrogate light chain complex as a receptor expressed on preB cell (a preB cell receptor). What then could be a ligand for this receptor? Since the complementation with p heavy chains carrying different V regions in RAG-deficient mice was capable of inducing the progression of proB cells to the small preB-I1 cell stage (Karasuyamaetal., 1994; Spanopoulou et al., 1994;Younget al., 1994; Rolink et al., 1994a),the function of the complex should be independent of

26

HAJlME KARASUYAMA ETAL.

the V region of p heavy chain. Indeed, the transgene encoding a truncated p heavy chain gene without VHDHJH induced allelic exclusion as well as B cell maturation (Corcoset al., 1991).In all preB cells, surrogate light chain is invariant. Therefore, an invariant ligand could be recognized by the surrogate light chain part of the preB cell receptor. However, it has recently been shown that two conventional light chain transgenes introduced into &-deficient mice could rescue normal B cell development in their bone marrow and the periphery (Rolink et al., 1996). In this case the transgenic light chains had to be expressed under the control of the p heavy chain gene enhancer (E,) to become expressed early, i.e., in the window of B cell development where surrogate light chain is expressed.The transgenic light chains under their own expression control appeared expressed too late to rescue B cell development in &deficient mice. These results make it less likely that there is a defined, structurally restricted ligand recognized by the preB cell receptor in its V-like regions of As and Vpme.However, it remains a possibility that the VPmBprotein carries the major ligand binding activity. It has been demonstrated in an in vitro transformed cell line that a set of proteins including Btk tyrosine kinase are constitutively tyrosine-phosphorylated independent of crosslinking of the preB cell receptor on the cell surface (Aoki et al., 1994a,b). This might suggest that there is no external ligand for the preB cell receptor. Thus, the issue of a possible ligand and the mode of action of the preB cell receptor remain unsolved.

B. MOLECULESINVOLVED IN SIGNAL TRANSDUCTION THROUGH THE PREBCELLRECEPTOR A heterodimer IgarlIgP (CD79dCD79b) is essential for cell surface expression of, and signal transduction through, the antigen-specific B cell receptor IgM (Sakaguchi et al., 1988; Hermanson et al., 1988; Reth et al., 1991; Pleiman et al., 1994).The heterodimer was also shown to be associated noncovalently with the preB cell receptor in murine and human preB cells grown in vitro or isolated ex uivo (Matsuo et al., 1991; Nishimoto et al., 1991; Iglesias et al., 1991; Chen et al., 1991; Nakamura et al., 1992). Cross-linking of either p heavy chain or Iga on preB cell lines with specific antibodies induced the elevation of intracellular Ca2+,indicating that the preB cell receptorlIgdIgl3 complex has potential to transduce signals (Takemori et al., 1990; Nomura d al., 1991; Nakamura d al., 1993). Interestingly, the crosslinking of the complex did not induce an incease of inositol phospholipid metabolism, in contrast to that of IgM on B cells (Takemori et al., 1990). The association with the IgdIgP heterodimer was shown to be essential for the preB cell receptor to exert its functions (Papavasiliouet al., 1995a).


27

Replacement of Tyr587 and Ser588 in p heavy chain by Val (YS :Wp) rendered p heavy chain incapable of associating with the IgdI@ heterodimer, though the mutated p heavy chain by itself was able to be anchored on the cell surface. When the transgene encoding this mutated p heavy chain was expressed in RAG1-deficient mice, the progression of proB cells to the small preB cell stage did not occur, in contrast to the effect of the intact p heavy chain transgene. The addition of the cytoplasmic tail of I@ to the YS :Wp (YS :Wp-I@) was sufficient to restore the proB to preB cell transition. However, further replacement of Tyr195 and Tyr206 in the ITAM motif of the cytoplasmic tail of the YS :Wp-I@ chimera with Phe (YS :Wp-I@ Y : F ) abolished this restoration. These results clearly indicate that the association with the I@ is essential for the preB cell receptor to deliver a signal for the progression of proB cells to the small preB cell stage. Phosphorylation of tyrosine residues in the ITAM motif of I@ appears important for mediating the signal, just as it is in B cell JH activation through the B cell receptor. Furthermore, the analysis of VHDH rearrangements in endogenous p heavy chain loci of normal mice with the mutated transgenes described above revealed that the signal for allelic exclusion also requires the association of the preB cell receptor with the I@ as well as the phosphorylation of tyrosine residues in the I@ tail. In addition, the expression of transgenes of mutated p heavy chains with cytoplasmic Iga tail gave comparable effects on preB cell maturation and allelic exclusion (Papavasiliouet al., 1995b).Thus, the I g d @ heterodimer is a critical signal mediator of the preB cell receptor. The I@-deficient mice showed a complete block in B cell development at the CD43' stage (Gongand Nussenzweig, 1996).Interestingly, however, this blockade appears to occur at an earlier stage of B cell development as compared to the blockade observed in h5-deficient mice or in mice where the transmembrane domain of p heavy chain has been disrupted. In the I@-deficient mice, VH to DHJH rearrangements were found to be severely disturbed, whereas DH to JH rearrangements were not affected. This indicates that I@ may play an important role in B cell development even before the preB cell receptor is formed. An in vitro kinase assay revealed that the preB cell receptor/Igd@ complex was associated with kinase activity which was indistinguishable from Src-family tyrosine kinases such as Fyn, Lyn, or Lck (Matsuo et al., 1993; Brouns et al., 1993). Furthermore, a number of Blk and Fyn Src homology 2 (SH 2) domain-binding phosphoproteins were detected in a preB cell line even without crosslinking of the complex on the cell surface, though there was no direct evidence that the signal through the complex is responsible for the phosphoxylation (Aokiet al., 1994a).These results may indicate that Src-family tyrosine kinases are involved in signal transduction

28


through the complex. The studies with genetically engineered mice revealed that the activation of Lck is critical and sufficient for T cell development (Abraham et al., 1991; Anderson et al., 1992, 1993; Molina et al., 1992; Levin et aE., 1993; Mombaerts et al., 1994). Such a dominant Srcfamily tyrosine kinase for B cell development has as yet to be identified. B cell development appears to be intact in mice deficient for either Fyn, Lck, Lyn, or Blk (Appleby et al., 1992; Stein et al., 1992; Texido et al., 1995; Hibbs et al., 1995; Nishizumi et al., 1995). It is known that the cytoplasmic tyrosine kinase Syk becomes associated with and activated by engagement of B cell receptor IgM. A homologous kinase, ZAP-70, was shown to be crucial in signaling through TCR as well as in T cell development in both human and mice (Arpaia et al., 1994; Elder et al., 1994; Chan et al., 1994; Negishi et al., 1995). Mice deficient for Syk suffer severe hemorrhaging as embryos and die perinatally (Turner et al., 1995; Cheng et al., 1995). Therefore, the ability of hematopoietic stem cells from these mice to differentiate to T and B cells was tested in radiation chimeras, i.e., by transferring their fetal liver cells to irradiated normal or RAG-deficientmice. In contrast to the relatively normal reconstitution of T cells, Syk-deficient stem cells failed to produce both conventional and B1 cells in periphery. Bone marrow of chimera mice contained normal numbers of CD43TD25-sIgM- proB/preB-I cells, but substantially reduced numbers of CD43-CD25'sIgM- preB-I1 cells and immature B cells. These results indicate that B cell development is impaired at the transition from the proB/preB-I to the preB-I1 cell stage as is observed in &-deficient mice. VHDHJH rearranged DNA fragments from Sykdeficient preB cells displayed a pattern in which the in-frame bands were underrepresented as compared with those from normal preB cells. This suggests a lack of clonal expansion of cells expressing a functional p heavy chain. Furthermore, the underrepresentation of RFII of DH-JH joints was not observed in Syk-deficient bone marrow preB cells, as in the case of As deficient mice. All the results strongly indicate that the Syk tyrosine kinase mediates signaling downstream of the preB cell receptor and of the DJsurrogate light chain-complex. Since no mature Syk-deficient B cells accumulate in the periphery, in contrast to &-deficient B cells, Syk may also be required for the transition of immature to mature B cells, their survival, or their migration to the peripheral lymphoid organs. B cell development is severely impaired at the early stage in patients with Bruton-type X-linked agammaglobulinemia characterized by severe depression of circulating B cells (Bruton, 1952). A recently identified Xlinked gene that encodes a cytoplasmic tyrosine kinase, Btk, has been shown to be mutated in the patients (Tsukada et al., 1993; Vetrie et al., 1993), suggesting that Btk may be involved in B cell development. By


29

contrast, a point mutation of mouse btk gene found in X d (CBNN) mice and a null mutation generated by homologous recombination lead to a much milder B cell deficiency in mice (Scher, 1982; Rawlings et al., 1993; Thomas et al., 1993; Khan et d.,1995; Kerner et al., 1995). Relatively normal numbers of proB/preB-I, preB-11, and immature B cells were detectable in bone marrow from Xid mice and Btk-deficient mice. However, in the presence of competition with Btk+ normal progenitor cells in chimera mice, Btk- progenitor B cells failed to expand at the transition from the proB/preB-I to preB-I1 stage, resulting in the relative lack of Btk- B cells (Kerner et al., 1995). Thus, Btk might be involved in the signal transduction through the preB cell receptor both in humans and mice, though it is not indispensable for B cell development in mice. It is presently uncertain whether HS1, a substrate of the antigen receptor-coupled tyrosine kinase, is involved in signahng from the preB cell receptor (Kitamura et al., 1989; Yamanashi et al., 1993). In HS1-deficient mice B cells appear to develop normally (Taniuchi et al., 1995). VI. olher Surrogab Light Chain-like Molecules

Recent studies have identified additional molecules besides As and VPmB which are associated with p heavy chain in preB cells. The Vp&3 (originally designated 8HS-20) gene isolated from a mouse preB cell line was shown to be selectively expressed in proB and preB cell lines as well as in bone marrow and, at low level, in spleen (Shirasawa et al., 1993). The gene encodes 123 amino acids including a leader sequence which forms an immunoglobulin domain-like structure and display 36% homology to V . In contrast to V reB1, V and A5 proteins, VpreB3protein is modifie by heterogeneityin isoelectricpoints and molecuglycosylationso &at it lar weights (13.5, 14, 15.5, and 16 kDa). All forms of the protein were found associated with p heavy chain in preB cell lines. Interestingly, they are expressed concomitantlywith VpreBl/A:,surrogate light chain in the same cell, although the surface expression of Vp&3 has not been demonstrated. 3 A5 are associated with The pulse chase experiments revealed that v p ~ B and p heavy chain at an early phase. As the assemblyproceeds, the association of v p e B 3 appears to decrease while the association of Vp,, becomes dominant (Ohnishi and Takemori, 1994). It remains to be elucidated whether VpEM and VpreBlare exchanged during assembly of the preB cell receptor or whether the preB cell receptors with either v p ~ B 3or VPrealhave different functions and fates in preB cells. A human counterpart of VpreBJ has not been identified. In some human preB leukemia cell lines, a germline JC, transcript was shown to be translated into a 15-kDa protein (Frances et al., 1994). The

SLES

rB1

30


protein was detected on the cell surface in covalent association with p heavy chain although the majority of the protein was found intracytoplasmically, not unlike Vp,$A5 surrogate light chain in B-lineage cells. The protein was also detected in preB cells from human fetal bone marrow. Any concomitant expression of jC, protein and surrogate light chain together with p heavy chain has not yet been investigated. It remains to be ascertained what roles these surrogate light chain-like molecules play in B cell development. The JC, protein seems not essential for B cell development, since the deletion of the K locus did not abrogate the production of A bearing B cells (Zou et al., 1993; Chen et al., 199313). VII. Concluding Remarks

The ordered recombination of the gene segments coding for variable region of antigen receptor during differentiation of cells is a common strategy of B and T cells to create the widely diversified T and B cell receptors. Therefore, it is not surprising to find striking similarities in the mechanisms regulating the development of B and T cells. Ample evidence has been accumulated that the expression of TCRP chain, which usually occurs prior to that of TCRa chain during T cell development in thymus, is essential for early development of thymocytes (Bosma et al., 1983; Schuler et al., 1986; Philpott et al., 1992; Mombaerts et al., 1992a; Shinkai et al., 1993; Mallick et al., 1993; Dudley et al., 1994). The expression of TCRP chain induces the transition of CD4-CD8- thymocytes to the CD4+CD8' stage, the positive selection and clonal expansion of productively VBDBJB-rearranged thymocytes, and the allelic exclusion at the TCRP chain locus. Thus, TCRP chain appears to be functionally equivalent to p heavy chain in the early states of lymphocyte development. This similarity is further evident in the fact that preT cells express a T cell analog of surrogate light chain, called preTa chain (pTa), which can pair with TCRP chain to form a preT cell receptor (Groettrup et al., 1993);pTa, a 33-kDa glycoprotein (gp33),is encoded by a gene on chromosome 17 near the MHC gene locus, which is selectively expressed in early stages of T cell development (Saint-Ruf et al., 1994).The critical role of pTa has been clarified by creating pTa-deficient mice. In these mice ap TCR+T cell development is severely impaired at the transition from the CD4-CD8to CD4+CD8' stage, resulting in a marked reduction of peripheral T cells (Fehling et al., 1995). Though the direct correlation of the preT cell receptor expression and RAG down-regulation has not yet been reported, the transient drop of RAG transcripts at the CD44-CD25-stage of thymocyte differentiation strongly suggested that the preT cell receptor mediates


31

allelic exclusion through down-regulation of RAG expression (Wilson et al., 1994). The developmental block in thymus of pTa-deficient mice phenotypically resembles that observed in bone marrow of the &deficient mice. It appears that B and T cells overcome the inefficient generation of p heavy chain+ and TCRP+ precursor cells based on the in- and out-of-frame random rearrangements of Ig heavy chain and TCRp chain loci, respectively, by means of selective proliferation of productively rearranged cells with signals given by the prelymphocyte receptors composed of p heavy chain (or TCRP chain) and surrogate chains. While we have made considerable progress in understanding why p heavy chain has to be produced prior to light chain during B cell differentiation and what molecules are associated with p heavy chain at the early stage of B cell development, much remains unknown about possible differential signalingthrough the preB cell receptor. The analysis of molecules involved in the signal transduction may identify novel genes responsible for primary immunodeficiencies. ACKNOWLEDGMENTS The Basel Institute for Immunology was founded by and is supported by F. HoffmanLa Roche Ltd, Basel, Switzerland.

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Snodgrass, H. R., Dembic, Z., Steinmetz, M., and von Boehmer, H. (1985). Expression of T-cell antigen receptor genes during fetal development in the thymus. Nature 315, 232-3. Spanopoulou, E., Roman, C. A., Corcoran, L. M., Schlissel, M. S., Silver, D. P., Nemazee, D., Nussennveig, M. C., Shinton, S.A., Hardy, R.R., and Baltimore, D. (1994).Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice. Genes Deo. 8, 1030-42. Stein, P. L., Lee, H. M., Rich, S., and Soriano, P. (1992). pp59fjm mutant mice display differential signaling in thymocytes and peripheral T cells. Cell 70, 741-50. Storb, U. (1995).Iggene expression and regulation in Ig transgenic mice. In Zmmunoglobulin Genes, 2nd Ed., (Honjo, T., and Alt, F.W., Eds.), pp 345-65. Academic Press, London. Takemori, T., Mizuguchi, J., Miyazoe, I., Nakanishi, M., Shigemoto, K., Kimoto, H., Shirasawa, T., Maruyama, N., and Taniguchi, M. (1990).Two t y p e s of p chain complexes are expressed during differentiation from pre-B to mature B cells. EMBO J. 9, 2493-500. Taniuchi, I., Kitamura, D., Maekawa, Y.,Fukuda, T., Kishi, H., and Watanabe, T. (1995). Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases. EMBO J. 14,3664-78. ten Boekel, E., Melchers, F., and Rolink, A. (1995). The status of Ig loci rearrangements in single cells from different stages of B cell development. Znt. Zmmunol. 7, 1013-9. Texido, G., Rajawsky, K., and Tarakhovsky, A. (1995).Targeted disruption of the B-cell specific protein tyrosine kinase blk in The 9th Znternational Congress of Zmmunology Abstrad No. 4042. Thomas, J. D., Sideras, P. Smith, C. I., Vorechovsky, I., Chapman, V., and Paul, W. E. (1993).Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261,355-8. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302,575-81. Tsubata, T., and Reth, M. (1990).The products of pre-B cell-specificgenes (A5 and VpreB) and the immunoglobulin p chain form a complex that is transported onto the cell surface. J. Exp. Med. 172, 973-6. Tsubata, T., Tsubata, R., and Reth, M. (1991). Cell surface expression of the short immunoglobulin p chain ( D p protein) in murine pre-B cells is differently regulated from that of the intact mu chain. Eur. J. Zmmunol. 21, 1359-63. Tsubata, T., Tsubata, R.,and Reth, M. (1992). Crosslinkingof the cell surface immunoglobulin (p-surrogate light chains complex) on pre-B cells induces activation of V gene rearrangements at the immunoglobulin K locus. Znt. Zmmunol. 4,637-41. Tsukada, S., Saffran, D. C., Rawlings, D. J.. Parolini, O., Allen, R. C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S.,et d.(1993).Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia.Cell 72,279-90. Turner, M., Mee, P. J,, Costello, P. S., Williams, O., Price, A. A,, Duddy, L. P., Furlong, M. T., Geahlen, R. L., and Tybulewicz, V. L. J. (1995). Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 378, 298-302. Urbanek, P., Wang, Z. Q., Fetka, I., Wagner, E. F., and Busslinger, M. (1994). Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pa5iBSAP. Cell 79, 901-12. Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A,, Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R.,Bobrow, M., et al. (1993).The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature 361,226-33.


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Weill, J. C., and Reynaud, C.-A. (1995). Generation of diversity by post-rearrangement diversification mechanisms: The chicken and the sheep antibody repertoires. In Immunoglobulin Genes (Honjo, T., and Alt, F. W., Eds.), pp. 267-288, Academic Press, London. Wilson, A., Held, W., and MacDonald, H. R. (1994). Two waves of recombinase gene expression in developing thymocytes. /. Exp. Med. 179, 1355-60. Winkler, T. H., Rolink, A., Melchers, F., and Karasuyama, H. (1995). Precursor B cells of mouse bone marrow express two different complexes with the surrogate light chain on the surface. Eur. 1.Immunol. 25, 446-50. Yamanashi, Y.,Okada, M., Semba, T., Yamori, T., Umemori, H., Tsunasawa, S., Toyoshima, K., Kitamura, D., Watanabe, T., and Yamamoto, T. (1993). Identification of HS1 protein as a major substrate of protein-tyrosine kinase(s) upon B-cell antigen receptor-mediated signaling. Proc. Natl. Acad. Sci. USA 90,3631-5. Yanagi, Y., Yoshikai, Y.,Leggett, K., Clark, S. P., Aleksander, I., and Mak, T. W. (1984). A human T cell-specific cDNA clone encodes a protein having extensive homology to immunoglobulin chains. Nature 308, 145-9. Yancopoulos, G. D., and Alt, F. W. (1985). Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell 40, 271-81. Yang, J., Glozak, M. A., and Blomberg, B. B. (1995). Identification and localization of a developmental stage-specific promoter activity from the murine A5 gene. 1. Immunol. 155,2498-514. Young, F., Ardman, B., Shinkai, Y., Lansford, R., Blackwell, T. K., Mendelsohn, M., Rolink, A., Melchers, F., and Alt, F. W. (1994). Influence of immunoglobulin heavy- and lightchain expression on B-cell differentiation. Genes Deu. 8, 1043-57. Zhuang, Y., Soriano, P., andweintraub, H. (1994).The helix-loop-helixgene E2A is required for B cell formation. Cell 79, 875-84. Zou, Y. R., Takeda, S., and Rajewsky, K. (1993). Gene targeting in the Ig K locus: Efficient generation of A chain-expressing B cells, independent of gene rearrangements in Ig K. EMBO]. 12,811-20. This article was accepted for publication on 22 January 1996.

ADVANCES IN 1MMUNOU)CY. VOL.63

CD4O and Its Ligand

1. Introduction

A. OVERVIEW OF T-DEPENDENT B LYMPHOCYTE ACTIVATION Antibody responses to soluble protein antigens usually require the collaboration of B lymphocytes with helper T (Th)lymphocytes. In contrast to Tindependent (TI) antigens, which are typically polysaccharides possessing highly repeated or polymeric epitopes, thymus-dependent (TD) antigens are monovalent or paucivalent proteins. The binding of TD antigens to specific IgM and IgD on the B cell surface does not result in extensive cross linking of these membrane immunoglobulin receptors (mIg). Therefore, unlike the binding of TI antigens, the signals transduced through mIg by TD antigens are unable to provide a major growth stimulus to B cells (DeFranco, 1993).This suggests that differences in the degree of receptor clustering by the antigen influences the biochemical response of the B cell. However, the Ig receptor signal transduction mechanisms responsible for these differing responses are currently unknown (Cambieret al., 1994). TD antigens are insufficient to induce B lymphocyte cell cycle entry, proliferation, or subsequent antibody production without the delivery of additional signals to B cells from Th. These include signals delivered via direct cell-cell contact between B cells and Th, and also through the elaboration of soluble cytokines by helper T cells (reviewed in Noelle and Snow, 1990).

B. RECOGNITION PHASE AND T CELLACTIVATION TD humoral responses are initiated by the presentation of antigen to helper T cells. Antigen bound via mIg to antigen-specific B cells is rapidly endocytosed and the internalized protein is processed into antigen-derived peptides which complex with MHC class I1 molecules in the late endosomes. Antigenic peptides, in association with MHC class 11, are then reexpressed on the cell surface where they initiate cognate interactions with antigen-specific helper T cells. Thus, the initial events of Th-dependent B cell activation, termed the recognition phase (Noelle and Snow, 1990), are MHC-restricted and antigen-specific. Engagement of the antigenMHC complex by a T cell receptor (TCR) of the appropriate specificity 43 Copyright 0 1996 by Academic Press,Inc. All rights of reproduction in any form resewed.

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triggers, in turn, a cascade of events critical to the reciprocal activation of both partners. Reciprocal activation requires the formation of stable physical conjugates between Th and B cells and also the sequential and interdependent induction or up-regulation of accessory membrane proteins such as CD40 ligand (GP39, gp39) on T cells and B7.1 and B7.2 on the B cells. The downstream signals transduced by the binding of these proteins to their respective ligands, CD40 on the B cell and CD28/CTLA4 on the T cell, result in the polyclonal, antigen-nonspecific and M HC-nonrestricted activation of both partners. Antigen-specific recognition by the TCR of the antigen-MHC complex and the binding of CD4 with nonpolymorphic domains of MHC class 11, results in a rapid up-regulation of cellular adhesion through the functional regulation of LFA-1, a member of the integrin family of adhesion molecules. TCR crosslinking rapidly and transiently increases the affinity of LFA-1 for its ligand ICAM-1 (Dustin and Springer, 1989),thereby directly up-regulating the avidity of the Th cell for the B cell and contributing to conjugate stability. These interactions are followed by increased expression of the Th membrane glycoprotein gp39, the ligand for CD40, and upregulated IL-2 production (Hirohata et al., 1988; Roy et al., 1993). GP39 in induced within 2-4 hr of T cell activation, with maximal expression reached within 8 hr, followed by a return to resting levels within 2448 hr. Thus, the recognition phase of T-B cell interaction results in T cell activation and the induction of T cell effector function, while B cells remain in a Go resting state. A N D B CELLACTIVATION C. EFFECTOR PHASE Induction of gp39 expression by activated Thleads to the interaction of GP39 with CD40 on the B cells. This phase of T cell help, termed the effector phase (Noelleand Snow, 1990),is Class I1 unrestricted and antigen nonspecific. Signaling through CD40 triggers B cell activation and progression through the GI-G2 stages of the cell cycle (Banchereau et al., 1991). Activated B cells also up-regulate the expression of their IL-2, IL-4, and IL-5 interleukin receptors (Grabstein et al., 1993; Gray et al., 1994) and become highly responsive to the growth and differentiative effects of these lympholdnes. In response to CD40-gp39 interactions and IL-4, B cells become fully competent antigen-presentingcells (APCs) by increasing the expression of B7.1 and B7.2 (Hathcock et al., 1994; Roy et al., 1994), cell surface molecule critical for the costimulations of T cells through the counterreceptors CD28 and CTLA-4, thereby preventing T cell anergy, as reviewed by (Linsley and Ledbetter, 1993). At this stage, the fully activated B cells are competent to progress through G2 and mitosis, produce soluble antibody, and undergo terminal differentiation.

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II. CD40 and Ligand Expression and Mdecular Struchmre

A. EXPRESSION OF CD40 The CD40 antigen was first identified by the generation of monoclonal antibodies (mAbs) that recognized a 50-kDa protein on the surface of B cells and certain carcinomas and provided costimulatory signals which primed B cells for growth, differentiation, and survival (Clark and Ledbetter, 1986). CD40 is constitutively expressed on a variety of cell types including B lymphocytes (Stamenkovic et al., 1989), interdigitating cells (IDC) (Caw et al., 1994), monocytes (Alderson et al., 1993), vascular endothelial cells (Hollenbaughet al., 1995),follicular dendritic cells (FDC) (Schriever et al., 1989),thymic epithelial cells (Galy et al., 1992),hematopoeitic progenitor cells (Uckunet al., 1990),and some carcinomas (Carbone et al., 1995, Paulie et al., 1989).

B. CD40 MOLECULARSTRUCTURE A cDNA sequence encoding CD40 was isolated in 1989 from a mammalian cell expression library prepared from the Burkitt lymphoma Raji cell line (Stamenkovicetal., 1989).The human CD40 gene maps to chromosome 20 qll-2-q13-2 (Ramesh et al., 1993), while the murine homolog is located on the syntenic region of chromosome 2 (Grimaldi et al., 1992).The gene sequence predicts a membrane bound protein consisting of a 171-aminoacid (aa) extracellular domain, a single 22-aa hydrophobic transmembrane domain, and a 62-aa cytoplasmic domain. It also encodes a 22-aa NH2terminal secretory signal sequence and two potential N-linked extracellular glycosylation sites. CD40 is classified as a member of the tumor necrosis factor receptor (TNF-R) superfamily based upon the presence of a type I extracellular binding motif composed of tandem cysteine-rich pseudo repeats. This family, which includes Fas, LNGFR, TNF receptors I and II,OX40, CD27, and CD30, interacts with a parallel family of related ligands (reviewed in Smith et al., 1994). Notably, many members of this family promote either programmed cell death upon ligation (Fas/Apo-1, TNF-R) or cell survival (LNGFR, CD40). In spite of the common structural motif of their extracellular domains, the cytoplasmic domains of TNF-R family members vary widely in length (46-221 residues) and share little homology. The 62-aa intracellular domain of human CD40 is not homologous to any previously identified molecules; however, human and murine CD40 molecules share 78% homology at the amino acid level, and the C-terminal 32 residues are completely conserved between the two species (Mallet and Barclay, 1991; Torres and Clark, 1992). The N-terminal cytoplasmic region of CD40 lacks tyrosine yet appears to be critical for signal transduction, as

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a deleterious Thr234-) Ala point mutation in this region has been identified which interferes with signaling (Inui et al., 1990). While CD40 is primarily expressed as a membrane-bound receptor, a soluble form (sCD40) released by proteolysis has been detected by specific ELISAs in the supernatants of tonsillar human B cells and EBV-transformed B cell lines (Bjorck et aE., 1994; van Kooten et a!., 1994). sCD40 is capable of binding to gp39 expressed on activated T cells and may function to regulate gp39 expression. OF gp39 (CD40 LIGAND) C. IDENTIFICATION The identification of the gp39 as a novel T cell surface molecule expressed on activated Th resulted from extensive in vitro studies which provided insights into the nature of cognate interactions between Th and B cells. Early studies using purified B cells and anti-TCR activated TI, clones demonstrated that T cells, once activated, were competent to induce the activation of resting B cells in the absence of cognate, genetically restricted T-B cell interactions (Julius and Rammensee, 1988).T cell effector activity was also shown to require the physical interaction of T cells with B cells, as physical separation of T and B cells abrogated B cell activation (Hirohata et al., 1988). Bartlett et al. (1989) further demonstrated that Th effector function was contact-dependent and not due to the production of soluble lymphokines by showing that an antigen-specific, IL-2-dependent Th clone unable to produce lymphokines could induce B cell activation (Bartlett et al., 1989). In later experimental systems, T-dependent activation of B cells was reconstituted in culture using plasma membranes derived from anti-TCR activated, but not resting, Th clones and the addition of soluble lymphokines (Gascan et al., 1992; Hodgkin et al., 1994; Schultz et aE., 1992; Noelle et al., 1991). It was further shown that Th effector activity in this system was not due to the increase of known cell surface markers such as CD3, CD4, LFA-1, or ICAM, and that the induction of effector function required de now protein synthesis (Bartlett et al., 1990). Collectively, these studies established that B cell activation triggered by activated Th membranes was polyclonal, antigen-nonspecific, and MHC-unrestricted, yet was critically dependent upon preactivation of T cells as well as physical contact between T and B cells. These studies also strongly suggested that activated Th express a previously uncharacterized, nonpolymorphic cell surface molecule responsible for the mediation of effector function and the initiation of TD humoral responses.

D. CLONING OF gp39 (CD40 LIGAND) The murine and human ligands for CD40 (CD40-L (Armitage et al., 1992a), gp39 (Hollenbaugh et al., 1992), TRAP (Graf et al., 1992)), a

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33- to 39-kDa protein expressed on activated T cells, were cloned and characterized by several independent groups in 1992. In early studies, the murine thymoma cell line ELA was observed to trigger the polyclonal activation of resting B lymphocytes (Zubler et al., 1987).Armitage and coworkers (1992a,b)further observed that a fusion protein consisting of the extracellular domain of murine CD40 linked to the Fc region of human IgGl could bind specifically to EL4 cells, a finding which led to cloning of the ligand. On this basis, EL4 cells were selected for high levels of CD40 ligand expression by several rounds of flow-cytometric cell sorting using biotinylated CD40-Ig. To clone gp39, mRNA extracted from sorted ELA cells was used to construct a mammalian cDNA expression library and radiolabeled CD40Ig was used as a probe to isolate a single cDNA clone capable of binding specifically to CD40-Ig. CVliEBNA cells were then transfected with the single open reading frame (ORF) sequence contained within this clone. This was followed by affinity precipitation of gp39 from the transfected cells with CD40-Ig (Annitage et al., 1992a). The amino acid sequence encoded by this clone predicted a novel type I1 transmembrane glycoprotein of 260 aa with significant homology to tumor necrosis factor (TNF). In functional studies, CVlEBNA cells transfected with recombinant gp39 were directly mitogenic for splenic murine B cells and purified human tonsillar B cells. IgE secretion was also induced in this system in the presence of IL-4. Thus, cell surface expression of gp39 on CVlEBNA cells induced functional activities previously ascribed to ligation of CD40 by a-CD40 mAbs, thereby confirming its identity (Armitageet al., 1992a). Additional studies in humans by Lederman and co-workers described a mAb that recognized a molecule on the surface of a mutant Jurkat cell line, referred to as TBAM, that activated human B cells. This mAb, 5c8, was ultimately shown to recognize the ligand for CD40 (Lederman et al., 1992a,b; Yellin et al., 1991). Noelle et al. (1992a)simultaneously isolated a hamster monoclonal antibody specific for gp39 based upon the production of mAbs which selectively recognized proteins expressed upon activated, but not resting, Thl clones. MR1, an anti-mouse gp39 mAb, was found to block in vitro murine B cell activation inducible by coculture with plasma membranes isolated from activated Th (Noelle et al., 1991). Both CD40-Ig and MR1 detected a molecule expressed on activated Th, and both were shown to immunoprecipitate the same molecule of approximately 39 kDa in lysates of activated Th.These results confirmed that MR1 and CD40-Ig were binding to the same protein, the ligand for CD40. To clone the human homolog of gp39, oligonucleotide primers based on the murine sequence were constructed and used to amplify a cDNA

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sequence from a cDNA library prepared from PHA-activated human peripheral blood T cells (Hollenbaughet al., 1992).The resulting PCR product was subcloned into an expression vector and used as a probe to isolate the complete human gp39 gene from the same activated T cell cDNA library by colony hybridization. The human ligand for CD40 was also independently cloned from activated human T cells by two other groups that same year, through binding to soluble CD40 constructs (Graf et al., 1992, Lane et al., 1992). E. CD40 LICAND EXPRESSION GP39 is inducibly expressed on normal, activated CD4' splenic and peripheral blood T cells, activated TJ and Th2 clones, and antigen-primed lymph node cells (Roy et al., 1993). A low percentage of CD8+ T cells also express gp39 following activation by PMA and ionomycin, but not by anti-CD3 (Lane et al., 1992; Roy et al., 1993). GP39 mRNA transcripts have also been detected by PCR in CD8' T cell clones, a TCR ytSt T cell clone, purified NK cells, monocytes, fetal thymocytes, and small intestine (Cocks et al., 1993). Human basophils and mast cells also express gp39 (Gauchatet al., 1993b) and low-level expression may be pharmacologically induced on eosinophils (Gauchatet al., 1995).The broad tissue distribution of GP39 implies that its function is not limited T-B cell collaboration. CD40-gp39 interactions may potentially regulate a complex network of in vivo physiological functions which are just beginning to be appreciated. CD40 ligand is detectable within 1-2 hr following in vitro T cell activation, reaches maximal levels at 6-8 hr postactivation, then declines to resting levels between 24 and 48 hr (Lane et al., 1992; Maliszewski et al., 1993; Roy et al., 1993). The kinetics of gp39 expression in vim, as determined by immunohistochemical examination of TNP-KLH immunized mice, revealed maximum frequencies of GP39' Th cells present in the periarteriolar lymphocyte sheaths (PALS)of the spleen at 3-4 days postimmunization, with a rapid decline to background levels by Days 5-6 (Van den Eertwegh et al., 1993). Immunohistochemical examination of human lymphoid tissues revealed that gp39+T cells are associated with B cells in the mantle and centrocytic light zones of germinal centers, as well as the splenic PALS region (Lederman et al., 1992a).

F. CD40 LICAND MOLECULARSTRUCTURE Murine gp39 is a type-I1 transmembrane glycoprotein of 260 residues; the human homolog consists of 261 aa. The murine and human molecules are highly homologous, sharing 83% identity within the coding region at the nucleotide level (Hollenbaugh et al., 1992). The human sequence consists of a 22-aa amino-terminal intracellular domain, a single trans-

CD40 AND ITS LIGAND

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membrane element consisting of 24 predominantly hydrophobic aa, and a carboxy-terminal extracellular (EC) domain of 215 aa. The EC domain of the mouse homolog consists of 214 aa. Each shares a conserved Nlinked glycosylation site and 4 cysteine residues within the EC domain. Differences between the predicted and observed molecular masses of this molecule (approximately 29 and 33 kDa, respectively, in humans) suggest that the protein is modified post-translationally by glycosylation. Based upon significant structural homology to TNF-a, gp39 is characterized as a member of the tumor necrosis factor cytokine family (Hollenbaugh et al., 1992; Smith et al., 1994). This family, which includes lymphotoxin (LT)-a and -P, and the recently cloned CD27-L, CD30-L, 4-1BBL, FasL, and OX40 ligands (Goodwin et al., 1993),exhibits considerable divergence at the amino acid level with sequence identity limited to approximately 150 residues in the C-terminal (receptor binding) domain. The C-terminal residues of soluble TNF-a and LT-a have been shown by X-ray diffraction studies to fold into P-pleated sheets and trimerize, forming a distinctive three-dimensional structure termed the TNF fold. Based upon the high degree of sequence conservation at critical folding interfaces it is likely that all ligands of this family fold in the same manner and activate their receptors as multimers (Smith et al., 1994). Structure-based sequence alignment studies of GP39 indicate that its amino acid sequence is compatible with the formation of a TNF-like fold and trimeric quaternary structure (Bajorath et al., 1995). Members of this family, with the exception of LT, exist as membranetethered cytokines which bind to specific cell surface TNF-R ligands, thereby mediating signaling in a contact-dependent manner. However, LT, TNF-a, and TNF-P also exist as soluble molecules and are capable of receptor signaling in a secreted form. Both human and murine gp39 contain residues at the junction of the extracellular and transmembrane domains which may be susceptible to proteolytic cleavage. Armitage et al. (1992b)found that the supernatant (SN) of sorted EL4 cells contained an activity stimulatory for human and murine B cells, which could be removed by preclearing the SN with CD40-Ig. This observation suggests that gp39 may also exist in a naturally soluble form with activity for human and murine B cells (Armitageet al., 1992b). Like TNF-a and TNF-P, soluble recombinant gp39 is predicted to exist in solution as a noncovalently linked homotrimer (Hollenbaugh et al., 1992). G. gp39 STRUCTURE IN HYPER-IgM SYNDROME Hyper-IgM Syndrome (HIM) is an X-linked immunodeficiency characterized by elevated serum IgM, reduction or loss of downstream isotype switching to IgG, IgA, and IgE, the absence of germinal center formation,

50


and an inability to mount immune responses to TD antigens. Consequently, afflicted males suffer from recurrent life-threatening bacterial infections. In a majority of cases studied, the molecular defect for HIM results from deleterious mutations of the gp39 gene (Allen et a!., 1993; Aruffo et a!., 1993; DiSanto et al., 1993, Korthauer et al., 1993; Bajorath et al., 1995; Hollenbaugh et al., 1994). These amino acid substitutions, usually point mutations, may result in the generation of premature stop codons, interfere with receptor binding, or prevent normal ligand expression. Normal B cell activationvia CD40-gp39 interaction apparently does not occur in affected individuals in duo, although B cells from these patients exhibit normal in vitro activation when cultured in the presence of a-CD40 mAbs and cytokines (Durandyet al., 1993).HIM also occurs in some patients expressing functional CD40 ligand but having B cells which respond abnormally to CD40 ligation. The data suggest that in the latter group, the defect is caused by faulty signal transduction through CD40 (Conley et al., 1994). 111. lntracellular Signal Transdudion through CD40

A. CHARACTERIZATION OF CD40-BINDING PROTEINS It is clear that CD40 mediates signal transduction pathways distinct from those triggered by the engagement of sIg. As CD40 lacks intrinsic enzymatic activity, signal transduction is likely to be mediated through the association of recently identified CD40 binding proteins (Cheng et al., 1995; Hu et al., 1994; Mosialos et al., 1995; Sat0 et al., 1995) with the cytoplasmic domain of CD40 as well as via the downstream activation of nonreceptor tyrosine kinases (Hasbold and Klaus, 1994; Knox and Gordon, 1993; Marshall et al., 1994; Uckun et al., 1991). A human cDNA encoding a novel protein that interacts directly with the cytoplasmic tail of CD40, designated CD40 binding protein (CD4O-bp) (Hu et al., 1994) [also LAP1 (Mosialos et al., 1995),CRAF-1 (Cheng et al., 1995), CAP-1 (Sato et al., 1995)],was recently isolated using the yeast two-hybrid screen. CD40-bp possesses several zinc-finger like domains (Sato et al., 1995) and an Nterminal RING finger motif common to DNA-binding proteins such as the V(D)J recombinase RAG1 (Hu et al., 1994). The N-terminus also possesses strong homology to a novel family of tumor necrosis receptor (TNF-R2) associated factors proteins, TRAFl and TRAF2 (Rothe et al., 1994),which associate with the cytoplasmic domain of TNF-R2 as homoor heterodimers (Cheng et al., 1995; Hu et al., 1994; Sat0 et al., 1995). The murine and human sequences of CD40-bp are 96% homologous. Binding to the cytoplasmic domain of CD40 and homodimerization of CD40-bp are mediated by the C-terminal region (Cheng et al., 1995; Sato et al., 1995).

CD40 AND ITS LIGAND

51

A direct role for CD40-bp in CD40 signal transduction is postulated based upon evidence that growth transformation of lymphocytes by Epstein-Barr Virus (EBV) is mediated through the direct interaction of the first 44 amino acids of EBV latent infection membrane protein 1 (LMPl)carboxyl-terminuswith CD40-bp (LAP1).The constitutiveinteraction of LMPl with CD40-bp appears to transform infected cells via constitutive activation of a CD40 signaling pathway (Mosialos et al., 1995). Overexpression of a CD40-bp C-terminal truncation mutant has also been shown to inhibit CD40-mediated up-regulation of CD23 (Cheng et al., 1995). The mechanisms by which CD40-bp mediates signal transduction remain enigmatic, as its structural characteristics suggest a direct capacity for DNA binding.

KINASES B. THEROLEOF NONRECEFTORTYROSINE Ligation of CD40 by plasma membranes isolated from gp39+ Th (Marshall et al., 1994; Kato et al., 1994) or a-CD40 mAb (Knox and Gordon, 1993) has been shown to induce tyrosine phosphorylation of a number of B cell substrates. Also, inhibition of protein kinase A suppresses proliferation induced by gp39+membranes (Kato et al., 1994).Stimulation of Daudi cells with a-CD40 results in a rapid increase in phosphorylation of the src kinase Lyn, as well as phosphorylation of phopholipase Cy2 and phosphatidylinositol (PI)-3-kinase (Rene et al., 1994). Rapid ( C 1 min) changes in tyrosine phosphorylation, including specific dephosphorylation of the protein tyrosine kinases (PTKs) Lyn, Fyn, and Syk and the transient phosphorylation of a 28-kDa protein, were observed in EBV-transformed Burkitt’s lymphoma cells and tonsillar B cells stimulated with a-CD40 mAb (Fans et al., 1994). Prolonged stimulation also resulted in increased tyrosine phosphorylation of 50- to 80-kDa proteins (Faris et al., 1994). Btk, a B-cell specific tyrosine kinase, has also been implicated in downstream signal transduction through CD40 based upon its role in development of the X-linked immunodeficiency, xid. B cells derived from CBA/N xid mice are unable to proliferate in response to ligation of CD40 via gp39+T cells, a-CD40 mAb, or gp39 fusion proteins in the presence of IL-4, resulting in abnormal antibody responses (Hasbold and Klaus, 1994). The defect responsible for xid has been determined to be a point mutation in the gene encoding Btk (Hasbold and Klaus, 1994). Collectively, these studies establish that signaling through CD40-gp39 interactions induces the activity of PTKs and protein kinase A. However, conflicting data have been reported for the involvement of other signal transduction pathways including elevation of intracellular calcium, CAMP,and inositol trisphosphate ( IP3)production (Francis et al., 1995). Further studies are needed to clarify and expand these observations.

52

LISA B. CLARK ETAL.

N. CD4O-gp39

Functional Inbradons

A. GENERAL A large number of recent studies have documented the role of CD40 as a signalingmolecule critical to the development of T-dependent humoral immunity. Briefly, signaling through CD40 is known to be required for B cell activation, proliferation,antibody class switching, germinal center (GC) formation, rescue of germinal center B cells from apoptosis, and memory cell generation (reviewed in Banchereau et al., 1994; Dune et al., 1994; Ochs et al., 1994).Antibody blockade of signalingthrough CD40 in murine models has also been shown to block the development of both primary and secondary immune responses to TD antigens in vivo (Foyet al., 1993). The critical nature of functional CD40-gp39 interactions in the development of TD humoral immunity in uivo has been underscored by recent advances in our understandingof the genetic defects underlying the development of hyper-IgM syndrome (see above). Recently, the development of both CD40- and gp39-deficient mice through genetic deletion has confirmed the functional roles ascribed to these molecules by previous studies. Significantly, CD40-deficient mice exhibit many of the same immunologic deficiencies as HIM patients, including lack of germinal center formation, inability to respond to TD antigens, and severely decreased IgG and IgE production (Castigli et al., 1994; Kawabe et al., 1994). In accordance with these findings, mice deficient for gp39 also fail to develop normal humoral responses and germinal centers in response to TD antigens (Renshaw et d., 1994; XU et d., 1994). The role of CD40-gp39 interactions in B cell proliferation, antibody responses, and the development of GC and memory cells will be discussed at length below. While signaling through CD40 in conjunction with the presence of appropriate cytokines is clearly an important requirement for each of these processes, the available data are consistent with the interpretation that these signals are not sufficient.

B. EARLY B CELLACTIVATIONEVENTS Signaling through CD40 has been demonstrated to exert a wide range of potent biological effects on B cells. Early activation events induced by mAb cross-linking of the CD40 receptor include blast formation (Valle et al., 1989)and up-regulated expression of a number of cell surface antigens including the low-affhity IgE receptor CD23 (Saeland, 1993),MHC Class I1 (Klauset al., 1994b),intercellular adhesion molecule-1 (ICAM-1, CD54) (Barrett et al., 1991), B7.1, and B7.2 (Ranheim and Kipps, 1995, 1993; Roy et al., 1994).CD40 ligation by soluble mAb also induces the homotypic aggregation of human resting B cells via ICAMLFA-1 and CD21/CD23

CD40 AND ITS LIGAND

53

(Bjorck et al., 1993; Bjorck and Paulie, 1993). Normal human B cells are induced to secrete IL6 in response to CD4 signaling (Clark and Shu, 1990). B cell activation also induces the heterotypic adhesion of B cells to endothelial cells via the very late antigen-4 (VLA-4) adhesion molecule (Flores et al., 1993). C. STIMULATORY EFFECTS ON NON-B CELLS The ligation of CD40 also exerts a variety of stimulatory effects on hematopoeitic non-B cells, indicating that it is pleiotropic in its activity. GP39 is stimulatory for resting peripheral blood T cells, up-regulating its own expression and IL-2 receptor (CD25) expression, suggesting a potential autocrine activation loop (Armitage et al.,1993b). CD40 expression of resting monocytes is highly up-regulated in response to IL-3, GM-CSF, and IFN-y. Ligation of CD40 on monocytes results in their adhesion to cells expressing gp39, induces secretion of IL-6, IL-8, and TNF, and results in increased tumoricidal activity (Alderson et al., 1993). Cross-linking of CD40 on human dendritic cells (DC) by gp39-transfected L cells results in profound phenotypicchanges which are thought to reflect the physiological, antigen-specific interactions which occur between DC and T cells in secondary lymphoid organs, where DC act as antigen-presenting cells (Caux et al., 1994). These changes include enhanced cell survival, increased dendritic development, up-regulation of MHC Class 11, CD58, CD80 (B7l), CD86 (B7-2),and CD25. In addition, CD40-activated DC are induced to secrete a variety of cytokines, including IL-la, IL-lp, IL-6, IL-8, IL10, TNF-a, and MIP-la (Caux et al., 1994). CD40 signaling is reported to up-regulate expression of the adhesion molecules E-selectin, VCAM1, and ICAM-1 on endothelium, and may play a role in the homing of lymphocytes (Hollenbaugh et al.,1995; Karmann et al., 1995). V. The Role of CD40 Signaling in B Lymphglte Proliferation

A. GENERAL Signals transduced through CD40 synergize with other contact-dependent signals and soluble cytolcines to induce the proliferation of mature human B cells. The recent availability of murine and human recombinant gp39 has permitted study of the contributions of signals delivered by CD40-gp39 interactions relative to those of other signals in triggering proliferation. In vitro studies using distinct constructs of soluble gp39 (,GP39), all of which bind to CD40 with high affinity, suggest that B cell responses to ligation of CD40 are hierarchicallydependent upon the degree of oligimerization of the ligand (Fanslowet al., 1994).A soluble, recombinant fusion construct, consisting of the extracellular domain of murine

54


gp39 linked to the extracellular domain of murine CD8 (,gp39-CD8), supports B cell proliferation only when cross-linked in culture by a-CD8 mAb, or in the presence of IL-4; these data suggest that GP39 requires the presence of costimulatory molecules to induce activation when CD40 receptor cross-linking is suboptimal (Kehry and Castle, 1994). sgp39 in trimeric form, in contrast to monomeric or dimeric ,gp39, exhibits the greatest activation potency presumably because it best mimicks the degree of receptor cross-linking provided by membrane bound ligand ( Fanslow et al., 1994). While there appears to be no strict requirement for ligand in a membrane-bound form, gp39 most efficiently induces proliferation when presented as a membrane-bound protein, suggesting the possible contribution of signals by other membrane-bound ligands.

B. CYTOKINE REQUIREMENTS Ligation of CD40 on human peripheral blood and tonsillar B cells by anti-CD40 mAb or via gp39 is reported to directly support limited cell proliferation (Armitage et al., 1992a; Hollenbaugh et al., 1992). However, optimal or long-term proliferation invariably requires the addition of exogenous cytokines or other costimuli (Table I). Resting tonsillar human B cells may be sustained in culture for at least 10 days when cocultured with anti-CD40 and a murine fibroblastic L cell line stably transfected with FcyRIUCDw32 (CDw32 L cell system), which present the anti-CD40 mAb89 to B cells (Banchereau et al., 1991; Rousset et al., 1991).Yet the establishment of long-term human, Epstein-Barr (EBV) virus-negative B cell cultures in this system requires the addition of IL-4. Among a panel of cytokines tested. IL-la, IL-4, IL-6, IL-10, and IFN-y all enhanced the uptake of [3H]thymidine by B cells cultured with CDw32 cells and aCD40. However, only IL-4 and IL-10 were found to significantly boost the a-CD40 induced expansion of B cells, as measured both by [3H]thymidine incorporation and cell enumeration (Rousset et al., 1991, 1992). When added simultaneously IL-4 and IL-10 displayed additive effects, as did combinations of IL-4 with IL-la or IFN-y. In related experiments, the monkey fibroblast CV-1/EBNA cell line was transfected with human gp39. The transfected cells, which express hgp39 as a membrane protein, were fixed in paraformaldehyde and added to highly purified tonsillar B cell cultures (>98% CD20') in the presence or absence of soluble cytokines. Under these conditions, proliferation induced by gp39 is strongly enhanced by the addition of IL-2, IL-4, or IL10 (Armitage et al., 1993a; Spriggs et al., 1992). This system also supports increases in cell number as well as enhanced [3H]thymidineincorporation. Furthermore, cytokines which were not stimulatory in the presence of

CD40 AND ITS LIGAND

55

hGP39 alone, such as IL-2, IL-5, IL-10, TNF-a, and IFN-y, enhanced the proliferative response in the presence of IL-4. Only the addition of transforming growth factor P (TGF-P) strongly suppressed proliferation (Armitage et al., 1993a). IL-13, in the absence of IL-4, has also been reported to significantly promote the proliferation of human B cells cultured with hGP39-transfected COS cells (Cocks, et al., 1993). A similar role for IL-4 in B cell proliferation has been demonstrated in the murine system. Soluble recombinant gp39 expressed on the surface of fixed CVU EBNA cells activates purified splenic mouse B cells and directly stimulates [3H]thymidineincorporation. However, the proliferative response is enhanced by the addition of IL-4, or IL-5, and together these cytokines provide strong costimulation (Maliszewski et al., 1993). In contrast to human studies, IL-10 in this system was not found to costimulate proliferation.

c. CDdO-DRIVEN PROLIFERATION OF B CELLSUBSETS

Most, but not all, subsets of mature B lymphocytes are reported to proliferate in response to GP39 and costimuli in vitro. Dense, resting, tonsillar B cells isolated by Percoll fractionation proliferate in response to GP39-transfected CVlmBNA cells, alone and in the presence of cytokines, demonstrating that proliferation is not restricted to preactivated cells (Armitage et al., 1993a). Similarly, purified (>98% CD19') sIgD+ and sIgDtonsillar cells proliferate in response to CDw32L cells and a-CD40, although sIgD+cells are reported to exhibit a higher rate of growth (Galibert et d., 1994). Germinal center B cells (CD38+CD10fCD39-CDw 75b"gh'IgGt) purified from human tonsils proliferate in response to a-CD40 and IL-4, with proliferation somewhat enhanced by the further addition of a-IgM Ab (Dono et al., 1993). Engagement of CD40 by its ligand or by a-CD40 mAb together with IL-4 is also reported to enhance DNA synthesis, proliferation, and survival of a number of malignant cell types including chronic lymphocpc leukemia (CLL) B cells, hairy cell leukemia (HCL), and Hodgkin's disease cell lines (Carbone et al., 1995; Crawford and Catovsky, 1993; Kluin et al., 1994). Purified CD5+ and CD5- B cell subsets isolated from human tonsils proliferate in response to costimulation with anti-Ig, immobilized a-CD40, and IL-4 (Defrance et al., 1992a; Dono et al., 1993).Dono and co-workers (1993)further characterized two distinct CD5- B cell subsets that vary in their proliferative response to a-CD40 mAb (Dono et al., 1993). Resting CD5- (sIgD'") B cells copurified from a 60% Percoll gradient along with responsive CD5+ (sIgD+) B cells failed to proliferate in response to aCD40 mAb in the presence of IL-4 and a-IgM (Dono et al., 1993). In contrast, less dense CD5- (sIgD+)B cells isolated from the 50% Percoll

56


fraction proliferated vigorously in response to the same stimuli. Upon depletion of cells bearing the activation markers CD69', CD23', CD25+, and CD71+,CD5- B cells from the 50% Percoll fraction did not proliferate in response to in uitro stimulation, suggestingthat in uiuo preactivation may prime this subset for proliferation in response to CD40. D. THEROLE OF sIg CROSSLINKING A number of studies have suggested that cross-linking of surface immunoglobulin (sIg)is also critical to the development of TD responses, particularly when the cross-linkingreagent is displayed to the B cell in a multivalent array. In functional studies, mouse or human B cells cultured with aCD40 (Paulieet d.,1989)or a mgp3O-mCD8 soluble fusion protein (Lane d al., 1993)were induced to proliferate only weakly, but when costimulated with anti-Ig, strong proliferation was observed. Coligation of CD40 and sIg, via a-CD40 mAb together with picomolar concentrations of mouse a-IgM and a-IgD mAbs of the y l isotype, results in potent stimulation of resting B cells when cocultured in the presence of F,RyII-transfected murine L cells (Wheeler et al., 1993). Costimulation is dependent upon multivalent presentation of the mAbs via interaction with the transfected F, receptors, as an IgG2a a-IgM mAb, which is not recognized by F,yRII, was 1000-fold less effective at costimulation of B cell proliferation. Similarly, cross-linking of sIg by low concentrations (pg/ml) of multivalent, dextran-conjugateda-IgM or a-IgD is strongly synergistic with stimulation through CD40 for murine B cell proliferation and Ig secretion, while divalent a-IgM or a-IgD induces little enhancement of B cell proliferation, even at ng/ml concentrations (Snapper et al., 1995). For each of these studies, it is worth noting that the costimulatoryactivity of sIg is measured by incorporation of [3H]thymidineby B cells grown in bulk culture. These studies do not address the fundamental question of whether costimulation through sIg acts to increase the precursor frequency of cells which proliferate in response to CD40 signaling, or alternatively, whether the increase in [3H]thymidineincorporation is due to the increased responsiveness of individual B cells already committed to proliferation in response to CD40.

E. A ROLEFOR ACCESSORYMOLECULES While the degree of CD40 receptor cross-linkingprovided by membrane bound gp39 is clearly important to the level of B cell response, the available data are consistent with the interpretation that COS cells, CVl/EBNA cells, and the L cells which present gp39 or a-CD40 mAbs in these experimental systems may represent a source of other, as yet unidentified, costimulatory molecules which act in synergy with gp39 to drive B cell

CD40 AND ITS LIGAND

57

activation and proliferation. Further, many CD40-based experimental systems rely on the use of B cell preparations which are not highly purified and may potentially be contaminated by accessory cells responsive to gp39, or employ tonsillar B cells, which may have received preactivation signals in vivo. Evidence from a number of studies suggests that signals provided by accessorycells contribute to CD40-dependent B cell activation. Membrane bound, recombinant gp39, expressed on fixed CVl/EBNA cells, is consistently less effective than activated, fixed Th at inducing antigen-specific B cell responses, suggesting that the transfected cells do not reconstitute all of the signals delivered by Th cells (Grabstein et al., 1993). Similarly, Ig production of human B cells cultured with IL-4 and optimal levels of aCD40 is strongly enhanced by coculture with activated CD4' T cell clones (Gascan et al., 1991b).It is important to note that murine Thcells activated with concanavalin A (Con A) exhibit maximal gp39 expression at 3 hr postactivation, yet membranes prepared from 3-hr-activated Th do not induce B cell proliferation as effectively as membranes prepared from Th activated for 6 to 9 hr. Therefore, the high level of CD40 ligand expression on recently activated T cells is not correlated with efficiency of B cell activation (Kehry and Castle, 1994). This observation lends support to the view that other membrane-bound ligands may be required to induce vigorous proliferation. Further evidence is provided by the observation that a-CD40 mAb immobilized on plastic culture plates induces B cell proliferation less effectively than a-CD40 presented by FcR-transfected L cells (Banchereau et al., 1991). COS cells transfected with hgp39-CD8 directly support weak proliferation of B cells in the absence of costimuli, while the same construct in soluble form does not (Hollenbaugh et al., 1992). Finally, proliferation of purified sIgD' tonsillar B cells cultured with a-CD3-activated T cell clones was significantly less inhibited by a-CD40 mAb than sIgD- cells cultured under the same conditions, suggesting that CD40-independent molecules may be contributing to T-dependent proliferation of the sIgD' subset (Blanchard et al., 1994). A variety of other stimuli are reported to enhance B cell proliferation in the presence of gp39. Soluble, recombinant hGP39-mCD8 is weakly mitogenic for human B cells in the absence of costimulation, yet triggers vigorous proliferation in the presence of PMA or a-CD20 mAb (Hollenbaugh et al., 1992). Prostaglandin E2 (PGE2),a compound secreted by activated macrophages, fibroblasts,and FDC, also enhances DNA synthesis of B cells cultured with a-CD40 and CDw32 L cells in a dose-dependent fashion in the presence or absence of IL-4 and IL-10 (Garrone et al., 1994). Similarly, synthetic lipoprotein analogs derived from Escherichia

58

LISA B. CLARK ETAL.

coli lipoprotein induce a synergistic and more rapid B cell proliferation response in the presence of a-CD40 and IL-4, but are not mitogenic in the absence of costimuli (Edinger d al., 1994). Some evidence suggests that costimulation by adhesion molecules also modulates proliferation. For instance, anti-LFA-1 mAb added to human B cell cultures abrogates aCD40-induced clustering and partially suppresses DNA synthesis (Klaus et al., 1994a). Together, these findings support the hypothesis that accessory proteins provided by activated Th or other cell types increase the effectiveness of CD40 ligation and may play an important role in regulating the humoral immune response. VI. The Role of CD40 in Immunoglobulin Production

A. SIGNALS REQUIREDFOR SWITCH RECOMBINATION AND Ig PRODUCTION Signaling through CD40 is essential for thymus-dependent production of all immunoglobulin isotypes and for switching to downstream heavy chain isotypes. The role of CD40 in Ig production closely parallels its role in proliferation, in that CD40 ligation is required, but not sufficient in the absence of costimuli, to dnve significant levels of Ig secretion. This probably reflects a requirement for rounds of cellular proliferation as a prelude to Ig production (Hodgkin d al., 1994). Cytokines produced by activated Th not only affect Ig levels, but also direct isotype switching, thereby regulating the course of the humoral response. A growing body of evidence obtained from diverse gp39-driven experimental systems indicates that distinct cytokines or cytokine combinations specifically modulate the production of distinct Ig isotypes in both a positive and negative fashion. These studies are summarized in Table 11. It is likely that cytokines control switch recombination through the specific activation of DNA-binding proteins. Evidence for this has been provided by Gauchat et al. (1990) who found an IL-4responsive promoter/enhancer element located 5’ to the CE switch region which regulates the production of CE germline transcripts via the binding of an IL-4-responsive nuclear factor (Gauchat et al., 1990). It is not clear whether signaling through CD40 is sufficient to induce the germline transcription of heavy chain isotypes in the absence of exogenous cytokines as experiments in a variety of experimental systems have yielded inconsistent results. Germline or sterile mRNA transcripts lack a rearranged constant heavy chain region (C,) coupled to the variable region encoding antigenic specifity. Therefore, they fail to encode exon (VHDJH) intact or “productive” Ig heavy chains (reviewed in (Snapper and Finkelman, 1993)). PCR and Southern blot analysis of Ig transcripts isolated

CD40 AND ITS LIGAND

59

from human peripheral and cord blood B cells have provided evidence that a single signal, stimulation with a-CD40, is sufficient for the sterile or germline transcription of most CH chain regions, including Cyl-3 and C a l and Ca2, while induction of y4 and E sterile transcripts in this system requires the further addition of IL4 (Jumper et al., 1994).In studies using plasma membranes from activated Th, C y l germ-line transcripts were also induced in the absence of added cytokines and CE transcription also required IL-4; however, TGF-P was required for C a transcription (Schultz et al., 1992).In contrast, human B cells produced CEtranscripts in response to stimulation with either IL-4 or a T cell clone (Gauchat d al., 1990). In the murine system, membranes from activated Th were not sufficient to induce Cyl transcription in the absence of IL-4 (Noelle et al., 1992b). It is likely that the engagement of CD40 alone may be sufficient for germline transcription of some isotypes, while cytokines enhance their production and both signals are required for class switching. The production of sterile Ig transcripts is closely correlated with switch recombination and the production of mature downstream Ig isotypes, yet does not necessarily result in Ig synthesis (Gauchat et al., 1990). It is also not clear whether germline transcription plays an active role in the process of switch recombination, The production of germline transcripts may simply reflect increased accessibilityof the downstream switch region, thereby targeting it for switch recombination (Snapper and Finkelman, 1993).PCR analysis has provided direct demonstration that the molecular basis of IgE production in response to CD40 ligation and IL-4 is a deletional E switch ( S ) regions (Shapira et recombination between the S ~ S genomic al., 1992). These studies demonstrate that CD40-dependent production of downstream isotypes is the result of class switching, and does not merely reflect the expansion of post-switched cells.

B. IgM IgM is the primary isotype produced in response to both TI and TD antigens. However, unlike TI humoral responses, the IgM component of TD responses in normal individuals is predominantly CD40-dependent. In vivo experiments, in which mice immunized with sheep red blood cells (SRBC) simultaneously received the blocking anti-gp39 mAb MR1, have demonstrated complete inhibition of the primary IgM response as measured by IgM anti-SRBC plaque-forming cell assays (Foy et al., 1993).Other studies support the existence of a minor pathway of thymusdependent IgM secretion independent of CD40-gp39 interactions. For example, primary IgM responses to the TD antigen DNP-OVA are not inhibited by the in vivo administration of a soluble, chimeric mouse CD40human Fcyl fusion protein, although IgG, and IgG%production are stron-

TABLE I B CELLPROLIFERATION IN RESFONSE TO CD40 LIGATION AND CITOKINES Experimental system

B cell subset

Cytohnes

Prolif. response

Reference

CDw32-transfected L cells and a-CD40 mAb

Human tonsils (>98% CD19')

IL4 IL4+IL-10 IL2+ILlO

Rousset et d.(1992)

cDw32-transfeted L cells and a-CD40 mAb

Human tonsils (>98% CD19')

IL4 ILl+IL-4 ILl+IFNy

Rousset et d.(1991)

cDw32-transfected L cells anda-CD40db

Human tonsils (98% CD20') Human spleen (97% CD20')

None IL-4 IL-4

Banchereau et d.(1991)

CDw32-transfected L cells and a-CD40 mAb gp39-transfected, fixed CV-l/EBNA cells

Human tonsils sIgD+ Human tonsils sIgDHuman tonsils (96%CD20+)

IM IL4 IL4

Galibert et al. (1994)

Murine spleen (95% IgM')

IL-4

Armitage et al. (1993a)

Human tonsils (>98% CDZO')

IL-2, IL-4, or IL-10 IL-4+IL-5, TNFa, or IFNy TGFB

gp39-transfected, fixed CV-1EBNA e l l s

spriggs et az. (1992)

r

@g-transfected, fixed CV-lmBNA cells

Murine spleen (99% sIgM+)

IL4 ILA+IW

gp39-transfected COS7 cells

Human PBLs (>98% CD20+)

None IL4 IL-13

(+)

hgp39-mCD8 (soluble) with PMA or a-CD20 hgp39-mCD8 transfected COS cells with PMA or a-CD20

Human PBLs

None

(+)

Human tonsils

None

(++)

(++/+ ++)

None

(+++)

Mouse spleen (95% B cells) Human PBLs (>98% B cells)

None None None None

(+++) (-) (+++) (+)

Soluble monomeric gp39 Soluble dimeric gp39 Soluble trimeric gp39

Human tonsils

None None None

(+)

a-CD40 mAb

Human tonsils (96-98% CD20')

None IL-4

(-) (+++)

mgp39-mCD8 (soluble) with a-IgM without a-IgM with a-IgD without a-IgD

Cocks et al. (1993)

(+++) (+++)

Hollenbaugh et al. (1992)

Lane et al. (1993)

(-1

Fanslow et al. (1994)

(++I Bjorck and Paulie ( 1993)

62

LISA 8 . CLARK ETAL.

gly inhibited by this treatment (Grayet al., 1994).This observation suggests

that an alternative signaling pathway may lead to IgM secretion in the absence of gp39. IgM is secreted in vitro in response to a relatively broad range of cytokines and CD40 signaling. Certain studies also suggest that triggering of CD40 and sIg together may support IgM production independent of exogenously added cytokines. Both sIgD+ and sIgD- human B cells cultured with the polyclonal activator Staphylococcus aweus Cowan (SAC), a-CD40, and CDw32 L cells secrete high levels of IgM without the addition of cytokines. However, IgM production in this system is strongly enhanced by the addition of IL-10 (Defrance et al., 1992a).In contrast, stimulation by a-CD40 mAb in the absence of presentation by L cells does not support Ig secretion from SAC-activatedhuman peripheral blood B cells (Splawski et al., 1993).In light of this report, it is not possible to rule out a potential contribution of either soluble or contact-dependent factors provided by L cells which may provide a costimulus critical for IgM production. CDw32 L cells, a-CD40, and IL-10 also support IgM production in the absence of SAC (Rousset et al., 1992),while the further addition of IL-2 or IL-4 to this system does not enhance IgM production. In the L cell system, sIgD' (naive)B cells, purified by immunomagnetic separation, produce IgM exclusively,while sIgD- (activated)cells may also produce downstream isotypes. IgM and IgG are the predominant isotypes produced by sIgD- cells in response to IL-10, SAC, a-CD40, and CDw32 L cells (Defrance et al., 1992b). Human tonsillar B cells cultured in the presence of human gp39 expressed on CVl/EBNA cells secrete high levels of IgM in response to IL-2 or IL-10, and relatively low levels in response to IL-4. In this system, IL-4 does not synergize with IL-2 or IL-10, but is somewhat inhibitory, and TGF-/3 is strongly inhibitory for IgM production (Armitageet al., 1993a).In contrast, IL-4 and IL-2 synergize in the CDw32 L cell system for the production of high levels of IgM (Rousset et al., 1991).Collectively,these studies suggest that IgM production is less tightly regulated than the production of downstream isotypes. Splenic human B cells and sIgM' B cell clones also produce IgM in response to IL-4 and CD4+ T cell clones (Gascan et al., 1991a,b). Fixed CVlEBNA cells transfected with mgp39 activate resting murine B cells to secrete moderate levels of IgM in the presence of IL-2 or IL-5, while a combination of IL-4 and IL-5 are strongly synergistic for IgM production (Maliszewski et al., 1993). Preactivated CD5-B cells also produce IgM in response to a-CD40 and IL-4 (Dono et al., 1993). In contrast, a subset of resting human CD5-B cells copurified by Percoll fractionation along with responsive CD5'B cells consistently failed to respond to a-CD40 and IL-4 by proliferation or IgM production.

CD40 AND ITS LIGAND

63

C. IgE A number of studies have characterized the control of switching to IgE, an isotype consistently induced in response to CD40 signals and IL-4. An IL-4 responsive DNA fragment located 5' to the switch recombination region of human CE has been identified which is responsible for the induction of germline transcripts by IL-4 via activation of an IL-4-induced DNA binding protein (Ichiki et al., 1993). In addition, IFN-.)Ihas been found to down-regulate IgE production by repressing CE germline transcription and switch recombination (Xuet al., 1994). Although both IL-4 and a T cell clone were found to independently induce germline E-mRNA transcription, neither signal alone was sufficient to induce IgE synthesis (Gauchatet al., 1990), suggesting that additional components are required for class switching and IgE production. a-CD40 and IL-4 together induce IgG and IgE secretion from a variety of B cell subsets, including neonatal cord blood B cells, IgD' naive adult B cells, and surface IgE- adult B cells (Bjorck and Paulie, 1993; Jabara et al., 1990; Splawski et al., 1993) (Table 11).Of a panel of cytokines tested, only IL-4 supports IgE and IgGl synthesis in purified splenic mouse B cells (>99% IgM' ) cocultured with recombinant mgp39-transfected, fixed, CVl/EBNA cells (Grabstein et al., 1993; Maliszewski et al., 1993). IL-5 together with IL-4 is also essential for the production of these isotypes in murine B cells cultured with plasma membranes from activated Th (Noelle et al., 1992b). While sterile CE transcripts may be induced in human B cells cultured with IL-4 or T cell clones, neither signal is sufficient to induce IgE synthesis, indicating that switching requires both signals (Gauchat et al., 1990). High levels of IgE production are also reported for human B cells cultured with IL-4 and soluble gp39 derived from EUO.9 supernatant (Armitage et al., 1992b);resting human peripheral blood B cells cultured with a-CD40 and IL-4 (Gascan et al., 1991a; Jabara et al., 1990; Shapira et al., 1992; Splawski et al., Fu, 1993);resting tonsillar B cells cultured with F,R-transfected CDw32-Lcells and IL-4 (Roussetet al., 1991)(reviewedin Banchereau et al., 1994); surface IgM' B cells cultured with activated CD4' T cell clones and IL-4 (Gascan et al., 1991a); and purified tonsillar B cells cultured with hgp39-transfected CVl/EBNA cells and IL-4 (Armitage et al., 1993a; Spriggs et al., 1992). IL-10 synergizes with IL-4 for IgE production in the gp39-transfected CVl/EBNA cell system, while TGFp has a strongly suppressive effect (Armitage et al., 1993).In contrast, IL10 alone does not support IgE secretion by CD40-activated B cells, but provides a potent costimulus for the production of other isotypes (Armitage et al., 1993; Rousset et al., 1992). Likewise in the murine system IL-5

64

LISA B. CLARK ETAL.

costimulates the production of secreted IgM, IgG1, and IgE (Hodgkin et

al., 1994; Maliszewski et al., 1993; Noelle et al., 1992b) but is insufficient

in the absence of IL-4. IL-4 appears to be a powerful stimulus for IgE secretion in most CD40based experimental systems, but apparently is not strictly required. Both IL-13, which exhibits significant homology to IL-4, and TNF-a are reported to stimulate IgE production by CD40-activated B cells (Gascan et al., 1992; Punnonen et al., 1993). While deletional recombination between switch regions in response to these cytokines has not been demonstrated, germline E transcripts are induced in response to a-CD40 mAb and IL13 (Punnonen et al., 1993). IL-4 and IL-13 together do not exhibit an additive or synergistic effect (Punnonen et al., 1993). IgE is secreted by B cells cultured with hgp39-transfected COS cells in the presence of IL13 and a neutralizing a-IL-4 mAb, demonstrating that isotype switching to IgE occurs independently of IL-4 (Cocks et al., 1993). IgE production by human peripheral blood B cells stimulated with aCD40 and IL-4 is costimulated by SAC (Splawskiet d.,1993),yet coculture of human B cells with SA, a-CD40, and CDw32-L cells, in the absence of IL-4, does not result in detectable IgE production (Defrance et al., 1992a). Endogenously produced IL-6 strongly up-regulates the IL-4dependent synthesis of IgE (Vercelli et al., 1989) and may be part of an autocrine feedback loop for B cells. IgE synthesis by human B cells cultured with a-CD40 and IL-4 has been shown to be strongly inhibited by the addition of a neutralizing a-IL-6 (Jabara et al., 1990). In summary, IgE is the predominant isotype secreted by all subsets of CD40-activated B cells in response to IL-4, including human tonsillar and peripheral blood B cells and splenic murine B cells (Table 11). While IL-4 or activation through CD40 may induce germline transcription of CE, IgE synthesis requires both signals (Gauchat et al., 1990). IgE secretion may be enhanced by IL6, IL-10, or SA and suppressed by TGF-P and IFN-.)I. IL-13 and TNF-a may also induce IgE secretion by an IL-4-independent pathway. D. IgA IgA is the isotype predominantly produced by the gastrointestinal tract and other mucosal surfaces. A number of studies have documented the role of TGF-/3 in directing heavy chain switch recombination of naive B cells through the induction of germline a-transcripts in humans and in mouse (Coffman et al., 1989; Islam et al., 1991; Lebman et al., 1990; also reviewed in Stavnezer, 1995).Naive, purified, sIgD'sIgM' B cells isolated from human tonsils produce high levels of IgA in response to coculture with a-CD40-presenting CDw32-L cells, TGF-P, and IL-10 (Defrance et al., 1992a). Significantly, limiting dilution analysis has demonstrated that

CD40 AND ITS LIGAND

65

TGF-/3 greatly increases the precursor frequency of naive, sIgD+ B cells secreting IgA in this system. CD40 ligation is essential, as crosslinking of sIg in lieu of CD40 in the presence of TGF-P and IL-10 does not support IgA secretion (Defrance et al., 1992b) IL-10 in the absence of TGF-fl is also reported to support moderate IgA secretion by B cells cultured with a-CD40-presenting CDw32-L cells and IL-10 (Defrance et al., 1992a; Rousset et al., 1992). In LPS-activated murine B cells TGF-P activates germline Ca transcription (Coffman et al., 1989; Lebman et al., 1990; Shockett and Stavnezer, 1991).TGF-P also induces very high frequencies of sIgAt murine cells in conjunction with anti-8 dextran, a gp39-CD8 fusion protein, IL-4, and IL-5 (McIntyre et al., 1995)and each component is required for the high-level production of sIgA+ cells. In this system, IFN-y strongly suppresses the appearance of sIgA+ cells. It is clear from these studies that multiple components are required to support IgA switching and high-level secretion. Under some circumstances, TGF-P may also act as a potent inhibitor of IgA synthesis. Activated sIgD- B cells secrete IgA in the CDw32-L cell system in response to coculture with SA and IL-10, but not in response to TGF-P, which is inhibitory for the secretion of IgA, IgG, and IgM by this subset (Defrance et al., 1992b).These findings are in agreement with previous studies which demonstrated IgA secretion from surface IgA-, but not surface IgA+ splenic human B cells cocultured with TGF-P and cloned, activated (but not resting) CD4+ cells, and pokeweed mitogen (van Vlasselaer et d.,1992). Further, IgA synthesis by sIgA- splenic B cells is completely inhibited when TGF-/3 is present beyond 24 h after the initiation of cultures, suggesting that a relatively short exposure to TGF-/3 is sufficient for isotype switching, while a longer exposure to TGFfl is inhibitory for proliferation and Ig secretion (van Vlasselaer et al., 1992). Tonsillar B cells cocultured with fixed, hgp39-expressing CVl/EBNA cells are reported to produce high levels of IgA in the presence of IL-10, moderate IgA levels in the presence of IL-2, and relatively low levels of IgA in the presence of TGF-/3 (Armitage et al., 1993a). In this system, the further addition of IL-4 is somewhat inhibitory in the presence of IL-2 or IL-10, but marginally enhances IgA production in response to TGF-P. Stimulation by a-CD40 mAb, with or without IL-4 and/or IL-2, is consistently ineffective at induction of IgA from peripheral blood B cells, unless the B cells are preactivated with SA (Splawski et al., 1993). Collectively, these data suggest that control of IgA secretion is under complex regulation, and may involve both TGF-&dependent and -independent pathways.

E. IgG Human CD40-activated B lymphocytes are reported to secrete IgG in response to CD40 ligation together with a variety of cytokines (Fig. 1).

TABLE I1 IMMUNOGLOBULIN IS~TWES SECRETEDIN RESFWNSE TO CD40 LIGATION AND CYTOKINES

Experimental system

+ SAC

B cell subset

Cytokines

Isotypes produced

Reference

(>W%CD19+)

IL-4 IL-2+IL-4 IL-4

Splawski et d.(1993)

Human P B L , sIgE-

IL-4 None

Jabara et d.(1990)

Anti-CD40 mAb plus CD4+ T cell clone

Human PBLs (99% sIgM+)

None IL-4 IL-4

Gascan d d.(1991)

Anti-CD40 mAb

Human P B L (95% sIgD+)

IL4

Fujieda et d.(1995)

Anti-CD40 mAb

Human tonsil

IL-4

IgE

Bjorck and Paulie (1993)

Anti-CD40 mAb

Human PBLs (>99% CD20+)

IL4 IM+TNFa

IgM, G, E Strong IgE

Gauchat et d.(1993)

mgp39-CD8 fusion protein plus anti-IgD dextran

Murine spleen

TGFP, IL4+IL-5 IL-4+IL-5

Strong IgA IgM, 1 6 1

McIntyre et d.(1995)

gp39-transfected,fixed CV-Y EBNA cells

Human tonsil

IL-2 IL-10 IL4 I L 4 +TGFB

IgM, G1, A IgM, G1, A I@, E Suppression of IgC4, E

Armitage et al. (1993a)

Anti-CD40 ascites

Human PBLs

Cord blood IgD’ AntiCD40 mAb-F(ab’)e

(+sort on CD20)

gl

0,

I

@g-transfected, fixed W-I/ EBNA cells

Human tonsil Murine spleen

IL-4 IL-4

IgE IgE

spriggs d d.(1992)

gp39-transfected, fixed cv-l/ EBNA cells

Murine spleen (99% IgM+)

IL4+IL5 IL-4

IgM, G1, G3, E IgGl, E

Mabszewski d d.(1993)

gp39-transfected, fixed W-I/ EBNA cells

Murine spleen (99% IgM+)

IL-4+IL-5 IL-4

IgM, G1, A, E IgE

Grabstein et al. (1993)

gp39-transfected cos 7 cells

Human PBL, sIgD+

IL-4 IL-13

IgM, total IgG, G4, E IgM, total IgG, G4,E

Cocks et d.(1993)

CDw32-transfected L cells and anti-CD40 mAb

Human tonsil (98% CD19')

IL-4 IL-4+IL2 IM+IFNy

IgM, G, E IgM, A IgM, G, A

Rousset d d.(1991)


Human tonsil (>98% CD19')

ILlO IL4+ILlO IL-2 +ILlO

IgM, G, A IgM, G, A, strong IgE IgM, G, A

Rousset d al. (1992)

CDw32-transfected L cells and a-CD40 mAb + SAC

Human tonsil, sIgD+ Human tonsil, sIgDHuman tonsil, sIgDt

ILlO ILlO IL-lO+TGFB

IgM IgM, G, A Strong IgA

Defrance et ul. (1992a)


Human tonsil, sIgDt Human tonsil, sIgDCord blood B cells

ILlO ILlO ILlO

IgM, IgGl, IgC3 IgG1, IgG2, IgG3

Briere et d.(1994)

68

LISA B. CLARK ETAL,

FIG. 1. A model of T-dependent B cell proliferation and terminal differentiation in secondary lymphoid tissues. (1) Naive B cells recirculate from the periphery to secondary lymphoid tissues. (2) Upon encounter with specific antigen, primary B cell blasts proceed along one of two pathways: migrating into primary follicles where they will form germinal centers as a result of cognate interactions with T cells, or (3)undergoing direct differentiation to primary plasmablasts which secrete low-affinity IgM antibody. (4) Activated B blasts within the GC differentiate into sIg‘” centroblasts which undergo rapid proliferation and hypermutation. Affinity selection of centroblasts via cross-linking of sIg by FDC immune complexes results in rescue from apoptosis and generation of ( 5 ) nonproliferating sIg+ centrocytes. Terminal differentiation of surviving centrocytes in parallel with immunoglobulin class switching results in production of (6)recirculating memory B cells and (7)secondary plasma cells which secrete large quantities of high-affinity isotype Ig of a downstream isotype. Memory cells and secondary plasma cells subsequently escape to the periphery.

CD40 AND ITS LIGAND

69

Ligation of CD40 on peripheral blood human B cells by a-CD40 mAb is reported to induce germline transcription of yl, 72, and y3 in the absence of exogenously added cytokines, while the production of germ-line y4 transcripts in this system required the further addition of IL-4 (Jumper et al., 1994). In another study, costimulation of peripheral blood B cells with IL-4 plus a-CD40 mAb mediated class switching from p to y l , y3, and y4, but not y2 (Fujieda et al., 1995).True in uitro class switching was demonstrated in this system by the generation of switch circular (SyISp) DNA and was accompanied by increased production of IgG1, IgG3, and IgG4, but not IgG2 (Fujieda et al., 1995). Unseparated tonsillar B cells (>98% CD19'), cultured in the CDw32L cell system, produce total IgG in response to IL-10 (Rousset et al., 1992) and IL-4 (Rousset et al., 1991).Tonsillar sIgD- cells, which highly express the germinal center markers CD20 and CD38 (Defrance et al., 1992b), produce little IgG in response to stimulation through sIg and a-CD4O in the L cell system; however, in the presence of IL-10, IgG is the predominant isotype secreted by this subset (Defrance et al., 1992b). Conversely, naive sIgD+ cells produce negligible IgG in response to IL-10, a-CD40, and CDw32-L cells, but are primed instead to produce high levels of IgM. Human peripheral blood B cells also secrete moderate levels of IgG in response to soluble a-CD40 mAb and IL-4 (Jabara et al., 1990; Splawsh et al., 1993), while the further addition of SA enhances IgG production (Splawski et al., 1993). Similarly, tonsillar CD5- B cells produce IgG in response to a-p-mAb, a-CD40, and 1L-4, while CD5+ cells produce little IgG under these culture conditions (Dono et al., 1993). Several studies have suggested that in addition to its role as a growth promoting and differentiation factor, IL-10 may function as a switch factor for human IgGl and IgG3 subclasses. Purified tonsillar B cells cultured with hgp39-expressing C V l E B N A cells secrete IgGl in response to IL2 and IL-10. In this system, IL-4 and TGF-/3 are somewhat inhibitory for IgGl secretion (Armitageet al., 1993a).Naive, tonsillar sIgD+,and neonatal cord blood B cells cultured in the L cell system secrete IgGl and IgG3, but not IgG2 or IgG4, in the presence of IL-10 (Briere et al., 1994). Unseparated B cells, which include sIgD- cells, also produce IgG2 in this system, suggesting a distinct role for IL-10 as a differentiation factor which induces isotype-precommitted cells to secret IgG1, IgG2, and IgG3 (Briere et al., 1994). Both IL-4 and IL-13 are reported to induce switching to IgG4 by CD40activated human B cells. IL-4 induces IgG4 production from purified peripheral blood B cells cultured with CD4' T cell clones (Gascan et al., 1991b) or with a-CD40 (Gascan et al., 1991a); and purified tonsillar B cells cultured with hgp39-expressing CVUEBNA cells (Armitage et al.,

70

LISA B. CLARK ETAL.

1993a). In contrast, TGF-P strongly suppresses the production of IgG4 from B cells cultured in the CVlEBNA cell system (Armitageet al., 1993a). IL-13 also induces IgG4 synthesis by naive, sIgD' human peripheral blood B cells cultured with activated CD4' T cells or PM" in an IL-4-independent manner; as demonstrated by the failure of a neutralizing a-IL4 mAb to inhibit the production of IgG4 (Punnonen et al., 1993). IgG subclasses in the mouse and human differ in their functional properties (reviewed in Snapper and Finkelman, 1993) and the production of specific subclasses appears to be regulated by different cytokines in each species. Studies in the murine system have shown that IL-4 primes purified mouse B cells for the production of IgGl in the presence of fixed, mgp39transfected CVUEBNA cells. In this system, IL-5 synergizes with IL-4 for IgG1, and also promotes IgG3 secretion (Maliszewski et al., 1993). This is in accordance with the finding that IL-5 is necessary for induction of Sp-Sy1 DNA rearrangement in murine B cells activated with IL-4 and anti-IgD antibodies conjugated to dextran (aSdex) (Mandler et al., 1993). While germline y l RNA could not be induced in crSdex stimulated cells by IL-5 alone, IL-5 stimulated productive y l rearrangement (Mandler et al., 1993). Cytokines which direct switching toward IgG2 and IgG4 in CD40-activated B cells have not been identified for the murine system. VII. The Role of CD40 in Germinal Center and Memory Cell Formation

A. OVERVIEW OF MEMORY B CELLRESPONSES The hallmark of the memory B cell response is the production of relatively long-lived plasma cells expressing high-affinity antibody generated through somatic hypemutation of Ig variable ( V ) region genes (reviews in Gray, 1993; Picker and Siegelman, 1993).Primary antigen contact gives rise to an enhanced secondary response based upon the antigen-induced differentiation of virgin B cells in the germinal centers (GC) of peripheral lymphoid tissues, resulting in the production of memory B cells. In response to secondary or prolonged immunization with a T-dependent antigen, memory B cells rapidly give rise to the high-titer production of predominantly non-IgM, high-affinity antibody. The process of memory cell differentiation is complex and multilayered, relying upon the ordered progression of activated B cells through distinct microenvironments in the secondary lymphoid tissues, where critical signals may be delivered sequentially. An in vivo model of memory B cell differentiation, based upon previous models and a synthesisof current studies, is summarized in Fig. 1(after Banchereau et al., 1994; Gray, 1993; Nossal, 1992; Picker and Siegelman, 1993; Van den Eertwegh et al., 1993).

CD40 AND ITS LIGAND

71

Several lines of investigation have recently demonstrated the critical role of CD40 and gp39 interactions in the production of germinal centers and memory cells. In vivo administration of a-gp39 mAb to mice immunized with TD antigens completely inhibits germinal center formation and the development of B cell memory (Foy et al., 1994). Similarly, CD40 liganddeficient mice fail to mount secondary humoral responses following immunizations with TNP-KLH (Renshaw et al., 1994). In humans, the absence of germinal center formation, isotype-switching, and secondary humoral immunity in hyper-IgM patients has been well documented (Allen et al., 1993; Aruffo et al., 1993; DiSanto et al., 1993; Korthauer et al., 1993). While these studies make it clear that the development of memory B cells is CD40- and TI,-dependent, the full range of signals, in addition to ligation of CD40, which control the transitions of mature B cells through the various stages of terminal differentiation remain to be defined.

B. ANTICENSPECIFIC T CELLACTIVATIONIN VIVO T D antigen introduced into the skin or mucosa initiates the TD-humoral response upon capture by dendritidlangerhans cells, which then migrate into T-cell-rich areas of the spleen and/or lymph nodes to serve as antigenpresenting cells (APCs).In the paracortical areas of the secondary lymphoid organs, the dendritic cells (now termed interdigitating cells, or IDC) present antigen-derived peptide fragments bound to MHC Class I1 molecules to antigen-specific T cells, resulting in Class II-restricted, antigen-specific T cell activation and up-regulation of gp39 on the T cells. Interactions between IDC and T cells are further strengthened by the binding of various adhesion molecule ligand pairs. Following antigen capture, IDC also express CD40 which interacts with gp39 on the T cells. This interaction drives increased IDC cytokine production, enhancing T cell activation and proliferation. T cells are further activated by the interaction of CD281 CTLA4 receptors with their ligands, B7.1 and B7.2 up-regulated on the IDC. The expression of gp39 and cytokines by activated T cells in turn drives the proliferation and differentiation of antigen-specific B cells through CD40 cross-linking. C. COGNATE INTERACTIONS BETWEEN T CELLS AND NAIVEB CELLSI N Vrvo Virgin B cells released into the periphery enter the secondary lymphoid tissues via the high endothelial venules (HEVs), or the marginal zone sinuses of the spleen (reviewed in Picker and Siegelman, 1993). During migration to the primary follicles, these cells traverse the T-cell-rich zone of the outer PALS (periarteriolar lymphoid sheath), where gp39+ Th are localized (Van den Eertwegh et al., 1994). In the absence of specific

72

LISA R. CLARK E T A L .

antigen, naive sIgD+sIgM’ B cells, which compose the major B cell subset in human peripheral blood (Klein et al., 1993), recirculate between the lymphoid tissues and bloodstream, periodically repopulating the primary follicles. In the presence of antigen-presenting IDC, naive B cells of the appropriate specificity are triggered to enter one of two possible differentiation pathways. One subset of B cells migrates to the primary follicles, where they will give rise to germinal centers and ultimately to secondary plasmablasts and memory cells. Others undergo primary blast formation in the T cell zone, where they differentiate into short-lived plasma cells which produce low-affinity, predominantly IgM, antibody. The recent availability of mAbs recognizing a wide range of B cell surface antigens has made it possible to separate B cell subsets into ordered maturational stages according to expression of a series of phenotypic markers (Fig. 1).Three major subsets of B cells may be distinguished in human tonsils by double staining for surface IgD and CD38, markers of follicular mantle cells and germinal center cells, respectively (Pascual et al., 1994). Mature, naive, B cells newly released into the periphery are small, resting, high-density cells which express high levels of sIgD+ and sIgM +,CD44, and cytoplasmic bcl-2. Naive B cells are also CD38-, CD10-, CD77-, IgG-, A-, and E-, and CD20Inw.This subset may be further characterized by variable expression of the activation marker CD23 (F,R&).Naive, sIgD’, CD38- tonsillar B cells are approximately30% CD23’ (Pascual et al., 1994), a subpopulation thought to represent naive cells at an early activation stage. D. SOMATIC HYPERMUTATION OF GERMINAL CENTER B CELLS

B cells recruited to tonsillar germinal centers are characteristically sIgDand CD38’. They also express high levels of PNA, CD20, CD10, and IgG, but are negative for CD39, CD23, and cytoplasmic bcl-2 (Pascual et al., 1994). In the germinal center microenvironment, B cells undergoing rapid clonal expansion form characteristic centroblasts, which compose the germinal center “dark zone” (Picker and Siegelman, 1993). GC dark-zone centroblasts are characteristically CD77’ (Pascual et al., 1994). It is at this stage that somatic hypermutation and the generation of antibody mutants takes place (Jacob, 1991). Sequence analyses of heavy chain V region mRNAs amplified from tonsillar B cells via PCR has shown that for the majority of naive, IgD+ IgM+ cells (both CD23’ and CD23- subsets), VII mRNA transcripts are 100% identical to their germline counterparts. In this group, most of those with mutations exhibited only single nucleotide changes, indicating a low mutation frequency (Pascual et al., 1994). In contrast, IgM transcripts isolated from germinal center subsets ( IgDCD38’ CD77+’-) had accumulated an average of 5.7 nucleotide substitu-

CD40 AND ITS LICAND

73

tions; IgG transcripts isolated from memory cells had accumulated nearly twice as many mutations.

E. AFFINITYSELECTION A N D MEMORY CELLPRODUCTION CD77' centroblasts give rise, in turn, to CD77- "light zone" nonproliferating centrocytes. These cells, which express mutated immunoglobulin, frequently of a switched isotype, are thought to undergo affinity selection based upon competition for binding to antigen complexes sequestered on the surfaces of GC follicular dendritic cells (FDC). Triggering of the antigen receptor promotes rescue of cells bearing high-affinity receptors from apoptosis (Lindhout et al., 1993; Liu et al., 1992). CD40 ligation is also required for survival of GC B cells and their entry along a proliferation pathway; however, it is not clear whether CD40 ligation by gp39-bearing T cells occurs within the GC light zone (Lagresle et al., 1995). While engagement of FDC antigen complexes by GC B cells is clearly pivotal to their survival and differentiation, it cannot be excluded that other ligandreceptor interactions between B cells and FDC also play an important role in these processes. Selected B cells give rise to either secondary plasmablasts or memory B cells. Memory B cells are small to medium sized, CD38- sIgD-, and express high levels of IgG, CD39, CD44 and cytoplasmic bcl-2; they are negative for CD23, CD10, CD77, and are CD20'"" (Pascual et al., 1994).Terminally differentiated memory cells and plasmablasts finally migrate from the germinal centers to recirculate or take up residence in other tissues. VIII. Summary

This review has focused on the role of CD40 and its ligand, gp39, as central players in the regulation of B cell growth and differentiation. Although much has been learned in the past 3 years, the role of this ligand-receptor pair in the regulation of B cell memory, somatic mutation, and the germinal center reaction still demands significant attention. More surprising is the evolving role of gp39 in mediating inflammation. The fact that anti-gp39 therapy can all but eliminate T-cell-mediated autoimmune diseases, like experimental allergic encephalomyelitis (Gerritse et al., 1996), autoimmune oophoritis (Griggs et al., 1996) acute GVHD (Dune et al., 1994a,b), and graft rejection (Parker et al., 1995) suggests another pivotal role of gp39 in the regulation of cell-mediated immune responses. The expanding tissue distribution of CD40 to endothelial cells, fibroblasts, and other nonimmune cells suggests a far broader role of gp39-CD40 than envisioned just a few years ago.

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LISA B. CLARK E T A L .

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ADVANCES IN IMMUNOLOGY, VOL 63

Human Immunodeficiency Virus Infection of Human Cells Transplanted to Severe Combined Immunodeficient Mice DONALD E. MOSIER hpmiment of Immunology, The Scripps Reseamh Inrfifvihrls, la M a , California 92037

1. Introduction

Since the first description of normal human cell transplantation to severe combined immunodeficient (SCID) mice (McCune et al., 1988; Mosier et al., 1988),there has been widespread use of these xenotransplant models for the study of human immune responses, autoimmunity, infectious diseases, and hematopoietic development. However, the original motivation in transplanting human cells to immunodeficient mice was to create a small animal model susceptible to human immunodeficiency virus (HIV) infection. It was quickly shown that SCID-hu mice bearing fetal thymus/ liver grafts could be infected with HIV-1 (Namikawa et al., 1988). Subsequently hu-PBL-SCID mice grafted with adult human peripheral blood mononuclear cells were shown to be infectable with HIV-1 and to undergo loss of human CD4+ T lymphocytes as a consequence of infection (Mosier et al., 1991).Thus the xenotransplant models were successful in generating a new way to study HIV infection. But what have they provided in terms of understanding virus function and pathogenesis? In addition, what are the biologic features of the human cells residing in SCID mice that may alter or limit the potential to understand HIV infection. For example, it has recently been suggested that all human T cells are “anergic” in the hu-PBL-SCID model (Tary-Lehmann et al., 1994, 1995); were this to be true, how would it impact our understanding of HIV-induced CD4 T cell depletion? This review will attempt to put current work on HIV infection of xenotransplanted mice into the context of the natural history of HIV infection of humans and simian immunodeficiency virus (SIV) infection of primates, and to underscore where the xenotransplant models differ from virus infection of natural hosts. II. Description of Human-to-Mouse Xenograft Models

A. THEhu-PBL-SCID MODEL Human peripheral blood mononuclear cells are injected intraperitoneally into SCID mice (Blunt et al., 1995; Bosma et al., 1988) to create what is most commonly called hu-PBL (peripheral blood leukocyte)-SCID mice 79 Copyright 8 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(Hesselton et al., 1993; Mosier et al., 1988; Smith et al., 1991; Torbett et al., 1991), although the same model has been termed SCID-hu-PBL (Ifversen et al., 1995) or hu-PBMC (peripheral blood mononuclear cell)SCID (Abedi et al., 1992). Recent variations in the model have included the use of NOD.SCID mice (Greiner et al., 1995; Shultz et al., 1995) or SCID.Beige mice (Chin et al., 1994; Mosier et al., 1993c) as recipients of

human peripheral blood cells. The NOD (nonobese diabetic) and beige mutations decrease the natural killer (NK) cell activity found in SCID mice (Dorshkind et al., 1985), and appear to result in improved human cell engraftment. There are drawbacks to the use of either of the double mutant strains, however. NOD.SCID mice have a high incidence of murine thymic lymphomas at 4-6 months of age (Shultz et al., 1995) which precludes their use in any long-term experiments. SCID.Beige mice are relatively difficult to maintain because the beige mutation affects granulocytes as well as NK cells (Roder and Duwe, 1979), and the double mutant mice show higher susceptibility to bacterial infections than SCID mice. Some groups have pretreated SCID mice with anti-asialo-GM 1 antibodies and irradiation to improve human cell engraftment (Murphy et al., 1992; Sandhu et al., 1994). Injection of PBL from HIV-infected donors into SCID mice results in activation of HIV-1 replication, a model that has been termed the HIV-hu-PBL-SCID mouse (Boyle et al., 1995). The number of human cells injected into SCID mice varies from 2 X lo7 (Mosier et al., 1991) to 2 X 10' (Ussery et al., 1995); the higher cell numbers are more likely to produce graft-versus-host disease (Duchosal et al., 1992; Hoffmann-Fezer et al., 1992; Kyoizumi et al., 1993; Murphy et al., 1992; Sandhu et al., 1995; Thirdborough et al., 1993). It is likely that recognition of xenoantigens by human T cells contribute to their activation, survival, and expansion, although there is ample evidence that CD4' T-cell-dependent, antigen-specific immune responses can be obtained in hu-PBL-SCID mice (Dickey et al., 1994; Gagnon et al., 1995; Hozumi et al., 1994; Ifversen et al., 1995; Kudo et al., 1993; Malkovska et al., 1994; Martensson et al., 1994; Mosier et al., 1988; Nonoyama et al., 1993; Pestel et al., 1994; Pistillo et al., 1992; Reason et al., 1994; Shelton et al., 1992; Smith et al., 1991; Somasundaram et al., 1995; Walker and Gallagher, 1994,1995).The survivinghuman T cells therefore must include cells that recognize nominal antigens as well as some cells that recognize xenoantigens, and not solely xenoreactive cells as claimed by some (TaryLehmann et al., 1994,1995; Tary-Lehmann and Saxon, 1992).The engraftment of human T cells leads to the appearance of activation markers (CD25, CD69) and enrichment of cells bearing the CD45-RO isoform found on activated memory T cells (Hasui et al., 1994; Hoffmann-Fezer et al., 1992; Martensson et al., 1994; Tary-Lehmann and Saxon, 1992;

4

k

,

Fig. 1 . CD46 stainiiig of himman cells in (A) spleen or (B) perithyinic lyinplm notles ofhn-PBI,SCID inice. Organs were recvvered 4 weeks after hunrai PBL injection arid htiinaii cells detected by staining with antibodies to human CD45. Original mitpification: 1 0 0 ~ .

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Torbett et al., 1991). Human cells injected into SCID mice populate the peritoneal cavity, where they may form small focal adhesions to the peritoneal serosa, and they also migrate to local lymph nodes, the liver, lung, and spleen (Duchosal et al., 1992; Hoffman-Fezer et al., 1992; Lade1 et al., 1993; Martino et al., 1993). Human cells are mostly confined to lymphatic channels and occupy the previously empty white pulp in the spleen (Fig. 1A). Human cells are not found in the murine thymus, as has been suggested in one report (Tary-Lehmann and Saxon, 1992), but do repopulate perithymic lymph nodes which are adherent to the thymic capsule (Fig. 1B). Human T cells are the major surviving population, and CD8' T cells predominate in the peritoneal cavity while CD4+ T cells predominate in repopulated lymphoid tissue (Hoffmann-Fezer et al., 1993; Kyoizumi et al., 1993;Torbett et al., 1991).Human B cells show a spontaneous CD4+ T cell-dependent differentiation to immunoglobulin-secreting plasma cells, resulting in high levels of human IgM and IgG (Abedi et al., 1992; Ambrosino et al., 1994; Chin et al., 1994; Duchosal et al., 1992; Hasui et al., 1994; Martensson et al., 1994; Mosier, 1990; Palladino et al., 1995). Human IgE synthesis is also observed in the model (Gagnon et al., 1995; Kilchherr et al., 1993; Pestel et al., 1994). Human cells persist for many months in hu-PBL-SCID mice, but the number of cells tends to decline at 6 months or more after engraftment and human immunoglobulin synthesis tends to become oligoclonal (Saxon et al., 1991). There is little or no evidence for replacement of human lymphoid lineage cells by progenitor or stem cells found in peripheral blood unless progenitors are differentially enriched prior to SCID mouse transplantation (Goan et al., 1995). Monocytes and human NK cells present in PBL show relatively poor survival in SCID recipients (Duchosal et al., 1992; Hesselton et al., 1993; Tary-Lehmann et al., 1994; Torbett et al., 1991). Numerous studies have documented both primary (Ihersen et al., 1995; Nonoyama et al., 1993; Reason et al., 1994; Sandhu et al., 1994; Walker and Gallagher, 1994, 1995) and secondary antibody responses (Chin et al., 1994; Duchosal et al., 1990; Hesselton et al., 1993; Kilchherr et al., 1993; Krams et al., 1989; Martensson et al., 1994; Mosier et al., 1988, 1993b; Pestel et al., 1994; Spiegelberg et al., 1994) in hu-PBL-SCID mice, and T cell responses to allografts and tumors (Alegre et al., 1994; Bestian et al., 1995; Malkovska et al., 1994; Rencher et al., 1994; Sabzevari and Reisfeld, 1993; Shiroki et al., 1994; Wecker et al., 1995) have also been seen. A significant level of immune function thus exists in this model despite the relative absence of human professional antigen-presenting cells. The use of Epstein-Barr virus (EBV)-infected donors to generate huPBL-SCID mice can result in the development of EBV-associated human B cell lymphoproliferative disease at 8 or more weeks after PBL engraftment

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(Mosier et al., 1990, 1988;Picchio et al., 1992; Rochford and Mosier, 1995; Rowe et al., 1991).This lymphoproliferative disease can be avoided by the use of EBV-seronegative donors, or by the choice of EBV-seropositive donors who do not give rise to B cell lymphoproliferation (Picchio et al., 1992).The proliferating human B cells are susceptible to HIV-1 infection [as are in vitro EBV-transformed lymphoblastoid cell lines (Montagnier et al., 1984)], but we have failed to detect viral pseudotype formation between EBV and HIV (Van Kuyk and Mosier, 1995), so rare doubly infected B cells are unlikely to produce phenotypically altered HIV. Lethal graft-versus-host disease is seen when one introduces adult human PBL into neonatal SCID mice (Dickey et al., 1994; Pflumio et al., 1993; Reinhardt et al., 1994), but not when cord blood cells are injected into neonatal SCID mice (Reinhardt et al., 1994). These findings suggest that humans become immunized to mouse xenoantigens through exposure to cross reacting antigens so that adults contain more xenoreactive cells than newborns. They also indicate that adult SCID mice have some natural resistance to engraftment of human cells that is not present in neonatal SCID mice. The view that all human T cells become anergic (Tary-Lehmann and Saxon, 1992) is difficult to reconcile with these observations, since graft-versus-host disease would require active xenoreactive T cells. Moreover, human T cells reactive to specific antigens have been recovered from hu-PBL-SCID mice (Hesselton et al., 1993; Somasundaram et al., 1995).The balance of the evidence would suggest that human cells maintain some level of immune competence in the hu-PBL-SCID model, although it is clear that immune function is not at the level of intact humans (Mosier et al., 1992, 199313). As a target for HIV infection, the hu-PBL-SCID model represents a subpopulation of mature, activated, memory T cells expected to be found in the course of an ongoing immune response in the adult.

B. THE SCID-hu thyAiv MODEL Human fetal thymus and fetal liver fragments from Week 18 to 22 gestational age embryos are transplanted under the renal capsule to create this model (McCune, 1991, 1992; McCune et al., 1989, 1988; Namikawa et al., 1990). The two tissue fragments are placed together so that they form a “conjoint organ” with the fetal liver supplying myeloid and lymphoid precursors and the fetal thymus supplying a supportive microenvironment for human T cell differentiation (McCune et al., 1988; Peault et al., 1991). Small numbers of human T cells are found in the circulation of such mice, and these T cells are functional (Krowka et al., 1991; Vandekerckhove et al., 1991).They are tolerant to mouse xenoantigens, as would be expected of T cells differentiating in the presence of the mouse cells which infiltrate


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the thyAiver graft (Aldrovandi et al., 1993; Krowka et al., 1991; McCune, 1992; Roncarolo and Vandekerckhove, 1992; Vandekerckhove et al., 1991). Exposure of the thymus graft to the bacterial superantigen staphylcoccus enterotoxin B either deletes developing T cells or renders them reversibly anergic (Scholset al., 1995),suggesting that normal mechanisms of negative and positive selection are operative. Some workers have modified the SCID-hu thyAiv model by placing multiple fragments of fetal thymus under both kidney capsules, and they report an increased number of human T cells in the peripheral circulation (Kollmanet al., 1994a,b).Postnatal thymus has been reported to repopulate SCID mice if NK cells are first reduced by treatment with anti-asialo-GMl antibody (Barry et al., 1991). Use of alternative immunodeficient strains, such as beige-nude-xid triple mutants, has been reported (Kollman et al., 1993), but most workers continue to use SCID mice as recipients of fetal tissue grafts. The thymus in SCID-hu thy/liv mice undergoes several rounds of T cell differentiation and reaches maximum size at 3-6 months after engraftment. Thymopoeisis rarely continues much past 6 months, presumably because of a depletion of available precursors (Kaneshima et al., 1994a; McCune et al., 1991b; Namikawaet al., 1990).The human thymocytes differentiating in this model recapitulate the stages of development seen in a normal thymus; the majority of human T cells are CD4'CDB' (double positive) and only a small minority are CD4- or CD8-single positive cells (Aldrovandi et al., 1993; Kaneshima et al., 1990; Krowka et al., 1991; McCune et al., 1988). B lymphocytes are not generated in this model, but islands of human myeloid differentiation often form at the junction between fetal liver and thymus grafts (McCune et al., 1989, 1991b; Namikawa et al., 1990). As a target for HIV infection, the SCID-hu thy/liv model seems to accurately represent late fetal-early neonatal human thymus development, and as such is highly relevant to maternal-fetal virus transmission and infection of human T cell progenitors (Aldrovandi et al., 1993; Bonyhadi et al., 1993; Stanley et al., 1993b). 111. Human Immunodeficiency Virus

A. THEVIRUS A brief review of the biology of HIV infection and the nature of the virus itself is necessary to understand the importance of the experiments performed in the hu-PBL-SCID and SCID-hu thy/liv models. HIV (then termed lymphadenopathy-associated virus, LAV) was first associated with the acquired immune deficiency syndrome in 1983 (Barre-Sinoussi et al., 1983), and was renamed human immunodeficiency virus in 1986 (Coffin et aZ., 1986; Gallo et al., 1988). HIV-1 is a lentivirus that apparently entered

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the human population by cross species transmission from primates (Chapman et al., 1995; Doolittle, 1989). It is closely related to HIV-2 and simian immunodeficiency virus (SIV), both of which infect primates as well as humans (Desrosiers et al., 1989; Gardner and Luciw, 1989; Khabbaz et al., 1994). The genomic organization of HIV-1 is summarized in Fig. 2. The genome codes for structural proteins required for viral assembly, enzymes required for viral replication, and regulatory proteins whose function is not fully understood [reviewed in (Levy, 1993b; Rosenberg and Fauci, 1991)l.Viral infection is initiated by the binding of the HIV-1 gpl20 envelope protein to CD4 on human cells (Dalgleish et al., 1984; Maddon et al., 1986), although some CD4-negative human cells are susceptible to virus infection at high multiplicities of infection (Levy, 1993b). The sequence of gpl20, particularly of the V3 loop in the middle of the molecule (see Fig. 3), is an important determinant for cell tropism (Chesebro et al., 1992; Cordonnier et al., 1989; Hwang et al., 1991; Westervelt et al., 1992). Cell tropism is categorized by the cell types that HIV-1 infects: most HIV-1 isolates are either monocyte/macrophage-tropic or T cell-tropic. Macrophagetropic virus replicates in primary cultures of human macrophages as well as activated peripheral blood T lymphocytes, but does not grow in estab-

FIG.2. Genomic organization of HIV-1. Adapted from a review by Levy (1993b).Viral transcripts and protein products are shown. Protein functions are indicated when known. LTR, long terminal repeat; gag, group-associated antigen (structural proteins); pol, polymerase; pro, protease; vif, viral infectivity factor; vpr, viral protein r; vpu, viral protein, u; rev, regulator of viral protein expression; tat, transactivating protein; nef, negative factor (misnamed); env, envelope, including surface (SU) and transmembrane (TM) components.


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lished T cell lines. T cell tropic-virus isolates replicate in established T cell lines and activated peripheral blood T cells, but not in primary macrophage cultures. Rare viral isolates replicate in both T cell lines and primary macrophages (Collman et al., 1992), and in such instances tropism may not map to V3 (Cheng-Mayer et al., 1990; Kim et al., 1995). Viral tropism is important in transmission of infection; macrophage-tropic isolates are much more frequently transmitted than T cell-tropic viruses (Gendelman et al., 1989; Korber et al., 1992; Massari et al., 1990). Macrophage-tropic isolates are also frequently recovered from the central nervous system, where they grow in tissue macrophages or microglial cells (Gendelman et al., 1989; Koenig et al., 1986; Poli and Fauci, 1992). Distinct HIV-1 isolates can also differ in their replication rate and cytopathic effects, including direct cellular cytotoxicity and syncpal induction (Castro et al., 1988; Cheng-Mayer et al., 1988; Evans et al., 1987; Tateno and Levy, 1988). Most macrophage-tropic isolates show lower rates of replication in macrophages than in primary T cells, and are noncytopathic (Cheng-Mayer and Levy, 1988; Cheng-Mayer et al., 1988; Gendelman et al., 1989; Mosier and Sieburg, 1994), although there are rare exceptions (Yu et al., 1994). In contrast, many T cell-tropic isolates show higher rates of replication and cytopathic, syncytial-inducingisolates are common. The appearance of syncytial-inducingHIV variants in approximately half of late stage patients is associated with accelerated loss of CD4’ T cells (Bozzette et aE., 1993; Connor et al., 1993). The broader range of biologic variability in T cell-tropic HIV isolates is accompanied by greater sequence diversity in the V3 region of gp120 (Chesebro et al., 1992; Korber et al., 1992; Westervelt et al., 1992). Envelope sequence and cell tropism are also linked to susceptibility to antibody neutralization; macrophage-tropic isolates and most patient isolates are more resistant to neutralization than “laboratory isolates” that have been passed repeatedly in T cell lines (Burton et al., 1994; Moore et al., 1995; Sattentau and Moore, 1995), which leads to the rapid selection of genotypes that often do not represent the dominant virus population in the patient (Mullins et al., 1991). In addition to the biologic properties of HIV-1 influenced by gpl20 envelope sequence, expression of the accessory gene vpu, 2Ypl; and nef contributes to infectivity and viral replication in subtle but potentially important ways (Balliet et al., 1994; Jabbar, 1995; Levy et al., 1995; Park and Sodroski, 1995; Willey et al., 1992). The importance of the nef gene is probably clearest and best understood (Aiken et al., 1994; Aiken and Trono, 1995; Bandres et al., 1995; Cullen, 1994; Littman, 1994; Miller et al., 1994, 1995; Ratner and Niederman, 1995; Sawai et al., 1994). A key observation was that a pathogenic clone of SIV-1is rendered nonpathogenic by deletion of nef (Kestler et al., 1991). The highly pathogenic phenotype

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of the SIV PBj/l4 isolate (Dewhurst et al., 1990) can be reproduced by introduction of the PBj nef sequence into a less pathogenic molecular clone (Du et al., 1995). As will be described below, nefdeletion mutants also show delayed kinetics of CD4' T cell depletion in the SCID-hu thy/ liv model (Jamieson et al., 1994) and the hu-PBL-SCID model (Mosier et al., submitted). It would appear that the xenochimeric human transplants are reproducing the obvious in vivo phenotype of the SIV nef-deletion mutant rather than the subtle in vitro phenotype of HIV-1 nefdeletion mutants. Mutational analysis of nef function have shown that myristoylation of the N-terminus of the protein is necessary for transport to the cell surface, and that overlapping but distinct domains of nef are important for CD4 down-regulation and infectivity, respectively (Cullen, 1994; Goldsmith et al., 1995; Miller et al., 1995; Niederman et al., 1993). Figure 3 shows the functional domains of nef. In addition to its effects on CD4 modulation and infectivity, nef has been reported to associate with a cellular protein kinase which could impact lymphocyte activation pathways (Sawai et al., 1994, 1995). Finally, the observations that some long-term survivors of HIV-1 infection have deletions in nef (Deacon et aZ., 1995; Kirchhoff et al., 1995) strongly implicate the nef protein as an important determinant of HIV-1 pathogenicity. The t y r gene product appears to impact nuclear localization of the viral preintegration complex and to arrest infected cells in the G2 phase of the cell cycle (He et al., 1995; Heinzinger et al., 1994; Jowett et al., 1995). cell tropi8m

CD4 binding I

r

v1 L

1 ) Z

..

v2'

V3 ' U

I

1

-

0 0 N

-

neut Ab determinants

~4

I ~5

n

IKI

I

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gp120 su

7

membrane DOIV. targeting moahic

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fusion

intracellular

7 0 0

-

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I

o

8

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-

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gp41 TM

--

CD4 downregulation infectivity

IZ

PKC site

I

l-

nef

FIG. 3. Functional domains of envelope and nefgenes of HIV-1. Adapted from Levy . Antibody binding studies indicate that the variable (1993b) and Goldsmith et ~ l (1995). (V) regions of envelope are exposed surfaces, and that V3 is a frequent but not unique target of neutralizing antibodies (see text).

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The latter effect may prolong the period of time during which infectious virus is produced and provide a temporary reprieve from apoptosis. The 2ypu gene encodes a cytoplasmic protein that appears to be involved in viral assembly (Strebel et al., 1989). Deletion of upr and y u of SIV-1 reduces pathogenicity in adult but apparently not newborn macaques (Baba et al., 1995; Hoch et al., 1995; Lang et al., 1993).

B. THEDISEASE HIV-1 infection of humans ultimately leads to loss of CD4' T lymphocytes and generalized immunodeficiency in more than 95% of infected individuals. While loss of CD4+T cells is an excellent predictor of progression to clinical disease (Veugelers et al., 1993), other consequences of HIV infection include loss of natural killer cell activity (Bonagura et al., 1992; Scott-Algara et al., 1992),alteration in cytokine production (Poli and Fauci, 1992), chronic B cell activation and a high risk of B cell lymphomas (Herndier et al., 1992; Rieckmann et al., 1991), and neurologic consequences of HIV infection, including HIV-associated dementia (Ho et al., 1989; Lipton et al., 1995; Portegies, 1994). Figure 4 illustrates the course of disease with respect to two important parameters: viral load and CD4+ T cell count. 107

1000 900

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700 105

600 500

104

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300 103

200 100

102

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6 years after infection 4

CD4 T cells

8

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plasma RNA copy number

----

FIG,4. Clinical course of HIV-1 infection in humans. This schematized course ignores considerable individual variation in time to progression to AIDS and viral burden. The high viral burden during primary infection may be biased by the selection of individuals with symptomatic infection.

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Primary infection involves transmission of HIV-1 to susceptible cells, infection of primary targets (e.g., macrophages in submucosal tissue or T cells in local lymph nodes), local spread of virus to other susceptible cells, and dissemination of virus infection by emigration of infected T cells and plasma viremia (Antonioli et al., 1995; Graziosi et al., 1993b; Henrard et al., 1995; Lamhamedi-Cherradi et al., 1995; Nielsen et al., 1993; Niu et al., 1993; Piatak et al., 1993; Pratt et al., 1995; Sinicco et al., 1993; van Gemen et al., 1993; Zaunders et al., 1995; Zhang et al., 1993; Zhu et al., 1993).Transmission and dissemination of virus is likely to be due to cellular transmission rather than spread of cell-free virus, since infected cells are several orders of magnitude more efficient at transmitting infection (Dimitrov et al., 1995, 1993). Early infection usually involves the expansion of a single molecular species of HIV-1 (Antonioli et al., 1995; Shpaer et al., 1994; Zhang et al., 1993; Zhu et al., 1993), with rapid mutation, selection, and emergence of diverse genotypes (quasi-species) coinciding with the onset of immune responses to the virus (Coffin, 1995; Pang et al., 1992). As illustrated in Fig. 4, very high viral burden may be achieved prior to control of infection (Daar et al., 1991), which is almost certainly due to the onset of an effective cytotoxic T lymphocyte response (Koup et al., 1994a; Lamhamedi-Cherradi et al., 1995; Safrit et al., 1994). Neutralizing antibody to HIV-1 infection may not appear for several months after the decline in viremia (Graziosi et al., 199313; Henrard et al., 1995; Levy, 1993a). Moreover, the more frequently transmitted macrophage-tropic HIV isolates are more difficult to neutralize than T cell-line propagated “laboratory strains” (Bou-Habib et al., 1994; Moore et al., 1995; Sattentau and Moore, 1995), suggesting that even if neutralizing antibody were present earlier, it might not be effective in resolving primary infection. The human thymus is a target for primary HIV-1 infection in fetuses, newborns, and adults (Burke et al., 1995; Papiernik et al., 1992; Rosennveig et al., 1994, 1993; Schuurman et al., 1989; Tremblay et al., 1990). Thymic infection appears to lead to deletion of CDltCD4+CD8+progenitor cells as well as disruption of the thymic epithelial microenvironment (Li et al., 1995; Numazaki et al., 1989; Schnittman et al., 1991). There remains a controversy over whether or not earlier CD34+ stem cells are infected by HIV-1 (Calenda and Chermann, 1992; Schnittman et al., 1990; Stanley et al., 1993a; Valentin et al., 1994). There are relatively few reports on the potentially important role of thymic infection on T cell production and turnover, and the development of the SCID-hu thy/liv model has provided a new experimental system for addressing the consequences of intrathymic HIV-1 infection. Following resolution of primary HIV-1 infection, there is a long, clinically asymptomatic stage of disease during which viral load is slowly increasing


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and CD4+ T cell numbers are slowly decreasing (Fig. 4). However, it is now generally recognized that chronic infection with a relatively high viral and cell turnover is a feature of the disease, and that a failure of the immune response to keep pace with the high rate of viral replication ultimately results in rapid disease progression (Coffin, 1995; Graziosi et al., 1993a; Ho et al., 1995; Pantaleo et al., 1993; Wei et al., 1995). A N D THE hu-PBL-SCID A N D C. HIV-1 DISEASE SCID-hu thyhv MODELS While there is evidence of functional competence of human lymphocytes in both the hu-PBL-SCID and SCID-hu thy/liv models (see above), there is no evidence to date for a primary immune response to HIV-1 infection. Secondary antibody responses to HIV-1 have been observed in hu-PBLSCID mice derived from gp160 envelope-vaccinated volunteers (Mosier d al., 1992) or from infected patients (Boyle et aZ., 1995); these antibody responses (which did not include neutralizing antibody) did not appear to alter the course of HIV-1 infection. As will be discussed below, extensive HIV-1 infection appears to lead to depletion of all or most target human cells in both models, and with the loss of targets, the viral burden drops (Aldrovandi et al., 1993; Bonyhadi et al., 1993; Mosier et al., 1993a; Su et al., 1995). These models thus resemble an unopposed primary HIV-1 infection occurring in the absence of a cellular or humoral immune response. As such, the models establish that antiviral immunity is not necessary for many features of HIV-1 pathogenesis, including depletion of CD4' T cells (Aldrovandiet al., 1993; Bonyhadi et al., 1993; Mosier et aZ.,1993a, 1991). This is not to say that immune responses may not contribute to some of the features of HIV-1 pathogenesis in humans; e.g., CTL responses in the central nervous system may contribute to AIDS neuropathy even though they are essential to controlling virus replication in peripheral lymphoid tissue. Several reports also suggest that autoimmune responses are frequent in HIV-1 infection and could potentially impact the development of disease (Ascher and Sheppard, 1988; Clerici et al., 1993; Dalgleish, 1993; Gougeon et al., 1993; Pinto et al., 1994; Stricker et al., 1987). It is important to underscore that the xenotransplant animal models are appropriate for some experimental questions regarding HIV-1 infection, but inappropriate for others. The replication of HIV-1 in human tissues in SCID mice could result in the formation of novel viruses arising from recombination, phenotypic mixing, or pseudotyping of HIV and endogenous xenotropic mouse retroviruses (Canivet et al., 1990; Cavallo et al., 1994; Lusso et al., 1990), or the pseudotyping of HIV-1 in passenger human herpesviruses [EBV, CMV, HHV6, HHV8 (Chang et al.,1994; Lusso et d., 1989; Lusso et al., 1993;

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Mocarski et al., 1993; Moore and Chang, 1995; Spector et al., 1990; Van Kuyk and Mosier, 1995)l.There is a clear risk to working with HIV-1 in an experimental animal (Milman, 1990),since any change in the cell tropism of HIV-1 could pose a significant new risk for transmission. Fortunately, extensive searches for HIV-1 with altered tropism in either the SCID-hu thy/liv or the hu-PBL-SCID model have been negative (McCune et ul., 1990b;Van Kuyk and Mosier, 1995).The risk of murine xenotropic retroviruses contributing to an HIV with altered cell tropism cannot be entirely dismissed, however, and handling of SCID mice bearing human grafts should always be performed under appropriate biosafety containment. IV. HIV-1 Infection in the hu-PBL-SCID Model

The hu-PBL-SCID model tests the ability of HIV-1 to infect and induce pathogenic changes in a graft consisting primarily of adult T cells with a high proportion of activated CD4’ and CD8’ cells. T cell replacement can occur only by proliferation of existing cells, and the primary immune responses to virus infection are either absent or delayed until after the peak of virus infection. At the outset of these experiments, it was difficult to predict whether activated T cells in hu-PBL-SCID mice would differ in any significant way from activated primary T cells in tissue culture (this argument was once crystallized by terming the hu-PBL-SCID mouse an “expensive furry test tube”). As will become clear, a unique biology of HIV1has been revealed in the hu-PBL-SCID model, with results paralleling in vivo studies of SIV infection in primates. A. STUDIESOF HIV-1 PATHOGENESIS

In the first description of HIV-1 infection of hu-PBL-SCID mice (Mosier

et al., 1991), it was found that the mice were susceptible to both cell-free

and cell-associated virus infection with the common laboratory isolate HIVlU1(then termed alternatively LAV-UBru or IIIB). The percentage of infected animals ranged from 100%at 3-4 weeks postinfection to 30-50% at 16weeks postinfection. The minimal infectious dose was 10 tissue culture infectious doses (TCID) of virus. It may be of historical interest that the HIV-lllIBvirus stock used for infection had the same animal infectious titer in chimpanzees (Arthur et al., 1989).Infection resulted in a transient elevation of human immunoglobulin levels and a loss of CD4’ T cells in some but not all mice. In retrospect, the use of very high virus input ( lo4 TCID) in most experiments probably led to the rapid depletion of CD4’ T cells and the failure to recover virus in some animals. We also failed to observe consistent CD4’ T cell depletion because hu-PBL-SCID spleens rather than peritoneal lavage cells (which were used for virus isolation)


91

were evaluated by flow cytometry. A consistent reduction of CD4+ T cells was later observed using human cells recovered by peritoneal lavage (Mosier et al., 1993a). The initial report also documented infection by three other HIV-1 isolates, MN, SF2, and SF13 (Cheng-Mayeret al., 1988; Levyet al., 1984; Mosier et al., 1991).These are all T cell-tropic laboratory isolates with extensive prior passage in T cell lines, but they are more representative of clade B HIV-1 than the LA1 isolate (Korber et al., 1992). HIV-1 virus stocks for these experiments were prepared using peripheral blood mononuclear cells (PBMC) activated with phytohemagglutinin (PHA) and interleukin-2 (IL-2); it was noted that HIV-lMN grown in the H9 T cell line was much less infectious for hu-PBL-SCID mice than the same virus propagated in PBMC. Koup and colleagues (Koup et al., 1994a) extended these findings of HIV-lLAI(IIIB) infection of hu-PBL-SCID mice. They employed endpoint dilution cultures, PCR, and p24 capsid antigen determination to more clearly show active viral replication and levels of virus comparable to those seen in acute primary infection or late-stage AIDS in humans. They also documented spread of HIV-1 infection to human cells in peripheral blood, lymph node, and bone marrow, as well as the previously reported spleen and peritoneal cavity sites of infection. Enhanced T cell activation was shown to accompany HIV-1 infection, with higher levels of soluble IL-2 receptors in infected compared to uninfected mice (Koup et al., 1994a). The elevation in human immunoglobulin seen in patients (Fauci, 1988; Lane et al., 1985) and the prior studies with HIV-1-infected hu-PBL-SCID mice (Mosier et al., 1991) could be secondary to this T cell activation. A more extensive survey of HIV-1 isolates and a focus on rates of CD4+ T cell depletion led to a surprising finding (Mosier et al., 1993a); two macrophage-tropic molecularly cloned viruses, HIV-lSF162and H1V-2~1 (Cheng-Mayer et al., 1990; Evans et al., 1988b), induced more rapid loss of CD4' T cells in infected hu-PBL-SCID mice than did the T cell-tropic HIV-1 isolates SF2, SF13, or SF33. These virus isolates were chosen for study because they differed in their biologic properties not only with regard to cell tropism, but also in their ability to induce syncyhal formation and cytopathicity in infected cells (Castro et al., 1990; Cheng-Mayer and Levy, 1988; Cheng-Mayer et al., 1990, 1988; Evans et al., 1988a; York-Higgins et al., 1990). It was expected that the SF33 isolate with its rapid replicative rate, high cytopathicity, and syncytial-inducing capacity would be the most pathogenic in hu-PBL-SCID mice. However, SF33 caused the slowest rate of CD4+ T cell depletion of any of the viruses studied despite its rapid replication in the model. Quantitative PCR analysis showed that both SF162 and SF33 gave proviral copy numbers ranging from 10' to 104/105 recovered human cells, and between 103and 105/105residual CD4' T cells

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DONALD E. MOSIER

(Mosier et al., 1993a). These numbers suggest that a high frequency of CD4' T cells may be infected (assuming only one proviral copy per infected cell, which may be an underestimate) with some viral isolates. However, infection with the SF2 and SF13 isolates resulted in a log lower proviral copy number and rates of CD4+ T cell depletion intermediate between SF162 and SF33. These data do not allow a clear distinction between death of only infected cells (direct mechanism) or death of uninfected cells (indirect mechanism), but they do suggest that a high proportion of cells may be infected during the 3-4 weeks of viral replication in the huPBL-SCID model. The results presented in this paper represent one of the first indications that the in uitro phenotype of HIV-1 might not predict its behavior in an animal model. The finding that a syncytial-inducing (SI) isolate was poorly pathogenic was at odds with the observation that patients who develop SI variants show a more rapid loss of CD4' T cells during the late stages of HIV-1 infection (Bozzette et al., 1993; Connor et al., 1993; Schellekens et nl., 1992; Tersmette et al., 1989). Two studies in the SCID-hu thy/liv model, each comparing one SI isolate with one non-syncytial-inducing (NSI) isolate, both reached the conclusion that the SI isolate caused more extensive viral replication and depletion of CD4'/8' T cells (Jamieson et nl., 1995; Kaneshima et al., 199413). In the study of Jamieson et al. (1995), the molecularly cloned viruses JR-CSF (Koyanagiet al., 1987) and N U - 3 were compared and N U - 3 was clearly more pathogenic. We have examined these isolates as well as many others in the hu-PBL-SCID model; representative results are shown in Fig. 5. In Fig. 5, HIV-1 (or HIV-2ucl)isolates are compared based on three properties: (1)replication rate in primary PBMC, (2) cytopathic effect on cultured T cell lines such as MT-4 (Tateno and Levy, 1988), and (3)rate of human CD4+ T cell depletion in the hu-PBL-SCID model. We find that HIV-lIR.CSF replicates poorly in the hu-PBL-SCID model and causes a slow rate of CD4' T cell depletion, in agreement with the findings in the SCID-hu thy/liv model (Jamieson et al., 1995). Conversely, the N U 3 isolate replicates well and causes as extensive CD4' T cell loss as the HIV-lLAIvirus. The primary isolate JR-CSF (Koyanagi et al., 1987) and the macrophage-tropic BaL isolate (Gartner et al., 1986; Gendelman et al., 1988) each show slower replication and less extensive CD4+ T cell depletion than three macrophage-tropic isolates with higher replicative capacity, SF162, UC1 (Mosier et al., 1993a; Mosier and Sieburg, 1994), and SF128A (Liu et al., 1990). Macrophage tropism thus is not an absolute predictor of pathogenic potential, and the replication of the virus isolate must also be taken into account. Two isolates from rapid progressors, WEAU (Li et al., 1991) and 89.6 (Collman et al., 1992; Kim et al., 1995;


I

-extent

of CD4 T cell depletion

I

93

I

-b

FIG.5. Characterization of different HIV-1 isolates for depletion of CD4+ T cells in the hu-PBL-SCID model, replication rate, and cytopathic effect for T cell lines. Replication rates in primary PBMC and cytopathic effects on T cell lines were measured in uitro, and depletion of CD4+ T cells was determined in hu-PBL-SCID mice. The scales are arbitrary units. The rate of CD4+ T cell depletion ranges from slow (95% depletion after 10 days of infection). Replication rates in tissue culture of primary PBMC seem to accurately predict replication rates in hu-PBLSCID mice.

Yu et al., 1994), induce the most rapid CD4' T cell loss observed in the hu-PBL-SCID model. These viruses differ significantlyin cytopathic effect, so the value of this biologic feature in predicting rate of CD4' T cell depletion is not dominant. That being said, it is also clear the the SF33 isolate is poorer than other T cell-tropic isolates in triggering CD4' T cell loss, a point to which we will return when nefdeletion mutants are discussed. The most obvious point from this analysis is that almost any HIV1 isolate is pathogenic, and those with higher replication rates are more pathogenic. In vitro analysis of biologic phenotype may or may not be predictive of pathogenic potential in the hu-PBL-SCID model, and the hu-PBL-SCID model may or may not be predictive of pathogenic potential in infected humans.

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Data for several deletion mutants we have studied in the hu-PBL -SCID model are also summarized in Fig, 5. Nef deletion mutants haje been studied in the context of three HIV-1 molecular clones; SF2 (provided by Jay Levy), NM-3 (provided by Didier Trono), and 89.6 (provided by Ronald Collman). In each case, nefdeletion slowed the rate of CD4’ T cell depletion compared to the unmutated virus. The impact of nefdeletion was greatest for SF2, where the parental virus has only average pathogenicity. SF2Anefvirus was poorly infectious and infected animals had low viral burden. NLA-3Anefvirus was highly infectious and replicated to near wildtype levels, but still showed little or no CD4’ T cell depletion. The highly pathogenic 89.6 clone was only moderately impacted by nef deletion; high virus loads were achieved, and CD4’ T cell depletion was slowed by 1 week (data not shown). These results confirm the important role of nef for in vivo pathogenicity originally observed in the SIV system (Kestler et al., 1991), but additionally suggest that other viral determinants of pathogenicity interact with nefto determine the outcome of virus infection. These results are in accord with those previously reported in the SCIDand upti hu thy/liv model (Jamieson et al., 1994). We also studied tpmutants of 89.6 (Balliet et al., 1994); these viruses were indistinguishable from unmutated 89.6 in the hu-PBL-SCID model. Boyle and co-workers (1995) have extended the hu-PBL-SCID model to the study of PBL from HIV-infected donors. This has the advantage that the population of viruses present in the naturally infected donor is reactivated in the transplanted cells, so that “relevant virus” rather than laboratory isolates is being studied. Asymptomatic donors with high CD4+ T cell counts (709-1043/mm3) and low or undetectable plasma viral RNA levels were used for these studies. A substantial increase in plasma viral RNA levels was seen in hu-PBL-SCID mice injected 7-12 days earlier with 50 X lo6PBL from these donors, with peak viremia above lo4copies/ ml. Recovered HIV-1 was sequenced following amplification of the V3 region of envelope, and preservation of the sequence diversity present in the donor was observed. This is a key observation in terms of the validity of the model, since it means that the selective replication observed in tissue culture ( Wain-Hobson, 1989) does not occur in the hu-PBL-SCID model. Mice also exhibited antibody reactive with viral antigens by Western blot, although this probably represents preservation of antibody formation initiated in the donor rather than the SCID mouse. Neither that antibody or passively administered autologous serum was able to block viral replication, which probably reflects the frequent finding that neutralizing antibody is relatively ineffective against autologous isolates (Burton et al., 1994). Treatment of the infected hu-PBL-SCID mice with fluorodideoxyadenosine reduced the rate ofvirus detection and reversed CD4’ T cell depletion.


95

A less decisive therapeutic benefit was seen with antibodies to tumor necrosis factor (TNF) a and p. The use of HIV-1 seropositive donors to generate hu-PBL-SCID mice has some clear advantages that are demonstrated in this paper. The use of donors with more advanced disease and fewer CD4+ T cells is problematic, however, since relatively large blood volumes are required to recover sufficient PBMC to generate meaningful numbers of mice for study. The finding that patients with relatively stable disease uniformly activated viral replication in hu-PBL-SCID mice suggests that some controlling immune component, e.g., CDB+ T cells, does not function as well in the SCID transplant as in the donor. One study has attempted to use a modified hu-PBL-SCID model to study the neuropathology of HIV-1 infection (Tyor et al., 1993).SCID mice were inoculated intracerebrally with PBMC and HIV-1. HIV-1 infection of T cells and some human macrophages ensued, some multinucleated giant cells were seen, and gliosis was striking. These effects were not seen in the absence of HIV-1 infection. The extent to which this model mimics natural infection of macrophages and microglial cells in human brains is unclear. A different model for HIV-1 pathogenesis in the brain may have more promise (Achim et al., 1993; Achim and Wiley, 1994). Achim and Wiley have prepared cultures of fetal neural cells which aggregate to form microspheres. These cultured microspheres have been cocultured with HIV-1 infected human macrophages, and transplants of these microspheres continue to replicate HIV-1 after transplantation to SCID mice. HIVinfected transplants contained multinucleated giant cells, and virus was recovered for up to 3 months after transplantation (Achim et al., 1993). Neuronal cell degeneration was observed at later time points. We have also helped develop a model which may have particular applicability in the study of pediatric HIV-1 infection (Reinhardt et al., 1994). Cord blood cells were introduced into neonatal SCID mice (because they failed to engraft adult SCID mice), and the resulting hu-CBL-neoSCID mice were found to be highly susceptible to infection with both laboratory and primary pediatric HIV-1 isolates. However, the naive phenotype of cord blood CD4+ T cells quickly converted to the CD45RO' activated/ memory phenotype in this model, and HIV-l-induced loss of CD4+ T cells was quite rapid compared to the adult PBL-SCID model. It is thus not clear that the cord blood graft mimics the activity of the newborn immune system. OF PROTECTIVE IMMUNITY B. STUDIES The ability to induce primary HIV-1 infection in a small animal model presents an opportunity to investigate both active and passive immunity to viral infection. Several studies have addressed the protective effect of

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passive antibody or CTL therapy on HIV-1 infection (Mosier et al., 1992; Parren et al., 1995; Safrit et al., 1993; van Kuyk et al., 1994), and we have studied active immunity induced by candidate HIV-1 vaccines (Mosier et al., 1993b). We collaborated with Larry Corey and Phil Greenberg in a study of volunteers receiving vaccinia expressing the gp160 envelope of HIV-lLAV.I/Bm(Hu et al., 1986) and subsequent secondary stimulation with a recombinant gp160 preparation from HIV-1IIIB (Lai) (Redfield et al., 1991). These donors were identified as candidates for hu-PBL-SCID studies because of their high T cell proliferative responses to HIV antigens, which were higher than comparable vaccinees receiving only recombinant gp160 (Cooney et al., 1993). PBMC were obtained from four of these donors, and transferred to SCID mouse recipients at periods of 4-146 weeks after the last injection of gp160. Mice were challenged with hornologous HIV-1 2-4 weeks after PBL reconstitution. The results of these experiments are summarized in Table I. They show that a highly significant proportion of mice (compared to the 393 control mice prepared from unimmunized, placebo-immunized, or recombivax-immunized donors) were protected from infection if recombinant gp160 had been administered to the PBL donor within the previous 10 weeks. Vaccination with the combination of vaccinia-gpl60 and restimulation with recombinant gp160 thus seemed to stimulate a transient state of viral resistance that could be adoptively transferred to SCID mice. Analysis of the available correlates of immunity from the donors (CTL assays were not available at the time) showed that T cell proliferative responses correlated with protection against TABLE I PROTECTION OF hu-PBL-SCID MICE FROM HIV-1 INFECTION HY PRIOR IMMUNILATION OF THE PBL DONOR (ADAPTEDFROM MOSIEHet a[., 1993b). Experiment/ donor 1/06

Do65

3/06 4065 1/078 DO78 3/078 4078 3/033 4/033 3/053 4/053

Boost

Weeks after boosting

1 1 1 2 1 1 1 2 1 2 1 2

4 41 66 5 10 47 72 5 48 4 72 5

Donor immune status

gp160 Ab

+ + t

++ +++ ++ ++

+++ ++

+++ t

+

neut Ab

T pro1

-

++ ++ ++ + +++ ++

+ -

+

-

?

++

++ ++ + +

% Protected

100 50 0

42 67 0 0 33 0

33 0 0


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infection in the hu-PBL-SCID model, but that antibody levels did not. Based on these encouraging preliminary results, we used the hu-PBLSCID model to evaluate a large number of vaccinees in a trial of recombinant gp160 alone. Unfortunately, these studies were performed at 6 months or more after the most recent immunization with gp160, and no difference was discernible between control and vaccinated donors. These studies raised interest in the old question of whether cellular immunity or antibodies would be more effective in preventing HIV-1 infection. Three studies have been reported in which passive administration of antibody has shown protective effects against HIV-1 infection in the hu-PBL-SCID model. In the first (Safrit et al., 1993), hu-PBL-SCID mice were administered 40 mgntg of the mouse monoclonal BAT123 neutralizing a humanized version of antibody directed at the V3 loop of H1V-lIIIBILAI), the same antibody, or a control mouse monoclonal antibody. Mice were then challenged with a low dose of the IIIB isolate, and infection was assayed 3 weeks later. Mice receiving either BAT123 or its humanized version were protected, while five of six control mice were infected. This demonstrated that very high doses of neutralizing antibody could protect against infection with a modest challenge dose of virus. Antibody half-life was 9-12 days, which was close to normal for mouse immunoglobulin. A subsequent study using the same BAT123 antibody (Gauduin et al., 1995) more closely examined the timing and dosage of antibody administration. It was found that a dose of 1 mgntg was protective at either 1 hr prior to HIV exposure or 4 hr after exposure. Later administration of the antibody was not protective. Protection was also found to be HIV-1 strain-specific, since infection with isolates other than IIIB (LAI) were not blocked by BAT123 antibodies. Passive administration of a broadly neutralizing human monoclonal antibody generated from a phage display library is also protective against HIV-1 infection (Parren et al., 1995). Both Fab b12 and IgG1reconstructed bl2, which recognize the CD4-binding domain of gpl20 (Burton et al., 1994), were tested. Antibody treatment was initiated prior to HIV-1 challenge in these experiments. A titration of the IgG1-b12 antibody (which had a much more favorable half-life) showed complete protection against HIV-lsF2challenge at 5 mg/kg and 50% protection at 2.5 mgntg. These results suggest that sterilizing immunity is attainable with relatively high concentrations of neutralizing antibody, concentrations that have not yet been achieved in any trials of candidate HIV-1 vaccines (Cooney et al., 1993; Kahn et al., 1994). These studies also show that at least 100-fold higher concentrations of antibody are necessary to neutralize virus in hu-PBL-SCID mice compared to tissue culture systems (Burton et al., 1994). The higher protective concentration probably reflects the difficulty in preventing viral spread within lymphoid tissue if even one cell

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becomes infected, whereas the much lower cell density of typical tissue culture systems would allow neutralizing antibody to continue to be effective in secondary rounds of viral replication. The hu-PBL-SCID system allows relatively rapid and simple screening of passive antibody protection, and should be useful as the search for neutralizing antibodies capable of blocking primary isolates and viruses from diverse origins (non-clade B) continues. Our studies of gpl60-immunized vaccinees suggested that cellular immunity might be protective against HIV-1 infection in the hu-PBL-SCID model (Mosier et al., 1993b), which concurs with the important role of CTL in controlling primary infection (Koup et al., 1994b; Safrit et al., 1994). We therefore initiated studies in which cloned, nef-specific, CD8+ CTL derived from an HIV-l-seropositive donor (Koenig et al., 1990) were passively transferred to hu-PBL-SCID mice reconstituted either with PBL sharing the HLA-A3.1 restriction element or with HLA-mismatched PBL (van Kuyk et al., 1994). Several findings emerged from this study. Passive CTL therapy had to be initiated on the day of HIV-1 challenge or 1 day prior to challenge to be effective. With only a single injection of lo7 CTL, partial protection (Y5challenged mice) against HIV-1 infection was observed and the protection was HLA-restricted. To achieve complete protection against infection, we had to administer CTL repeatedly every 2-3 days beginning prior to HIV-1 challenge and continuing for 2 weeks after challenge. Under these conditions, protection was achieved with both HLA-matched and HLA-mismatched PBL grafts. Moreover, partial protection could be achieved by HTLV-1 tax-specific CTL clones with this high dose (5-10 X lo7 total CTL) regimen, so the protective effect was neither antigen-specific nor HLA-restricted. These results suggest that two mechanisms were operative in the antiviral activity of human CTL clones: (1)HLA-restricted CTL-mediated killing of infected cells, and (2) production of antiviral activity when large numbers of any activated CD8+ T cells were introduced into the hu-PBL-SCID mice. This latter activity is probably associated with the known antiviral activity of CD8+ T cells in vitro (Baier et al., 1995; Castro et al., 1991;Cocchi et al., 1995; Mackewicz and Levy, 1992; Mackewicz et al., 1995, 1991; Walker et al., 1991a,b, 1989), and suggests that cytokine or chemokine production by CTL may be as important for the control of HIV-1 infection as killing of infected cells. The rapid turnover of infected cells in patients and the high viral load (Coffin, 1995; Ho et al., 1995; Wei et al., 1995) both suggest that virus-specific CTL must have some other means of controlling the infection in addition to cytolytic activity, since few cells would be killed early enough in the viral replication cycle to prevent release of infectious virions from the lysed cell.


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V. HN-1 Infection in the X l D h u thy/lii Model

Engraftment of human fetal thymus and liver under the kidney capsule (or subcutaneously) provides a model for intrathymic HIV-1 infection in the context of ongoing T cell development in an organ microenvironment. This model allows one to address questions regarding infection of precursor cells versus mature T lymphocytes, and to examine the effect of virus infection on macrophages and thymic epithelial cells. Since the thymus is known to be infected in both pediatric and adult HIV-1 infection (Burke et al., 1995; Papiernik d- al., 1992; Rosenzweig et al., 1994, 1993; Schuurman et al., 1989; Tremblay et al., 1990), studies in this model are highly relevant to thymopoiesis. Their relevance to deletion of mature CD4+ T cells in peripheral lymphoid tissues is open to question, but the concurrence between viral pathogenicity in humans and in the SCID-hu model underscores its potential importance. The first report of HIV-1 infection in the SCID-hu thy/liv model appeared in 1988 (Namikawa et al., 1988). The molecularly cloned HIV-l,, CSF isolate (Koyanagi et al., 1987) was injected directly into fetal thymus or lymph node grafts. Infection was monitored by in situ hybridization (ISH) with a probe for viral RNA and by immunohistochemical staining with a polyclonal anti-HIV antibody. The number of infected cells detectable by ISH increased between 2 and 8 weeks postinfection, and they were localized primarily in the medulla. More ISH-positive cells were found than cells staining for HIV-1 proteins, suggesting that some cells may have been abortively infected. When the same protocol was used with the H I V - l l I I B isolate, ~ ~ I ~ no infection resulted (McCune et al., 1991a), leading to the conclusion that the SCID-hu thymus graft was susceptible to primary but not laboratory-adapted strains of HIV-1. In these early studies, the cell types infected and the effect of infection on CD4' T cells was not characterized. A subsequent report (McCune et al., 199Oc) documented that azidodeoxythymidine (AZT, Zidovudine) reduced DNA proviral copy number and the number of ISH-positive cells at 2 weeks after infection of SCID-hu thy/liv mice with either the JR-CSF or the closely related JR-FL isolate, Virus was detected in mice that had AZT administration stopped after 2 weeks and were assayed 4 weeks later. In a second study, postexposure prophylaxis with AZT was demonstrated if drug was administered within 2 hr of HIV-1 injection (Shih et al., 1991). These findings heralded the use of either SCID-hu or hu-PBL-SCID mice for HIV drug development, but neither model has found widespread use in this application because the expedited drug discovery rules for antivirals targeted at HIV do not require any preclinical efficacy data in animal models (Ruprecht et al., 1990; Ussery et al., 1995).

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DONALD E. MOSIER

The SCID-hu thy/liv model was augmented by implanting fetal lymph nodes either under the renal capsule or subcutaneously. Intravenous rather than direct injection of HIV-l,R.CSF was used to infect mice, and HIV-1 could be detected within 2 weeks by ISH in the lymph node but not the thymus graft (Kaneshima et al., 1991). Plasma viremia was detectable by RNA PCR, although p24 capsid antigen was not. The 50% animal infectious dose of virus by the iv route was 12,000 TCID. As in the thy/liv model, HIV-lIIIR(Lhl) was not infectious at titers up to lo6 TCID, but 10 primary isolates from patients were found to be infectious. Both CD3+ T cells and esterase-positive macrophages were detected by ISH. The infectivity of primary isolates and the finding of infected macrophages suggests that macrophage-tropic HIV-1 may be more efficient in primary infection in the SCID-hu model just as it is in patients. More insight into the pathogenesis of HIV-1 infection in the SCID-hu thy/liv model was gained by findings reported in two papers published simultaneously in 1993 (Aldrovandi et al., 1993; Bonyhadi et al., 1993). The critical feature of both reports was the use of flow cytometry to analyze changes in thymocyte subpopulations induced by HIV-1 infection of fetal thymus grafts. In the study of Bonyhadi et nl. (1993), SCID-hu thy/liv mice were infected with a series of primary isolates of HIV-1 as well as JR-CSF and analyzed 3-8 weeks postinfection. Uninfected control mice showed the expected distribution of CD4+/CD8+(87%),CD4+(8%),and CD8+(5%)cells in the thymus, and recovery of 184 2 33 x lo6cells per graft. The most pathogenic primary isolate, SM, induced a substantial loss of first CD4+/CD8' double-positive cells and later CD4 single-positive T cells, with a sparing of CD8 single-positive T cells. Thymus cellularity was reduced by 96% by 6 weeks postinfection. Other isolates also induced significant loss of CD4+/CD8' cells, and more variable loss of CD4 singlepositive cells. The JR-CSF isolate was slower than other tested isolates in inducing cell loss. A second important observation in this paper was that much of the cell loss in infected thymus grafts appeared to be due to apoptotic cell death by morphological criteria and staining of cells with propidium iodide. Immunohistochemical staining of thymus grafts for viral p24 antigen showed foci of infected cells in the thymic cortex as early as 1 week after infection with the SM isolate, and p24 content in the graft peaked at 3-4 weeks after the infection and declined rapidly thereafter. The study by Aldrovandi et al. (1993) used similar methods to reach similar conclusions. SCID-hu thy/liv mice were infected with HIV-1 isolates JR-CSF, NL4-3, or a pool of five primary pediatric isolates. Quantitative proviral PCR analysis showed viral load to peak in the range of lo4proviral copies/105 cells at 3 weeks postinfection. The pediatric pool of viruses caused the most rapid loss of CD4CD8 double-positive cells as well as

HIV INFECTION OF HUMAN CELLS IN SClD MICE

101

CD4 single-positivecells. CD8 single-positive cells and CD4CD8 doublenegative precursor cells were spared, and increased in relative representation. Infection with JR-CSF or N U - 3 isolates caused somewhat slower loss of thymocytes, but a major decline in both CD4+/CD8+and CD4+ cells was seen by 6 weeks postinfection. Histological examination of grafts showed pyknotic nuclei in infected samples, which are consistent with but not definitive evidence for apoptotic bodies. These two studies thus showed an HIV-1 isolate-dependent induction of CD4+ T cell depletion, which appeared to begin with the more immature CD4CD8 double-positive precursor. Staining of infected cells in the first study (Bonyhadi et al., 1993) was consistent with deletion of cells by direct infection, as was the high proviral copy number detected in the second study (Aldrovandi et al., 1993), but neither of these observations was quantitative enough to eliminate death of some cells by indirect exposure to virus. A study by Stanley et al. (1993b) examined the effect of HIV-1 infection on the thymic epithelial cell microenvironment of fetal thymus grafts in the SCID-hu thy/liv model. Mice were infected with either the JR-CSF or SM isolates of HIV-1 as described in Bonyhadi et al. (1993). Extensive ISH and histochemical analyses of infected thymus grafts were performed. ISH showed relatively high numbers of infected cells in both the cortex and the medulla, and foci of infected cells were evident. By both in situ staining and analysis of sorted cells, both CD4+ and CD4- (CD8+)cells were infected, Rare thymic epithelial cells, identified by cytokeratin staining, were also infected. The kinetics of thymocyte loss were much faster for the SM isolate than the JR-CSF isolate. Two patterns of cellular infection and pathology were seen, sometimes involving separate areas of the same thymus graft. In the first pattern, infected thymocytes appear to die, leaving an intact thymic epithelial cell network now separated by empty spaces. In the second pattern, HIV is seen in thymic epithelial cells and epithelial cell degeneration occurs with sparing of adjacent lymphocytes. Some thymic epithelial cells showed degenerative changes in the absence of any detectable virus. The techniques did not allow distinction between productive infection of thymic epithelial cells or endocytosis of virus. Nonetheless, it was clear that HIV-1 infection induced not only T cell loss but also disruption of the epithelial cell microenvironment in the thymus graft. The persistence of CD8 single-positive cells infected by JR-CSF suggests that less pathogenic isolates of HIV-1 may infect CD4CD8 double-positive cells and, in the absence of a cytopathic effect, allow differentiation into CD8 single-positiveprogeny. The failure to detect HIV-1 in adult peripheral blood CD8' T cells argues that this is either a rare event or confined to the thymus.

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DONALD E. MOSIER

Kollman et al. (1994b) altered the SCID-hu thy/liv model slightly by implanting more fragments of fetal thymus and liver under both kidney capsules (Kollman et al., 1993; 1994a), resulting in higher numbers of human T cells in the circulation and peripheral lymphoid tissue than the original model. Peripheral blood, spleen, and lymph nodes of SCID-hu extra thy/liv mice showed approximately 5, 25, and 35% human cells, respectively,with CD4:CDS ratios greater than 2 : 1.The grafts were shown to contain a random representation of T cell receptor Vp usage by antibody staining. Mice were infected with a pediatric primary isolate of HIV-1, and infection monitored by limiting dilution cultures of grafted cells or PCR for HIV RNA and DNA. Either direct injection of HIV-1 into a graft under one kidney capsule or intraperitoneal injection led to a systemic infection of all sites of human cell engraftment. Intravenous injection did not lead to infection. Virus recovery was significantlylower in the peripheral blood and spleen than the thymus grafts, but relatively high in the contralatera1 kidney thymus graft of mice injected intrathymically. Cytokine expression was determined by a semiquantitative reverse transcriptase PCR method (RT-PCR) to determine cytokine mRNA. There were high levels of cytokine mRNA for TNF-a, TNF-P, IL-2, IL-4, IL-6, and IFN-y in the thymus grafts of both infected and uninfected mice. HIV-1 infection led to an increased fraction of mice with these cytokine mRNAs detectable in the spleen and lymph nodes, although little change was seen in IL-4 and IL-6. The data are consistent with HIV-1 induction of T cell activation and cytokine production, particularly in the thymus. The relative pathogenicity of syncytial-inducing HIV- 1 isolates was addressed in two studies using the SCID-hu thy/liv model to compare rates of CD4/CD8 double-positive cell loss after infection with different HIV1 isolates. In the first study (Kaneshima et al., 1994), two sets of patient isolates obtained before and after conversion from NSI to SI phenotype (Conner et al., 1993) were injected directly into thymic grafts. The two NSI isolates had low replicative ability in tissue culture and in SCID-hu thy/liv mice, while the two SI isolates had higher replication rates in both tissue culture and SCID-hu thymus grafts. At 8 weeks after infection, only the SI isolates had induced loss of CD4CD8 double-positive cells, although only one SI isolate (A7/88) caused substantial loss of T cells. It was also shown that the A7188 isolate caused a substantial increase in thymocytes apparently undergoing apoptosis by the TUNEL assay (Gorczyca et al., 1992). In a second study (Jamieson et al., 1995), the kinetics of T cell depletion following infection with JR-CSF and NL4-3 HIV-1 molecularly cloned isolates were compared. By serial biopsies of the same thymus graft and quantitative PCR, it was possible to follow viral replication kinetics in the first longitudinal study of infection in the SCID-hu model. As might


103

be expected from earlier studies (see above), the N U - 3 isolate replicated to higher levels earlier in the course of infection, and led to more rapid loss of both CD4CD8 double-positive cells and CD4 single-positive cells. Virus recovered after 6 weeks of infection was sequenced, and little or no mutation was seen in the V4 domain of envelope. This is consistent with the lack of immune selection which seems to drive mutation in infected humans (Lukashov et al., 1995). The results of these two studies do not resolve the NSI versus SI issue since in each case the less pathogenic NSI isolate also replicated to a much lower extent in the SCID-hu thy/liv model. Everyone would appear to agree that a certain virus load must be obtained to trigger CD4+T cell depletion. The fact that some 50% of AIDS patients die without developing a virus with the SI phenotype establishes that SI viruses are not essential for rapid T cell loss (Gruters et al., 1991; Koot et al., 1992). The issue of HIV-1 variability and pathogenicity in the SCID-hu thy/liv model was further confounded by a study of several commonly used HIV1 isolates in the multiple graft model (Kollman et al., 1995). The HIV-1 isolates studied were two primary. macrophage-tropic pediatric isolates (28 and 59), the macrophage-tropic isolates ADA, Ba-L, JR-FL, and SF162, the often employed JR-CSF isolate, and the T cell-tropic laboratory isolates IIIB ( M I ) and RF. Only the latter two strains of HIV-1 are syncytial inducing. A large number of mice were followed for up to 6 months after infection with the two primary isolates. High virus recovery was obtained from the grafts, but loss of either CD48 double-positive cells or CD4 single-positive cells was observed in only a few animals, and this loss did not correlate with virus load. The same inconsistent effect on CD4+ T cells was observed in the peripheral blood at 6 months after infection with HIV-128.The remainder of the viral isolates were studied 3 months after either intraperitoneal (ip) or direct injection into the graft. In the case of ip injection, only one animal receiving HIV-1IIIB (LAI) showed depletion of CD4'/8+ T cells. Direct implant injection led to significant loss of cells in one of two animals inoculated with IIIB (LAI) and one animal inoculated with RF virus. Viral load was higher in animals infected with macrophagetropic BaL, SF162, and JR-CSF isolates than in mice with thymocyte depletion, The informative experiment in this study consisted of only one or two animals per group, but it suggests that macrophage-tropic isolates may persist in the thymus without causing major damage, while T celltropic isolates can wreak havoc. To put this study in context, however, it should be remembered that the same IIIB (LAI) isolate of HIV-1 was noninfectious in the early studies of McCune and co-workers (McCune et al., 1991a; Namikawaet al., 1988).The long duration of infection (3months)

104

DONALD E. MOSIER

and the necessity of viral spread to different thymic fragments are variables that may contribute to the unique findings of this study. The SCID-hu thy/liv model allows analysis of HIV-1 infection in an organized lymphoid environment. Although hu-PBL-SCID mice show repopulation of lymph nodes and splenic white pulp with human T cells (see Fig. l),the local organization of T cells and antigen-presenting cells within lymphoid tissue is not reproduced. An important question to be addressed in both models is the mechanism of CD4' T cell depletion. Several recent papers have suggested an indirect mechanism of CD4+ T cell death which depends upon cell-to-cell contact between infected and uninfected cells (Maldarelli et al., 1995; Nardelli et al., 1995). It has also been postulated that many uninfected lymphocytes are undergoing apoptosis based on histological examination of HIV-l-infected patient lymph node biopsies and SIV-I-infected monkey lymph nodes (Finkel et al., 1995). The issue of direct (death of infected cells) versus indirect induction of cell death was addressed in an important study in the SCID-hu thyAiv model (Su et al., 1995). It is clear that HIV-infected cells die by apoptosis and that uninfected T cells from HIV-1 infected patients are prone to apoptosis (Ameisen and Capron, 1991; Terai et al., 1991), so assays of apoptosis do not settle this issue. An informative result would be the detection of many more cells undergoing apoptosis than are demonstrably infected by HIV-1. Su et al. (1995) infected SCID-hu thy/liv mice with HIV-lNM.:I and followed the occurrence of apoptosis by the TUNEL assay, which uses terminal deoxynucleotidyl transferase (TdT) to insert biotinylated dUTP at sites of double-stranded DNA breakage (Gorczyca et al., 1992). Fluorescein-conjugated avidin is then used to detect sites of biotin-dUTP insertion, and stained cells detected by flow cytometry. HIV-1 infection of fetal thymus grafts increased the percentage of TUNEL+cells (inappropriately termed TdT+ in this paper; prothymocytes express TdT which allows encoding of N regions in T cell receptor junctions) from less than 2% in control grafts to greater than 50% in grafts infected for 3-4 weeks. Double staining for TUNEL and CD4CD8 showed that apoptosis was occurring in all populations of lymphocytes, including CD8 single-positive cells, but was most prominent in CD4 single-positivecells. It was estimated from quantitative PCR that about 10% of thymocytes were infected by 3-4 weeks postinfection. This excess of apoptotic cells over the calculated number of infected cells provides the argument for indirect cell killing. Unfortunately, the technical quality of the TUNEL assay on human thymocytes is poor and gives much higher numbers than might be expected. For example, steroid treatment of mice leads to a rapid induction of apoptosis with up to 50% of cells becoming TUNEL+,as was demonstrated in control experiments in the report of Su et al. (1995). However, these apoptotic


105

cells are rapidly cleared by thymic macrophages and are detectable for only a few hours (Surh and Sprent, 1994). Under the best conditions, the TUNEL assay as employed gives 8- to 10-foldbrighter staining of apoptotic cells compared to viable cells (Gorczyca et al., 1992). The data in Fig. 1D of Su et al. (1995) show a pattern of TUNEL staining that is inconsistent with this level of sensitivity. It is also clear from light scatter profiles that a large proportion of recovered cells were dead and that only a small number ofviable cells were within the light scatter gates analyzed. Additionally, 62% of CD8 single-positive cells are scored as TUNELt, although CD8+ cells are spared the consequences of HIV-1 infection (Aldrovandi et al., 1993; Bonyhadi et al., 1993).Although these technical considerations may not allow dismissal of the data, they do raise enough concern to question the ratio of apoptotic to infected cells, and, therefore, a major conclusion of the paper. If 10% of thymocytes are infected and the halflife of an infected cell is approximately 2 days (Ho et al., 1995), and new cells continue to be infected, then direct infection could lead to the observed 90% loss in thymus graft cellularity within a few days without proposing indirect induction of cell death. This paper more clearly demonstrates that a population of CD3-, CD4+, CD8- progenitor cells (Galy et al., 1993) is infected and deleted in thymic grafts. Three-color analysis, cell sorting, and PCR detection of virus in sorted cells showed that up to 50% of this progenitor population was infected with the N U - 3 isolate, whereas infection with two primary isolates led to selective depletion of more mature thymocyte populations. One important determinant of pathogenicity thus is the ability to infect CD4' progenitor cells as well as more mature lymphocytes. Two studies have addressed the impact of mutations in nef, u p , wpr, and uif on infectivity and CD4+ T cell depletion in the SCID-hu thy/liv model (Aldrovandi and Zack, 1996; Jamieson et al., 1994). Nef-deletion or frameshift mutants of HIV-1 JR-CSF and N U - 3 isolates were used to infect thymus grafts in SCID-hu mice (Jamieson et al., 1994). These mutants were indistinguishable from wild-type viruses in primary PBMC cultures. However, nef mutants showed delayed replication kinetics in uiuo. By quantitative PCR, wild-type JR-CSF replicated to 30-fold higher level than the nef frameshift mutant at 6 weeks postinfection. Infection with the nef deletion mutant of N U - 3 caused an even greater difference in replication kinetics, with very low proviral copy number at 6 weeks postinfection with the mutated virus. As might be expected from the low virus load, no depletion of any thymocyte population was seen with the N U - 3 nefdeletion mutant at 6 weeks postinfection. However, 1 of 3 thymus grafts evaluated 9 weeks after NU-3Anefinfection did show a decline in CD4/ CD8 double-positive cells. In a follow-up study (Aldrovandi and Zack,

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1996), deletion mutants of nef, zypr, vpu, and uifgenes of the NL4-3 isolate (Gibbs et al., 1994) were introduced into SCID-hu thy/liv mice and viral replication and CD4+cell depletion determined 3 or 6 weeks later. Deletion of the vpr gene had little effect on virus replication or depletion of CD4/ CD8 double-positive cells. Deletion of opu, v$ or nefgenes reduced viral replication and CD4+ T cell loss. However, if the dose of virus inoculated was increased 10-fold (to 1000 infectious units) and only the 6 week postinfection time point was considered, the deletion mutants showed a less attenuated phenotype. Virus load (by quantitative PCR) was in the same range as wild-type N U - 3 for thymus grafts infected with all mutants, although some individual animals receiving either oif or nef-deletion mutants had low viral load. The recovery of CD4/CD8 double-positive cells correlated with viral load; mice receiving either the vif- or nef-deletion mutants had variable depletion of T cells with some grafts containing near control values. With the exception of vpr, which seemed to be dispensable for in vivo infection, mutation of other accessory genes led to an attenuated but not apathogenic phenotype. This finding is consistent with the pathogenicity of SIV-1 accessory gene mutants in neonatal macaques (Baba et nl., 1995), where an immature immune system probably leads to the same absence of immune response as in the SCID-hu system. Their results also agree with the analysis of accessory gene mutants in the hu-PBL-SCID model (see above), except that deletion mutants of zypu did not show a phenotype distinct from wild-type virus in those experiments. This is probably due to the use of the 89.6 molecular clone of virus, which is more pathogenic than N U - 3 (Fig. 5). The results of studying accessory gene mutants in both models point to three important conclusions: (1) nef is the most essential “accessory” gene; (2) accessory gene mutants show delayed replication and pathogenicity, but they still eventually cause loss of CD4’ T cells; and (3) attenuated live virus vaccines (Daniel et al., 1992) should be considered with great caution. A third modification of the SCID-hu thy/liv model has been reported in which fetal lung tissue is also implanted (Cesbron et al., 1994; Grandadam et al., 1995). These mice are susceptible to intraperitoneal infection by a number of primary HIV-1 isolates, and infected cells were detected within the lung grafts. Relative high doses of virus (20,000 TCID) were required to infect all animals, and viral replication was most easily detected in the thymus graft. Peak virus load by quantitative PCR was 50,000 proviral copies/lO‘ cells, which is slightly lower than the viral load seen after direct intrathymic infection (see above). Infection led to a depletion of CDl’, CD4’, CD8+ T cells within the thymus graft. The infection of alveolar macrophages within the lung graft may allow future studies on tissue-


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specific pathology associated with HIV-1 infection, but this modified model has yet to contribute new insights into pathogenesis. VI. Conclusions

When the hu-PBL-SCID and SCID-hu thyAiv models were first developed, it was anticipated that they would be very useful as efficacy screens for the development of antivirals (McCune et al., 1990a; Mosier, 1990) and probably less useful as models to understand the pathogenicity of HIV-1 infection. This viewpoint has proven to be not entirely correct. The models have not been used extensively in drug development but have aided our understanding of HIV-1 infection. Several general conclusions derived from studies in both models can be drawn. First, the primary determinant of viral pathogenesis and CD4+ T cell death is HIV-1 itself, and not the immune response to it. The differences between primary and laboratory-adapted HIV-1 isolates, macrophage-tropic and T cell-tropic isolates, SI and NSI isolates, and wild-type versus accessory gene mutants are clear in these models, and all relate to properties of the virus itself. Second, virus replication is rapid and continuous in both models, and a relatively high proportion of cells are infected by 3-4 weeks after introduction of HIV-1. We have observed plasma RNA copy numbers of lo5/ ml in the hu-PBL-SCID model (D.E.M., unpublished observations), and proviral copy numbers in the SCID-hu model achieve similarly high levels. Third, HIV-1 infection is difficult to prevent and more difficult to reverse once established. Adoptive transfer studies of antibody or CTL in the huPBL-SCID model suggest that once a few cells are infected, it is impossible to prevent the spread of HIV-1 infection. These observations suggest substantial obstacles for HIV-1 vaccine development. Finally, most findings in the two model systems are compatible, and there does not seem to be any major qualitative difference between HIV-1 infection of fetal thymus grafts and of adult PBL grafts. The choice of models is dictated by the experimental question to be addressed. The insight into HIV-1 pathogenesis gained by the use of both the huPBL-SCID model and the SCID-hu thyAiv model points to their importance in future studies. As but one example, we have found that introduction of HIV-1 infected macrophages yields quite different results than infection transmitted by infected CD4' T cells. Clearly we have much more to learn about cell-to-cell transmission of virus and efficiency of the first round of infection. I believe that it is premature to abandon the use of the two SCID models for drug development; in particular, the use of hu-PBL-SCID mice generated from HIV-1 seropositive donors to evaluate multidrug

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resistance could have a major impact on the design of' increasing complicated clinical trials.

ACKNOWLEDGMENTS The author thanks Dr. Jerry Zack and Dr. Michael McCune for providing data prior to publication. The work on the hu-PBL-SCID model in our laboratory has involved productive collaborationswith the followinginvestigators:Jay Ley,Stephen Spector, Deborah Spector, Stephen Baird, Hans Sieburg, Didier Trono, Ronald Collnian, Scott Koenig, Lawrence Corey, and Phil Greenberg. Individuals within our laboratory who have contributed to the work include: Richard Gulizia, Jacqueline Glynn, Ying Lin, Robert van Kriyk, Gastcin Picchio, Bruce Torbett, Barbara Reinhardt. Rosemary Rochford, David McElligott, lsabella Atencio, Denise McKinney, Andrew Beeniink, Brian Kent, Rebecca Sabbe, Jennifer Lentz, Matthew Kohls, and Brile Chung. Much of the work on HIV-1 infection in the hu-PBL-SCID iiiotlel has been supported by National Institutes of Health Grant A129182. The assistance o f Bonnie Towle in the preparation of this review is gratefully acknowledged. This is Publimition 9825-lmm from The Scripps Research Institute.

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Tersmette, M., Gruters, R. A,, de Wolf, F., de Goede, R. E., Lange, J. M., Schellekens, P. T., Goudsmit, J., Huisman, H. G . , and Miedema, F. (1989). Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: Studies on sequential HIV isolates.J Virol63,2118-2125. Thirdborough, S. M., Chamberlain, J. C., Swift, S. M., James, R. F., and London, N. J. (1993).The kinetics of SCID-hu reconstitution: A comparison between human splenocytes and peripheral blood leucocytes. Cell Transplant 2,339-343. Torbett, B. E., Picchio, G., and Mosier, D. E. (1991). hu-PBL-SCID mice: A model for human immune function, AIDS, and lymphomagenesis. lmmunol Reo 124, 139-164. Tremblay, M., Numuaki, K., Goldman, H., and Wainberg, M. A. (1990). Infection ofhuman thymic lymphocytes by HIV-1. J Acquir Immune Defc Syndr 3,356-360. Tyor, W. R., Power, C., Gendelman, H. E., and Markham, R. B. (1993). A model of human immunodeficiencyvirus encephalitis in scid mice. Proc Nut1 Acad Sci USA 90,8658-8662. Ussery, M., Broud, D., Wood, O., Kunder, S., Black, P., Dailey, P., Wilber, J.. Yang, L., Piatek, M., and Lifson, J. (1995). Antiviral agents reduce viral load and delay the detection of infected cells in vivo in the HIV-infected hu-PBMC SCID mouse model. Antioiral Res 26, A238. Valentin, H., Nugeyre, M. T., Vuillier, F., Boumsell, L., Schmid, M., Barre-Sinoussi, F., and Pereira, R. A. (1994). Two subpopulations of human triple-negative thymic cells are susceptible to infection by human immunodeficiency virus type 1 in vitro. 1 Virol 68,3041-3050. van Gemen, B., Kievits, T., Schukkink, R., van Strijp, D., Malek, L. T., Sooknanan, R., Huisman, H. G., and Lens, P. (1993). Quantification of HIV-1 RNA in plasma using NASBA during HIV-1 primary infection. Virol Methoh 43, 177-187. Van Kuyk, R., and Mosier, D. E. (1995). Lack of pseudotype formation between human immunodeficiency virus type 1 and Epstein-Barr virus in productively coinfected B lymphoblastoid cell lines. Virology 209, 643-648. Van Kuyk, R., Torbett, B. E., Gulizia, R. J.. Leath, S., Mosier, D. E., and Koenig, S. (1994). Cloned human CD8+ cytotoxicT lymphocytes protect human peripheral blood leukocytesevere combined immunodeficient mice from HIV-1 infection by an HLA-unrestricted mechanism. J lmmunol 183, 4826-4833. Vandekerckhove, B. A., Krowka, J. F., McCune, J. M., de Vries, J. E., Spits, H., and ROnCdrOlO, M. G. (1991). Clond analysis of the peripheral T cell compartment of the SCID-hu mouse. J Immunol 146, 4173-4179. Veugelers, P. J.. Schechter, M. T., Tindall, B., Moss, A. R., Page, K. A,, Craib, K. J., Cooper, D. A., Coutinho, R. A,, Charlebois, E., Winkelstein, W., Jr., and van Griensven, G. J. P. (1993). Differences in time from HIV seroconversion to CD4+ lymphocyte end-points and AIDS in cohorts of homosexual men. Aids 7, 1325-1329. Wain-Hobson, S. (1989). HIV genome variability in vivo. AIDS 3 (Suppl. I), S13-Sl8. Walker, C . M., Erickson, A. L., Hsueh, F. C., and Levy, J. A. (1991a). Inhibition of hunian immunodeficiencyvirus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism. J Virol 65, 5921-5927. Walker, C. M., Moody, D. J., Stites, D. P., and Levy, J. A. (1989). CD8 T lymphocyte control of HIV replication in cultured CD4 cells varies among infected individuals. Cell lmmunol 119,470-475. Walker, C. M.. Thomson-Honnebier, G . A., Hsueh, F. C., Erickson, A. L., Pan, L. Z., and Levy, J. A. (1991b).CD8+ T cells from HIV-1-infected individuals inhibit acute infection by human and primate immunodeficiency viruses. Cell lmmunol 137,420-428. Walker, W., and Gallagher, G . (1994). The in vivo production of specific human antibodies by vaccination of human-PBL-SCID mice. Immunology 83, 163-170.


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Walker, W., and Gallagher, G. (1995). The development of a novel immunotherapy model of human ovarian cancer in human PBL-severe combined immunodeficient (SCID) mice. Clin Exp Immunol 101,494-501. Wecker, H., Grusby, M. J., and Auchincloss, H., Jr. (1995). Effector cells must recognize antigens expressed in the graft to cause efficient skin graft rejection in SCID mice. Transplantation 59, 1223-1227. Wei, X., Ghosh, S., Taylor, M., Johnson, V., Emini, E., Deutsch, P., Lifson, J., Bonhoeffer, S., Nowak, M., Hahn, B., Saag, M., and Shaw, G. (1995). Viral dynamics in human immunodeficiency virus type I infection. Nature 373, 117-122. Westervelt, P., Trowbridge, D., Epstein, L., Blumberg, B., Li, Y., Hahn, B., Shaw, G., Price, R., and Ratner, L. (1992). Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo. 1 Virol 66, 2577-2582. Willey, R. L., Maldarelli, F., Martin, M. A., and Strebel, K. (1992).Human immunodeficiency virus type 1 vpu protein regulates the formation of intracellular gp160-CD4 complexes. J Vitd 66,226-234. York-Higgins, D., Cheng-Mayer, C., Bauer, D., Levy, J., and Dina, D. (1990). Human immunodeficiencyvirus type 1cellular host range, replication and cytopathicity are linked to the envelope region of the viral genome. J Virol64,4016-4020. Yu, X., McLane, M. F., Ratner, L., O’Brien, W., Collman, R., Essex, M., and Lee, T. H. (1994). Killing of primary CD4+ T cells by non-syncpum-inducing macrophage-tropic human immunodeficiency virus type 1. Proc Natl Acad Sci USA 91,10237-10241. Zaunders, J., Cam, A., McNally, L., Penny, R., and Cooper, D. A. (1995). Effects of primary HIV-1 infection on subsets of CD4+ and CD8+ T lymphocytes. Aids 9,561-566. Zhang, L. Q., MacKenzie, P., Cleland, A,, Holmes, E. C., Brown, A. J., and Simmonds, P. (1993). Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection. J VimZ67,3345-3356. Zhu, T., Mo, H., Wang, N., Nam, D. S., Cao, Y.,Koup, R. A,, and Ho, D. D. (1993). Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science 261, 1179-1181. This article was accepted for publication on 22 January 1996.

ADVANCES IN IMMUNOLOGY, VOL. 63

lessons from Immunological, Biochemical, and Molecular Pathways of the Activation Mediated by 11-2 and 11-4 ANGEUTA REBOUO,JAWERG ~ M E ZAND , CARLOS MART~NEZ-A.' h p a m t of immunology and Onmhgy, Centm National de Bm~nobgh,

Uniwrsidod Aut6noma de Madrid, 28049 Madrid, S p i n

1. Introduction

Cytokines participate in the control of immunologically relevant events such as lymphocyte activation, differentiation, maturation, proliferation, apoptosis, and acquisition of effector functions. Two salient features characterize the cytokines: their capacity to exert a large number of different biological effects (pleiotropism) and, in most systems, individually nonessential complex functions such as cellular differentiation or inflammation (redundancy). Although essentially pleiotropic in their in vitro physiology, a number of mechanisms allow cytokines to exert local, circumscribed, and well-balanced effects (Kroemer et al., 1990; Kroemer and Martinez-A., 1992). Clonal proliferation of T lymphocytes is initiated by the specific interaction of antigen peptide/MHC complex with T cell antigen receptor, which triggers the expression of interleukin-2 (IL-2) and its receptor. Subsequent IL-WIL-2R interaction allows T cells to undergo proliferation. The IL-2/ IL-2R system is probably the best characterized in terms of biochemistry and function. However, the transducing molecules involved in signaling from the cytoplasmic membrane to the nucleus are still not well defined. IL-2 plays an important role in regulating lymphocyte proliferation after interaction with its specific receptor expressed on the cell surface (Smith, 1988; Waldman, 1989; Taniguchi and Minami, 1993). IL-WIL-2 receptor interaction triggers various intracellular signaling events including protein tyrosine phosphorylation and subsequent induction of nuclear protooncogenes (Taniguchi and Minami, 1993; Minami et al., 1993a,b; Kono et al., 1993). In this chapter, we summarize the data produced over the last few years, focusing on the IL-WIL-2R system and illustrating the signaling molecules participating in cytokine signal transduction. Several studies have contributed to the delineation of a signaling pathway structured in three independent channels, which for the sake of clarity we

'

To whom correspondence should be addressed at Centro Naciond de Biotecnologia, Campus Cantoblanco, Universidad Authoma, 28049 Madrid, Spain. Fax:34-1-3720493. 127 Copyright 0 1996 by Academic Press, Inu AII nghts of reproduction in any form reserved

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will designate channels 1, 2, and 3. As a general overview, these three channels serve as major landmarks: Lck-fosljun (channel l),Syk-myc (channel 2), and a pathway leading to actin organizationlbcl-2expression (channel 3). The detailed hierarchical organization of these three channels will be presented throughout this review and the models depicted in the figures. Although the IL-2 receptor has a variety of intracellular signaling molecules, the evidence suggests that a similar heterogeneous group of signal transduction pathways can be used by other cytokine receptors and specific references will be made throughout the review. II. Cell Surface Receptors for 11-2 and 11-4

A. IL-2 RECEPTOR The high-affinity IL-2R consists of at least three subunits identified as a,p, and y chains or p55, p70, and p64, respectively; this trimolecular complex binds IL-2 with high affinity (lo-" M ) (Fig. 1) (Hatakeyama et al., 1989a,b;Takeshitaet al.,1992;Wang and Smith, 1987).The fly complex binds IL-2 with intermediate affinity (lo-' M ) and the ay complex binds M ) (Hatakeyama et al., 1989a,b). None of these with low affinity subunits possesses any known catalyhc activity. Either the trimolecular complex (a,p, y ) or the fly complex can be responsible for the IL-2induced growth signaling (Minami et al., 1993a,b; Hatakeyama et al., 1989a,b; Kawahara et al., 1994; Nakamura et al., 1994a,b; Nelson et al., 1994). Expression of IL-2Ra is inducible in T cells by activation through the TCR (Greene and Leonard, 1986).The cytoplasmic region of IL-2Ra chain has 13 amino acids, including serine and threonine residues as potential phosphorylation targets. Deletion of this region does not affect the capacity of the high-affinity IL-2R to transmit the IL-2-mediated proliferative signals (Hatakeyama et al., 1987). The extracytoplasmic region of IL-2Ra can also be detected in soluble form after T cell activation in vivo (Rubin et al., 1985; Osawa et al., 1986). Release of soluble IL-2Ra is important in the regulation of immune response, controlling the number of highaffinity IL-2R expressed on the cell surface. High levels of this soluble form indicate the presence of possible autoimmune diseases or neoplasia (Ishida et al., 1987; Tsudo et al., 1987). Regulation of IL-2Ra expression is controlled by the activity of transcription factors such as SP1, SRF, NF-KB,and UE-1, which are bound to the 5' region of the gene (Greene et al., 1989). Recent reports have characterized the IL-2 and IL-1 response elements in the proximal promoter region of the IL-2Ra gene (Sperisen et al., 1995; Soldaini et al., 1995).In addition,

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FIG.1. The high-affinity IL-2 receptor. The picture shows the three subunits: a (p55).

p (p70),and y ( p a ) ,with the respective number of amino acids in each region (extracellular, transmembrane, and cytoplasmic) of each subunit. p and y chains belong to the superfamily

of cytokine receptors, characterized by four conserved cysteines (Cys-rich)and the juxtamembrane WSXWS motif (WS).In the cytoplasmic regions, the y subunit shows an SH2 domain and the /3 subunit exhibits three distinctive regions, the serine-rich (S), the acidic (A), and the proline-rich (P)domains. Also represented are the protein tyrosine kinases that are known to associate to fl and y subunits upon IL-2 binding: Lck associates to the acidic region of p chain, Syk and Jakl bind to the serine-rich region of p chain, and Jak3 interacts with the carboy-terminus of the y subunit. Jaklgak3 are postulated to initiate a signaling pathway that diverges in two pathways mediated by Lck and Syk (channels 1 and 2, respectively),while an unidentified protein tyrosine kinase is supposed to trigger another independent pathway (channel 3).The role of the a chain in this signaling radiation is unknown.

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IL-1 and IL-2 control a chain expression in immature thymocytes (Wilson et al., 1994). Although the a chain of the IL-2R has been related only with increased IL-2 binding affinity, several reports have shown that intermediate and high-affinityIL-2R use different pathways for signaling (Pitton et al., 1994; Rebollo et al., 1995; Nemoto et al., 1995). Interaction of intracytoplasmic receptor domains with signaling molecules depends on the binding affinity of both molecules. It is therefore possible that the a chain, in addition to its role in increasing IL-2 binding affinity, modifies the binding affinity to transducer molecules. Transgenic and knockout mice are proving to be powerful tools for revealing unknown physiological or pathological functions of cytokines in vivo. Young mice lacking the IL-2Ra chain show normal T and B cell development. However, as adults, these mice develop massive enlargement of peripheral lymphoid organs associated with polyclonal T and B cell expansion; older IL-2Ra chain-deficient mice also develop autoimmune disorders. Thus, IL-2Ra participates directly in the regulation of both the size and the content of the peripheral lymphoid compartment (Willerford et al., 1995). IL-2Ra chain is also expressed on precursors and immature B cells in the bone marrow. Its expression is initiated by functional rearrangement and expression of I g c ~heavy chain gene and is down-regulated when B cells mature and express IgM (Chen et al., 1994b). Thus, the different levels, stages, and regulation of IL-2Ra expression may suggest a differential function of IL-2Ra during B cell development. The IL-2Ra chain is a useful marker to distinguish pre-BI, which do not express IL2Ra, from pre-BII cells which do express it (Rolink et al., 1994). The P subunit has a long cytoplasmic domain of 284 amino acids with significant homology with the cytoplasmic domain of the erythropoietin receptor. This subunit is included in the cytokine receptor family, in addition to other molecules such as IL-3, IL-4, IL-5, IL-6, gp130, granulocytemacrophage colony-stimulatingfactor (GM-CSF),IL-7, and IL-9 receptors (Cosman et al., 1990). IL-2RP is constitutively expressed in CD8+ T cells (Hatakeyama et al., 1989a). Analysis of the IL-2RP chain expression during human thymic differentiation reveals its presence at very early stages of development. Interaction with IL-2 at this stage triggers in these thymocytes a number of events associatedwith IL-2 production and up-regulation of the IL-2RP chain or the differentiation into y6 T cells (Toribio et al., 1989; Barcena et al., 1991). The transcription factors which interact with the IL-2RP chain gene promoter region are AP1, AP2, and SP1. This promoter region lacks a TATA or CAAT box, which are specific to constitutively expressed genes. Differences in the regulation of IL-2Ra and IL-2RP chain expression are


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related to their functions: induction of IL-2Ra requires antigen activation prior to clonal expansion, while IL2RP expression can be modified by I L 2 or IL-4 in the absence of antigen. The critical role of some intracytoplasmic areas of the IL-2RP chain was demonstrated using deletion mutants of this subunit. Using this approach, it was discovered that the serine-rich region of the P chain is critical for the transmission of IL-2-induced proliferative signals, and the acidic region is required for Lck binding (Fig. 1) (Hatakeyama et al., 1989a). In mice lacking the IL-2RP chain, T cells are spontaneously activated, resulting in exhaustive differentiation of B cells into plasma cells and the appearance of autoantibodies which cause hemolytic anemia and death of the animal. Thus, IL-2RP is required to keep the T cell activation programs under control and to prevent autoimmunity (Suzuki et al., 199513). The IL-2Ry chain is a glycoprotein of 347 amino acids with an 86 amino acid cytoplasmic domain. The critical role of this subunit was suggested by the fact that mutants which lose the y chain, or IL-2 mutants that do not bind y chain, also lose the ability to proliferate in response to IL-2 (Arima et al., 1992; Zurawski et al., 1990). The function of the cytoplasmic region of the IL-2Ry chain was shown by the observation that expression of a mutant of IL-2R-y chain lacking most of its cytoplasmic domain blocks the IL-2-induced proliferative signals (Kawaharaet al., 1994).These results clearly suggest that IL-2 signaling requires functional cooperation between the cytoplasmic domains of IL-2RP and -y chains. In fact, it has been shown that ligand or antibody-induced heterodimerization of IL-2RP and -y chain cytoplasmic domains triggers cellular proliferation (Kawahara et al., 1994; Nakamura et al., 1994a; Nelson et al., 1994). In addition, IL-2Ry chain was shown to play a critical role in the thymic maturation of T cell precursors. Finally, nonexpression of the IL-2Ry chain, which disrupts Jak3 association to IL-2R (Leonard et al., 1994), results in human X-linked severe combined immunodeficiency (XSCID) (Nopchi et al., 1993a,b; Leonard et al., 1994). This disease is characterized by absent or greatly reduced T cell numbers and severely diminished cell-mediated and humoral immunity (Conley, 1992). Mice lacking IL-2Ry chain expression have hypoplastic thymuses, Splenic T cells were diminished at 3 weeks of age but CD4+ T cells increase by 4 weeks and the B cell population was greatly reduced. These findings underscore the importance of the IL-2Ry chain in lymphoid development. Moreover, differences in humans and mice lacking IL-2Ry chain expression indicate species-specific differences in the roles of y-dependent cytokines or the existence of redundant pathways. These mice provide an important model for studying the pathophysiology of and therapy for human XSCID (Cao et al., 1995; DiSanto et d.,1995).

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The essential nature of IL-2/IL-2R for the generation of a normal immune response is readily demonstrated by the systemic deficiencies generated by the lack of either IL-2 or IL-2 receptor expression. Immunodeficient athymic mice lack the capacity to produce IL-2 but could respond to it when supplied exogenously (Gilis et al., 1979).When administered by means of a recombinant vaccinia virus which serves as an autonomously replicating device, IL-2 induces autoimmune disease in nude mice (Gutierrez-Ramos et al., 1992) and neonatally thymectomized mice (Andreu et al., 1991),but has no effect on normal euthymic mouse strains. The proautoimmune effect of IL-2 has been observed in athymic mice by the activation of T cells carrying a forbidden repertoire enriched in Vp gene products which, due to their unwanted reactivity with self-antigens, are normally eliminated during intrathymic T cell differentiation in normal mice (Kroemer et al., 1991a). IL-2 also abrogates the anergic state of nondeleted self-reactive T cells favoring the development of autoimmune diseases. The overall consequence is the development of inflammatory, presumably autoimmune lesions (Kroemer et al., 1991a,b). B. IL-4 RECEPTOH The molecular cloning of cDNA encoding murine and human IL-4R (Harada et al., 1990; Idzerda et al., 1990; Galizzi et al., 1990) indicated that it is constituted of a single chain of 140 kDa. It has been shown that the IL-2R7 chain is associated with IL-4R in the presence of IL-4, and its presence contributes to an increase in binding affinity (Russell et nl., 1993). A single class of high-&nity binding sites (& 20-300 pM) for IL4R has been detected on a wide variety of hematopoietic and nonhematopoietic cell types at levels ranging from 100 to 5000 sites/cell (Ohara and Paul, 1987; Park et al., 1987; Lowenthal et al., 1988). IL4R contains the characteristic cysteine residues and the WSXWS motif found in the cytokine receptor superfamily, and bears one of the largest cytoplasmic domains, composed of over 500 amino acids (Cosman, 1993). Like other members of this receptor family, the IL-4R contains no consensus sequences characteristic of tyrosine or serinehhreonine kinase. However, activation of IL-4R induces tyrosine phosphorylation of its receptor (Izuhara and Harada, 1993; Wang et al., 1992). The critical region for signal transduction and proliferation is located between amino acid residues 437 and 557, numbering from the carboxyl terminus (Izuhara and Harada, 1993; Harada et al., 1992; Keegan et al., 1994; Pernis et al., 1995).This region is highly conserved between human and mouse IL-4R, but lacks homology with other cytokine receptors.

SHARINC AMONG DIFFERENT CYTOKINE RECEPTORS C. SUBUNIT Some of the IL-2R chains are shared by other receptors: the IL-2Rpy chains are also components of the IL-15R (Gin et al., 1994; Grabstein et


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al., 1994), whereas the IL-2Ry chain is a subunit of the IL4R, IL-7R, IL-9R, and probably IL-13R (Gin et al., 1994, Kondo et al., 1993, 1994; Russell et al., 1993; Noguchi et al., 1993; Zurawski et al., 1993).

There are some controversial results regarding IL4R and IL-13R. While Vita et al. (1995) suggest that the IL-13 receptor may be constituted by a subset of the IL-4 receptor complex associated with at least one additional protein, Obiri et al. (1995) indicate that IL-13 interaction with IL-4R does not involve the y chain and that IL-13R itself may be a novel IL-4R subunit. Worthy of interest is that both IL-4 and IL-13 induce tyrosine phosphorylation of the IL-4R (Smerz-Bertling and Duschl, 1995). The contribution of the IL-2Ry chain to the IL-4 and IL-13 receptors has now become a matter of controversy: using two novel mAb to distinct mouse y chain epitopes, it has been shown that the y chain interacts with the IL-4R in a manner distinct from its role in the IL-2R. In addition, the B9 plasmacytoma cell line, which proliferates in response to I L 4 and IL-13, does not express the y chain. These results suggest that there are two distinct IL-4R, one of which in independent of the y chain, and that the y chain is not a required subunit of the IL-13R (He et al., 1995; He and Malek, 1995). 111. From Nonreceptor Protein Tyrosine Kinases to Adaptor Molecules

Phosphorylation of proteins on tyrosine is crucial in signal transduction from extracellular milieu uia transmembrane receptors through the cytoplasm to the nucleus which regulates proliferation, cell cycle control, differentiation, and programmed cell death (Cantley et al., 1991; Ullrich and Schlessinger, 1990; Williams, 1989; Lewin, 1990; Klausner and Samelson, 1991). Below, we describe the better characterized members of the nonreceptor protein tyrosine kinases families known to participate in hemopoietic cell signal transduction. A more extensive description is presented for those which participate, directly or indirectly, in cytokine responses.

FYNAND LCK A. SRCFAMILY: The protein kinases specific for tyrosine residues (PTK) can be divided into two groups: receptor-type PTK and non-receptor PTK. The latter group includes the Src-family PTK, which consists of at least 10 protooncogene members (Eiseman and Bolen, 1990): c-src, c-yes, fyn, yrk, c-fgr, lyn, lck, hck, blk, and rak. The architecture of all Src-PTK follows the same basic plan (Fig. 2A): a unique NH2-terminus (in which a glycine in position 2 is the target for modification by a myristic or palmitic acid residue), followed by Src-homology 3 (SH3) and SH2 domains in one or several copies, the PTK domain (including the catalytic domain and the

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A

PROTEIN TYROSINE KINASES

Fic:. 2. Basic stnictiire of three families of protein tyrosine kinases and four adaptor molecriles. (A) The domain structures of Src, Syk, and Jak families of tyrosine kinases are shown. The N-terminal catalytic domain of Jak kinases is supposed to be inactive. (B) The lower part of the figure represents the domain structures of the adaptor molecules Grb2, Shc, Vav, and Sos. Shc exhibits a collagen-homologydomain and Vav shows an acidic region and a CDC24-like domain, where Sos is constituted by an N-terminal conserved region, a central catalytic domain with guanine niicleotide exchange activity, and a C-terminal prolinarich region.

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ATP binding site), and finally, a highly conserved C-terminal tail (Schultz, 1985; Buss and Sefton, 1985; Buss et nl., 1984; Sefton et al., 1982; Marchindon et al., 1984). The SH3 domain binds to proline-rich sequences or to other SH3/SH2 domains (Cichetti et al., 1992; Ren et al., 1993; Rozakis-Adcock et al., 1993; Ye and Baltimore, 1994; Zhou et al., 1995) and the SH2 domains bind to phosphotyrosine within a specific sequence in other proteins and/ or within themselves (Cantley et al., 1991; Escobedo et al., 1991; Rotin et al., 1992; Kazlanskas et al., 1992). 1. Fyn

Fyn was described for the first time in T lymphocytes (Kawakami et nl., 1989; Katagiri et al., 1989; Tsygankov et aE., 1992; da Silva et al., 1992) and was coimmunoprecipitated with the TCWCDS complex (Tsygankov et al., 1992; Gassmann et al., 1992). Fyn also associates with CD2, CD5, Thyl, BCR, IL-7R, and CD36 (in platelets) (Burgess et al., 1992; Campbell and Sefton, 1992; Stefanovtiet al., 1991; Schieven et al., 1992;Venkitaraman et al., 1992).The developmental expression of Fyn in B cells may contribute to the regulation of unresponsiveness and tolerance susceptibility of immature B cells (Wechsler et al., 1995). Several groups have described the generation of knockout mice for Fyn. In the absence of Fyn, there was no demonstrable compromise of the sIgM-coupled signaling events such as tyrosine phosphorylation, inositol phospholipid hydrolysis, and Ca2+flux,suggesting that other PTKs are able to compensate for the absence of Fyn. As a whole, data based on Src family FTK knockout mice are consistent with the hypothesis that, at least in some cells, these kinases are able to compensate for the loss of the other related kinases (Kiefer et al., 1994; Lancki et al., 1995; Miyakawa et al., 1994, 1995; Sillman et al., 1994; Stein et al., 1994; Hedin et al., 1995; Grants et al., 1994; Humemory et al., 1994). However, Fyn deficiency affects the amplitude of the CD3-mediated CD4+CD8+and CD4+CD8thymocyte responses. Most of the SrdFyn negative double mutants die perinatally, while a substantial proportion of Fyn/Yes double mutants are viable but undergo degenerative renal changes. Fyn activity is regulated by the phosphatase CD45 (Shiroo et al., 1992), suggesting that CD45-mediated dephosphorylation and activation of this kinase are important for signal transmission (Mustelin and Bum, 1993).

2. Lck The T cell-specific kinase Lck was described independently in 1982 by two laboratories (Gacon et al., 1982, 1983; Casnellie et al., 1982, 1983). The lck gene is expressed at a high level only in T lymphocytes, NK cells,

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and most T cell lines (Trevillyan et al., 1986; Koga et al., 1986). Lck is expressed in all T lymphocyte subsets very early during ontogeny in the thymus (Veillette et al., 1989).A few B cell lines and some colon carcinomas also express Lck (Veillette et al., 1987). Lck is physically associated with the cytoplasmic domain of CD4 and CD8 glycoproteins in T cells (Rudd et al., 1988; Veillette et al., 1988a,b; Barber et al., 1989; Shaw et al., 1989; Turner et al., 1990). More recently, Lck has been found to associate with many other surface glycoproteins: TCWCD3, CD5, CD2, CD16, BCR (Burges et al., 1992; Carmo et al., 1993; Cone et al., 1993; Salcedo et al., 1993; Campbell and Sefton, 1992). More surprising is the association of this kinase with Thy1 and CD48, since these glycoproteins are bound to the cell surface through a GPI anchor (StefanovB et al., 1991; Garnett et al., 1993). Several groups have described the physical and functional association of Lck with the IL-2R (Hatakeyama et al., 1991; Minami et al., 1993a; Horak et al., 1991). The N-terminal region of Lck interacts with the acidic domain of the /3 chain of IL-2R (Fig. 1) (Hatakeyama et al., 1991). This domain is required for the induction of c-fos and c-jun protooncogene expression (Hatakeyama et al., 1992), but is dispensable in IL-2-induced proliferation and c-myc expression (Shibuya et al., 1992; Taniguchi and Minami, 1993). These two events are mediated by the serine-rich region of IL-BRP, which is also responsible for Lck activation. However, Lck is not an absolute requirement for IL-2-mediated signaling, since an Lck negative mutant of the IL-2-dependent T cell line CTLL2 is still responsive to IL-2. The phosphotyrosine phosphatase CD45 was described as the physiological regulator of Lck. No Lck activation was detected in CD45-negative mutants of murine lymphoma cell lines (Mustelin et al., 1989). In more detail, the C-terminal Tyr 505 is dephosphorylated by CD45 (Mustelin and Altman, 1990) and this dephosphorylation is important for Lck activation (Ostergaard et al., 1989). In addition, Lck is also regulated by the tyrosine kinase Csk. Support for interaction of CD45 with Lck comes from the observation that a fraction of Lck can be coimmunoprecipitated with CD45, although the stoichometry is very low (Schraven et al., 1991, 1992, Koretzky et al., 1993). Anti-phosphotyrosine immunofluorescence data suggest that the CD45-associated Lck is unphosphorylated (Guttinger et al., 1992). Activation of T cells through TCWCD3, CD2, calcium ionophores plus phorbol esters, or IL-2 also causes, in addition to tyrosine residue phosphorylation, serine phosphorylation of Lck (Marth et al., 1989; Danelian et al., 1989; Winkler et a/., 1993; Watts et al., 1993).

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Lck-mediated signals are not sufficient to induce cell proliferation, since transfection of an activated Lck into an IL-2-dependent T cell line does not abrogate its IL-2 dependency (Taichman et al., 1992). Other PTK, such as Fyn and Lyn, can be activated by IL-2R (Kobayashi et al., 1993). The ability of Lck to mediate IL-2 promoter induction is strongly irnpaired upon deletion of NH2-terminal domain. Thus, the unique domain is not required for the intrinsic Lck kinase activity; however, it influences substrate preferences and contributes to the physiological function of this Src-family tyrosine kinase (Carrera et al., 1995). In addition, Src PTK are directly or indirectly involved in the regulation of Ras, Ras-GTPase activating protein p120, guanine nucleotide exchange factors Vav and Sos (Bustelo d al., 1993; Margolis et al., 1992; Gulbins et al., 1993), and the adaptor molecules Shc and Grb2 (Pelicci et al., 1992; Rozakis-Adcock et al., 1992; Lawenstein et al., 1992; Simon et al., 1993). The first Lck and/or Fyn cellular substrate identified was the 5 chain of the TCWCD3 complex. Another molecule which has been suggested to be phosphorylated by Lck and Fyn is the phosphatidylinositol 3 kinase (Fukui and Hanafusa, 1991).Additional analyses indicated that Lckinduces the subsequent tyrosine phosphorylation of PLC-y2. Interestingly, the regulatory effects of Lck on ZAP70, Syk, and PLC-y2 could not be replaced by overexpression of either Fyn or Src (Ting et al., 1995; Nakamura et al., 1994b). Lck has been implicated in the regulation of early thymocyte differentiation and of allelic exclusion at the TCRP locus. Using mice overexpressing an activated Lck transgene and mice with an Lck gene disruption it has been demonstrated that Lck participates in a pathway which regulates the expansion of the CD4'CD8' thymocyte pools to wild-type levels. In addition, Lck may be involved in the down-regulation of the putative preTCR on CD4TD8' thymocytes.

B. SYKFAMILY: SYK,ZAP70 In addition to the Src family, other PTK have been shown to associate with IL-2R or activated by IL-2: the SykEAP70 PTK. Figure 2A summarizes the structure of this PTK family. While Syk and ZAP70 are structurally homologous, they have distinct tissue distributions: Syk is expressed in T and B cells, myeloid cells, and platelets, whereas ZAP70 expression is restricted to T and natural killer cells (Asahi et al., 1993; Chan et al., 1994). The serine-rich region of the IL-2RP chain can recruit Syk (Fig. 1)and this kinase is activated upon IL-2 stimulation (Minami et al., 1995). ZAP70 is also suspected to be IL-2R associated. The mechanism of SykfLAP70 activation by IL-2 is not known. Syk activation by IL-2R does not require

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the acidic region of the IL-2RP chain, indicating that Lck activation is not required for Syk activation in this system (Minami et al., 1995; Couture et al., 1994). However, nothing is known on the in uiuo Lck-Syk regulation. Kinetic studies of Syk and Lck activation following IL-2 stimulation reveal that IL-2-induced activation of Syk precedes activation of Lck. A recent report examining Syk regulation and its interaction with Lck concludes that Lck may be regulated by Syk through physical association and N-terminal tyrosine phosphorylation (Couture et al., 1994). Lck is much more active in the presence of Syk than when expressed alone and Lck and Syk pathways are linked to the induction of distinct protooncogenes, since Lck activation results in the induction offosljun protooncogenes and Syk activation results in the induction of c-myc. In spite of the association and regulation of Lck by Syk, the characterization of Syk-/-lymphoid cells showed that Syk mutation disrupts most signaling from the pre-BCR complex preventing the clonal expansion and maturation of pre-B cells. In addition, Syk-deficient mice showed impaired development of thymocytes which use the Vy3variable region (Chen et al., 1995; Turner et al., 1995). ZAP70 is constitutively bound to a phosphorylated TCR 5 chain in thymocytes and is not phosphorylated or activated until the TCR is stimulated (Van Oers et al., 1994).ZAP70 is regulated by CD45, since in CD45negative T cells, ZAP70 is constitutively phosphorylated on tyrosine. In resting, wild-type CD45' cells, ZAP70 was mainly unphosphorylated, but was rapidly phosphorylated on tyrosine after treatment of the cells with anti-CD3 antibodies or phosphatase inhibitors (Mustelin et al., 1995). ZAP70 plays several important roles in T cell differentiation and function (Hivroz and Fischer, 1995). The role of ZAP70 in T cell development and TCR signal transduction has been shown to be critical using ZAP70deficient mice. (Negishi et al., 1995). In addition, mutation in ZAP70 results in a combined immunodeficiency syndrome with a CD8' peripheral T cell deficiency and a TCR signal transduction defect in peripheral CD4+ T cells. However, in these ZAP70-deficient patients, Syk may partially compensate for the loss of ZAP70 (Gelfand et al., 1995).

c. THEJAK-STAT PATHWAY: J A K ~ JAK2, , JAK3,

TYK2 Recent reviews have focused on the role of Jak kinases in cytokine activation and we refer the reader to those texts (Briscoe et al., 1994; Ihle et al., 1994, 1995; Ziemiecki et al., 1994). Figure 2A shows the structure of this family of PTK. Evidence for the involvement of the Jak kinases in cytokine signaling was provided initially by genetic complementation experiments in a mutant cell line in the response to IFN-a, -p,or -y (Velazquez et al., 1992; Hunter,


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1994). In those systems, Jakl and Jak2 are required for the IFN-y response, while Tyk2 and Jakl are required for I F N - d p response (Watling et al., 1993; Muller et al., 1993). The involvement of the Jak kinases in the response to other cytokines was quickly identified. Thus, in the case of IL-2R, p and y chains recruit Jakl and Jak3, respectively (Fig. 1) (Miyazaki et al., 1994). These interactions occur throughout the serine-rich region of the IL-2RP chain and the COOH-terminal region of the y chain, respectively (Johnston et al., 1994; Miyazaki et al., 1994). The mechanism of Jak PTK activation through cytokine receptors is not clear. Jakl and Jak3 bind simultaneously to the receptor complex, while Jak2 binds to receptors alone. The Jak recruitment sites of many cytokine receptors correspond to the regions required for proliferative signal transmission (Ihle et al., 1994). In fibroblasts expressing apy IL-2R and endogenous Jakl, IL-2-induced proliferation required the additional expression of Jak3 (Miyazaki et al., 1994). In corroboration of those results, Jak3-deficient mice showed profound reductions in thymocytes and severe B and T cell lymphopenia, similar to severe combined immunodeficiency disease (SCID); the residual T and B cells are also functionally deficient. B cell development in these mice is blocked at the pre-B stage; peripheral T cells do not proliferate and secrete small amounts of IL-2. These data demonstrate that Jak3 is critical for B cell development and for functional competence of mature T cells, as well as for y signaling (Thomis et al., 1995; Nosaka et al., 1995; Russell et al., 1995). Transfection of a mutant Jak3, which can still associate to IL-2Ry chain, inhibits the IL-2 response. Similarly, expression of a mutated Jak2 form inhibits the erythropoietin response. In both cases, the mutation corresponds to the predicted phosphotransferase motif within the carboxyterminal kinase domain of Jak2 and Jak3 (Zhuang et al., 1994). The receptors for IL-4, IL-7, IL-9, and IL-15 induce tyrosine phosphorylation and activation of Jak3 and, to a more variable extent, Jakl, upon interaction with the corresponding cytokine (Johnston et al., 1995). IL-3, IL-5, and GM-CSF utilize ligand-specific a-chains and a common P chain and activate primarily Jak2 upon receptor binding (Silvennoinen et al., 1993). IL-6, IL-11, oncostatin, LIF, and CNTF utilize a common gp130 signaling subunit, and this group of cytokines activates Jakl, Jak2, and, to a lesser extent, Tyk2 (Luttcken et al., 1994). IL-12 induces activation of Jak2 and Tyk2, and IL-13 induces tyrosine phosphorylation and activation of Jakl. Finally, it is still not known which Jak family member is activated by IL-10 (Ivashkiv, 1995). Table I summarizes the Jak kinases and Stat proteins activated by different cytokine receptors.

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TABLE I ACTIVATION OF JAK KINASESA N D STAT PROTEINS BY EXTRACELLULAR LICANDS Receptor

Activated Jaks

Activated Stats

IFN-a IFN-.), IL-2 IL-4 IL-6 IL-7 IL-9 IL-10 IL-12 IL-13 IL-15

Jakl, Tyk2 Jakl, Jake Jakl, Jak3 Jakl, Jak3 Jakl, Jak2, Tyk2 Jakl, Jak3 Jakl, Jak3 Unknown Jak2, Tyk2 Jakl Jakl, Jak3

Statl, State, Stat3 Statl Stat3, Stat5 Stat6 Statl, Stat3 Stat5 Stat3 Statl, Stat3 Stat3. Stat4 Stat6 Stat3, Stat5

Other activated proteins

4PS 4PS 4PS

The event following Jak kinase stimulation is the activation of a family of latent transcription factors termed Stat proteins (signal transducers and activatorsof transcription) (Damell et al., 1994;Wen et al., 1995; Gronowski et al., 1995; Horvath et al., 1995; Stahl et al., 1995). In addition, direct or indirect tyrosine phosphorylation of cytokine receptors by Jak kinases creates binding sites for the SH2 domains of some signaling molecules such as Shc, Vav, Grb2, and the p85 subunit of PI3 kinase (Ihle et al., 1994,1995). The Stat proteins vary in size from 734 to 851 amino acids, with the principal size differences occuring at the carboxyl terminus. The most highly conserved region is the SH2 domain, localized in the carboxyl half of the protein. Within the SH2 domain, there is a central motif completely conserved in all the known Stat proteins. The SH2 domain appears to play a critical role in the recruitment of a given Stat factor for each receptor. In addition to the SH2 domain, there is a region somewhat similar to the SH3 domain (Cichetti et al., 1992; Koch et al., 1991) although this region is much less conserved among the Stat proteins. The Stat proteins lack an identifiable motif associated with DNA binding. However, highly conserved blocks of homology exist within the amino terminal region of the proteins which are probably involved in DNA binding (Damell et al., 1994; Horvath et al., 1995; Ihle et al., 1994, 1995). Stat protein activation requires protein tyrosine phosphorylation, presumably as a direct result of Jak kinase activation. The nonphosphorylated form of Stat usually exists as a monomer, whereas the phosphorylated form exists as a dimer (Shuai et al., 1994; Ihle and Kerr, 1995) required for DNAbinding activity and also presumably for migration to the nucleus. This mechanism is believed to occur through the phosphorylation site of one

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molecule and the SH2 domain of a second molecule (Shuai et al., 1993; Akira et al., 1994). It has recently been shown that maximal activation of Statl and Stat3 transcription requires both tyrosine and serine phosphorylation, possibly mediated by the MAP kinases (Zhang et al., 1995), suggesting that serine kinases are involved in Statl and Stat3 activation and in linking Stat and Ras pathways (Wen et al., 1995; Zhang et al., 1995). The specificityof the activation for each cytokine depends on the particular coupling of Stat to the intracellular domains of the receptors. Different Stat proteins are activated by different receptors: IL-2 activates Stat3 and Stat5 in resting peripheral blood mononuclear cells (Fuji et al., 1995; Nielsen et al., 1994; Beadling et al., 1994), but activates Stat3 and a protein that is probably a different Stat5 isoform in cells preactivated with phytohemagglutinin (Lin et al., 1995).The cytoplasmic region of the IL2RP chain required for Stat5 activation mapped within the 147 carboxylterminal amino acids. This region is not essential for IL-2 induced cell proliferation (Fuji et al., 1995). According to these data, one may suspect the existence of a sequence motif required for Jak-mediated phosphorylation and Stat recruitment. Another explanation is that the SH2 or other domains within Stat proteins may contribute to the selective interaction of these domains (Ihle et al., 1994). In addition to the known Stats, a variety of studies have demonstrated that various cytokines activate GAS DNA-binding proteins serologically related to Statl. Several analyses have shown that IL-3, Epo, and GMCSF induce GAS-binding activity ( h e 1 et d.,1993). More recently, IL-4, IL-6, EGF, and CSF have been shown to induce GAS-binding activity that is functionally similar to that of Statl (Kotanides and Reich, 1993; Schindler et al., 1994; Rothman et al., 1994). One of the variations of the GAS sequence of considerable interest is the SIE found in the cfos gene (Wagner et al., 1990), which can be bound by Statl (Sadowski et al., 1993) and Stat3 (Zhong et al., 1994).

D. ADAPTERMOLECULES:VAV,G R B SHC, ~ , sos Tyrosine phosphorylation creates binding sites for the SH2 domains of cytoplasmic signaling proteins (Anderson et al., 1990).The SH2 domain is a sequence of -100 amino acids which binds to phosphotyrosine residues flanked by short sequences of specific amino acids (Cantley et al., 1991; Escobedo et al., 1991; Kdauskas et al., 1992; Rotin et al., 1992).The SH2 sequences were originally identified in protein tyrosine kinases such as Src, Abl, Syk, and Csk (Pawson, 1992; Koch et al., 1991).The SH3 domain is a short region of -50 amino acids which plays a role in directing

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protein-protein interactions (Koch et al., 1991; Clark et al., 1992). The binding regions to the SH3 domains are proline-rich sequences (Cichetti et al., 1992; Ren et al., 1993; Rozakis-Adcock et al., 1993).In addition to their positive regulatory role, SH3 domains may exert a negative regulatory role (Jackson and Baltimore, 1989; Seiden-Dugan et al., 1991). SH2 and SH3 were originally described as conserved, noncatalytic regions in cytoplasmic tyrosine kinases and are present in many signaling molecules such as phospholipase C y,the p85 subunit of PI3 kinase, Ras-GTPase activating protein (GAP), and protein tyrosine phosphatases (Montminy, 1993). A set of molecules which contains SH2 and/or SH3 domains are the adapter proteins (Fig. 2B), which act as molecular plugs between signaling proteins, Some examples of this group are Crk, Grb2, Vav, Shc, and Nck. In addition, Sos is an adaptor molecule without SHWSH3 domains, but with proline-rich sequences. Recently, Vav has gained considerably increased relevance in lymphokine signaling. The product of the protooncogene uau, p95, is specifically expressed in hematopoietic cells (Katzav et al., 1989), which contains one SH2 and two SH3 domains, two proline-rich regions and nuclear localization signals (Koch et al., 1991; Katzav et al., 1989; Coppolaet al., 1991).Vav is a signal-transducing molecule with an important role in hematopoiesis (Coppola et al., 1991) and in development of hematopoietic cells from totipotent cells (Wulf et al., 1993; Zmuidzinas et al., 1995). Its deletion by homologous recombination (Zmuidzinas et al., 1995; Zhang et al., 1995; Fischer et al., 1995) shows that Vav is essential for an early developmental step which precedes the onset of hematopoiesis. Moreover, deletion of the N-terminal region produces an oncogenic form of Vav. In Vav knockout mice, T and B cells are hyporeactive, suggesting a role for Vav in the control of T and B cell development and activation. In addition, Vavdependent signaling pathways regulate maturation during T cell receptormediated positive selection of immature precursors into mature T cells. Moreover, there is a direct link between the low proliferative response of Vav-deficient B and T cells and the reduced number of these cells in peripheral lymphoid organs (Tarakhovsky et al., 1995). Vav may also be recruited to the receptor complex through phosphotyrosine residues on Jaks (Matsuguchi et al., 1995). Vav is phosphorylated in T cells stimulated through the TCR or the CD28 costimulator molecule (Nunes et al., 1994; Ramos-Morales et al., 1994), and in a T cell line stimulated through the IL-2R (Gbmez et al., 1996b). Vav shares homology with guanine nucleotide-releasing factors (GRF) which activate GTP-binding proteins of the Rho/Rac family (Puil and Pawson, 1992). Vav associates with Grb2 either through an unusual dimerization of intact SH3 domains of each partner or through SH2 do-

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mains. This association could also recruit Shc and Sos (Ye and Baltimore, 1994; Ramos Morales et al., 1995). Shc, an SH2-containing adaptor protein, is tyrosine phosphorylated (Zhu et al., 1994; Pelicci et al., 1992; Rozakis-Adcock, 1992; Pronk et al., 1993) and associates with the IL-2RP chain after IL-2 stimulation (Ravichandran and Burakoff, 1994). The phosphorylation of Shc correlates with IL-2dependent proliferation. Tyrosine phosphorylated Shc was found to associate with Grb2, a cellular protein composed of two SH3 domains flanking a central SH2 domain (McGlade et al., 1992b; Rozakis-Adcock et al., 1992; Pronk et al., 1993; Egan et al., 1993). The SH3 domain of Grb2 binds to the proline-rich domain of Sos (Li et al., 1993; Egan et al., 1993). Recent studies have shown that the SH3 domains of Grb2 can interact and regulate other proteins (Gout et al., 1993). These data raise the possibility that Grb2-SH3 domains can couple to effector molecules other than Sos and Grb2 may thus function as an adapter molecule in more than one signaling pathway. Finally, Sos is known to play a role in Ras activation in mammalian cells by stimulating the GTP/GDP exchange (Lowenstein et al., 1992; Aronheim et al., 1994). In T cells, Shc interaction with Grb2 regulates association of Grb2 with Sos following TCR stimulation (Ravichandran et al., 1995). These adaptor molecules might play a crucial role in IL-2 response. Thus, we have shown that Vav, Grb2, and Shc are phosphorylated after IL-2 stimulation, while only a fraction of the Sos pool becomes phosphorylated. In addition Vav, Grb2, Shc, and Sos are part of binaxy complexes which appears in an IL-2 dependent fashion. These complexes may directly link tyrosine phosphorylation in Ras activation and may be a critical step in IL-2-induced proliferation (G6mez et al., 1996b). IV. From Ras to he MAPK Pathway

A. RAS Ras involves a family of 21-kDa proteins highly conserved among a wide variety of species (Hall, 1993;Wassarman et al., 1995; Kayne and Sternberg, 1995) which exhibit CAAX consensus sequences for membrane anchoring through a process of farnesylation, proteolysis, and carboxymethylation. All Ras proteins have a guanine nucleotide-binding region and no kinase motifs. The primary role of these molecules is the control of signaling pathways which drive cellular proliferation; this is supported by the fact that ras gene mutations are related to tumor progression (Hall, 1993). In mammals, at least three Ras isoforms (H-, K-, and N-Ras) and two Ras-related proteins (R-Ras and TC21) regulate mitogenic responses triggered by multiple growth factors in different tissues (McCormick, 1995).

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Ras proteins effect their function by switching between an active (GTPbound) and an inactive (GDP-bound) state. Two intracellular signaling pathways have been suggested to lead to Ras activation: a lymphocytespecific PKC-mediated pathway (Downward et al., 1990; Izquierdo et al., 1992b) and a ubiquitous protein tyrosine kinase-mediated pathway (Satoh d al., 1990, 1992; Torti et al., 1992). These events stimulate Ras activation through the membrane targeting of GDP/GTP exchange-promoting factors (Quilliam et al., 1994), such as the 170-kDa proteins Sos 1 and 2, which are mammalian homologs of the Drosophilu son of seuenless gene product (Bowtellet al., 1992; McCormick, 1994),and the guanine nucleotide releasing factor cdc25. Ras protein inactivation occurs via intrinsic GTPase activity, which is enhanced by GTPase activating proteins (GAPS) such as pl20RasGAP and the type 1 neurofibromatosis gene product ( N F l ) , neurofibromin (Marshall, 1993; McCormick, 1995). Although little is known regarding GAP regulation, it has been suggested that Ras activation by PKC in T cells takes place by phosphorylation and inhibition of Ras-GAP (Downward d al., 1990; Graves et al.. 1991; Izquierdo et al., 1992a). GAPS have also been proposed as Ras effectors (Marshall, 1993). The best identified Ras effector is the serine-threonine protein kinase Raf (Koide et al., 1993),which is activated through Ras-dependent recruitment to the plasma membrane (Stokoe et al., 1994; Leevers et al., 1994), since a chimeric protein consisting of Raf fused to the Ras membrane localization signal ( Raf-CAAX) shows constitutive activation. Other molecules have been proposed as Ras effectors in certain systems (Marshall, 1995), such as PI3 kinase in the rat pheochromocytoma cell line PC12 (Rodriguez-Vicianaet al., 1994),the cisoform of PKC in Xenopus lueuis oocytes (Diaz-Meco et al., 1994), the Ras-dependent extracellular signal-regulated kinase/mitogen-activated protein kinase kinase stimulator (REKS) in Xenqus oocytes (Shimizu et ul., 1994), the Ras-related Ral protein guanine nucleotide dissociation stimulator (RalGDS) (Hofer et al., 1994), the RalGDS-like protein RGL, and the 53-kDa protein Rinl (Han and Coticelli, 1995). These last three molecules have been identified as Ras-binding proteins by means of yeast two-hybrid and GST fusion assays but it has not been shown whether they are present in vim. Tyrosine-phosphorylated adapter proteins such as Shc or p36 may couple to the adaptor protein Grb2 and the guanine nucleotide-releasing factor Sos (Buday et ul., 1994). This association recruits Ras molecules to the receptor complex, where Ras is activated, promoting the entry of the primed T lymphocyte into the G1 stage of the cell cycle and the expression of the IL-2 gene (Rayter et al., 1992; Woodrow d al., 1993). Ras proteins are essential for signal transduction pathways involved in both thymocyte


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selection and TCR-triggered T cell proliferation (Swan et al., 1995). Crosslinking of the clonotypic T cell receptor/CD3 complex induces Ras activation through PKC and protein tyrosine kinase-mediated pathways (Downward et al., 1990; Izquierdo et al., 1992b, 1995). Although both TCWCD3 and CD4 are capable of triggering Ras activation, the PKC-mediated pathway may be restricted to TCWCD3 crosslinking, since a dominant negative Shc mutant is able to inhibit CD..VLck-mediated, but not TCW CDSmediated, Ras-dependent signaling (Baldari et al., 1995). This suggests that, in the latter case, the tyrosine kinase/Shc pathway may be replaced by the PKC pathway to achieve Ras activation. In addition, p36 may substitute for the function of Shc in TCWCD3-triggered Ras activation, which can also constitute a bypass of Shc phosphorylation (Buday et al., 1994; Izquierdo et al., 1995; Nel et al., 1995). In the second phase of T cell activation, which involves G1-S phase progression and mitosis, IL-2 binding to its surface receptor triggers the accumulation of Ras in its GTP-bound state (Graves et al., 1992; Izquierdo et al., 1992b) through activation of protein tyrosine kinases (Izquierdo and Cantrell, 1993) and the association Shc-Grb2-Sos (Bums et al., 1993; Ravichandran and Burakoff, 1994; Zhu et al., 1994). Two intracytoplasmic domains of the IL-2 receptor chain are required for Ras activation, the serine-rich and the acidic regions (Satoh et al., 1992). These domains have been identified as being involved in Lck-mediated induction of the protooncogenes c-fos and c-jun (Hatakeyama et al., 1989b; Minami et al., 1993a; Miyazaki et al., 1995),which suggests that Ras may be implicated in the IL-2-induced fosljun expression pathway. We have recently found that IL-2 promotion of cell growth requires Ras activation, since transfection of the Ras dominant negative mutant Serl7Asn (N17), which has reduced affinity for GTP, blocks IL-2-induced DNA synthesis in the murine T cell line TSlaP (G6mez et al., 1996d). However, the precise nature of the downstream effectors of Ras in the responses to IL-2 has not yet been determined. The IL-4R seems to be a surprising exception in the Ras dependency of mitogenic signaling pathways. While it is widely accepted that Ras activity is an absolute requirement for proliferation in different cellular systems, several reports have shown that triggering of the IL-4R does not increase the amount of GTP-bound Ras (Satoh et al., 1991; Duronio et al., 1992; Welham et al., 1994b), does not induce Shc or Sos modification (Welham et al., 1994a),and does not activate ERKs (Welham et al., 1994a). This major characteristic of the I L 4 R signaling mechanisms seems even more striking considering the similarities of the IL4R with other Rasactivating receptors. Thus, the y chain of the IL-2 receptor is present within the IL-4 receptor complex and the IL-4R shares some features with

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the insulinhnsulin-like growth factor ( IGF) receptors. These similarities include a region of homology in the intracellular domain (the 14R region) (Keegan et al., 1994), the multiple tyrosine phosphorylation of a 170-kDa substrate (insulin receptor substrate-1 ( IRS-l), possibly the 190-kDa IRS-2 and 4PS) which can serve as a docking molecule for cytoplasmic kinases and adaptors (White et al., 1988; Sun et al., 1991; Wang et al., 1992, 1993; Araki et al., 1994; Tobe et al., 1995). Insulin stimulation induces IRS-1 and Shc tyrosine phosphorylation, formation of a Grb2-Sos complex, association of Grb2-Sos with IRS-1 or Shc, and Ras activation (White and Kahn, 1994). The functional similarity between IRS-1 and 4PS (Wang et al., 1993a) allowed the comparison of the signals delivered through insulin and IL4R in the same L6 myoblast cellular model, showing that IL-4 also triggers tyrosine phosphorylation of IRS-1 and IRS-l-Grb2-Sos association, while it induces neither Shc tyrosine phosphorylation nor Shc-Grb2 association (Pruett et al., 1995). This suggests a correlation between Shc phosphorylation and Ras activation. Insulin receptor mutants have recently been used to further support the hypothesis of a direct relationship between insulin-induced Shc phosphorylation and Ras activation in the absence of IRS-1 phosphorylation and IRS-1Grb2 complex formation (Ouwens et al., 1994). The fact that some IL-4-responsive cell lines, such as the murine CTLL-2, can grow in an IL-4-dependent manner for only short periods of time, has been suggested to reflect the inability of the IL4R to induce Ras activation. However, other cell lines, such as the murine LD8 and TSlaP, proliferate indefinitely in response to IL-4. In the latter cell line, we have confirmed that signaling through the IL4R does not activate Ras, and that transfection of the dominant negative mutant Ras N17 has no effect on IL-4-induced proliferation (Gbmez et al., 1996e).Thus, sustained cell growth may take place in the absence of detectable Ras activation, and TS lap cells may exhibit two alternative proliferative mechanisms: the first, IL-2-triggered, proceeds through Ras-dependent signals and the second, stimulated by IL-4, either bypasses Ras activation or uses other signaling pathways. Recent results have uncovered a new role for Ras in cell functions, namely an involvement in signaling pathways controlling apoptosis. Ras has been implicated in cell death upon nerve growth factor deprivation of PC12 cells (Ferrari and Greene, 1994), and Fas-triggered T lymphocyte apoptosis is proposed to be mediated by ceramide-induced activation of Ras (Gulbins et al., 1995). In concordance with the latter result, ceramidemediated pathways have been associated with the onset of apoptosis. Ceramides have also been proposed to activate P K C , which mediates both mitogenesis and actin cytoskeletal organization in IL-2-triggered pathways


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( G m e z et al., 1995a,b). Thus, at least in some cases, proliferative and apoptotic pathways seem not to be easily distinguishable, which may support the evidence that, in a number of cellular systems, initial stimuli can determine a final outcome of cell growth or death, depending upon the cellular status or the concommitant activation of complementary pathways. A dual role in mitogenesis and apoptosis had been suggested previously for the product of the protooncogene c-myc, which mediates proliferation but also activation-induced apoptosis in T cell hybridomas (Shi et al., 1992). In IL-2-mediated activation, Ras activation prevents apoptosis, as demonstrated by the fact that blocking Ras function by transfection of a dominant negative mutant form of Ras induces apoptosis in IL-2-stimulated cells (Gbmez et al., 1996d). Strikingly, Ras activation is also required for the triggering of apoptosis in lymphokine-deprived cells (Gbmez et al., 1996e). This observation is supported by reports showing that an H-Rasactivated oncogene confers susceptibility to apoptosis in fibroblasts (Fern h d e z et al., 1995), and that an activate form of the Ras-related protein R-Ras enhances apoptosis caused by IL-3 withdrawal in IL-3-dependent cells (Wang et al., 1995).

B. RAFKINASE Raf is a serinekhreonine kinase discovered as an oncoprotein. Receptors which regulate Raf include members of the cytokine receptor family that regulate intracellular protein tyrosine kinases. The essential inducer of Raf is the activated form of Ras, Ras-GTP, which is related to activated receptors by adapter proteins such as Grb2, Shc, and Sos (Schlessinger, 1993). An early indication that Raf might be an important signaling element downstream of Ras was the observation that only Raf could overcome the blocking of mitogenesis induced by anti-Ras antibodies (Smith et al., 1986). In addition, Raf-Ras association (Moodie et al., 1993; Van et al., 1993; Zang et al., 1993; Warne et al., 1993; Koide et al., 1993; Vojtek et al., 1993) is disrupted by the destabilization of the Raf-Hsp9O complex (Schulte et al., 1995),and stabilization of the active form of Raf is believed to require association of Raf-Hsp50-Hsp9O-14-3-3 proteins (Morrison, 1994; Wartmann and Davis, 1994). Additional regulatory mechanisms may be required for Raf activation, since Ras-GTP does not stimulate in vitro Raf kinase activity. The role of phosphorylation in Raf activation is still not well defined; however, the results suggest that Raf requires serinekhreonine phosphorylation for kinase activity after IL-2 and IL-3 activation (Kovacinaet al., 1990; Fabian et al., 1993). Furthermore, stimulation of CTLL2 cells with IL-2 induces tyrosine phosphorylation of Raf (Turner et al., 1993).These findings, how-

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ever, appear to be specific to CTLL2, since they were not observed in human primary T cells. In addition, Raf can probably be activated directly, bypassing Ras by stimulation of PKC (Kolch et al., 1993), and can also be activated by the 14-3-3 family of proteins (Lin et al., 1995). Substrates of Raf include proteins belonging to the pathway of mitogenactivated protein kinases (MAPKs),which define a cascade of phosphorylation events from MAPKKKs or MEK kinases (MEKKs), which include Raf and MEKK1, to the dual-specificity kinases MAPKKs (or MEKs) and the serine-threonine kinases MAPKs (de Vries-Smits et al., 1992; Marshall, 1994, 1995). A unique proline-rich sequence of MEK kinases is required for Raf binding and regulation of MEK function (Catling et al., 1995). Raf has been proposed as a connecting point between mitogenic signal transduction and the cell cycle machinery, since it is also responsible for phosphorylation and activation of the cdc25 tyrosine phosphatases. These phosphatases are involved in dephosphorylation of the cyclinB/cdc2 complexes that regulate the cell progression along the GUM transition (for a review of cell cycle control in mammalian cells, see GraAa and Reddy, 1995). PROTEIN KINASES C. MITOCEN-ACTIVATED Mitogen-activated protein kinases, MAPK, are serinekhreonine kinases which rapidly become activated by phosphorylation on tyrosine and threonine residues (Erickson et al., 1990; Anderson et al., 1990). The MAPK pathway defines a cascade of phosphorylation events from MEK kinases such as Raf and MEKKl to MEKs (MAPKKs) and finally to MAPK. MAPK family is composed of two groups, the extracellular signalregulated kinases (ERKs) and the stress-activated protein kinases (SAPKs) or Jun kinases (JNKs),which phosphorylate the nuclear transcription factor Jun. Both groups define different signaling pathways, since Raf is responsible for ERK activation through MEKs, while MEKKl activates SAPKs through the MEK-related protein kinase SAPUERK kinase-1 (SEK1) (Yan et al., 1994). Activation of JNK and p38 and concurrent inhibiton of ERK are critical for induction of apoptosis in PC-12 pheochromocytoma cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis (Xia et al., 1995). Recent results suggest that stimulation of MAPK pathway is inhibited by CAMP as a result of Raf inhibition (Erhard et al., 1995). Lck plays a critical role in the activation of MAPK and Raf as well as MEK-1 in Jurkat T cells. However, in Lck-deficient Jurkat mutants, it was possible to activate Raf-1, MEK-1, and MAP kinases by treatment with phorbol esters. These


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results demonstrate the presence of a PKC-dependent pathway which functions independently of Lck in MAP kinase activation. V. From the GTP-Binding Protein Rho to Control of the Cytoskeleton

A. RHO Organization of actin structure is controlled by the Rho protein family (Paterson et al., 1990;Wiegers et al., 1991; Nobes and Hall, 1994), a group of molecules belonging to the Ras superfamily of low-molecular-massGTPbinding proteins. This family of proteins, known as RhoA, B, and C, participate in the polymerization of actin (Bertoglio, 1994). Bacterial toxins such as C3-like exoenzymes from Clostridium botulinum and other bacterial species (Aktories et al., 1989; Sekine et al., 1989; Aktories et al., 1992)which induce specific ADP-ribosylation and block Rho activity, cause depolymerization of actin. In addition, Rho proteins are involved in cell motility (Lang et al., 1992), regulation of cellular adhesion (Ridley and Hall, 1994) and malignant transformation (Avraham and Weinberg, 1989; Perona et al., 1993). Rho may also play a role in the regulation of apoptosis (JimBnez et al., 1995). In addition, a defect in a putative Rho/Rac guanine nucleotide exchange factor has been identified as the genetic abnormality responsible for faciogenital dysplasia or Aarskog-Scott syndrome (Pasteris et al., 1994). The sequence of the cascade from Rho to the cytoskeleton is presently unknown, although recent reports suggest that Rho-induced reorganization of the actin cytoskeleton could be mediated by the Ras mitogenic pathway, since the Rho-related protein Rac has been shown to be essential for Ras transformation (Qiu et al., 1995).It could be mediated as well as by the downstream MAP kinase cascade (Vojtek and Cooper, 1995; Coso et al., 1995; Hills et al., 1995; Minden et al., 1995). Rho might also regulate the reorganization of the actin cytoskeleton through the formation of phospholipid metabolites (Janmey, 1994) by acting on several enzymes, including PI3 kinase, phospholipase D, and some genistein-sensitive tyrosine kinases (Stossel, 1993; Salmon, 1989; Fishkind and Wang, 1994; Zang et al., 1993; Janmey, 1994; Ridley and Hall, 1994). We have shown that Rho inhibition by Clostridium dificile toxin B affects IL-2-stimulated PI3 kinase activity. This suggests a signaling pathway for the control of actin organization stimulated by IL-2 which derives the signal from Rho to PI3 kinase and the lisoform of protein kinase C (JPKC) (Fig. 3) (Gbmez, et al., 1996d).

B. PHOSPHATIDYLINOSITOL 3 KINASE PI3 kinase is a heterodimer composed of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (Carpenter et al., 1990; Shibasaki et al.,

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Fic:. 3. Comparative scheme of the proximal signaling events stimulated by 11,-2 and IL-4. Both receptors trigger tyrosine phosphorylation, although the tyrosine kinases activated may be different in each case. IL-2 receptor induces Ras activation (channel 1). whereas IL-4 receptor does not. Since both lymphokines elicit Crb2-Sos association, the ability to activate Ras may be related to the differential capacity to induce tyrosine phosphorylation of 4PSflRS-l-like molecules. Grb2-Sos binding to tyrosine-phosphorylated Shc may trigger Ras activation, while phosphorylated 4PS may sequester the Crb2-Sos complexes and thus prevent Has activation, even if Shc is also phosphorylated. In addition. PI3 kinase is activated through both receptors, via Rho activity and tyrosine phosphorylation of pR5 in the case of IL-2 (channel 3) and via 4PS in the case of IL-4. CPKC is activated in both cases, yet this activation is essential only for IL-2 responses.

1991; Fry et al., 1992; Otsu d al., 1991; Morgan d al., 1990). Both subunits have been characterized at the cDNA and protein level (Hiles et al., 1992; Skilnik et al., 1991).At least two distinct 85-kDa proteins, p85a and p85/3, have been identified, but neither of these isoforms has PI3 kinase activity (Escobedo et al., 1991; Otus et al., 1991). Analysis of the p85 primary sequence reveals a protein with one SH3 and two SH2 domains (Pawson and Schlessinger, 1993; Pawson and Gish, 1992). The SH2 domains of p85 bind to phosphotyrosine-containingproteins (Otsu et al., 1991; Klipped et al., 1992; Hu et al., 1992; McGlade et al., 1992a). The role of the SH3


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domain in binding proline-rich sequences is unclear, although it may mediate interactions with the cytoskeleton (Koch et al., 1992). In addition, p85 contains a region between the SH3 domain and the first SH2 domain with homology to the C-terminal region of the Bcr protein (break-point cluster region), a molecule of the Rho-GTPase activating protein family (Heisterkanpet et al., 1985). The presence of these distinct functional domains suggests that p85 may have multiple interactive and regulatory roles. Three pllO isoforms are known today: a,p, and y (Carpenter et al., 1990; Shibasaki et al., 1991; Hunter, 1995; Fry et al., 1992; Hiles et al., 1992).pllO is homologous to Vps34, a yeast PI3 kinase involved in vacuolar protein sorting (Herman and Emr, 1990) and to TOR2, another putative yeast PI3 kinase required for G1 progression (Kunz et al., 1993). pllOa, pllOp, pllOy, and Vps34 all have intrinsic PI3 kinase activity, although they have distinct substrate specificities (Hunter, 1995). Phosphatidylinositol3 kinase has emerged as a critical signal-transducing molecule activated by a large variety of protein tyrosine kinase receptors in different cell types (Panayotou and Waterfield, 1993; Frant et al., 1992; Valius and Kazlauskis, 1993). PI3 kinase phosphorylates the D3 position of the inositol ring of PI and its phosphorylated derivatives to produce PIphosphate, bisphosphate, and trisphosphate. The D3-phosphorylated lipids appear not to be substrates of known isoforms of phospholipase C and thus may themselves play a direct second messenger role, possibly as activators of P K C (Cantley et al., 1991; Ha and Exton, 1993). PI3 kinase may be regulated by a series of events, such as translocation to the cell membrane with access to substrates, and thus to subsequent phosphorylation by protein tyrosine kinases (Panayotou and Waterfield, 1992). Evidence for physical translocation comes from Zhang et al. (1992) and Susa et al. (1992) who have shown that, after thrombin or plateletderived growth factor (PDGF) stimulation of platelets and fibroblasts, respectively, there is an activation of membrane-bound PI3 kinase. PI3 kinase was found to associate physically with several tyrosine kinases such as pp6CY" (Fukui and Hanafusa, 1989), polyoma middle T antigen-pp60'" complexes (Whitman et al., 1995), PDGF receptor (Coughlin et al., 1989; Williams 1989), CSF receptor (Varticovski et al., 1989), EGF receptor (Bjorge et al., 1990), and c-kit (Lev et al., 1991). PI3 kinase becomes tyrosine phosphorylated after IL-2 stimulation (Mkrida et al., 1991;Augustine et al., 1991),PDGF (Auger et al., 1989; Coughlin et al., 1989; Kaplan et al., 1987) or CSF (Varticovski et al., 1989) and is also activated through costimulatory membrane proteins during antigenic activation of T and B lymphocytes. Serine and threonine phosphorylation of the components of the PI3 kinase complex may play a role in the regulation of the enzyme. Recent reports have shown that PI3 kinase

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copurifies with an associated protein serine kinase activity (Carpenter et al., 1993)which phosphorylates this enzyme on serine residues. In apparent contradiction to these observations, Dhand et al. (1994) claimed that PI3 kinase pllO subunit has intrinsic serine kinase activity with unique specificity for p85 which regulates its own enzymatic activity. However, the in vivo role of this dual specificity kinase activity is unknown. Upon PKC activation, the pllO subunit complexed to p85a becomes rapidly phosphorylated on serine residues. In addition, PKC activation results in a rapid increase in phosphorylation of the p85p subunit on threonine residues, suggesting that PI3 kinase can be a substrate for serine/ threonine kinases (Reif et al., 1993). We have shown functional association of P K C to the PI3 kinase pllO subunit after IL-2 stimulation. This association appears to be necessary serine-phosphorylated PI3 kinase activity since P K C release from PI3 kinase abolishes the phosphoserine PI3 kinase activity (G6mez et al., 1996~).Serine-phosphorylated PI3 kinase activity is maintained for longer periods than tyrosine-phosphorylated PI3 kinase activity. Our data suggest that an initial pool of PI3 kinase is activated by IL-2-induced tyrosine phosphorylation. releasing D3-phosphorylated products which activate P K C . This PKC isoenzyme would then complex to a second pool of PI3 kinase, which is activated through pllO-PKC association and serine phosphorylation of p85 (Fig. 4). It has been described that increase in PI3 kinase activity in thrombinstimulated platelets is dependent on the GTP-binding protein Rho, which participates in reorganization of the actin cytoskeleton (Zhang et al., 1993). We have shown that Rho is required for PI3 kinase activity in a pathway which connects these two molecules and P K C to the organization of actin cytoskeleton (G6mez et al., 1996a). However, the biochemical mechanism by which Rho participates in PI3 kinase activation is unknown. Blocking of PI3 kinase recruitment to the plasma membrane in some cell types prevents Ras activation. This could be explained by the fact that the GAP-like domain of p85 competes with GAPS for binding to the Has effector domain, thereby inhibiting GTPase inactivation of Ras. Ras and Rho might modulate the activity of different PI3 kinase species or, alternatively, Rho and Ras may cooperate to regulate PI3 kinase activity (Feig and Schffhausen, 1995; Valius and Kazlaus, 1993; Satoh et al., 1993). Recent studies have established that after CD28 ligation, the cytoplasmic domain of this molecule can bind to the 85-kDa subunit of PI3 kinase, which may be important in CD28 signaling.Treatment of cells with phorbol esters dramatically inhibits the PI3 kinase activation and its association with CD28 (Hutchroft et al., 1995).

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FIG.4. Mechanism of biphasic activation of PI3 kinase through the IL-2R (channel 3). A first pool of PI3 kinase (pool I ) is activated by a receptor-associated tyrosine kinase (PTK), that phosphorylates the p85 regulatory subunit. This activation event, which also requires the mediation of the small GTP-binding protein Rho, delivers a transient signal through the generation of D3-phosphorylated phosphoinositides. These lipidic metabolites induce activation of the 6 isoform of PKC, that complexes to a second PI3 kinase pool (pool 11). Association of SPKC to the pllO catalytic subunit and serine phosphorylation of p85 trigger activation of this PI3 kinase pool, which delivers a sustained downstream activation signal.

The serinekhreonine kinase known as protein kinase B (PKB) has been suggested to be a downstream effector of PI3 kinase activated by PI3 kinase products. However, Ras stimulation is not sufficient to activate PKB. As Ras interacts with the pllO subunit of PI3 kinase and is involved in stimulating this lipid kinase in intact cells, it would be expected that Ras function would be necessary for PKB activation by growth factors

154


(Downward, 1995; Burgerin and Coffer, 1995; Franke et al., 1995; Hunter, 1995; Kodaky et al., 1994; Burgerin et al., 1993). Other proteins regulated by the PI3 kinase products include some members of the PKC family and the S6 ribosomal protein p7Wfik(Toker et al., 1994). Finally, PI3 kinase belongs to the family of kinases which transfer phosphate groups to the phospholipid phosphatidylinositol that also includes FRAP, which binds rapamycin (Sabatini et al., 1995), Tor2 (Cardenas and Heitman, 1995), ATM (Savitsky et al., 1995), MEC-1 (Hari et al., 1995), and FRP1. Tor2 and FRAP have been shown to possess associated PI4 kinase activity. FRAP is also presumed to mediate IL-2-induced elimination of ~ 2 7 ~ (Keith P' and Schreiber, 1995). This group also includes the large catalytic subunit of the double-stranded DNA-dependent protein kinase (DNA-PK), which lacks a conventional protein kinase catalytic domain, but has a PI3 kinase domain (Hartley et nl., 1995). DNA-PK behaves as a conventional protein kinase and can catalyze phosphorylation of several proteins in uitro. DNA-PK is a heterotrimeric enzyme whose activity is stimulated by the ends of double-stranded DNA molecules and that only phosphorylates substrates when they are bound to DNA. Formal evidence is lacking that the PI3 kinase domain of DNA-PK is responsible for its protein kinase activity, but this seems a reasonable supposition, since DNAPK activity is inhibited by wortmannin. In addition to DNA-PK, several of the recently described members of the PI3 kinase family have roles in DNA damage response and repair (Zakian, 1995). KINASEC C. PROTEIN Activation of conventional protein kinase C is one of the earliest events in the signal transduction pathway cascades which lead to a variety of cellular functions such as cell growth, differentiation, and gene expression (Nishizuka, 1984a,b). Several studies suggest that PKC also plays an important role in lymphocyte activation. This is indicated, first, by the ability of TCR triggering to activate PKC and induce its translocation from the cytosol to the particulate fraction (Altman et al., 1990) and second, by the ability of PKC inhibitors (Juszczak and Russell, 1989) or PKC depletion by prolonged phorbol ester treatment (Valge et al., 1988; Abraham et al., 1988) to block lymphocyte signaling and activation. PKC was originally described as a serinekhreonine kinase whose activity was calcium- and phospholipid-dependent (Takai et al., 1979). Molecular cloning experiments have established that PKC molecules constitute a protein family which can be classified in four groups: conventional PKCs (a,PI, 011, y ) , which are calcium- and diacylglycerol-dependent; novel PKCs (6, E , 0, q),which are calcium-independent; atypical PKCs (5, L, A), which are not activated by calcium, phorbol ester, or diacylglycerol; and

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finally, the unique isoform PKC p, which has a long N-terminal region with a potential transmembrane domain not found in other PKCs (On0 et al., 1988, 1989; Mischak et al., 1991; Tang and Ashendel, 1990; Osada et al., 1992; Lisovitch and Cantley, 1994). Sequence analysis of cDNA clones has shown very closely related structures for all these isoforms. There are contradictory data on the activation of PKC as an essential event in IL-2-induced proliferation. Previous reports made the observation that PKC activation seems to be crucial in the proliferative response to IL-2 (Clark et al., 1987; Farra and Anderson, 1985; Gbmez et al., 1994, 1995a). In contrast, other reports concluded that activation of this enzyme is not involved in the growth-promoting action of IL-2 (Valge et al., 1988; Mills et al., 1988; Redondo et al., 1988). The approach used to conclude that PKC activity was not obligatory for IL-2-induced proliferation was the down-regulation of the enzyme by longterm culture in the presence of phorbol esters such as TPA. However, some TPA-resistant cell lines appear to contain normally responsive PKC and may have abnormal responses to PKC activation or altered PKCindependent effects of TPA (Forsbeck et al., 1985; Lefwich et al., 1987). In addition, some TPA-resistant mutants have been reported to retain the PKC protein levels, exhibiting a decreased TPA response as indicated by decreased translocation of PKC from the cytosol to the membrane, downregulation of phorbol ester binding, and decreased TPA-induced protein phosphorylation (Homma et al., 1986). In addition, TPA-resistant mutants developed by Colburn et al. (1984) contain normal amounts of PKC, as measured by phorbol ester binding. Finally, TPA effects can only account for the phorbol ester-responsive PKC isoforms, whereas the phorbol esterunresponsive isoforms are not affected by TPA treatment. To conclude that PKC was necessary for IL-2-induced proliferation, several PKC inhibitors were used (Clark et al., 1987; Kase et al., 1987; Hall et al., 1988; Watson et al., 1988). However, some of these agents have significant toxic effects and are not very effective in intact cells. In addition, some are not strictly PKC-specific. We employed the bisindolylmaleimide GF109203X, a compound structurally related to staurosporine, which is a very potent PKC inhibitor and displays a high degree of selectivity. This compound inhibits PKC activity exclusivelyvia the ATP-binding site (Toullec et al., 1991). Specific antisense oligonucleotides were used as a complementary approach to show the importance of PKC in signal transduction (G6mez et al., 1995a). Using these two techniques, we have identified 5 and E as the PKC isoforms involved in IL-2-mediated proliferation, but not for IL-4 or IL-9, suggesting that these lymphokines use different pathways than IL-2 (Gbmez et al., 1994). These results exclude a significant role per se for the common y

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chain of IL-2R, IL-4R, and IL-9R in the activation of PKC through IL2R. Since @KC is an atypical, phorbol ester-unresponsive isoform, its involvement in IL-2R signaling could not be detected using the TPAinduced down-regulation approach. Several PKC isoforms have been reported to translocate to the plasma membrane and the nucleus upon activation. However, their cellular distribution has remained unclear, as the experimental method used to address this issue has been restricted to subcellular fractionation (Kare et al., 1994; Kiss and Anderson, 1994; Baumgold and Dyer, 1994; Ribin and Steinberg, 1994; Chen, 1993; Ha and Exton, 1993; Church et al., 1993). Increased nuclear PKC levels are associated with cell proliferation and decreased levels are associated with differentiation. Nuclear PKC targeting may be mediated by sequences in the catalytic domains (James and Olson, 1992). Nuclear PKCs could be responsible for changes in the phosphorylation state of proteins such as AP-1 (Boyle et al., 1991). This last factor, in turn, may cause rapid expression of additional proteins with a regulatory role in transcription (Karin, 1990; Shimizu et al., 1992). Another potential nuclear effect of some PKC species may be the phosphorylation of DNA topoisomerase I1 with subsequent effects on DNA structure and function (DeVore et al., 1992). Some PKC isoforms could translocate to the cytoskeleton (Gregorio et al., 1992; Kiley et al., 1992). We have detected the association of @KC, after IL-2 stimulation, with actin cytoskeletal structures and have implicated P K C in maintaining the integrity of the actin cytoskeleton (Fig. 5 ) .

FIG.5. IL-2-induced actin organization is mediated by lPKC (channel 3). The figure shows immunofluorescence micrographs ofTSlap cells stimulated with IL-2 in the presence of sense (left) or antisense oligonucleotides (right) against LPKC. Cells were fixed and stained with anti-actin antibodies plus a fluorescein-coupled secondary antibody. Sense oligonucleotide-treated cells show a normal pattern of actin staining, whereas antisensetreated cells exhibit a collapsed actin cytoskeleton, forming chaotic granulous structures.


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One likely explanation of these results is that P K C is involved in actin reorganization by phosphorylating proteins which regulate cytoskeleton integrity or its structural changes throughout the cell cycle. However, P K C is not involved in the actin cytoskeleton organization when cells are maintained in IL-4-containing medium (G6mez et al., 1996~).This corroborates previous results showing that P K C is not involved in IL-4induced signal transduction (G6mez et al., 1994, 1995a). Interestingly, Thl clones generated in vitro in the presence of IL-2 show major changes in cell shape, resulting in irregular and polarized morphologies, whereas IL-ktimulated Th2 clones maintain a spherical shape. These morphological dissimilarities may be related to the differential ability of the subsets to migrate through the endothelium. These observations suggest a possible role for P K C in Th subset differentiation. P K C has been detected associated with the mitotic apparatus of the shark rectal gland, suggesting a function for this kinase isoform in cell division (Lehrich and Forrest, 1994).More interestingly, P K C activity was associated with increased neurite extension in PC12 cells, a phenomenon related to changes in the actin cytoskeleton. Finally, association of P K C with the pleckstrin homology domain of Rac has been reported, which is clearly relevant to cytoskeletal organization (Konishi et al., 1994). Our results strongly suggest that, in addition to its implication in intracellular signaling that triggers cell proliferation, this PKC isoform plays an important role in cell structure organization during cell cycle progression. VI. Nuclear Transcription Factor NF-KB

The inducible nuclear transcription factor NF-KB was initially described as a heterodimer composed of a 50-kDa subunit ( N F - K Bor ~ p50) and a 65-kDa subunit (RelA or p65) (Kawakami et al., 1988; for reviews see Finco and Baldwin, 1995; Thanos and Maniatis, 1995; Israel, 1995; Kopp and Ghosh, 1995). Two additional proteins were subsequently included in the NF-KB family, RelB and p52 (NF-KBB)(Schmid et al., 1991; Ryseck et al., 1992; Bours et al., 1992; Mercurio et al.,1992).All these molecules, which form homodimeric or heterodimeric complexes, share a region of homology with the c-rel protooncogene product. This region is essential for dimerization, binding to the DNA recognition sequence, nuclear localization, and association to the inhibitor proteins IKB. Both p50 and p52 proteins are expressed as larger precursors exhibiting ankynn repeats, a domain implicated in protein-protein interactions. The mechanism of NF-KBfunction is unique among the diverse families of nuclear transcription factors (Fig. 6). In the absence of a stimulatory signal, NF-KB is a cytoplasmic resident complex whose nuclear localization

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FIG.6. Nuclear responses induced by IL-2. The figure shows the mechanisms of activation of the nuclear transcription factors NFAT and NF-KB.The serinekhreonine phosphatase cdcineurin dephosphorylates the cytoplasmiccomponent NFATc/N FATp. The dephosphorylated protein translocates to the nucleus and associates with the nuclear component AP1, giving rise to the active NFAT complex. On the other hand, a putative IKB kinase phosphorylates the IKB protein complexed to the NF-KB factor. This phosphorylation targets IKB for uhiquitination. Finally, NF-KB-hound uhiquitinated IKB is cleaved by the multisuhunit proteasome complex. Free NF-KBis able to translocate to the nuclear compartment, where it induces transient expression of certain genes. The best characterized genes in the IL-2 response are the protooncogenes c-fos, c-jnn, c-rnyc, and bcl-2.

signals are masked by the association to the 36-kDa inhibitor IKB (Beg et

al., 1992; Ganchi et aZ., 1992; Zabel et aZ., 1993) (Fig. 8). So far, four IKB ~ et al., 1991), IPBP isoforms have been characterized, termed I K B(Haskill (Thompson et al., 1995), IKBR (Ray et al., 1995),and Bcl-3. However, the nuclear localization of the last protein and its ability to coadjuvate p50 or

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p52 homodimer transcriptional activity suggest that its in vivo role may differ from that of the other family members (Fujita et al., 1993; Bours et al., 1993). IKBproteins also possess ankynn repeats, which mediate IKB interaction with NF-KB.The function of IKB proteins is to sequester NFKB in the cytoplasmic compartment, although this can also be done by the NF-KB precursor proteins p105 and p100, since the uncleaved molecules are unable to translocate to the nucleus (Finco and Baldwin, 1995). I K Bis~phosphorylated upon triggering of stimulatory signals. According to the current models, the putative IKB kinase may be activated by the 5 isoform of PKC (Lozano et nl., 1994; Diaz-Meco et al., 1994), although E PKC has also been suggested to induce NF-KB activation (Genot et al., 1995; Hirano et al., 1995) and another report shows that P K C overexpression does not alter NF-KBactivity (Montaner et al., 1995).Phosphorylation of IKB, however, seems to be insufficient for its dissociation from NF-KB (Finco et al., 1994; Miyamoto et al., 1994; DiDonato et aZ., 1995; Alkalay et al., 1995; Lin et al., 1995). Proteolysis of phosphorylated IKB is also required for full NF-KB activation and nuclear translocation. This process takes place through targeted ubiquitination of phosphorylated IKB, followed by in situ degradation by the multisubunit ATP-dependent 26s (1500 kDa) proteasome complex (Chen et aZ., 1995). Active NF-KB may then translocate to the nucleus and activate transient transcription of its ~ and the genes encoding the target genes, among which are the I K B gene NF-KB subunits (Finco and Baldwin, 1995). The subsequent increase in IKB levels may serve as a form of negative autoregulation, restoring the basal cytoplasmic levels of inactive NF-KB-IKB complexes by nuclear translocation of IKBand sequestering of DNA-bound NF-KB (ArenzanaSeisdedos et al., 1995; Finco and Baldwin, 1995). Several inducers have been shown to trigger NF-KB activation, namely TNF, IL-1, lipopolysaccharide (LPS), phorbol esters, okadaic acid, and serum growth factors (Baeuerle and Henkel, 1994), while it is inhibited by glucocorticoids (Scheinman et al., 1995). NF-KB activation in T cells is proposed to follow biphasic kinetics determined by TCWCD3 and TNF (Pimentel-Muiiios et al., 1995).A receptor-associated protein kinase called IRAK is suggested to mediate NF-KB activation by IL-1 (Croston et al., 1995), whereas a G-protein-coupled pathway mediates platelet activating factor (PAF)-induced NF-KB activation (Kravchenko et al., 1995). Nitric oxide (NO)induction of NF-KB activity is mediated by direct Ras activation (Lander et al., 1995). Mice deficient in RelB show multiorgan inflammation and impaired development of thymic medulla and dendritic cells (Burkly et al., 1995; Weih et aZ., 1995), while disruption of the p50 gene leads to multifocal immune response defects (Sha et al., 1995). Disruption of the relA gene

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causes embryonic lethality at 15-16 days of gestation, concommitant with massive hepatic cell death by apoptosis (Beget al., 1995),while overexpression of the gene in transgenic mouse thymocytes results in increased I K B ~ levels (Pkrez et al., 1995). NF-KB has been identified as a crucial factor in the expression of the I L - ~ R cchain N gene (Pierce et al., 1995; Sperisen et al., 1995), as well as in the CD28 induction of the IL-2 gene (Lai et al., 1995). The IL-2R induction of NF-KB is rapamycin sensitive, suggesting that it proceeds through a PI3 kinase-mediated pathway (Rebollo et al., 1995). Through this pathway, Rho has been found to be a requirement for PI3 kiiiase activation (G6mez et al., 1996a). Since PI3 kinase products activate P K C , NF-KB activation through the IL-2R may occur via a Rho-PI3 kinasePKC-IKB kinase pathway. VII. Target Genes in 11-2 Signals

A. c-jun,

C ~ Q S

The jun andfos protooncogenes are members of the class known as cellular immediate-early genes. In the majority of cell types, these genes are expressed at low levels but they are induced rapidly and transiently by extracellular signals. Induction ofjun andfos seems to be medited by a tyrosine kinase via the activation of Ras. As in other cell types, c-jbs is the earliest identified gene induced during T cell activation (GranelliPiperno et nl., 1987; Kumagai et al., 1987). Fos and Jun may contribute to the coupling of short-term signals elicited by cell-surface stimulation to long-term alterations in cellular phenotype by regulating expression of specific target genes. The situation is complex due to the existence of several fos- and jun-related genes. When expressed continuously at high levels, c-jun and c+is can cause transformation. Only a limited domain of Fos is sufficient for cellular transformation (Okuno et al., 1991). In addition to cell growth, they are implicated in cell differentiation and development. Although the jun and fos oncogenes were isolated independently, their protein products function cooperatively as components of the transcription factor AP-1. The AP-1 binding site is recognized by c-Jun homodimers and c-Junk-Fos heterodimers, Homodimer and heterodimer formation is mediated through the leucine zipper (O’Shea et ul., 1989; Halazonets et al., 1988).AP-1 binds to enhancer sequences responsive to PKC-activating agents (Angel et al., 1988). c-jun-deficient mice show generation of normal T and B lymphocytes and normal levels of proliferation, probably due to the fact that other members of thejun family contribute to the bulk of the activator protein 1 (AP-1) in normal T cells (Chen et al., 1994a,b). IL-2, upon interaction


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with its receptor, triggers cfos induction which is mediated by IL-2RP chain and being the SRE (serum response element) in the cfos promoter the target for the signal (Trouche et al., 1991; Hatakayama et al., 1992). c-fos-deficient mice generated using the gene knockout technique were comparable to wild-type mice in their thymocyte development pattern and in the ability of their peripheral T cells to proliferate and produce cytokines in response to TCR stimulation. This suggests that otherfos family members may be capable of substituting functionally for cfos during T cell development (Jain et al., 1994). In adddition, several genes known to contain functional AP-1 sites are expressed normally in cfos-deficient mice (Hu et al., 1994). The cfos protooncogene is also implicated in the control or execution of apoptosis. Studies in mouse embryogenesis (Smeyne et al., 1993) suggest that sustained expression of cfos coincides with regions that undergo apoptosis. At present it is unclear whether such cfos expression is part of a cell-autonomous death program or whether it indicates stimulation of affected cells by some external signals.

B. C - ~ Y C c-myc protooncogene encodes a DNA-binding protein whose expression is elevated or deregulated in virtually all tumors (Spencer and Groudine, 1991). c-Myc protein is a transcription factor with an amino-terminal domain with transcriptional activating activity and a carboxy-terminal DNAbinding/dimerization basic helix-loop-helix leucine zipper (Amati et al., 1992, 1993, 1994). The myc family includes at least five different and genetically unlinked functional members: c-, N-, L-, S-, and B-myc. c-myc is expressed in virtually all proliferating cells in embyronic and adults tissues. The rest of the members of the family are expressed in a restricted set of tumors. The c-, N-, and L-myc genes have all the same three-exon and two-intron structure (DePinho et al., 1991). Max acts as a partner protein and dimerizes with c-, N-, and L-myc to form a specific DNA-binding complex. Max may serve as a cofactor for Myc in transcriptional activation acting as both activator and suppressor of Myc transcriptional activity (Amati et al., 1993; Blackwood et al., 1992). These complexes regulate genes involved in cellular growth and differentiation (Schreiber-Agus et al., 1993). The interaction between Myc and Max constitutes a sensitive switch controling entry and exit form the cell cycle in response to extracellular stimuli (Amati et al., 1993). Finally, it is suggested that the reduced variability in Max expression preserves dimerization capabilitywith products ofmyc and related genes (Atchleyand Fitch, 1995).

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Expression of c-myc (or another member of the inyc gene family) appears to be necessary and, in some cases, sufficient for cell proliferation (Evan and Littlewood, 1993). Deregulated c-myc expression is associated with inability to withdraw from cell cycle (Eiler et al., 1991; Evan et al., 1992) and suppression of differentiation (Freytag et al., 1990). It seems that c-myc promotes cell proliferation and suppresses growth arrest by modulation of appropriate growth-related target genes. In fact, induction of c-myc correlates with the efficient induction of certain cell cycle genes that seem to be critical for cells to enter GWM phase, including cyclin A and B and cdc2 kinase (Kim et al., 1994). c-myc has also been recognized as an inducer of cell death. Transgenic animals whose lymphocytes express deregulated c-Myc exhibit increased sensitivity to induction of apoptosis (Neiman et al., 1991). High levels of expression of c-Myc correlate both with increased proliferative rate and with increased sensitivity to apoptosis (Evan et al., 1992). Identical regions of c-Myc protein are required for both growth promotion and induction of apoptosis. Dimerization with the heterologous partner protein, Max, is necessary for transforming and apoptotic functions of c-Myc (Kato et al., 1992; Ayer et id., 1993; Zervos et al., 1993). Induction of apoptosis by c-myc may respond to one of two alternative hypotheses: c-myc may induce either cell growth or death depending upon concomitant external signals, or otherwise c-myc may simultaneously induce both proliferation and apoptosis, the latter blocked by the action of mitogenic or survival factors (Harrington et nl., 1994; Wyllie, 1995; Amati and Land, 1994). Activation through IL-2R tirggers c-myc expression and the IL-2RP chain appears to be critical in such induction. Cells expressing the IL-2RP chain mutant lacking the serine-rich region critical for proliferation fail to induce c-myc upon stimulation with IL-2. On the contrary, cells expressing IL-2RP mutants which retain the serine-rich region still induce c-nayc expression and proliferation (Minami et nl., 1993a). C. bcl-2 bcl-2 was originally identified as an oncogene which is amplified in human follicular B cell lymphomas (Tsujimoto and Croce, 1986;for reviews see NhAez et al., 1994; Cory et nl., 1994; Boise et al., 1995; Korsmeyer et nl., 1995; Korsmeyer, 1995). The protein encoded by the bcl-2 gene is a transmembrane 26-kDa product located in the outer mitochondrial membrane, perinuclear membrane, and smooth endoplasmic reticulum (Monaghan et nl., 1992;Jacobson et al., 1993; Krajewski et al., 1993).A hydrophobic C-terminal domain serves as membrane anchor, with the major part of the molecule oriented toward the cytosol. Two conserved domains, BH1 and BH2, seem essential for Bcl-2 function (Yin et al., 1994).


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Bcl-2 may act as a survival signal for positive selection of T and B cells (Strasser et al., 1994; Chan et al., 1993; Hogquist et al., 1994; Liu et al., 1991a; Nliiiez et al., 1991). The role of bcl-2 in regulation of apoptosis is in agreement with the developmental regulation pattern of the bcl-2 gene during lymphocytic maturation. bcl-2 expression is reduced at developmental stages when lymphocyte selection is supposed to occur and when susceptibility to cell death is maximal, namely the pre-Bhmmature IgM+IgD- for B cells and CD4+CD8+ for T cells (Gratiot-Deans et al., 1993; Merino d al., 1994; Haury et al., 1993; Veis et al., 1993). The cellular functions of Bcl-2 have been extensively studied in the context of apoptosis inhibition induced by different stimuli, such as growth factor deprivation, y-irradiation, glucocorticoids, genotoxic drugs, or antiFas antibodies (Korsmeyer, 1992; Collins and Rivas, 1993; Vaux, 1993; Schwartz and Osborne, 1993; Weller et al., 1995). Although the precise aspects of Bcl-2 function are still obscure, inhibition of apoptosis may take place through the regulation of an antioxidant pathway (Hockenbery et al., 1993; Kane et al., 1993),or it may prevent oxidative damage to cellular components (Korsmeyer et al., 1995). A possible role for Bcl-2 in regulating intracellular calcium fluxes has also been suggested (Barry et al., 1993; Lam et al., 1994). Alternatively, it may modulate the function of some apoptosis effector molecules. The regulation of Bcl-2 function is exerted by heterodimerization with the Bcl-2-homologous 21kDa Bax protein (Oltvai et al., 1993). A new bcl-2-related gene, bcl-x, gives rise to two different proteins by alternative splicing, BcI-xL (long) and Bcl-xs (short). Another different unspliced transcript encodes the Bcl-xp protein (or Bcl-xATM in mice), which lacks the hydrophobic carboxy terminus (Boise et al., 1993; GonzdezGarcia et al., 1994; Fang et al., 1994). In contrast to Bcl-2 and BcI-xL, the Bcl-xs protein promotes cell death by inhibiting Bcl-2 function. The high embryonic expression of Bcl-xLand its similarities to Bcl-2 in function and subcellular distribution may explain the normal development of Bcl-2deficient mice (Nakayamaet al., 1993; Veis et al., 1993). However, mice die from lymphopenia at 2-5 weeks of age, and show fulminant metanephric apoptosis, abnormal kidney development, and hair hypopigmentation (Sorenson et al., 1995; Nakayama et al., 1994). These data indicate that Bcl-2 may be involved in the regulation of the mature immune system rather than its development. As expected, mice with a targeted disruption of the bcl-x gene suffer from massive death of immature hematopoietic cells and neurons (Motoyama et al., 1995). On the other hand, expression of a transgenic bcl-2 may predispose mice to a severe autoimmune disease resembling human systemic lupus erythematosus (Cory et al., 1994).

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Bcl-2 has been shown to prevent cell death triggered by the tumor suppressor protein p53 (Marin et al., 1994), a molecule known to induce apoptosis at the G1 phase when transit to either DNA replication or cell death is programmed (for reviews, see Chiarugi et al., 1994; Haffner and Oren, 1995). Furthermore, p53 is proposed to down-regulate Bcl-2 and up-regulate Bax expression (Miyashita et al., 1994). Recent results provide evidence for the importance of bcl-2 protooncogene expression in IL-2 signaling and cell cycle progression. A decrease in Bcl-x protein levels precedes apoptosis after IL-2 withdrawal in T cells and transfected bcl-2 promotes survival after IL-2 withdrawal (Broome et al., 1995).This suggests that, in addition to its function in inhibition of apoptosis, bcl-2 plays a role in promoting cell proliferation (Miyazaki et al., 1995).The control ofbcl-2 expression is suggested to be exerted through a Ras-dependent pathway in the IL-3/IL-3R system (Kinoshita et al., 1995; Wang et al., 1995); however, this appears not to be the case in the IL2R pathways. VIII. The Counterpart of Protein Kinarer: The Phorphoprotein Phosphatases

A. CD45 The fact that T cell activation and proliferation involve tyrosine phosphorylation of specific functional proteins suggests that it necessarily requires protein tyrosine phosphatases to revert these effects. Moreover, protein tyrosine phosphatases (F'TPases) appear to have an essential role not only in the regulation of receptor-linked signal transduction pathways, but also in cell mitosis regulation and maintenance of normal cell physiology. Among the PTPases involved in regulation of T cell activation, one transmembrane PTPase, CD45, may represent a unique prototype of a signal-transducing molecule. CD45, also known as leukocyte common antigene or T200, is a family of cell surface molecules found in all nucleated hematopoietic cells (Thomas, 1989). The CD45 isoforms arise from differential splicing of a single gene. Their heterogeneity resides entirely in the extracellular domains of the members of this PTPase family, with all members sharing identical transmembrane and cytoplasmic regions (Mustelin et al., 1989; Streuli et al., 1987). CD45 is a tyrosine phosphatase expressed at high levels on lymphocytes and all other hematopoietic cells, with the exception of erythrocytes and platelets. The molecule consists of three distinct regions: a large cytoplasmic carboxy-terminal region with two domains with PTPase activity, a single membrane-spanning region, and an amino-terminal extracellular glycosylated region which probably functions as a ligand-binding domain (Thomas, 1989; Trowbridge and Thomas, 1994; Trowbridge, 1991).


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The regulation of CD45 is far from clear since different leukocyte subpopulations express different CD45 isoforms (Trowbridge and Thomas, 1994). Expression of CD45 is required for TCWCD3-induced tyrosine phosphorylation and enhanced inositol phospholipid turnover, suggesting the src-family PTK as possible CD45 substrates in T cells (Mustelin and Bum, 1993). There is no evidence for CD45 participating directly in signal transduction from TCR or BCR. Rather is seems that CD45 acts on Lck and Fyn maintaining a considerable fraction of the Src-family PTK dephosphorylated and potentially active (Mustelin and Bum, 1993). Loss of CD45 correlates with a decrease in Lck and/or Fyn activity (Ostergaard et al., 1989; Hurley et al., 1993; Shiroo et al., 1992; Turka et al., 1992). CD45 selectively regulates Fyn and Lck pools associated with TCR and CD4 at the cell surface. Activation by CD45 of these receptorassociated kinase pools correlates with the ability of TCR and its coreceptors to couple to intracellular signaling pathways (Biffen et al., 1994). CD45defficient CD4+CD8+cells fail to proliferate in response to antigen or CD3 cross-linking. Treatment of CD45- T cells with PMA and ionomycin results in proliferation, indicating that activation of PKC, in addition to intracellular calcium increase, rescues the defect caused by CD45 deficiency. It suggests that CD45 is required for the activation of tyrosine kinase activity at the time of or prior to transmembrane signaling (Pingel et al., 1994). IL-2 stimulation of a cytolytic T cell line resulted in serine phosphorylation of CD45 by protein kinase C, but did not alter its PTPase activity (Valentine et al., 1991). Pingel and Thomas (1989) showed that a CD45deficient murine T cell clone did not proliferate in response to antigen, but this clone proliferated after exposure to exogenous IL-2. These results indicate that CD45 is essential for engagement of the TCR, leading to cellular growth, but that CD45 was not essential for the transmission of all growth signals to T cells.

B. PTP-1C Many members of the cytokine receptor superfamily initiate intracellular signaling by activating members of the Jak tyrosine kinase family. But cytokine receptors are also negatively regulated by hematopoietic cell phosphatase (HCP), also termed PTP-1C or SH2-PTP (Yi et al., 1992; Shen et al., 1991; Matthews et al., 1992; Plutzky et al., 1992). HCP contains a carboxyl catalytic domain specific for phosphotyrosine and two SH2 domains, the first of which facilitates HCP recruitment to activated receptors (Cyster and Goodnow, 1995; Yi et al., 1993, 1995). PTP-1C recruitment to the Epo or IL-3R is associatedwith dephosphorylation of Jak2 (Klingmuller et al., 1995; Yi et al., 1993, 1995)and termination of proliferative signals,

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suggestingthat Jaks are critical substrates. HCP is expressed predominantly in hematopoietic cells, although it is also detected in some epithelial cells. Lack of functional HCP expression causes hematopoietic abnormalities (Tsi et al., 1993; Shultz et al., 1993) and mutations in this gene are responsible for the motheaten phenotype in mice; the lethal form of motheaten represents a naturally occurring gene knockout of HCP (Shultz et al., 1993; Tsi et al., 1993). Little is known about the role of PTPs in early T cell signaling. HCP is basically phsophorylated on serine in resting T cells. Upon CD4 or CD8 stimulation, HCP becomes tyrosine phosphorylated lymphoma cell lines overexpressing Lck. The two tyrosines which are directly Lckphosphorylated in uitro are also phosphorylated in uiuo, and one of them is phosphorylated in response to Lck activation, suggesting that this phosphorylation can play a role in early T cell signaling (Lorenz et al., 1994). SH2 domain of HCP has been suggested to autoinhibit the phosphatase activity of the PTPase domain. The SH2 domains probably interact with the PTPase domain, driving it into an inactive conformation (Pei et al., 1995). In contrast, the SH-PTPY PTP-lD/Syp phosphatase is a positive signal transducer (Sun and Tonks, 1994; Stahl et al., 1995). SH-PTP2 contains two SH2 domains which allow it to bind to the receptors for IL-6 and other cytokines (Stahl et al., 1995).This phosphatase is tyrosine phosphorylated, although it is not clear whether Jak kinases are responsible for this phosphorylation. In addition, SH-PTP2 is required upstream of MAPK for early Xenopus development (Tang et al., 1995). AND OTHER PIIOSPHOSERINE/ C. CALCINEURIN PHOSPHOTHREONINE PHOSPHATASES There are four major classes of serinekhreonine phosphatases: PP-1, PP-2A, PP-2B, and PP-2C. All are formed by a catalytic and a regulatory subunit. The serinekhreonine phosphatase calcineurin, also known as phosphoprotein phosphatase 2B or PP-2B, has been identified as an active participant in signal transduction and attenuation pathways. Calcineurin, a heterodimer of a catalytic and a regulatory subunit, is a calcium/calmodulindependent phosphatase (Liu et al., 1991b, 1992; Fruman et al., 1992; Mckeon, 1991; Schreiber, 1992; Liu, 1993)which has been demonstrated to be an important component of the TCR signal transduction network (Clipstone and Crabtree, 1992; Liu et al., 1992; O’Keefe et al.,1992; Fruman et al., 1995; Paliogianni and Boumpas, 1995; Frantz et al., 1994). Biochemical, pharmacological, and genetic studies have led to the consensus that calcineurin activity is critical for propagating calciumdependent T cell signaling pathways. One potential substrate of this phosphatase is the transcription factor NFAT, which is required for optimal


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transcription of the gene encoding IL-2. NFAT is a multimeric complex composed of a nuclear component (AP-1) and a cytoplasmic component (NFATdNFATp)which translocates to the nucleus upon a calciumdependent dephosphorylation (Fig. 6) (Flanagan et al., 1991; Jain et al., 1992; McCafrey et al., 1993). Translocation of this component to the nucleus is prevented by the calcineurin inhibitors cyclosporin A and FK506 (Flanagan et al., 1991; Jain et al., 1992), suggesting that it may be a direct calcineurin substrate (McCaffrey et al., 1993). Although calcineurin is the best characterized serinelthreonine phosphatase, the role of these phosphatases in signaling through IL-2R is not well defined; calcineurin inhibitors block the IL-2dependent expression of NF-AT, suggesting its implication in signaling through IL-2R (Rebollo et al., 1995).

D. DUALTH R E O N IN E ~ Y R O PHOSPHATASE S IN E PAC-1 Progression through the cell cycle requires the expression of a series of genes which are induced within minutes after ligand-receptor binding. One of them, the phosphatase of activated cells (PAC-l), is transiently expressed during the GI phase in mitogen- or antigen-activated T cells (Zipfel et al., 1989; Kelly et al., 1992). PAC-1 is an immediate mitogen-inducible threonineltyrosine phosphatase localized in the T cell nucleus (Rohan et al., 1993). It is encoded by a gene composed of three exons. The C-terminus of the protein is homologous to the closely related phosphatases 3CH134 and VH2 (Gerondakis et al., 1995).This dual phosphatase has high substrate specificity for MAPK (Rohan et al., 1993; Ward et al., 1994) and is homologous with several protein tyrosine phosphatases in the most highly conserved region among tyrosine phosphatases. The PAC-1 gene encodes a nuclear 32-kDa dual phosphatase which dephosphorylates ERK-1 and ERK-2. Constitutive expression of PAC- 1 in uiuo leads to inhibition of MAPK activity normally stimulated by EGF, PMA, or TCR cross-linking. Given the role of phosphatase PAC-1 in inhibition of MAPK pathway (Ward et al., 1994), activation of PAC-1 by IL-2 may be of great interest in Ras signaling pathway triggered by IL-2. IX. Specific Signaling Molecules for 11-4, 11-9, 11-13, and Insulin Receptors

A. IRS-1 AND 4PS

IL-4 and IL-9 share many biological functions (Paul, 1991; Renaud et

al., 1993).Both cytokines have growth-promoting activity for T lymphocytes and mast cells (Uyttenhove et al., 1988; Hultner et al., 1990; Renauld et al., 1993). Also, IL-4 and IL-9 share similar activities for B lymphocytes,

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such as induction of specific immunoglobulin isotype secretion (Coffman et al., 1993). IL-4 is a cytokine produced by T cells (Howard et al., 1982), mast cells (Plaut et al., 1989), and basophils (Seder et al., 1991). Since it is associated to the appearance of a Th2-like immune response pattern, its effects are clearly detectable in B cells, including the development of the activated phenotype (up-regulation of MHC class I1 molecules and CD23) and the switch to IgGl and IgE isotype secretion (Conrad et al., 1987; Vitetta et al., 1985; Cofman et al., 1986). Activation of IL-4R induces a rapid tyrosine phosphorylation of a 170kDa protein called 4PS (Isford and Ihle, 1990; Wang et al., 1992), as well as phosphorylation of the receptor itself (Wang et al., 1992; Izuhara and Harada, 1993). IL-4, upon interaction with IL4R, induces tyrosine phosphorylation of residue 497 of IL4R within the 14R region that would create a site to which 4PS can dock directly or through an adaptor molecule (Myers et al., 1994). Phosphorylated 4PS would interact with p85 PI3 kinase, Grb2, or other presently unidentified molecules to initiate the IL-4-induced signaling pathways. The association of the p85 subunit of PI3 kinase with 4PS is reminiscent of PI3 kinase association with IRS-1 (Sun et al., 1991). However, it has been reported that IRS-1 is a cytosolic protein (White et al., 1987), while the majority of 4PS is membrane associated, implying that these substrates might not be identical. Finally, IL-4 also induces the association of the tyrosine kinase Jakl with IL4R and 4PS/IRS-l. These data support the hypothesis that Jakl and 4PS/ IRS-1 may be pivotal in the IL-4- and IL-9-mediated signaling pathways. However, unique pathways for each cytokine may still exist, since IL-4 and IL-9 induce distinct patterns of tyrosine phosphorylation. The insulin receptor is composed of two a-subunits, each linked to a P-subunit and to each other by disulfide bonds. This receptor is phosphorylated on tyrosine, serine and threonine residues in response to phorbol esters, CAMPanalogs, and insulin itself (Takayamaet al., 1988). The intracellular region of the insulin receptor /3 subunit is essential for signal transmission and contains a tyrosine kinase domain and at least one tyrosine autophosphorylation site. IRS-1 was initially detected in insulin-stimulated hepatoma cells as a tyrosine-phosphorylated 185-kDa substrate (Rothenberg et al., 1991; Keller et al., 1991, 1993; Sun et al., 1991; Araki et al., 1993; Nishiyama et al., 1992; Myers et al., 1994; White, 1994). IRS-1 contains at least 20 potential tyrosine phosphorylation sites and over 30 serinehhreonine phosphorylation sites. It is strongly phosphorylated on serine before insulin stimulation, and this stimulation increases the amount of phosphoserine and phosphothreonine (Sun et al., 1992).


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Serine phosphorylation could be of regulatory importance either to inhibit tyrosine phosphorylation or to block subsequent binding of SH2-containing proteins. IRS-1 acts as a docking protein to regulate the activity of signaltransducing molecules containing SH2 domains (Sun et al., 1991). The fact that IRS-1 undergoes tyrosine phosphorylation at multiple sites suggests that it may bind to many copies of PI3 kinase, causing signal amplification, or it could interact with several distinct SH2 domain-containing enzymes to regulate divergent pathways. Tyrosine phosphorylated IRS-1 also associates to Grb2. However, these interactions do not seem to trigger Ras activation, since the Ras-GTP complex formation appears to proceed via Shc-Grb2 association. Finally, since disruption of the gene encoding IRS-1 in mice is not lethal (Araki et al., 1994) and Shc seems to be functionally distinct from IRS-1, there must be other molecules which the insulin receptor can use to regulate critical metabolic pathways. The IRS-1 related molecule 4PS seems to be a likely candidate in hematopoietic cells (Myers et aZ., 1994). IRS-1deficient mice by targeted gene mutation show no evidence of IRS-1 phosphorylation or association to PI3 kinase. However, the appearence of a new phosphorylated protein, IRS-2, which binds to PI3 kinase, provides evidence for IRS-l-dependent and IRS-l-independent pathways of insulin signaling and for the existence of an alternative substrate for these receptor kinases (Araki et al., 1995). The novel insulin receptor kinase substrate (IRS-2, p190) can bind both PI3 kinase and Grb2. Hence, induction of p190 tyrosine phosphorylation may be one of the compensatory mechanisms that substitute for IRS-1 in deficient mice (Tobe et al., 1995). In addition, mice with targeted IRS-1 disruption show growth retardation and milk insulin resistance (Tamemo et al., 1994). IL-13 has many functional effects similar to those of IL-4. As mentioned above, IL-4 induces tyrosine phosphorylation of 4PS and, interestingly, IL-13 also induces the phosphorylation of this molecule; however, the Jak kinases phosphorylated are different in each case. These data suggest that IL-4 and IL-13 transmit the signals in a similar manner, via Jak 1 and 4PS activation. However, IL-13 does not activate Jak3 (Keegan et al., 1995), which has been raised as an argument to refute the utilization of the IL-2R common y chain by the IL-13 receptor (Welham et aZ., 1995). X. Cooperation of the Three Il-2-Induced Signaling Pathways

In agreement with previous reports by other authors (Miyazaki et al., 1995),we propose the existence of at least three distinct signaling pathways triggered through the IL-2R. Our results broad previous observations and

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Fic:. 7 . Thrre-channel model of the I L 9 R signaling system. The figire depicts initiation of the signal delivery by a variable intensity device, reflecting the fact that the mitogenic responses are proportional to the amount of lymphokine provided within a certain range of concentrations. Proximal transduction events are divided into JakllJak3-dependent and independent signals. The former rapidly branches into two independent pathways. channels 1 and 2. The Jakl/Jak3-independent signal dirrctly triggers channel 3. Channel 1 proceeds through activation of Src-family tyrosine kinases such as Lck and Fyn, connects to Ras


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outline in further detail some of the crucial events along the signaling pathways (Fig. 7). The three pathways, which we have referred to as channels 1, 2, and 3, respectively, are: (1) Channel 1: A pathway triggered by LcWJak which leads to Ras activation and, consequently, to c-jun and cfos induction. (2) Channel 2: A pathway triggered by the SyVJak which leads to c-myc induction. (3) Channel 3: A pathway triggered by still uncharacterized mediators which, upon activation of Rho, PISK, and P K C , controls the actin cytoskeleton and leads to bcl-2 expression and possibly to NF-KB activation

Activation of the three channels is required for cell proliferation in TSlaB cells, whereas only activation of the Ras pathway (channel 1)plus any one of the other two pathways is required for cell survival, suggesting that these three pathways cooperate with one another to ensure full signal transmission by IL-2. In other cellular systems such as BAF/BO3, a combination of only two of the three pathways is sufficient to promote cell growth. Still, the role of the JaWStat pathway within this scheme is not clear. It has recently been shown that a dominant negative Jak3 mutant suppresses both cell proliferation and activation of channels 1 (Lck-RasfosJun) and 2 (Syk-myc) (Kawahara et al., 1995). This mutant abrogates activation of endogenous Jakl and Jak3, suggesting crosstalk between both Jak isoforms. Given that Jakl associates to the serine-rich region of the IL-2RP chain, that this region is required for IL-Zinduced proliferation, and that it is also necessary for Lck activation but not association, a plausible hypothesis would be that Jak3-dependent activation of Jakl could be involved in the upstream activation of Lck and Syk. Thus, the strict requirement of the serine-rich region for IL-2-driven mitogenesis would be exactivation through adaptor molecules (ShclGrbWSosNav),and powers the MAPK pathway, rendering induction of c-fos and c-jrtn expression. Channel 2 is largely unknown, streaming from Syk activation down to expression of c-myc. Finally, channel 3 is initiated by a postulated link between tyrosine kinases and Rho activation cooperating to activate PI3 kinase. The signal flow falls down from PI3 kinaselPPKC complexes and distributes to a number of systems, including actin architecture and probably NF-KB activation and bcl-2 expression. Two final cellular programs may be triggered by the interplay between the three channels, cell proliferation, and suppression of apoptosis. Differences seem to exist among cellular systems regarding signal requirements for determining the final outcome. While some cell lines need only any two active channels to proliferate, others require all three signals. The model reflects the situation observed for the TSla& murine T cell line: two signals protect from apoptosis, being one of them channel 1, whereas three active channels promote proliferation.

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plained by the interaction of this domain with a molecule responsible for the initiation of two of the three signaling channels. This hypothesis would also explain the Lck activation requirement of a region that does not interact physically with Lck. Other IL-2-induced signaling events remain to be defined, and a number of molecules still need to be tested for localization within the three-channel model. There is evidence for Jak kinase activation as immediate-proximal signals prior to branching of the three channels. The involvement of the Stat proteins in this putative step is also still far from being conclusive. In addition, the role of Stat is outside of the scope of this review; these proteins probably modulate cell program execution by these signals. Finally, we have previously shown the implication of EPKC in the proliferative response to IL-2, although its localization in the three-signal scheme has not yet been addressed. We described that inhibition of EPKC prevented IL-2-induced proliferation in TS l a p cells. Considering that only 6- and EPKC isoforms were found to mediate the mitogenic effect of IL-2, and that we have already located P K C in channel 3, the model would predict that EPKC should act at some step in channel 1 or 2. As mentioned above, functional interplay between the signals delivered by the three channels determines the final cellular response. Two fundamental programs are performed by the cell in response to the stimuli provided: cell division or cell death. According to current models, a decision point exists at the G1 stage of the cell cycle when the cell may be activated to execute either the mitogenic or the apoptotic program. The system might be more complex, since it is not binary. Simultaneous execution of the two programs also seems possible, at least in a number of cellular systems and conditions. Thus, signal transduction through a truncated GMCSF receptor defective in Ras activation results in cell death, although cells exhibit concommitant DNA synthesis (Kinoshita et al., 1995).Also, the SEB-triggered apoptotic pathway in T cells carrying the Vpy receptor gene product requires previous proliferation (Gonzalo et al., 1993). This possibility, if extrapolated to the IL-2R, could in fact be explained from the model considering multiple signal integration. The fact that activation of three channels is required for cell growth may be interpreted as signal addition, i.e., all signals are interchangeable and act in acumulative fashion. This obviously does not apply to activation of intracellular mediators, but cannot be ruled out in the sense of minimal essential signals that can be redundantly switched on and determine a qualitative response through quantitative increases. Hence, there is a chance that modulation of the strength of a particular signal could overcome the threshold that is surpassed by an additive effect under physiological conditions. This hypothesis would apply to the differences observed concerning number of channels


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required for mitogenesis in different cellular models. It is conceivable, due perhaps to greater expression of an intracellular mediator or transducer acting at a step at which downstream signal amplification occurs, that a cell line could reach the mitogenic response signal strength threshold by activation of only two channels. Nevertheless, since channel 1 seems qualitativelyunique as essential for suppression of death, if the two channels triggered are 2 and 3, a dual proliferative/apoptotic response could be obtained. This hypothetical case would match the observations of Kinoshita et al. (1995), including the lack of Ras activation. However, further work is needed to examine such a possibility for the IL-2R. XI. Concluding Remarks

An outstanding feature of the immune system is that different cytokines can act on the same cell type to mediate similar effects, a phenomenon known as functional redundancy. This may reflect an evolutive advantage to ensure functionality under conditions in which the immune response could be subverted by pathogens that, rather than eluding or suppressing immune effector functions, are able to redirect the response in a way that does not interfere with their replication. Hostlpathogen coevolution selects adequate mechanisms to maintain an equilibrium between both species. Thus, immune system evolution may arrive at such a degree and quality of functional redundancy that the pathogen itself may select the appropriate alternative response. This issue has been raised due to the increasing number of studies in which a gene thought essential for the development of immune functions is shown to be mutated or eliminated by homologous recombination. In most cases, phenotypic impairments were much less severe than expected from deduced physiological functions of the gene product involved. On some occasions, subsequent studies have identified the alternative mechanism substituting for the altered gene. In the paradigmatic case of IL-2, the molecular cloning and characterization of IL-15 has explained, a posteriori, the relatively benign nature of the IL%deficient phenotype. Once this rule can be extended to other IL systems, it is possible to predict or postulate the existence of new lymphokines based solely on the results observed in knockout mice. A different case is receptor chain sharing by different cytokine receptors. Several receptor subunits have been mutated or partially deleted, revealing direct implications of specific domains in triggering activation of particular kinases or signaling effector molecules. Evidence argues against this fact, indicating that it is another form of functional redundancy. If this were the case, responses triggered through different receptors sharing common subunits should be functionally equivalent. However, and as an example,

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the common y chain is shared by two groups of receptors which direct the differentiation of distinct and excluding subsets of effector cells, namely the IL-WIFN-y group for T h l cells and the IL-4 group for Th2 cells. This statement implies that the signals linked exclusively to the y chain are likely to be nonspecific with respect to differentiation. Yet they could be specific in terms of proliferation, delivering signals required for mitogenesis which can be complemented by other receptor-specific signals to particularize the responses. Thus, sharing of common chains would rather be a strategy for genetic economy, avoiding unnecessary diversification when common signals are essential. Nevertheless, signal specificity with regard to the sole induction of cell growth is not required conceptually. As mentioned above, the requirement for various signaling pathways can be interpreted in terms of an additive effect for reaching a response threshold. This would be a clear case of functional redundancy of receptor chains giving qualitatively equivalent minimal additive signals. This would be the only mission of common chains, whereas unique chains would, in addition, deliver the effector-specific or differentiation-specific signals. A single cytokine can elicit a distinct range of biological effects in various tissues and cells, revealing a wide spectrum of functional pleiotropism. This phenomenon may be easily explained by the expression of tissue- or lineage-specific signaling molecules, which can lead to the expression of different genes, and thus result in phenotypic changes and particular effector functions. Results obtained using the TSlapy cell system (Fig. 8) show that IL-2R signaling is a complex process involving a large number of molecules different from those used for signaling through IL-4R. These two lymphokines participate in the differentiation of the T h l and Th2 helper subsets, respectively.A phenotypic feature associated with the development of each of these populations is the appearance of morphological changes in Thllike clones, which acquire irregular and polarized shapes corresponding to cytoskeletal reorganizations, while Th2 clones remain spherical. Interestingly, TSla,Py cells cultured in the presence of IL-4 exhibit a rounded shape, whereas stimulation with IL-2 induces morphological changes. Thus, TSlapy may resemble a Tho-like cell line which behaves as a Th2 when routinely maintained in IL-4, although retaining the capacity to revert and acquire Th 1-like characteristics when stimulated with IL-2. Further work will determine the validity of the use of TSlaPy as a Tho-like cell line. The availability of such model would allow the study of the specific intracellular processes which drive the differential development of Th subsets. The possibility of directing phenotypic differentiation in vitru by exogenous interference, regardless of the lymphokine provided, would give rise to great expectations in the field of therapeutic immunomodulation. The use of drugs acting at the level of message delivery is a potential approach

I.ESSONS FROM ACTIVATION MEDIATED BY IL-2 AND 11,-4

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Fic:. 8. The TS1 cell system. The murine T cell line TS1 is able to proliferate independently in the presence of IL-4, IL-9, or insulin. Transfection with a,P, or a+p chains of human IL-2 receptor rendered the TSlay, TSlPy, and T S l a P y variants. The T S l a y transfectans d o not proliferate in response to IL-2 while TSlPy o r T S l a P y do. Transduction pathways utilized by T S l p y cells in response to IL-2 are not affected by immunosuppressants due to the presence of a rescue pathway which is absent in TS1aP-y cells. The IL-2Ra chain, while deprived of a biological role on its own, appears to be a critical molecule that mntrols the transmission of intracellular signals in T cells.

which may prove to be interesting, especially if cell-type specificity can be achieved i n uiuo by coupling selective targeting molecules to the desired drug. Therapeutic uses of these kinds of strategies would include immunopathologies in which disease is a consequence of a pathogen-induced erroneous choice of immune response. In this review, we have discussed several molecules that are attractive candidates as transducing molecules involved in signaling processes and that mark the difference between signaling through IL-2R and IL-4R. As far as we know, TSla& is the unqiue cellular model able to proliferate

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in response to IL-2, IL-4, or IL-9, allowing us to dissect the pathways used by these lymphokines for signaling. The current challenge is to identify the downstream signal transduction molecules and to target genes which respond to the activation of different kinases. ACKNOWLEDGMENTS We thank Drs. S. Fischer, J. C. Gutierrez-Ramos, A. Grandien, and A. C. Camera for critical reading of the manuscript and C. Mark for editorial assistance. This work was partially supported by grants from Comision Interministerial de Ciencia y Tecnologia (CICyT),European Union and Pharmacia. J.G. is supported by a fellowshipfrom the Comunidad Autdnoma de Madrid. The Department of Immunology and Oncology was founded and is supported by Pharmacia.

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ADVANCES IN IMMUNOLOGY. VOL. 63

B Lymphocyte Development and Transcription Regulation in vivo

1. Introduction

B lymphocytes develop through an intricate interplay with the environment. Molecules on the surface of progenitor cells receive signals from the outside, which are translated via complex pathways into changes in pattern of gene expression. The change in molecular makeup implies a change in function, which is impressed upon the environment, allowing further maturation. In the last few years, targeted mutation and ectopic or “forced” expression assays in uivo have helped to identify genes that encode factors necessary for normal B cell development. An important driving force in this area of investigation is the search for a “master switch” for hematopoiesis and, further on, for B lymphocyte development. Genes may exist whose products are indispensable for the initiation of major changes in overall gene expression. A cascade of reactions would follow expression of such genes, turning uncommitted cells into B lymphocytes only. The idea of a master switch is based on the current understanding ofpattern formation and organogenesis in the embryo. For instance, eyeless in Drosophila (highly homologous to murine pax 6 and human Aniridia, a mutation associated with eye defects) encodes a transcription factor that is expressed in the developing eye. Loss-of-function mutation of eyeless blocks eye development. Forced expression of eyeless in imaginary disc primordia other than the one in which eyeless is normally expressed leads to full ectopic eye development (86).The studies in murine hematopoiesis have not yet advanced to an equivalent stage, but the progress in this direction is impressive. Section I1 gives a birds eye view of the context in which transcription factors operate. In somewhat more depth, spatial aspects of transcription regulation are highlighted, as these are crucial to the understanding of how well-balanced specific changes in gene expression are achieved in uiuo. In the third section the current knowledge of B cell development in the fetal, neonatal, young adult, and older mouse is reviewed, with an emphasis on changes in gene expression patterns. Advances in understanding the marrow organ as inductive microenvironment for postnatal B cell development are also highlighted. The fourth section reviews how the experiments on transcription regulation enrich the insight in pathways of B cell genesis. 197 Copynght 0 10% by Academic Press, Inc All nghts of repmdiichon in any form resewed

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II. Transcription Regulation in VVO

The first few paragraphs serve as a quick introduction for the immunologist who is interested in catching up with the subject area of Section IV yet is not regularly concerned with the biochemistry of control of gene expression. Rapid advances have been made in this field in the last decade. A sketch of events upstream and downstream of transcription is followed by a brief description of mechanisms of transcription regulation, including a note on common motifs in transcription factors. Environmental influence is exerted sometimes by a substance that can diffuse through the plasma membrane and binds with intracellular receptors. More often, ligands interact with cell surface receptors, creating a signal which is transduced to the cytoplasmic portion of the receptor molecule or associated membrane molecules. Cytoplasmic signal transduction pathways are engaged. Various pathways exist, e.g., those engaged after cytokine stimuli are unlike those engaged after antigen binding (22, 109, 127). They all represent a balanced action of protein kinases and phosphatases (107). Receptors themselves may possess kinase activity, e.g., c-kit, a cell surface (“receptor”) protein tyrosine kinase expressed early in hemopoietic development, for which the steel factor functions as ligand. CD45 is an example of a cell surface protein tyrosine phosphatase (PTP), expressed in particular isoforms (B220) from an early stage in B cell development onwards. CD22 may act as a ligand for CD45 through recognition of its carbohydrate side chains (207).Cytoplasmickinases and phosphatases which appear crucial for B cell development, but which will not be discussed in this review include Btk (encoded by a gene mutated in Xid mice), Vav, Abl, Pim-1, and PTPlC encoded by Hcph which is mutated in moth-eaten mice. Somewhere down the line, proteins translocate from cytoplasm to nucleus. These can be activated protein kinases or cytoplasmic DNA binding proteins activated to translocate (100, 126, 127). Once in the nucleus they may enhance, indirectly or directly, the initiation of transcription of specific genes. All stages (including the initial one; see for review (94)) involve the formation and dissociation of macromolecular complexes, multiple copies of the same molecule or heterocomplexes with activator or inhibitor elements. This level of complexity on the one hand helps achieve specificity. On the other hand, factors are often members of a family of proteins with partially overlapping functions, creating a redundancy which makes a cell more robust in case of gene inactivation events. While in most cases the initiation of transcription is the most important stage of regulation, post-transcriptional mechanisms may operate to further control protein levels. Immature transcripts may be processed in a cell

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type- and differentiation stage-specific manner, resulting in proteins that differ in structure and function. Transport of mRNA from the nucleus is another regulatory step. A change in mRNA stability, affecting steady state levels of mRNA, is a rapid way of, in particular, down-regulation. Control of the decay rate of a specific mRNA can be achieved through trans-acting factors binding to cis-elements of mRNA (see (17) for recent review). Rapid switches in protein level are achieved through control of translation initiation based on a similar principle, of sequence-specific transacting factors binding cis-regulatory elements in the 5’ and 3’ untranslated regions of mRNA (47, 96, 173). This type of regulation is often used by cells early in development (283). The amount of a specific protein available is regulated by protein degradation mechanisms (102).Lastly there are many ways of regulating the activity of a protein. Transcription regulation in the strict sense refers to the action of proteins (transcription factors) that bind to gene control regions. General transcription factors are those required for RNA polymerases to start transcription. They are involved in the assembly of the transcription initiation complex at the core promoter of all genes (for those essential for the action of RNA polymerase I1 see Refs. 29 and 140). Rate of initiation of transcription of genes in eukaryotes is under the control of proteins interacting with short stretches of DNA of defined sequences that lie outside the core promoter. The latter cis-regulatory elements occur in gene-specific combinations. Gene control regions may extend up to thousands of nucleotide pairs away. They may act on the promoter directly, after looping of the DNA to bring them in close proximity. Alternatively, factor binding to such distant control regions results in changes in chromatin structure over a long distance, indirectly affecting transcription initiation (see later in this section). Differences between cells in patterns of gene expression are mediated by differential expression and activity control of proteins that bind to specific motifs in the gene control region outside the promoter. Protein domains have evolved that recognize and bind specific DNA sequences with high affinity. They commonly interact with the major groove of the DNA double helix. Examples are helix-turn-helix and zinc finger motifs. The leucine zipper is a DNA binding motif generated by dimerization of two proteins via coiled-coil formation of their a-helices. In the helix-loop-helix motif, one helix mediates protein dimerization while the other interacts with DNA. Transcription activator proteins often operate by increasing the rate of assembly of the transcription initiation complex. DNA binding and transactivation abilities of such proteins usually reside in separate domains. Selective repression of transcription can be achieved by proteins in the

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cytoplasm that prevent DNA sequence-specific activators from gaining access to the nucleus (e.g., inhibitors of E2A and of NF-KB). In addition, transcription factors can act directly as repressors of RNA polymerase I1 transcribed genes. Such factors lack transactivating domains and may compete with transcription activators for a particular DNA sequence. Others can repress by masking the transactivating domain of transcription activator proteins bound to the DNA, or by direct interference upon DNA binding with the transcription initiation complex (118). A particular type of protein can act as repressor or activator. Alternative arrangements of its binding sites in cis-regulatory domains in different genes may determine their action. The outcome may also depend on occupation of other cisregulatory motifs nearby. Furthermore, expression levels influence function. Often, a treshold level is operative. Another example is the transcription factor Krtippel in Drosophilu, which can act as an activator (at low levels, in monomer form) or a repressor (at high levels, as a dimer) (233). Repression of transcription over longer stretches of DNA covering more than one gene can be achieved by regulation of chromatin structure (142) (see also below). A. ARCHITECTURAL ASPECTS OF TRANSCRIPTION REGULATION

As is true for cells, a gene cannot be considered separate from its “microenvironment.” Spatial aspects such as the topographical arrangement of cis-regulatory motifs and the 3D structure of DNA influence transcription (79, 274, 300). 1 . Chromatin Structure Each cell type and differentiation stage is unique in the accessibility of gene control regions; only accessible cis-regulatory motifs can function in transcription regulation. Accessibility of cis-regulatory elements of a gene is highly dependent on the chromatin structure, with the nucleosomes as basic building blocks and higher-order structures imposed on it (25). For instance, heterochromatin is a highly condensed form that renders it inactive in terms of transcription. This is possibly caused by special proteins packaging the DNA. Chromatin structure can be modulated in multiple ways, and such modulators (e.g., the high-mobility group proteins which induce a bend in the DNA (79)) may exhibit cell-type specific expression patterns. As a consequence, a particular gene may be open to transactivation in one cell type but not another. Relevant for Section IV is the notion that expression assays using target gene regulatory elements not in their native chromatin context are of limited value in predicting in uiuo mechanisms. This applies not only to in vitro assays, but also the in uivo assay of

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transgenesis, where the site of integration in the host genome (a “random” event) influences the activity of the transgene (position effect). 2. Nuclear Scaflold Another notion of importance is that the nucleus contains a protein scaffold, the nuclear matrix, linked to lamin proteins of the nuclear membrane which in their turn are linked to the cytoskeleton. The nuclear matrix is defined as the structure that remains after DNase and RNase treatment and high salt extraction, leaving relatively insoluble proteins. The nuclear matrix harbors many transcription regulatory proteins, such as sequencespecific DNA-binding proteins (95), deacetylation enzymes capable of nucleosome reorganization, and components of the basal transcription machinery; thus providing a scaffold for the spatial organization of these components. Also, particular AT-rich stretches of DNA can bind to the nuclear matrix, called matrix (or scaffold) attachment regions, which may play a role in higher order chromatin structure and changes therein. The nuclear matrix can provide a crucial link between the environment of a cell and pattern of gene expression (25). Ligand-cell surface receptor binding induces receptor clustering and generates biochemical signals. These by themselves may not be propagated properly. Cytoskeleton filaments associated with the cytoplasmic domains of receptor (-complexes) are modified and reorganize (e.g.,242), and through this evoke architectural changes in the nucleus via its association with the nuclear matrix. These mechanical changes would allow a proper interpretation by the nucleus of the biochemical signals generated. Brought together in a spatially meaningful way, incoming signalling molecules, transcriptional regulators, histone deacetylases, and the basal transcriptional machinery would promote the assembly of a functional transcriptional complex on the gene. Nuclear organization may best be viewed as highly dynamic, with enzymes (often kinases) capable of modifjmg proteins in such a way that their subnuclear distribution or function changes. Such modifjmg enzymes themselves exhibit differential patterns, sometimes cell cycle stage-specific,of association with the nuclear matrix (e.g., casein kinase 2 (270)). 3. Topographical Distribution of Transcription Regulator Proteins in the Nucleus

Nuclear factors may be compartmentalized within the nucleus, not only in terms of matrix attachment (see above), but also in terms of “domains.” A high degree of spatial organization for the components involved in pre-mRNA processing (splicing domains) has been described (95). These domains are visualized in situ by confocal laser scanning microscopy combined with identification (using labeled antibodies or riboprobes) of specific

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proteins and small nuclear RNA species. Comparable in situ information on the topography of transcription factors is rather limited. Those studied, when in the nucleus, show a rather widespread distribution pattern when identified by specific antibody and regular fluorescence microscopy (e.g., c-fos (286) and the p65 subunit of NF-KB (154) in NIH 3T3 cells; human CATA-3 in T- and neuroblastoma cell lines (304); E2A in cultured rat BM cells (112)).One exception is c-myc, localized in splicing domains in human colon adenocarcinoma (262), but appearing rather diffuse in the nucleus of NIH 3T3 cells (286). Another remarkable exception is the product of pml, which is located in discrete spheres, 0.3-1 pm in diameter, that are associated with the nuclear matrix (35). Ptrd has been identified first as one of the genes involved in the t( 15;17) chromosomal translocation of acute proEyelocyte leukemia; the other gene involved encodes retinoic acid receptor a (24, 48, 162). While wild-type PML is located in nuclear domains, cells which produce wild-type and PML-RARa fusion proteins show a disruption of this pattern. Pml consists of nine exons and by alternative splicing generates multiple isoforms, all of which are predicted to contain DNA binding and protein dimerization domains at the N terminal, and one or more phosphorylation sites at the C terminal region (61, 78, 149, 305). Pml is transcriptionally activated by interferons a,p, and y (150). At least two other proteins normally colocalize with PML: SplOO/ND, with homology to various viral transcription factors, and NDP52, with multiple protein-protein interaction motifs (141, 265). The number and size of PML domains is cell type- and differentiation stage-specific (147) (Fig. 1). For instance, in the B lineage, weakly p+ pre-B cells express a small number of domains, mature small B lymphocytes in a moderate number, and plasma cells in a moderate to low number. PML domains, however, are undetectable in germinal center blast cells (Fig. lb). The function of PML domains in the nucleus is not known. PML can perform as a transcription repressor, assessed by cotransfection with CAT reporter plasmids fused to promoters of a limited number of genes (190). Transfection experiments using transformed cell lines show an ability of PML to suppress proliferation (139, 160, 190). However, among populations of cells in normal organs with a high growth index, PML domain levels vary widely (147). In uiuo, cells respond quite rapidly to environmental factors such as hormones by a change in number and apparent size of PML domains (139, 147, 272). Disruption of the domains is associated with pathological conditions (virus infection, heat shock) (141, 169, 170, 223). Fibroblasts from PML mutant and wild-type mice are equally resistant to viral infection upon interferon-a treatment (150),even though pml

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203

FIC.1. Cell-type and differentiation-stage-dependent expression of PML domains. Imrnunoperoxidase staining of rat frozen organ sections using HIS55 mAb (147).Counterstain: hematoxylin. (a) BM. Note the variation between cell types in size and number of immunoreactive nuclear foci (the more intensely stained appear as black rings with a clear center due to phase contrast illumination (large arrows)). Arrowheads point to cells with an apparently low level of foci. Small arrows indicate foci that remained attached to chromosomes in mitotic cells. X1120. (b) Spleen. Note absence of nuclear foci in germinal center (GC) blast cells. Corona (c) cells were richer in foci than lymphocytes in the periarteriolar lymphocyte sheath. A: arteriole. Brightfield microscopy, x280.

is an interferon-responsive gene. Further phenotypic information about PML loss-of-function mutant mice has not yet been published. It would be valuable if more extensive data become available on the subnuclear distribution pattern of the currently known transcription regulator proteins in various normal tissues and in response to stimuli. Other components of the PML domains may be identified, and possible other topographical entities related to transcription regulation may be revealed. A transcription factor worth following-up for colocalization with PML is c-myb, localized in discrete domains in the nucleus of human T cell lines (60). 111. B Lymphocyte W o p m e n t in Vim in the Normal Mouse

Different approaches have been used to study the role of transcription factors in B cell genesis. Through a variety of in vitro assays, potentially important factors have been identified. In recent years the technology for creating mice with mutations in specific genes has allowed study of their role in vivo. This section is an update on those aspects of B cell development in normal mice that appear immediately relevant for the assessment of mice with a loss-of-function mutation in transcription factors (Section IV).

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DAVINA OPSTELTEN

Such aspects include genesis of B cells in ontogeny and in adults, with an emphasis on gene expression patterns. The marrow organ, postnatal site of B cell genesis is considered in depth for its structure, and for its function as supportive microenvironment. Other fascinating issues, such as the regulatory mechanisms governing Ig gene rearrangement and expression and Ig repertoire development, are not dealt with here. A. B LYMPHOCYTE GENESIS I N THE MOUSEEMBRYO

1 . Sites of Origin Embryonic stem cells derived from the inner cell mass of blastocysts (Embryonic Stage Day 4 (E4)) are totipotent. The developing mouse embryo has been examined for the earliest cells that are restricted in their potential to hemopoiesis including B cell genesis. Determination or commitment may occur before any known sign of differentiation is overt. Hemopoietic or B cell committed cells are identified on the basis of their anatomical location and surface molecule profile. Dissection, and/or fluorescence activated sorting of cells from dissociated embryos or embryonic tissues, is followed by functional assays. The adequacy of functional assays (in vitro development and transplantation) is a complicating factor in trying to assess the earliest stage of determination for hemopoiesis. A positive outcome (the generation of hemopoietic cells including B lymphocytes) is indicative of functional ability, but need not imply that the cells assayed actually do develop (only) in that direction in vivo. Absence of potential can reflect true functional restriction, but may also be a consequence of suboptimal assay conditions. Assays have been refined to the point that B cell generative potential can be demonstrated experimentally in embryonic stem cells by subjecting them sequentially to different culture conditions and transplantation (201). Information on hemopoietic commitment in postimplantation embryos up to E7.5 is lacking. The issue of determination from the four pairs-ofsomites stage (early E8) up to E12, when overt signs of B cell genesis have long been recognized in the liver (Table I), has been addressed by various studies in the last few years (see also (130) for review). Cells with B precursor activity have been identified in these studies by a variety of assays (45, 167,217). One assay measures the ability of single cells dissociated from a particular embryonic site to generate mature B cells in vitro, using B cell development-supportive culture conditions (stromal cell lines plus cytokines which at least include IL7). When done under semisolid conditions, the response of single cells is assessed. Readouts include the number of cells with B phenotype (B220, sIgM) and ability to secrete Ig upon LPS stimulation (45, 46, 217). Also assessed are intermediate signs

TABLE I OF MURINEB LYMPH~CV~E DEVELOPMENT IN VNO:EARLIEST STAGE OF DETECTION IN FETALLIVER^ HALLMARKS

E 10 Colonization by hernopoietic precursors (ckit+)(218)

Ell

El2 AA4.1tSca-l+cells(129) that can generate Blineage cells in oitro when cultured on strorna (or IL-11 + SCF(129) + IL7(46) B220' cells(177) IgH gene: DJ rearrangement(46)

Ikaros mRNA(71)

El3

p + ~cells( 1%)

IgH gene: VDJ rearrangements (45) RAG-1 mRNA(167, 189) A5 mRNA(167, hlx I ~ R N A ( G ) ~ 189)

El4

E 15

El6

El7

Sterile IgH K gene transcripts(152) transcripts(152) IgK gene: VJ Surface IgM' cells rearrangements (124, 148) (45, 46, 226) BSAP ( p d ) mb-1 mRNA(ll)b mRNA(2) B29 mRNA(1l)b PU.1 I ~ R N A ( ~ ~ c-rnyb ) ~ mRNA(GO)* CD19 mRNA(2)

B cell development in fetal liver occurs in a single wave; identity of cells expressing transcripts other than those of Ig genes has not been established Earlier time points not tested.

206

DAVINA OPSTEIJEN

of B cell development such as Ig gene rearrangement. Another technique is grafting dissected but intact tissue under the kidney capsule of IgM congenic SCID mice, reading out the donor IgM levels in serum 2-3 months later (for review see (167)). In the yolk sac, hemopoietic precursors are detected from early E8 (4 somite pairs) (43, 217). The issue of whether hemopoietic potential is retained by cells in the embryo proper independent from the yolk sac appears unresolved. The question can only be reliably assessed before the establishment of blood connection between yolk sac and embryo, according to Dieterlen-Lievre et aZ. (73, 74) at 7-8 pairs of somites, according to Cumano et aZ(43)at the stage of 8-10 somite pairs (E8-8.5), and according to Palacios and Imhof (217) after 12 pairs of somites (E9). Using cells suspended from whole embryos at 3-12 pairs of somites stages, Palacios and Imhof have not detected lymphoid differentiation potential in the embryo proper. Dissecting the paraaortic splanchnopleura or the splanchnic mesoderm from which it arises (for a schematic drawing of the location of this tissue see (73)), hemopoietic progenitors capable of generating B lineage cells have been detected in the 10-18 pairs of somites stage ((74); B1 cells only) and in later studies, in the stage of 10 pairs of somites (73) and even in the stage of 4 pairs of somites (43). Using the grafting technique, at E8.5-9 (10-18 somite pairs), the paraaortic splanchnopleura is the only intraembryonic site with capacity to generate cells secreting IgM (74). This has been confirmed by an in vitro limiting dilution technique to quantify B progenitors (73). If it turns out that, at the stage of 4 pairs of somites, the splanchnic mesoderm is the only site in the embryo proper where B cell-generative potential can be demonstrated, yolk sac-independent determination for hemopoiesis is highly likely to occur. Around E l 0 (28-32 pairs of somites), the liver is colonized by hematopoietic precursors, presumably derived from the yolk sac and paraaortic splanchnopleura (192). This organ sustains hemopoiesis up to 2 weeks after birth, with a predominance of erythropoiesis and myelopoiesis. Table I shows that signs of B cell genesis become overt in the liver from E12. Subsequently, also due to hemopoietic stem or precursor cell migration, a variety of sites appear to be-mostly transient-locations of B generative potential (for review see (210)),such as the placenta and peripheral blood (E10-13) (178,179),and the omentum (E13-14) (257).In placenta, development appears not to proceed to the stage of p chain synthesis, as p + ~ cells have not been detected at that site (124).In fetal thymus, B generative capacity is detected at E12, rapidly declining afterwards. At E15-16, the thymus still contains cells transformable by virus with a B lineage phenotype (133). In the spleen, c p + cells are detected from E l 5 (210). At E15-16,

B LYMPHOCYTE DEVELOPMENT IN VrVO

207

vascularization of BM starts and the organ is colonized by B progenitor cells (204).

2. Origins of B1 Cells and B2 Cells The first sites where B1 (IgMhi(IgD'")CD5+) cells have been detected in normal development are in the neonate, in the mesenteric lining of the small intestine (N7), peritoneal cavity (N8), and spleen (peak at N9) (87). Cells expressing CD5 on their surface are undetectable in fetal liver, but low levels of CD5 mRNA are detected by RT-PCR (91). B2 cells (sIgM'CD5- cells, a population which increases in expression of IgD upon further maturation) normally first arise in fetal liver at E16-17 (45, 210). Many lines of evidence suggest that B1 and B2 cells arise in development via independent pathways (for review see (167) and (92); (276)).Additional evidence comes from mice with mutant A5, which exhibit a much more severe delay in B2 cell genesis than in B1 cell genesis (136).Other examples of differentially affected B1 and B2 cell genesis in mutant mice are given in Section IV of this review. The embryonic site where B cell genesis is initiated, rather than limitations intrinsic to early stem cells, may be a lineage-determining factor. For instance, in the grafting assay the paraaortic splanchnopleura gives rise to B1 cells only (167). Conventional B cells are absent in the host periphery. The recipient spleen contains near normal levels of (donor) IgM plasma cells, compatible with other lines of evidence that these arise mainly from B 1cells (74). However, when assayed for their potential in uitro, cells dissociated from the paraaortic splanchnopleura generate both B1 and B2 cells (73). This suggests that the microenvironment of this early site is particularly inducive to B1 cell generation. Postnatally, BM cells gradually lose the capacity to generate B1 cells, as assessed by cell transfer into adult hosts. How they would perform in a fetal or neonatal environment is unknown (92). 3. Molecular Hallmarks of Prenatal B Cell Genesis Table I summarizes the current understanding of the onset of B cell genesis as it occurs in a single wave in the liver of mice before birth. It also includes gene expression data that come from studies discussed in Section IV. The methodologies that have been used are highly varied and have improved in sensitivity and resolution over the years. Some markers analyzed with methods of low resolution and sensitivity (e.g., Northern blot (152))may require reassessment. Using semiquantitative RT-PCR, ckit and RAG-1 mRNA are detected in paraaortic splanchnopleura from E9-11. In liver c-kit mRNA is detectable from E10, the day of colonization with hemopoietic precursors, and RAG-1 mRNA from E12. In omentum c-kit and RAG-1 mRNA are expressed at E13-14. A5 mRNA is detected

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DAVINA OPSTELTEN

from E l 3 in liver and E l 4 in omentum (167). The lack of detectable A5 mRNA in paraaortic splanchnopleura is compatible with the notion of a AS-independent pathway early in ontogeny for the generation of B1 cells (see above), AA4.1 is expressed by intraembryonic hemopoietic stem cells at E8.5 (10 somite stage) in the absence of Scal (44). In E l 2 liver, B generative capacity on stroma plus IL7 is among the populations that are AA4.1+ScalSB220- (H chain gene in germline) and AA4.1'ScalSB220+ (H chain gene in DJ rearrangement) (46). B220' B precusor cells in fetal liver are similar to those in adult BM with regard to AS, Vpre-B, Rag-1, and Rag-2 expression (see below in section on adult mouse), but lack expression of TdT and precursor lymphocyte-regulated myosin-like light chain (for review see (91)),and MHC class I1 (146). Data on the onset of CD43, HSA, BP1, and IL7R expression, used to classify precursors in adult BM (see below), in fetal B cell development appear to be lacking. IN THE YOUNGADULTMOUSE B. B LYMPHOCYTE GENESIS 1 . Molecular Hallmarks of B Cell Genesis in the BM The work of many investigators has contributed to a now rather unified model of B lymphocyte genesis in BM of mice aged 5-12 weeks (59, 91, 161, 181, 212, 236, 239). Table I1 uses Hardy's model as the guiding scheme, because it has been used most often to evaluate the mutant mice discussed in Section IV. Especially for fractions beyond stage A, markers other than CD43, HSA, and BP1 (e.g., CD25) have their particular advantages for initial characterization of BM B cell development (181,237). The model is of course a simplification, as not all cells in each fraction have the same phenotype, transition stages must exist, and not all cells need to follow an identical gene expression program. Many of the genes signifylng B cell development in ontogeny and adults have been tested for their role in vivo by generating loss-of-function mutant mice (Table 11). In Section IV, this information is used to interpret the phenotype of mice mutated for transcription factors that may regulate the expression of the B cell developmental genes. In the analysis of BM, many assays are performed at single-cell level, in particular for protein expression on the surface and in the cytoplasm (flow cytometry) and more recently also for the status of Ig H and L chain gene rearrangements (56, 271). With regard to specific mRNA detection in BM, single-cell analysis is not yet commonly applied (the nearest is RTPCR analysis of FACS-sorted cell subsets). Yet, it is often important to know, particularly in the context of transcription regulation, whether genes are transcribed to a high steady state level of mRNA in a few cells or to a low level in many cells. We have optimized in situ hybridization (ISH)

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209

for high-resolution, sensitive detection of specific mRNA from single-copy genes in unstimulated cells isolated from organs of normal animals (208). Figure 2 illustrates the power of this technique when applied to cells isolated from BM. Section IV will show that, for initial characterization of mutant mice, two-color flow cytometric analysis of cells suspended from the marrow of long bones is used most frequently. Sophisticated multiparameter analysis based on four fluorochromes, and/or staining of sorted subsets, would be required to resolve all subsets. The simplified version based on surface Ig and B220 reveals the incidence of B-progenitor cells (fractions A-D) and B cells (fractions E-F). Further subdivision using CD43 identifies B220tCD43+sIgM- cells as earliest progenitor population (fractions A-C). Fractions D-F normally contain B lineage cells only. Fractions A-C', while including all B progenitors, may also contain precursors of other lineages, e.g., the NK lineage (236). In BM of mutant mice, the presence of B220 and CD43 is thus not sufficient to draw conclusions on B progenitor incidence. One other reason to follow up the nature of such cells is that mutations may have dysregulated the expression of these two markers. Morphology as revealed by light microscopy of cytocentrifuged, MayGrunwald-Giemsa-stained cells from whole marrow or FACS-sorted cells, or by combined phase-contrast and fluorescence microscopy of BM cell suspensions double-labeled for B220 and CD43, may give valuable insight; however, it is rarely used. Morphological hallmarks of rodent hemopoiesis are known in detail (279). Informative additional assays are those assessing Ig gene rearrangement status, transcripts from germline and rearranged Ig genes, and Ig heavy chain protein expression in the cytoplasm. Genes, the expression of which is closely related to the processes of Ig gene rearrangement and expression, include RAG-1 and -2, surrogate light and heavy chains, and proteins that make up the B cell antigen receptor complex, i.e., mb-1 (Ig-a) and B29 (Ig-P). Expression of these proteins in B220t cells may be taken as confirmation of their B lineage nature. TdT is a helpful additional marker for fractions B-C, but not all B cells arising in adult BM may have gone through the expression of this enzyme. Functional assays such as growth in uitro under normally B generative conditions, or repopulation of SCID or RAG-2 -/- mice, may confirm B lymphocyte forming capacity among populations thought to contain B precursors. 2. B Lineage Population Dynamics The population kinetics of B lineage cells are well described ((211,212); Table 11). In the B220' population, four cell divisions are estimated to occur before the stage of p chain expression in the cytoplasm, and one division in the large cells that express cytoplasmic p chains in the absence

TABLE I1 B LYMPHOCYTE DEVELOPMENTAL STAGES I N YOUNG ADULTMOUSEBOXEMARROW Parameter

B

A

C

C'

D

E

F

~~~

E 0

B22Ob CD43 HSA' BP-ld Ig heavy chain gene (55, 56, 271)b Ig heavy chain protein Ig light chain geneb Ig light chain protein A5(125, 181, 252, 299)b' Vpre-B(125) Mbl RAG-1,-2(91,155, 297)b

Tdp

Size Cell cycle status IG7Ra(261)' Responsiveness to stromal cells + IL7g Responsiveness to IL-7 c-kit(59, 230) AA4.1(175)

LOW

LOW

LOW

GL

+

+ +

High

cpGL &A-

-

High

High

LOW Intermediate Intermediate High LOW to negative Intermediate to low Low to negative High High to intermediate Intermediate Intermediate

GUDJNDJ DJNDJ'

+

VDJ

+

cpGL

cc1GL

Partially cp+ GL

VJ

+ +

+ +

-

&A-

&A-

+ +

+ +

KIA+/+/-

+

High

High

HigMow LOW

Large Cycling

Large Cycling

Large Cycling

LOW LOW

+ + +

-

LOW

+

+

+ +

+

+

-

Large Cycling

+

LOW

-

VDJ

VDJ

cF'sIgM&A-

+

Lowhigh Small

Postmitotic

+

-

VDJ

sIgM+D-

sIgM 'IgD'

VJ

VJ

-

-

&A+

dA+

+

Very low

+

-

Small Postmitotic -

-

Small Postmitotic

-

-

CD4(155)b + and High Low bcl-2b MHC class I1 (93, 146, 269)b CDW(IL-2Ra)(37, 237) CD40". ' CD191

+

+

-

-

-and+

-and+

-

+

+

Largely -

+

+ +

+

+

+

+

+

+ +

~

N

c-

This table is based on (59,91, 161, 181, 212,236, 239) and references therein. Relevant studies that have been published since are referred to in this table and the footnotes below. Rough indications of frequency in normal mouse BM are: 5% for fraction A-C'. 10% for fraction D, 10% for fraction E, and 5%for fraction E; for detailed information on each parameter with regard to frequenciesof expressing cells in each fraction and levels of mRNA and protein expression, see original references. Overview (1994 and earlier) of references on targeted mutation of gene in (26-28); reviewed in (52, 82, 180, 221). Targeted disruption of HSA gene results in a modest inhibition of early B cell development (199). Targeted disruption of BP-1 gene results in no obvious disturbance of early B cell development (158). mRNA detected by RT-PCR (28);protein detection is complicatedby (i)variable performance of specificantibodies and (ii) induction of surface expression in oitro.

fTargeted disruption of IL7Ra gene results in severe block in early B cell development (220). g IL3 can play a role similar to IL-7 when starting culture with B220+c-kit+ cells purified from BM (298). Targeted disruption of CD40 gene results in defects in germinal center formation and Ig isotpe switching (34, 128). ' Reported in (236) but original references not identified. J Targeted disruption of CD19 gene results in lack of formation of B1 cells, and deficienciesin mitogen and T cell-dependent responses of B2 cells (58, 229). Fraction C is enriched for cells with 2 nonproductive VDJ rearrangements (56).

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DAVINA OPSTELTEN

of Ig light chains. This would represent a daily output of 3.5 X lo7 cells in the whole marrow organ. Descendants of large cytoplasmic p+ cells exhibit a smaller cell size and exit the cell cycle. It is estimated that only one quarter of the cells produced in the precursor B compartment survive and proceed via a phenotype of postmitotic, small, cytoplasmic p+ to that of newly formed B cell (fraction E). The rest is lost from the marrow, most likely by apoptosis in situ. Changes in these dynamics may have profound effects on the function of the immune system, such as Ig repertoire development and propensity to tumor development (253).SCID mice with defective B cell development from fraction D onward still exhibit normal proliferation rates up to the stage of the defect (216), suggesting that B cell developmental stages beyond fraction C, and their factors, do not normally exert simple feedback. However, the production rates are quite sensitive to macrophage-mediated environmental influence. Mutant mice may be assessed in various ways for perturbations in kinetic parameters. The easiest approach is a measurement of the proportion of cells in DNA synthesis (S-phase index) or with replicated DNA (S G2), as an indication of whether growth indices of normally proliferating populations (fractions A-C') are unusual. More elaborate kinetic techniques, such as stathmokinesis and continuous infusion of a labeled DNA precursor, are required to assess production rates in proliferating populations, and transit times through normally postmitotic compartments such as small pre-B cells (fraction D) and B lymphocytes (fractions E-F) (42, 65, 165, 212, 213, 215, 224). Kinetic measurements should include total cell number recovered from BM of multiple long bones. This allows stress assessment, but also the calculation of production rates in terms of absolute numbers, which is important to know as the periphery depends on the total marrow output.

+

3. Histophysiology of the BM Organ B cell genesis is initiated in fetal development without the proximity of bone. However, from 2 weeks after birth the cavities of bone are the only site of B cell formation, while erythropoiesis and myelopoiesis also proceed in red pulp of spleen. In mice with mutations that cause a severe reduction in marrow space (osteopetrosis, as found, e.g., in c-fos loss-of-function mutant mice; see Section IV), extramedullary sites appear not to compensate for the reduction in B cell genesis. To understand how B cell genesis is regulated, insight in the structurally and functionally complex marrow organ is vital. Marrow-containing ossicles can be generated subcutaneously, with influx from local cells, by transplanting devitalized demineralized bone matrix (21, 227), indicating that bone matrix is a guiding element in marrow

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213

formation. In ontogeny, the marrow organ develops as cavities expand during bone growth and remodelling. Marrow stromal development occurs in concert with hematopoietic cell entry and development (3, 174). In fully formed marrow (156, 211, 214, 293), an intricate network of venous sinusoids organizes parenchymal tissue into irregular cords (Figs. 3a, 3b). Hemopoietic cells are generated in parenchyma and leave the marrow through sinusoidal walls. Many components contribute to the hemopoietic inductive microenvironment. Stromal elements in parenchyma include bone-lining cells, reticular cells, adipocytes, macrophages, mast cells, and vascular walls. Sinusoidal walls, the barriers that regulate hemopoietic cell entry and egress, consist of adventitial reticular cells, basement membrane, and endothelium (see also (1)). Hemopoietic cells themselves are likely to play an active role in their own genesis and that of other lineages, through direct interactions or through their “instructions” to the stroma. Stroma may transform as a consequence of abnormalities in hemopoietic cells (76, 216). Extracellular matrices, associated with cell surfaces, are built up from molecular contributions by the stroma, hemopoietic cells, and the blood. Matrices consist of locally produced and systemic growth factors (including hormones such as insulin-like growth factor-1 (IGF-1) (39)) and inhibitors (e.g., sex hormones, for review see (135))trapped in networks of extracellular matrix proteins, proteoglycans, and glucosaminoglycans. Innervation can also be considered a microenvironmental component. In rat BM noradrenergic fibers enter along blood vessels, and branch sparsely into marrow parenchyma (62-64). Many different microclimates may exist in BM as a consequence of the spatial organization of the various types of relatively immobile stromal cells (see, e.g., (163, 241)). However, with mobile cells contributing to the microenvironment, such “niches” may be of transient and flexible nature in terms of particular function. Several studies (51, 307) indicate that certain stromal cell types in BM are rather homogeneous with regard to their functional potency, and that their selective action depends on external signals. The location of /3l integrin at the point of physical contact between stromal cells and undifferentiated cells in oivo (113,301)may suggest that polarization is one mechanism creating microenvironmental flexibility. The spatial distribution of newly formed B cells and their precursors in mouse and rat BM has been described (15,97, 114,214). A preferred site for B cell progenitors is the subendosteal area of marrow. Newly formed B cells occur scattered throughout the parenchyma. Thus, B lineage cells appear to move away from the periphery during maturation (115).They exit the marrow via sinusoidal endothelium. Before newly formed B cells are discharged into the blood stream, they may reside for a while in the sinusoidal lumen (the “sinusoidal loading” phenomenon).

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DAVINA OPSTELTEN

The bone-lining cells (endosteum) (Figs. 3a, 3b), being a unique feature

of marrow, may be a most crucial fostering factor for progenitor B cell

proliferation and differentiation. Morphological studies in dog ( S O ) and man (110) and studies of primary cultures established from endosteal cells in rat (187) and man (266) indicate that a majority of cells are (progenitors of) osteoblasts and osteoclasts. Cultured human osteoblasts contain mRNA for several cytokines. They produce functional G-CSF (266), and through that may support myelopoiesis, the other hemopoietic lineage with precursors preferentially located near the bone. We are currently characterizing the endosteum of mouse and rat for the expression of genes encoding proteins potentially involved in B cell genesis, using ISH and immunohistology on sections of intact long bones. The endost of mouse femora strongly binds an antisense riboprobe of BP1 (Fig. 3c; Veuger and Opstelten, in progress), the surface metalloprotease best known for its expression in B precursor cells. As BP1 would act to enhance B precursor proliferation by inactivatinginhibitors (295),endosteal BP1 expression may be an important factor in the B cell genesis-favorable microclimate adjacent to the bone. With regard to cytokines, we are in the process of locating the cells in intact marrow that synthesize IL-7, proved to play a crucial role in vivo in B cell genesis (77, 220, 261). It will also be of interest to indentify the source in BM of other molecules described to support pre-B cell growth in vitro, such as c-kit ligand (also known as steel factor, stem cell factor, mast cell factor), IGF-1 (39,72),BST-1 (a surface molecule with homology to CD38) (122), and PBSF, a soluble mediator (193). With the current insight in the BM organ, histological analysis of BM (Figs. 3a and 3b) is particularly helpful as an initial assessment of possible microenvironmental abnormalities, often suspected in mutant mice such as those discussed in Section IV. The fact that BM histology is technically and analytically demanding may have prevented common use of this approach so far. Other features that may be detected initially in situ, are alterations in proliferation indices (Fig. 3b) and death rates of hemopoietic cells (Fig. 4a; (116,216,253)).A high steady-state level of cell death may be associated with the blocks in heinatopoiesis and/or B cell development that are observed in several of the mutant mice discussed in Section IV. As already indicated above, when combined with antibodies or probes detecting specific protein or mRNA (Fig. 3c), respectively, BM histology becomes a particularly valuable tool. Perfusion of radiolabeled antibody may be used for detection of surface antigens in situ (15, 114, 216). Techniques for immunohistology of sections from frozen femora of mouse (and rat) have been described in detail (98) (further applied in (97) and (99)), as have those for ISH (208). Among others, insight in transcription

Fig. 2. Analysis of cells suspended from BM for morphology and specific mRNA expression. Digoxigenin (DIG)-labeled riboprobes generated from a 900-bp PstI fragment of murine Cp, alkaline-phosphatase anti-DIG antibody, and NBTBCIP as chromogedsubstrate (208)were used to detect IgM heavy chain mRNA in cytomntrifuge preparations of BM cells isolated from a 4week-old mouse. Phase contrast microscopy at high power was used to visualize both the reaction product (appearing intensely and shining brown) and the morphological features. (a) High levels of antisense riboprobe binding were evident in the subset with lymphoid morphology (in a frequency corresponding to that of IgM heavy chain protein-expressing pre-B cells, B lymphocytes. and plasmablastd-cells, not shown). Note that the apparent level of pmRNA in larger lymphoid cells (large pre-B cells; large arrows) is similar to that in small lymphoid cells (small pre-B cells and B lymphocytes; small arrows). Insets: (x) a positive large cell in mitosis, presumably a representative of the dividing large pre-B cell subset; (y) a strongly positive cell with plasmablast morphology; (2) a strongly positive cell with plasmacell moThology. (b) Sense riboprobe at matched concentration did not bind to any of the BM cells. M,cells from the myeloid lineage; L, lympoid lineage cells: E, erythroid lineage cells; Mit. cell in mitosis. Original magnification: ~ 8 7 5 Courtesy . of A. Y. H. Kwong.

Fig. 3. BM separately analyzed in situ for morphology and specific mRNA. A femur of a 6-weekold mouse, fived ex sttu in paraformaldehyde. decalcified, and embedded in paraffin (our routine procedure for ISH). (a) and (b): A 4ym-thick section stained with hematoxylin and eosin. (a) With low-power microscopy, major features evident are the bony cortex (B), the endosteal lining (arrowheads), and in the marrow: the central vein (C), sinusoids (large arrows), and megakarycocytes (small arrows). Adipcytes are rare at this age. Some mechanical damage always arises from the preparation of the organ for histology,in this case a crack across the central vein and partial dissociaton of marrow from the endost. ~ 7 0(b) . A highpower photograph of subendosteal manow, showing the endosteal lining (arrowhead), arterioles (*), sinusoids (S),lymphocytes (large arrows) some of which are in a sinusoid, a cell in mitosis (curved arrow), a megakaryocyte (M), and myeloid lineage cells (small arrows). This subendosteal area is poor in morphologically recognizable erythmid lineage cells. ~5.25.(c) Detection of transcripts from the BP1 gene, using a DIG-labeled antisense riboprobe generated (208) from a 3.5-kb EcoRI cDNA fragment covering the f d coding region (a kind gift of Dr. M.D. Cooper, HHMI,Birmingham, AL). Note the strong staining of the cytoplasm of endosteal cells (arrowheads point to their nuclei). P, parenchyma: S, sinusoid; B, bone. Original magnification: ~ 8 7 5Courtesy . of M. Veuger.

Fig. 4. Detection of apoptosis in situ in BM. A mouse was sacrificed 6 hr after ip injection of an antibody against mouse fas (from Pharmingen, U S A . ) . The mouse was moribund, with maqsive apoptosis in the liver (as has been reported (203)).(a) In BM the incidence of apoptotic bodies was, as normal, low (arrow points to the only convincing sign of nuclear fragmentation in four sections, near the endost (arrowheads)).~ 8 7 5 (b) . At that same time, many cells in the subcapsular area of thymus were apoptotic. Those engulfed hy phagocytes (arrows; arrowhead points to the nucleus of a phagocyte) stained strongly in a reaction (orange-red color) detecting double-stranded DNA breaks (264). IC, inner cortex. Counterstain: hematoxylin. Original magnification:x875. Courtesy of J. Bendoor.

I3 LYMPHOCYTE DEVELOPMENT IN VlVO

215

Factor expression in the organ stroma and hemopoietic cells, information that is now often lacking (see Section IV), should result from the application of these techniques. GENESIS IN NEWBORN A N D JUVENILE MICE C. B LYMPHOCYTE As evident later in this review, many mutant mice are assessed for dysregulation of B cell development in the first few weeks of life. This developmental stage of nornial mice is least well studied, with regard to cell surface marker profile and correlation with Ig gene rearrangement status and Ig gene expression. Gene expression patterns may still differ considerably from that in adult BM. For instance, B lymphocyte populations in the first month of life gradually increase in the proportion expressing MHC class I1 molecules (93, 146, 269), a feature of all sIgM' B cells and most pre-B cells in adult BM. At birth, the BM is already a rich source of cells with characteristics of fractions D-F in adult mice (B22OtCD43-) (146), but the B precursor nature of the sIgM- subset in young mice (e.g., p chains in the cytoplasm) has not been confirmed. The liver declines rapidly as a site of B cell genesis. - are still detectable, as foci separated by In the first 2 weeks, p + ~cells liver parenchymal cells, and not mixed with erythroid or myeloid cells (80). These foci appear to be clonal (244). In the peritoneal cavity at N10, a very low number of B220'sIgM- cells are detected, rapidly declining in frequency and also total numbers in the first month (146). The spleen of newborn mice is rich in progenitors of the B lineage as tested using in uitro assays (146, 238). Splenic B220+sIgM- cells peak in numbers at Day 10 after birth (NlO), declining to insignificant numbers beyond 1 month of age (146). However, it is unclear to what extent B lymphocytes are actually generated in that organ, vs immigration of newly formed B cells from the BM. By N7, very few cells containing IgH p chains in the cytoplasm only are detected in the spleen (121). D. B LYMPtIOCYTE GENESIS IN OLDER MICE The fiinction of the immune system changes substantially with aging. With regard to the B lineage, from 4 to 32 weeks of age the frequency of total B220' cells is reduced from 50 to 22%, and of B220'c-kit' cells from 3 to 0.4%. Correspondingly, by 6-8 months of age the frequency of clonable B precursors in BM is reduced to 5-10% of that found in the neonate (238),yet by 2 years of age such cells are still detected in BM. Age-related reduction in IL-7 responsiveness has also been reported by others (for review see (240)). RAG-1 gene expression in BM declines after 5 months to a minimal level by 10 months of age (18, 19). The Ig repertoire also changes with ageing, with less usage of 5' Vh gene families (for review

216

DAVINA OPSTELTEN

see (103)). Hodes further reviews the evidence that T cell-dependent responses are reduced with age in titer and affinity. This may be explained by the recent findings of a lack of B7-2 costimulatory molecules and somatic hypermutations in histologically normal germinal centers formed by ageing mice in response to T D antigen (103, 184). IV. Transcription Factors in 8 Lymphocyte Genesis

The transcription factors acting on the genes listed in Tables I and I1 must exhibit differentiation-stage dependent activity, and this may be (but need not be; see Section 11) a consequence of regulated expression levels of these factors. For some of the transcription factors to be discussed, onset of expression in fetal liver is known (Table I ) . However, the types of expressing cells have not been characterized. Expression patterns of transcription factors in normal BM B lineage cell subsets (Table 11) are virtually unknown. The interesting situation has thus arisen where importance of such factors for B cell development in uivo has been discovered often without knowing yet at which stages in development of B cells these factors are expressed, and what their target genes are in uivo. Only in a rare case (e.g., pax-5, operating on CD19 and A5 genes), the phenotype of transcription factor loss-of-function mutant mice may be understood from that limited knowledge. Transcription factors selected for review are those for which loss-offunction mutant mice have been generated that exhibit defects in B cell development and function. Such defects may arise from cell-autonomous abnormalities in the B lineage, as well as stromal defects affecting B cell genesis. In several types of mutants it is not yet clear to what extent an abnormality has arisen from defects intrinsic or extrinsic to the B lineage. Studies on forced (ectopic- and/or over-) expression of normal or mutated genes encoding transcription regulators are included when done by transgenesis. The phenotype of mutant mice is discussed in depth, with a summary in Tables IIIA and IIIB. Several reviewers have recently highlighted different and overlapping aspects of the area of research dealt with here (12, 52, 54, 84, 132, 195, 256). Techniques used to create mutant mice and the variations that have been designed to circumvent lethality or to regulate the expression of the mutation in time and space are discussed elsewhere (14,31,36,38,68,82, 221,235,285).Useful overviews of loss-of-function mutant mice generated through 1994, and their phenotype, are given in (26-28), and in (40) (on mutations that result in prenatal death).

B LYMPHOCYTE DEVELOPMENT I N

vrvo

217

A. TRANSCRIPTION FACTORS NECESSARY FOR B CELLGENESIS 1. GATA-2 GATA-1, -2, and -3 proteins each contain two zinc fingers that mediate DNA binding. They display distinct but overlapping patterns of expression (for review see (294)).The GATA proteins recognize a consensus sequence found in many genes including the globin locus. GATA-2 is expressed widely, but particularly high levels appear in subpopulations of murine BM enriched for hematopoietic stem cells (209). GATA-2 -/- mice die in utero at E10-11, with severe anemia due to a defect in primitive and definitive hemopoiesis (279). Such early death prevents an analysis of the role of GATA-2 proteins in other hemopoietic lineages. To circumvent this, ES cells have been generated with a null mutation in both alleles of the GATA gene. Injection of GATA-2 -/- ES cells in wild-type blastocysts generates chimeric mice. A PCR assay to distinguish wild-type and mutant alleles, performed on DNA isolated from organs, indicates a selective impairment in the ability of GATA-2 -/- ES cells to contribute to the hemopoietic system, from the fetal liver stage into adulthood. Further evidence that lymphocytopoiesis is defective has been obtained by RAG-2deficient blastocyst complementation, a technique that allows lymphocyte progeny analysis at single-cell level (36, 38). RAG-2 -/- mice fail to develop mature T and B cells. When injecting RAG-2 -1- blastocysts with RAG-2 +/+ ES cells that carry a mutation in both alleles of a particular gene (in this case GATA-2), any mature T or B cells developing in such chimeric mouse must derive from the ES cells. The ability of GATA-2 -/- ES cells to generate mature B and T cells in RAG-2 -/GATA-2 -/- chimeric mice is minimal (278). GATA-2 therefore appears to be a transcription factor that is indispensable for the development of all lineages, most likely by affecting pluripotent stem cells in their proliferation and differentiation. The question of which role GATA-2 plays in later stages of hemopoiesis including B cell development may be addressed by tissue-specific gene targeting.

2. PU.1 PU. 1 has initially drawn the interest of developmental immunologists because of its highly restricted pattern of expression in the hemopoietic system. PU.l is a transcription factor encoded by the PU.l/Scf)pi-l gene, a member of the ets family. The ets homology domain, located in PU.l in the carboxy-terminal region, mediates DNA binding and protein-protein interactions. The ets domain exhibits no homology to other known DNA binding motifs, There is limited homology to the D N A binding domain of the myb family. The DNA target recognized by the ets domain is a purine-

TABLE IIIA SUMMARY OF PHENOTYPE OF MICE WITH Loss OF FUNCTION MUTATIONSIN TRANSCRIFTON FACXOR-ENCODING GENES Mutated gene Expression and/or function of normal gene produds

!2

GATA-2

PU.1

lkaros

50x4

EBF

P-5

High levels in BM stem cells

Expmssed in E l 4 FL; targets include several genes active in hemoporesis

Transient expression in early F 1 targets include T lineage-

Detectable in BM, high lwels in thymus.low in mature T cells

Regulator of mb-l gene expression

Activates IgH and lgLr transcnpbon

inbibitor of IRF1; transcription repressor independent of IRF-1: I L 7Ra gene as target?

(a) E17.5 ( b ) N1

956 &e

El4

30% die before 4 weeks of age

Expressed from E l 4 FL; isoform BSAP i) inbibits NF-uP, an activator of the IgH gene; ii) is required for LPS-induced proliferation of mature B cells 95% die between birth and 3 weeks of age

Most die in first week after

Abnormal brain development; failure to thrive Not obvious

severe growth retardation

Death rate in first 8 months higher than wild h p G n e r a l fragdity

Lethality (developmental stage)

EIO-11

Abnormalities beyond bemopoiesis Immunodeficiencies

Enlarged pericardial sac

Not obvious (b) Highly susceptible to infection

specific genes

between birth and 4 week of age F d u r e to thnve after birth

Cardiac defea

Growth retardation

Su-pbble to mfecbon

EW

birth

stelile care improves growth and sunival rate

LCMV inoculation is Ietbal although

Spleen increzsingly degenerated

Reduced 6 of IL3 responsive

Slightly abnormal

Reduced % of CSF-I responsive cells

Hemopoietic

Efimid

Myeloid

Double Imock+ut chimeras: GATA-2 -1ES cells not mnbibuting to hemopoiesis

(a) Thymus hpcellu lar (b) BM and tbymus hpcellular

Null mutant mice: severe anemla at death

(a) Variable anemia: S&Cti"e

reduction of more mature stages (a) E16.5 FL: myelopoiesis uninitiated: rnegakarpcyte development present

BM and thymus 5ewrely

hpcellular: enlarged spleen: no LN and Peijer's patches *regulated but not blocked

*regulated but not blocked

E l 3 FL mUs from -1- mice proteci lethally irradiated host against death

H.pxeUular spleen at 1-2 weeks of age

E l 3 FL cells generate e f i m y t e s in a host

Number severely reduced in spleen

E l 3 FL cells generate granuloqtes. mon-trs and platelets i n a hat

Normal 6 m spleen

Number severely reduced in spleen

IRF-2

mnfectlon is

cleared

Cells

T-lymphoid

B-l)mphoid

Phenotype

Function

GATA-2 -/RAG2 -Ichimeric mice: no mature T cells

GATA-2 -IRAG-2 -/chimeric mice: no mature B cells

(b) N4 blood. small number of neutrophils (a) E16.5 thymus. T cell development uninitiated (b) No mature T mlls

(a) E16.5 FL: B CeU

development uninitiated (b) N1 BM: B220' cells present: no mature B cells

At 2 weeks of

age: Tby-1. cell development uninitiated: no NK cell activity in spleen

E l 3 FL cells generate thymocytes and mature T cells in a host

At 2 weeks of age: BM B220* cell 46 severely reduced: no sIgM* cells in BWspleen

El3 FL e l l s deficient in generating B lineage cells beyond fraction A in BM of host: respond poorly in a B lineage donogenic assay: LPS responsiveness can be acquired in uitm

Normal CDUCD8 profile in thymus and hlmd

T cell number in spleen relatively undected

Increasingly abnormal thymus

Thymus: normal: BM.reduced % of T C R W mlls

Bl and 8 2 cells absent, in BM only fraction A type of e l l s , erpressing germline p, TdT and ILiR tcdnscnpts, lacking DJ rearrangements; heteraygotes: moderate duction m B lineage

1-2 weeks of age: in BM a severe redudlon in 8220' cell number. CD4X and s1gM' subsets most strongly affected; in spleen and LN: drastic reduction in B cells. adult follicles in spleen absent: B1 cells undetectable in peritoneal cavity

Young adult BM normal % of 8220' sIgMcells. reduced % sIgM+ cells; spleen and LN: normal % of B e l l s

Serum IgM lev& below detection level

Adult Yrum Ig levels below detection

In FL and in BM. spleen at 2 weeks after birth B220* cells. IgH DJ rearrangements undetectable. at 5 weeks 1" BM B220' ells present but DJ rearrangements undetectable, in spleen few sIgM' cells present, El85 FL of hetemzygotes modrrate reduchon in 8220' cells

BM: r e d u d 9E of IL7 responsive cells, LPS responsive cells; spleen: redued % of

LPS

rrsponsive cells. serum Ig levels: reduction in

R I Gb

SUMMARY OF PHENOTWE OF

LOSS OF

TABLE IIIB FUNCTION MUTATIONSIN TRANSCRIFTION FACTOR-ENCODINGGENES

bmi-1

oct-2

Negative replator of

Lymphoid-restricted transcription factors, regulated by tisuespecific mactivators? target genes include IgH

50% cannibalized

Death within a few hr after birth

Abnormalities beyond hemopoiesis

Posterior transformations. gmwth retardation. neurological abnormalities

Not

Immunodeficiencles

Mutated gene Expmsion and/or fundion of normal gene produds

Lethality (developmental stage)

(c)-myb

MICEWITH

High levels in BM stem (PIIS, immature T. acbvated mature T and B cells; CD4. CD34 as target genes (a) c-myh -I- mice: El5 (b) transgenic mice generated with a construct of a dominant negative allele of c-myb w t h the mntrol region of human CD2: viahlr

(a) Multipotmtial

pmgenitor 4t reduced in FL

P50

Rel-B

c-fos

Stimulator of cell proliferation?

Binder of rB sites (as homo- or h e t e r d m e r l in a vanety of genes active in immune system. including

Binder of M - 1 sites as hetemdimer with jun or others: expressed in B m ' cells generated m BM culture

Viable

Viahle

High levels in dendritic cells of t h p u and possibly spleen and LN: ronstitutive hinder of r B sites in thymus Viable at birth hut ill health increacing with age

Higher than normal stomach weight

Not obvious

Inflammation of lung. liver

Suniving mice retarded in gmwth fmm N10: hone malfomahon. subtle alterations In newow s y t e m

Opportunistic infechons in moribund mice

Normal health

Susceptible to infections when housed mnventionally

Inflammation appean not induced by common pathogens

AdieF-rich ma-! at very y u n z a g e normal response to IL-3 In adult BM

Spleen lachng

tun genes?

hox- 1 1

IgLr between birth and N 3 rest me before 20 week5 of age

ohvious

H>perplashr.BM: increased splmir

weight

Many me on day of birth

Marrow cavi? space r e d u d due to hone formation and -resorphon ahnomalitwa

EFhrotd

(a) Absent

in FL

N4 blood: epihrocwes mth nuclear fragments

Anemia in m n h u n d mtce

Mwloid

T-l\mphoid

rb) Thynus at 3-6 weeks of age severely

hpxvllular: mature T mll number in spleen reduced

El4 5 FL and BM swerely reduced responsiveness to MCSF. in BM, cells with myloid phenotype r e d u d in number less than thr B lineage ThImus cellularit)-at brth half, at 2-3 months 1% of wild type: splenic T cell numbers spwrely redud

I n c r r d numbers in hlood

Neonatal thymus normal in ellulanty and p h e n o 3 i c profile

Normal thy-yte Th\mcx)tr and phenotpz. blood TI splenic T rvll B raho normal hut number and numben incre-d phmohpir profile normal: abnormal respinsr of T cells to shmulahon in ritm

B-hmphoid Phenotype

ch) Splenic slg'cell number reduced

BM 82.20' cells d u m d . the B 2 W subset most

reverelv: splenic B r d l numbers rpverelv r e d u d

Funchon

E14.5 FL and adult B M unresponsive to IL-i: LPS responsiveness normal in newborn spleen. p i in adult spleen and B M . hetem%otrr. red"CVd

rrrponsivmrss

E l 6 5 FL normal R of B W . f i + wlls. n&rn spleen m r r e d rahon BPl'/fi* cells. also m heteroqoter

E16.5 FL normal 91: of IL-i-responsive mlls. newhorn I n ~ r andspleen % o f LPS responsive mlls reducvd. also in h~trro~gotrs: clones f o d in r q o n s p to LPS r r d u d Ig wrehon

FL and B M not studied: hlood B K ratio normal hut numbers increased

BM and splenic B lineage cell number and p h m o t y i r profile normal

LPS response severely defettiw: anti-IgM response moderately aITrcted. anhhody response to NP & r e d . change an w r i s t m e to particular pthogrnr: SPIPNIII Ig levels rrdirnd cxcvpt IgM

I ) R r e d % in BM: i n c r e d splenic rrythrcpoiesa: normal R B C cuntent in blood Hperplasia in BM and spleen: lrukoqiosis in blood

Epihropoiesis in spleen rather than BM. normal R B C mntmt in blood

Thvmus: variable degree of medulla? atrophy. reduced % of dendnhc cells. spleen: R Ihv-l+ cells redurmg with

Thymic atrophy

=R BM phenohpc profile of BZII' mils normal. spleen R of BZII' cells reduced. normal IgMIlgD raho

M!~lopotests~n spleen

rather than BM: R in hlood i n c r r d

rorrelahng wth degree of ill health: at 4-10 weeks. defective response of mature T cells to T cell receptor stimuli

Normal R BZO' cells in FL and prritoneal cavity. redurrd % in spleen at 2 Weeks Of age. agedrprndent B I?mphoprnia in

blood

Splmqtrr.d T m w IL-i responswenrrs. generahon of B W l ' cells in Irthdh irradiated wild-hpr host

222

I)A\'INA

0PSTEI.TEN

rich sequence. The targets of PU.l share the sequence GAGGAA. PU.l is an activator of transcription, with the relevant domain in its N-terminal region (for review see (166, 290)). Ets binding sites are in regulatory regions and in core promoters of many genes active in the hemopoietic system. Target genes for PU.1 include Ig H and L genes, J chain and mbl, the (Y chain of the GM-CSF receptor (104), the receptor for M-CSF, CDllb, CD18 (243), IL-1p and p-globin (see (256) for review), Recent preliminary reports add human btk (101) and BP3 (53).Ets proteins can interact with members of their own family as well as of other transcription factor families. PU.l may act in concert with another transcription factor, PU.1 interacting protein (Pip, a member of the interferon regulatory factor ( I R F ) family), which has been detecqed in an RNase protection assay in mouse BM, spleen, and thymus (57). PU.l expression has been detected by Northern blot as a single 1.4 kb transcript in high levels in adult murine spleen and BM-derived inacrophages, and in low levels in tlie thymus (137). In this same study, many other organs in the adult have undetectable levels of mRNA. Expression of PU.1 as studied by low-resolution ISH (69) is detected in liver as early as E14. At E19, other organs that are also positive include marrow of rib and pelvis, thymus, spleen, testis, and spinal vertebrae. After birth, at N17, the spleen contains high levels of transcript in the white pulp, and much lower levels in the red pulp. Liver at that stage also reacts in ISH, in a pattern that could represent Kuppfer cells. Higher-resolution ISH is required to see to what extent hemopoietic cells and stromal elements of the various organs express PU.l mRNA. In femora from adult mice, immunohistology shows the PU.l protein to be confined to the nucleus, most prominently to that of immature erythroid and myeloid blasts and immature eosinophils (105). BM macrophages and mature megakaryocytes, as well as an occasional cell with lymphoid morphology, also react with the antibody. Mitotic cells that strongly bind the antibody have been observed as well. From the complex pattern of expression of PU.l in adult BM, theories on its role in hernatopoiesis are hard to come up with. One suggestion put foward is that down-regulation of PU.l is required for erythroid and myeloid maturation, a hypothesis not yet tested. Testing the extent to which PU.l is indispensable for mouse development, two different groups have created mutant mice. Remarkably, tlie resulting phenotypes appear to differ. The first type has been generated by targeted mutation of exon 5 of the gene which encodes the DNA binding domain of PU.l (250). Heterozygous mutant mice show no abnormality, suggesting that mutant proteins, if at all synthesized, do not interfere with transcription pathways. Homozygous mutant mice, present at the expected

B LYMPHOCYTE DEVELOPMENT IN VlVO

223

Mendelian frequency up to E16.5, do not survive beyond E17.5. The cause

of death has not been established, but may be related to hemopoietic

defects as no other abnormalities have been detected. Mutant mice display on average a reduced hematocrit in aortic blood at E16.5, but with great variability. The liver of one severely anemic mutant mouse at E16.5 shows a relative reduction in reticulocytes and erythrocytes as compared to earlier stages in development such as proerythroblasts and erythroblasts. PU.1 appears dipensable for P-globin gene expression in early development: using ribonuclease protection assay on total RNA, P-globin mRNA levels as compared to p-actin mRNA levels are not drastically different in anemic mutant and wild-type mice. Megakaryocyte development seems unperturbed, with megakaryocytes present in liver, and normal blood platelet content in mutant embryos. None of the 14 mutant mice analyzed at E16.5 have a detectable percentage of B220' cells in the liver. Using RT-PCR, no transcripts of rearranged Ig H and K genes, germline H genes, RAG-1, RAG-2, mbl, B29, and Vpre-B genes are detected either. Thus, B cell development appears uninitiated by this stage of development of mutant mice. At E16.5, the thymus is hypocellular and Thy-l+ cells and DP cells are severely reduced in incidence. There is no sign of myeloid development either, from the earliest in vitro clonogenic precursors onwards. The most obvious explanation for the defects observed in PU. l-deficient mouse embryos is that PU.l is required for initiating lymphocytopoiesis and myeloid differentiation but not for initiating erythrocytopoiesis and megakaryocytopoiesis in the developing mouse. Whether any of the currently known targets of PU.l are involved in this early defect is unknown. The second PU.l loss-of-function mutant mouse has been reported only preliminarily (7,176).These mice are born but succumb within 48 hr after birth to bacterial infections. Leukocytes are absent from the blood at that time. When the life span of the mice is prolonged with antibiotics to 96 hr, a small number of neutrophils are found in the blood. In culture, neutrophils develop but monocytes and macrophages do not. BM and thymus are hypocellular. In contrast to the mutant mice reported by Scott et al. (250), B220' cells are present, but mature T and B cells have not been detected. In an attempt to assess the extent to which the lymphocyte developmental defect observed is cell-autonomous or due to environmental abnormalities, SCID mice have been reconstituted with fetal liver cells from the second type of PU.1 mutant mice. Donor-derived cells are detected in BM, thymus, and spleen. Further analysis as to their nature is required to judge the differentiation potential of the donor cells in the SCID environment.

224

DAVINA OPSTELTEN

To understand the differences observed between the two types of 11111tants, a detailed report on the type of mutation in the latter is required, as is information on the genetic background of the mice in both studies. In light of the phenotype of the second mutant mouse, it is worth testing in mice with the type of mutation reported by Scott et u1. (250),whether early embryonic cells from various sites, including paraaortic sphnchnopleura have any developmental capacity in the direction of B cell development (B1 and conventional B) when transferred to, e.g., RAG-deficient hosts, or when cultured. The liver of the first reported PU.1-deficient mice may be a particularly unfavorable host to lympho-myelopoiesis. It would therefore be informative to study the mutants for appearance of B precursor cells (B220' cytoplasmic pt) in organs other than liver where they normally appear before E16.5, i.e., the spleen and BM.

3. Iknros In hemopoiesis, Ikaros proteins, belonging to the zinc finger family of transcription factors, appear to selectively play a role in development of lymphocyte lineages. Ikaros proteins are expressed in B and T cell lines representing various stages of development, but absent in a plasmacytoma (85).In the developing embryo, low-resolution in situ hybridization (ISI1) using a probe that detects all isoforms (see below) (71) shows that the Ikaros gene is expressed in fetal liver from the time of hematopoietic activity (E9.5-10.5), the level of expression declined by E14. In thymus Ikaros mRNA is detected from the time of colonization by lymphoid cells (E12). In spleen, low levels of Ikaros mRNA are detectable at late gestation stage, at the time of entry of mature T cells. In E l 9 BM, no ISH signal has been detected, which might suggest absence of Ikaros mRNAt cells. Sensitive high-resolution ISH would be required to detect any cells expressing low levels of Ikaros mRNA, particularly if they are scattered throughout the marrow. Ikaros expression in prehepatic sites of hemopoiesis (yolk sac, paraaortic splanchnopleura) has not yet been reported. Later in development, only the T lineage appears to maintain high levels of expression. Adult mice express high levels of Ikaros in thymus and peripheral T cells of spleen, as shown by Northern blotting (NB). Beyond the hemopoietic system, Ikaros mRNA' cells are detected by ISH in the brain at E12, E16, and E l 9 (71). Several proteins can be generated from the Ikaros gene by alternative splicing, and they differ in number and arrangement of amino-terminal zinc finger modules (85,188).Using quantitative RT-PCR, the alternatively spliced mRNAs appear to be expressed differentially in development, with, e.g., Ik-1 and -2 mRNA more readily detectable in E l 4 liver than Ik-3,4, and -5 mRNA, and Ik-4 mRNA at higher level in E l 4 thymus than in

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E l 6 thymus (188). In vitro transient expression assays show differential subcellular localization of the isoforms. Ik-1 and -2 are mainly in the nucleus, and Ik-3 and -4 mainly in the cytoplasm (188).The latter two lack exon 5 which may contain a nuclear localization signal. Alternatively, there may be active retainment of Ik-3 and -4 in the cytoplasm by other proteins. The DNA binding specificities and affinities of the isoforms, and the ability to activate transcription from reporter gene constructs, differ significantly (85, 188).There is no evidence that Ikaros proteins bind to DNA as a complex with other proteins. Actual binding and transcriptional activation has been demonstrated for Ik-1 and the murine CD3S enhancer (this was the sequence on which Ikaros was discovered (71)), as well as for Ik-1 to -4 and the human IL2Ra promotor (188).Many lymphocyte-specificgenes are potential targets of Ikaros proteins, as they contain sequences in their control regions which have been shown in vitro to represent high-affinity binding sites for Ikaros. Examples are the genes encoding murine CD3 members, TdT, TCR members, CD2,CD4, MHC Class 11, IL-2, h5, Vpre-B, and mb-1 (85, 188). In light of the differences in specificity and expression pattern of the various Ikaros isoforms, and the overlap of Ikaros binding sites with that of NFKB,a very complex regulatory mechanism must exist during in uivo development of lymphocytes. Ikaros heterozygous and homozygous mutant mice have been created by targeted disruption of the Ikaros gene in part of exon 3 and exon 4, encoding the first three (N-terminal) zinc finger modules which mediate the high-affinity sequence-specific DNA binding of Ik-1 to -4 (70). An impairment of DNA binding ability and transcription modulation of these four isoforms is therefore expected. The mutation does not affect the survival of mutant mice in utero. However, homozygous but not heterozygous mutant mice fail to thrive after birth, and 95% die within the first 3-4 weeks, many cannibalized by the mothers. The major cause of death appears to be infection, often associated with liver necrosis. It is unclear whether abnormalities other than those in the immune system, e.g., in the brain where Ikaros is also expressed in ontogeny, contribute to the failure to thrive as well. In the vast majority of homozygous mutant mice, only a thymic rudiment with very few lymphoid cells (less than 0.1% of normal) is present. At 2 weeks of age, these cells lack any phenotypic characteristics of thymocytes beyond the very early stage. In the few mutant mice surviving up to 12 weeks of age, a peripheral pool of mature T cells does not develop. TCRyS cells are absent from the epidermis. The mutant mice lack identifiable LN and Peijer’s patches. In femoral marrow of 2-week-old homozygous mutant mice, only 10% of normal cell numbers are present. Low frequency or

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absence of B220'" cells, and absence of IgM' cells in BM as well as spleen, indicates that that these organs are not generating B cells. Whether B cell development in FL, and development of the B 1lineage are also disturbed has not been reported. Natural killer cell activity is undetectable in spleen. Erythropoiesis and inyelopoiesis are not blocked but severely dysregulated. In blood of homozygous mutant mice, RBC counts are within the physiolo@calrange, but unlike in normal mice, nucleated erythrocyte precursors (orthochromic normoblasts) are present. An increase in proportion of polymorphs and basophils is seen with age, and these leukocytes appear clustered over bacteria. In the hypocellular BM, GM-CSF colony forming cells occur in a frequency almost %fold that of normal. By surface marker analysis, the erythroid (E) over inyeloid ( M ) lineage ratio is 2.5 times higher than that in normal BM. The mutant mice show age-related changes in E/M ratios in BM which are more dramatic than those in wild type. Between 2 weeks of age and over 3 weeks of age, the E/M ratio drops from 2.5 to 1/3 (wild type: from 1to 1/2).In contrast to the hypocellularity of BM, spleens of homozygous mutant mice appear 1.5-3 times larger than normal; exact cell numbers are not reported. GM-CSF colonies are present in spleen of mutant mice in 10-fold higher frequency than normal. By surhce antigen analysis, E and M lineage cells are detected, in ratios changing with age (E/M ratio drops from 7 to U2.5). These observations indicate that poor development of the BM cavity is a major defect in Ikaros -1- mice. In cases of BM hypocellularity, the spleen tends to compensate for erythropoiesis and myelopoiesis (see Section 111). Histology of BM would help identify abnormalities in marrow organ formation in the mutants. The block in lymphocyte and NK cell development niay have directly caused the severe hypocellularity and dysregulation of E and M lineages. The lymphoid/NK lineages may also play an important role in preparing the BM stroma for full support of hemopoiesis. Alternatively, Ikaros gene mutation directly affects BM stroma, or osteoclasts involved in bone resorption. The severe immunological stress experienced by mutants postnatally may also contribute to BM abnormalities. Cell transfer experiments may give further insight. Transplantation of FL and BM cells from Ikaros mutant mice to normal mice would reveal hemopoietic cell-autonomous abnormalities. Inoculation of newborn mutant mice with wild-type hemopoietic cells may show the influence of Ikaros-deficient nonhemopoietic cells on postnatal BM organ formation. A detailed expression study in the various hemopoietic lineages during normal development and in adults is also required to understand the function of Ikaros.

4 . sox-4 Sox-4 encodes a protein containing an N-terminal high-mobility group (HMG) box which exhibits sequence specificity in DNA binding, and a


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serine-rich C terminus. Sox-4 was originally cloned from a mouse embryo cDNA library through its homology with SRY (84), the mammalian sex determining gene. The HMG box and the C terminal serine rich region are highly conserved between man and mouse. The DNA sequence-specific binding of the HMG box occurs in an unusual fashion, in the minor groove, inducing a strong bend in the DNA helix. Many members of the large HMG family may play an architectural role rather than a direct activating role on target genes (16, 164). sox-4 can transactivate transcription through an AACAAAG concatamer, and the serine-rich C terminus is indispensable for this. The AACAAAG motif is found in enhancers of, among others, CD3-E and CD4 genes. sox-4 proteins are likely to exert their transactivating action in cooperation with other factors (281). Sox-4 consists of a single exon (248) yielding a major RNA species of 5 kb. Northern analysis of human and murine cell lines has shown expression in those representing maturing thymocytes, but absence in those representing the earliest T progenitors. In normal adult mice, thymus is rich in sox4 mRNA, while LN and spleen, organs which contain many mature T cells, have a low and undetectable level of sox4 transcripts, respectively (281). In the B lineage, cell lines representing early stages express sox-4 mRNA, but those representing late stages do not (281). In BM of adult mice sox-4 transcripts are detectable, but it is not currently known in which cell types (Schilham, personal communication). Gonads express high levels of sox-4 mRNA, while heart and lung express low levels (281). In development, neonatal brain exhibits moderate levels of sox-4 mRNA detected by Northern blotting, which rapidly decline with age. The tissue distribution of sox-4 protein has not been reported. Sox4 null mutant mice die in utero at E l 4 because of a heart defect (247). At the time of death, no histological abnormalities are detectable in organs other than heart, such as the CNS which normally expresses high levels of sox-4 mRNA. To reveal possible abnormalities in lymphopoiesis, the developmental potential of E l 3 FL cells has been assessed in various ways. First, transplantation into 5- to 7-week-old lethally irradiated MHC disparate mice shows that homozygous mutant donor cells are similar to wild-type and heterozygous mutant mice in their ability to protect the host against death, and to generate erythrocytes and platelets. Also, normal levels of granulocytes and monocytes are detected in the blood of the host. A serious defect in B cell development is observed 8 weeks later only in those hosts which received fetal liver cells from homozygous mutant mice. In BM, the frequency of cells with the phenotypic characteristics of fraction B-E B lineage cells (see Table 11)is extremely low in mutant cell recipients as compared to that in recipients of wild-type cells, while the frequency of fraction A (large B220fCD43+HSA'""cells) of donor origin is similar.

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Some donor-derived sIg+ cells are detectable in BM, blood, spleen, and LN of mutant cell recipients, which suggests that development is not completely blocked. Second, when assessed in vitro, a developmental defect in E l 3 FL is evident as well. Using S17 feeder cells, IL-7, and IL11, the frequency of responsive cells (which include early B precursors; see Table 11) is very low in FL of mutant mice, and they grow at a reduced rate. Two weeks after start of the culture, the mutant colonies do not respond to LPS by detectable Ig secretion; however, they are able to do so 4-6 weeks later. Thus, sox-4 appears not necessary for B cell activation by LPS. Although sox-4 is expressed in the thymus of normal mice, abnormalities in T cell development are not obvious in the transplantation assay: at 8 weeks, the thymus and periphery of the host are reconstituted to the same extent by E l 3 FL ofwild-type, heterozygous mutant, and homozygous mutant mice. The observations suggest a selective defect in the genesis of B cell precursors in sox-4 mutant mice, intrinsic to hemopoietic cells as they appear in E l 3 liver. An analysis of E l 3 FL from mutant mice for signs of initiation of B cell development, such as IgH gene rearrangement (Table I), may give further insight in the nature of the defect. It may be an expansion rather than a differentiation defect, since sox4 -/- FL cells are not unable to generate mature B cells. For instance, sox4 -/- cells may not express the necessary array of growth factor receptors. It would also be of interest to assess other hemopoietic sites in the early mutant embryo for their capacity to reconstitute both the B1 and the B2 lineage. 5. Pax-5

Pax-5 is a member of the Pax family, sharing the paired domain DNA binding motif (259, 288). Multiple isoforms generated through tissuespecific alternative splicing have been reported (2, 30, 260). One isoform is the B cell lineage specific activator protein (BSAP), originally identified in the human B cell line BJA-B (13) as a factor homologous to a tissuespecific activator protein (TSAP) of the sea urchin, which can bind to promoters of the four late histone H2A-2 and H2B-2 genes. Through biochemical purification from BJA-B nuclear extracts followed by microsequencing, and cDNA cloning and sequencing (2),BSAP has been identified as an O-glycosylated product of pax-5, with (in BJA-B) an apparent MW of 50 kDa, and a mRNA length of about 10 kb. BSAP cDNA codes for an N-terminal paired domain, an octapeptide conserved among pax genes, and a partial homeodomain. The paired domain appears both necessary and sufficient for DNA binding. Human and mouse BSAP cDNA are more than 90% identical (2).


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Among hemopoietic cells, BSAP activity appears restricted to the B lineage, as shown by extensive analysis of BSAP activity in cell lines of murine and human origin (13).Cell lines representing stages from pro-B to mature B cells, but not those representing plasma cells, exhibit BSAP activity. Using RNase protection analysis, a similar expression pattern in a panel of murine and human cell lines has been detected, suggesting that BSAP activity is regulated at transcriptional level (2). RNase protection analysis in adult mouse tissues has detected BSAP mRNA in spleen, lymph node, and testis (but not in ovary, liver lung, kidney, brain, heart muscle, salivary gland) (2). In adult mouse lymph node and spleen, expression as assayed by ISH (see below) is restricted to the follicles (2). In organs of normal rat, BSAP activity is detectable in spleen but not in thymus, liver, or brain (13). In developing mouse embryos, an early wave of BSAP mRNA detected by RNase protection analysis starts at E10, and peaks at E12.5. ISH shows that the early transient expression of BSAP mRNA is in the CNS (8,258). Later, from E l 4 onward, the liver exhibits, by RNase protection analysis, increased expression of BSAP mRNA, in parallel with CD19 mRNA (2). This correlates with the maturation and expansion of the B lineage population in this organ. Targets of BSAP include the genes encoding CD19 (144),Vpre-B1, and A5 (205). In the IgH gene, BSAP binds to several CHgene intronic regions and sites 5’ to and within the IgH3’a enhancer ((287) and references therein). A role for BSAP late in B cell development is indicated. In B lymphoma cell lines, the binding of BSAP to the IgH3’a enhancer affects the downstream binding of NF-aP, a transcriptional activator of IgH (196, 197). NF-aP is present in cell lines representing B cell stage and plasma cell stage, but the NF-aP site is only occupied in plasma cell lines, where BSAP is absent. Transfection of B cell lines with a triple-helix forming oligonucleotide to inhibit BSAP activity results in higher levels of IgH transcripts, in particular when those cell lines synthesize IgH isotypes other than p. Apart from a repressor role in IgH transcription, BSAP may play a role in proliferation of B cells (171, 287). Antisense oligonucleotideinduced BSAP suppression in splenic B cells of 4- to 8-week-old mice results in inhibition of LPS-induced proliferation. Transfection of BSAP cDNA followed by LPS stimulation increases BSAP levels and proliferation rate of splenic B cells. Beyond the immune system, a role for pax-5 in proliferation and tumorigenesis is suggested by the deregulated expression of pax-5 in human medulloblastoma (143). In desmoplastic medulloblastoma, Pax-5 mRNA level correlates positively with cell proliferation and inversely with neuronal differentiation. It is a phenomenon common to

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the pax gene family that ectopic expression leads to an increase in proliferation, and that they appear to play a causative role in turnorigenesis (260). To study the role that pox-5-encoded proteins may play early in development, mutant mice have been generated by targeted disruption of exon 2 which encodes the paired domain (280). Homozygous mutant offspring are born at the expected Mendelian ratio, but exhibit severe growth retardation and most die in the third week after birth, with a body weight 1/3 of normal littermates. The immediate cause of death may be an inability to wean. Injection of newborn mutant mice with BM cells from wild-type mice does not prevent early death. Provided the transplants have "taken," death may thus not be caused by hemopoietic abnormalities. Abnormal morphogenesis of the posterior midbrain and anterior cerebellum, obvious by 3 weeks of age, is first detectable histologically at El6.5, about 4 days after the onset of pax4 gene expression. The testis of the few surviving males appears normal, and they are fertile, suggesting that the mutation does not affect the development of this organ. BM of 1- to 2-week-old homozygous mutant mice contains one-third the normal cell number, a degree of reduction that correlates with the lower than normal body weight. B220'sIgM' cells are selectively reduced, to less than 1% of normal numbers. B220'sIgM- cell number is reduced, mainly because of an absence of the small CD43- subset. Large B220fCD43+ cells occur in a frequency roughly equal to that found in normal mice. In the few mutants surviving into adulthood, B220' cell frequency in BM is severely reduced as well, but no further analysis of B precursor stages has been reported. At 1-2 weeks of age, the spleen of homozygous mutant mice exhibits a dramatic difference in size (2- to 5fold smaller) and cell number (on average 10-fold lower) as compared to wild type. Follicles are rarely detected. The population most drastically reduced is sIgMf cells, with numbers below 0.1% of normal. Splenic B220'sIgM- cells, not further assayed for B precursor status in mutant or normal mice, are 2.2% of normal numbers. In LN of 2-week-old hoinozygous mutant mice, the incidence of sIgM' cells is 0.1% (28% in wild type). In serum of 3- to 7-month old homozygous mutant mice, levels of IgM, IgG, IgA, K, and A are below detection. The sIgM' (CD5' and C D S ) B cell populations in the peritoneal cavity, as assessed in a 4-month-old mutant mouse, are undetectable. It thus appears that in the absence of normal pax-5 gene products, the formation of B1 and B2 cells and their progeny ( Ig-secreting cells) is severely blocked in development. There is no indication that the dependency on intact pax-5 gene products decreases with age. Concerning the IgH and -L chain gene rearrangement status in B220tCD43' cells of mutant mice, DJ but not VDJ rearrangements have been detected; with a phenotype of c-kit' HSA+ IL-7Rt and proliferating

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in vitro in response to stroma and IL-7 (unpublished data in (30)),a block in the transition of fraction C to fraction D (Table 11) is indicated in pax 5 -/- mice. While the thymus of mutant mice has not been analyzed, the T lineage

(CD4+and CD8’ single positive cells) in spleen are reduced in number to one-third and one-half of normal, respectively. This reduction may be a consequence of the growth retardation or stress as a consequence of ill health which may affect the output from thymus. However, a direct role for pax-5 in T cell development cannot be excluded. In adult rat thymus, pax-5 expression is not detected, but a study of expression in the developing mouse thymus has yet to be reported. With regard to erythroid and myeloid lineages, dysregulation is observed in spleen. Myeloid cells characterized by Mac-l+Cr-l+are reduced to onefifth of normal numbers. The “rest” cell population in spleen of mutant mice (likely to include myeloid precursors and erythroid lineage cells) is 12% of normal numbers. The cause of such dysregulation may be solely extrinsic, e.g., relatively more “space” in BM for the development of these lineages. Spleen size of the few surviving adult mutant mice is noted to be similar to that of adult normal mice. Spleens from adult mutant mice do not contain follicles. It is unclear at present which cell types do make up the adult mutant spleen. Discussing how absence of functional pax-5 gene products leads to the selective B cell developmental arrest observed, CD19 transcripts are mentioned to be undetectable in mutant mice (280), while the level of transcripts encoding A5 would be normal (30); information is lacking on cell samples used for this assay. An analysis of Vpre-B transcripts in pax5 -/ - mice has not been reported. CD19 null mutant mice show a developmental block in B1 cells; B2 cells develop normally but exhibit defects in mitogen- and T cell-dependent responses (58, 229). Thus, the inability of pax-5 mutant mice to generate B1 cells may be explained by the defect in CD19. In man, CD19 appears required for IL-7-induced downregulation of RAG (20); this action of CD19 may be compensated for in the genesis of B2 but not of B1 cells. The transcription of IgH may also be affected in a direct way; currently, it is unknown whether any of the multiple pax9 gene products bind to Ig regulatory regions early in development, More detailed information is required on expression of pax-5 in the early B lineage in normal developing and adult mice. Another question of interest is whether pax5 proteins directly affect cell cycle regulation in early stages of the B lineage. Preliminary findings indicate normal proliferative behavior of the B220+ cells from mutant mice (unpublished data in (30)). A consequence of a block in an early stage of development may be a change of fate to an inescapable death and/or shift in differentiation pro-

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gram. The latter could be tested by analyzing the contribution of mutant andwild-type B220+CD43' cells from BM (and B220+cells from E l 3 FL) to other hemopoietic lineages after transplantation to congenic (normal) newborn mice.

6. E2A The mouse E2A gene can generate two proteins of the basic helix-loop-

helix (bHLH) family by differential splicing of bHLH containing exons, named E l 2 and E47. ITFl (E2-5) is a N terminal-truncated form of E47.

The HLH domain is required for protein dimerization, while a basic region is responsible for DNA binding. Target sequences for E2A gene products are, among others, the E boxes as found in enhancer regions of Ig hcavy and kappa light chain genes (for review see (12)).DNA binding requires homodimerization or heterodimer formation with other bHLH family members. DNA binding activity as E2A homodimers may be restricted to the B lineage ((306) and references therein). In normal rat, expression of the E2A gene (also referred to as pnri in this species) as studied by ISH (234) is widespread, with higher levels in inany epithelia and in certain areas rich in cell division. Among lymphohemopoietic organs, relatively high levels are detected in thymus and marrow of bone at E l 8 (with levels in liver not reported), and in the thymus and germinal centres of spleen in adult rat (with other organs not studied). In adult thymus the pattern of expression appears ubiquitous; thymocytes and/or thymic epithelium have not been judged separately for expression. In long-term rat BM cultures supporting B cell development, E2A proteins appear before the expression of Ig heavy chain genes. At that early stage E47 (Pan-1) proteins rather than E l 2 (Pan-2) proteins are active Ig enhancer binding components (112). Similar expression studies of E2A in mouse have not been reported. The human E2A gene product E47 when overexpressed in a mouse preT cell line induces germline Ig heavy chain gene transcription and DJ rearrangement, suggesting a direct, initiating role in B cell genesis (249). A crucial role for E2A in the early stage of B cell development has been confirmed in E2A mutant mice (11, 306). Two different strategies have resulted in the generation of mice lacking normal E l 2 and E47 proteins; the phenotypes of these mice are in line with each other. Homozygous mutant mice are born at normal frequency. No gross niacro- and microscopic developmental abnormalities are detected at E 18.5.However, postnatally their growth is severely retarded and most die in the first week. Females are even more affected than males. Sterile care does improve, but does not normalize, the growth and survival rate.

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In the liver at E15.5 (ll),IgH VDJ rearrangements, while abundant in wild type, are undetectable in homozygous mutant mice. Using RT-PCR, transcript levels that may be taken as signs of initiation of B cell development at that stage in development (sterile transcripts of the IgH chain gene, RAG-1, pax-5, CD19, and mb-l) appear low or undetectable in the liver of mutants. Surprisingly, the level of transcripts encoding B29, a component of the Ig receptor complex, is not much reduced. No information is available on the cell types that express this gene in E15.5 FL of mutant or normal mice. At E18.5 (306), the liver of mutants still shows no sign of B cell development, as B220' cells and AA4.1' cells are below detection level in flow cytometric analysis, and DJ rearrangements are not detected. In mutant mice surviving into the second week of postnatal life (306), B220' cells and DJ rearrangements are still undetectable in BM, and the spleen lacks B220' cells. At 5 weeks of age (ll),a homozygous mutant mouse contains very few sIgM+ cells in the BM. B220' cells are detected, with low levels of B220 and at a much lower incidence (6%) than in wildtype mice (32%). Half of those found in mutant BM are CD43-, a stage where VDJ rearrangements are normally present. (V)DJ rearrangements nevertheless are below detection level in DNA prepared from total mutant BM. In the spleen of this E2A -/- mouse, 13%of splenocytes are B220' (compare with 68% in +/+ littermates). The large majority of B220+ cells in mutant spleen are sIgM-; the nature of these cells has not been further studied. SIgM' cells (half of which appear B220-) are detected (5%in a mutant vs 66% in a normal littermate). The data may indicate that B cell development may become less dependent on E2A gene products with age. Serum Ig levels in E2A mutant mice of increasing age have not been studied. An interesting question left open is whether the genesis of B1 cells in E2A mutant mice is as seriously affected as that of conventional B cells. The phenotype of E2A homozygous mutant mice may be explained by an absolute requirement for E2A gene products, particularly in early life, to initiate Ig heavy chain gene rearrangements. It is currently unknown whether E2A gene products act directly on other genes, such as pax-5 and RAG, required for B cell development at such early stage. Not seeing a dramatic abnormality in the T lineage of E2A homozygous mutant mice, such as a block in T cell receptor gene expression, might suggest that E2A gene products do not regulate RAG-1 expression in T cell development, but this is worth actually testing. A cell-autonomous element in the B developmental abnormality is indicated by preliminary transplantation experiments (306). However, E2A mutation may have affected hemopoiesis in other ways as well. The genesis

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of myeloid cells in BM of mutant mice seeins not completely normal. The thymus becomes abnormal in number and proportion of subsets. The spleen shows a severe degeneration in the first few weeks of life. All this may be secondary to the observed growth and viability defects. In addition, E2A proteins may normally contribute to a supportive microenvironment by regulating gene expression in stroinal cells of BM, thymus, or lymphoid organs. Zhiiang et n2. (306) observed that at E18.5, the proportion of B220+ cells in liver of heterozygotes is half of that found in wild type. As the cellularity of FL has not been recorded, such difference can be accounted for by a relative increase in B220- cell numbers, or a decrease in B220+ cell numbers. Assuming that it is actually the B220+ population that is reduced in size in FL, the early stage of B cell development seems very sensitive to gene dosage. No such difference is noted in BM a few weeks after birth. Inhibitor proteins may operate, which could bring E2A protein level below functional treshold levels in a heterozygote fetus. Levels of inhibitor proteins for bHLH transcription factors indeed regulate B cell development (263). The family of Id proteins can form heterodiiners with class I (or A) bHLH proteins (to which E2A belongs) that are inactive in DNA binding (12). Id proteins are expressed highly in murine cell lines representing early B lineage cells, and much lower in those representing pre-B and mature B cells. No data are available on expression of Id proteins in normal B cell development of mouse. It is therefore unknown whether in normal B cell genesis Id levels decline during maturation. A construct of Idl, with a 250-bp promoter region of mb-1 and IgH intronic enhancer as regulatory elements, has been used to generate transgenic mice. Id1 transgene product levels should be elevated throughout the mb-l+ stages of the B cell developmental pathway, but this has not been tested. Transgene expression is reported for the thymus, suggesting a deviation of the transgene expression pattern from that normally found for mb-1. Transgenic mice are viable, show no gross abnormalities, but are prone to infections. Thymocyte phenotype appears normal, indicating that the transgene does not interfere with T cell development. At 3-8 weeks of age, low-density cells from Id1 transgenic mouse BM are poor in B220t cells, with the degree of reduction dependent on transgene copy number. The phenotype of this residual B220' population in transgenic mice is predominantly B2201"CD43+.There is no information on the number of total cells and the density-separated fraction recovered from the BM of transgenic versus wild-type mice. Alterations in cellularity and/ or cell density may have contributed to the observed abnormalities. When testing DNA from fractionated BM for IgH and K gene rearrangements, 3-week-old mice with two transgene copies exhibit levels below that found


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in wild type, and below that expected on the basis of the reduced B220' cell proportion in that population, indicating that many of the residual B220+ cells in that BM fraction have not engaged in rearrangements. In line with this is the severe reduction in transcripts from rearranged IgH and K genes, and in I,, a sterile transcript from germline and rearranged IgH, assessed by quantitative RT-PCR. The germline IgH transcript F')is not much reduced, suggesting that this early event in B cell development is not affected in transgenic mice. Density-separated cells from spleen show a transgene copy number-dependent reduction in B220tsIgMt cells which appears less drastic with increasing age. Since RAG-1 and -2 transcripts and h5 transcripts are at a level much lower than expected on the basis of reduced proportions of B220' cells, Id1 transgene phenotype may be caused by a defect in transcription of Ig genes as well as other genes required for B cell development. The Id1 transgenic mice data indicate the importance of normal levels of functionally available class I bHLH proteins for early B cell development, and are compatible with the observations in E2A -/- mice. The challenge is to dissect the sequence of events leading to the observed B cell developmental abnormality; as suggested by Sun, the phenotype of mice transgenic for combinations of Id1 and genes found severely reduced in their expression in Id1 transgenic mice will be highly informative.

7. Zntelferon Regulatorrj Fuctor 2 An abnormality in B cell development appears present in mice defective

in a transcription regulator which is best known for its involvement in antiviral responses. Type I interferon genes and many interferon-inducible genes (e.g., in man, the genes encoding MHC class I heavy chain (119) and VCAM-1 (194)) exhibit a cis-regulatory motif to which interferon regulatory factor (IRF) -1 and -2 can bind. Transfection studies have indicated that IRF-1 activates such genes, while IRF-2 interferes with the action of IFR-1 ((90);see also (168) and references therein). IRF gene family members (also including ICSBP, ISGFSy, and Pip) share a highly conserved amino acid sequence in the N-terminal region, in which the DNA binding specificity is located. In the limited number of cell types studied (which include mouse thymus and spleen but not BM), IRF-1 and -2 gene expression as detected by Northern blotting is low. Rapid up-regulation can occur in response to virus infection, or mitogen (e.g., the response of mouse splenocytes to Con A (185)). Human ZRF-I has been found deleted or inactivated in several cases of myelodysplasia and leukemia (296), suggesting that a lack of IRF-1 contributes to uncontrolled hemopoietic cell growth. A role for IRF-1

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and -2 in regulating cell division is also suggested by transfection studies in NIH 3T3 cells. IRF-2 exhibits oncogenic potential, an action that IRF1 can reverse (88, 198). One target for IRF-2 is the human histone €I4 gene which exhibits cell cycle stage-dependence in transcript levels (284). Interestingly, the human IRF-2 gene has an IRF binding site in its promoter (89), suggesting autoregulation. A clue to a role for IRF in hematopoiesis comes from E, enhancerdriven human IRF-I transgenic mice, which express the transgene in high levels in BM and thymus. The mature B cell population is depleted while T lineage development appears undisturbed. The BM lacks cells that grow in vitro under normally B-generative conditions, which include IL-7 (302). Thus, forced expression of IRF-1 in the B lineage suppresses B cell genesis at an early stage. A potential target of IRF is the gene encoding the (Y chain of the receptor for IL-7 (CD127) (222), which has an IRF-binding motif in its promoter. ZRF-I and ZRF-2 mutant mice have been generated (168), primarily to judge the role of IRFs in in vivo immune responses and in susceptibility to cancer. They also reveal a role in lymphocytopoiesis. Mice with an inactivating mutation of the DNA binding domain of IRF-1 and kept under specific pathogen free conditions are healthy (observed for 60 weeks). They are thus not prone to develop tumors at a young age spontaneously, such as that seen in p53 -/- mice. However, primary embryonic fibroblasts derived from mutant mice are more susceptible than those from wild type to activated rs-induced transformation, and more resistant to activated ras-induced apoptosis (268). Young adult ZRF-1 mutant mice appear undisturbed in the B cell compartment, indicating that in the B lineage, IRF-1 does not normally play a role, or that other factors can replace its action. They do show a defect in the generation of TCRaPtCD4-CD8' cells, with a 10-fold reduction in frequency in thymus and periphery (see also 228). Further analysis of the thymus at 3 weeks of age suggests that IRF-1 plays a crucial role in the generation of this T cell subset from the CD4'CD8+ progenitor pool. This appears not to be caused by dysregulation of one of the known target genes, MHC class I. Expression of MHC class I genes appears normal in mutant mice. IRF-I mutant mice clear a LCMV infection normally, indicating that they can still mount an effective cytotoxic response. However, ZRF-I mutant mice are impaired in their response to various other infectious agents. Encephalomyocarditis virus resistance is reduced, probably because of a lower interferon-mediated capacity to inhibit viral replication ( 134).The response to Mycobactmiurn hovis infection is impaired, most likely because IRF- 1 is indispensable for the activation of the inducible nitric oxide synthase gene in macrophages (123). ZRF-IIZRF-2 double mutant mice have been


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generated, and used to demonstrate that the abnormalities in ZRF-1 mutant mice are not dependent on normal IRF-2 (134, 268). In contrast to the finding that IRF-1 is dispensable for B cell genesis, mice with inactivated ZRF-2 display B cell defects and wider abnormalities. They are born at a frequency expected on the basis of normal Mendelian inheritance. However, they die before 8 months of age at a frequency higher than wild-type mice. Female mice often die after giving birth. Older mice frequently develop skin erosions. Flow-cytometric analyses of young adult mutant mice show abnormalities in BM but not in thymus, spleen, and lymph node. In the BM, the frequency of Thy-l+TCRa@+cells is reduced to less than half the normal value. This T lineage population may normally be generated in the BM itself (49), rather than in the thymus. Among the population of B220+ cells in BM, the proportion of cells that expresses sIgM is reduced to 30-70% of the normal value. Also reduced in BM are the frequencies of cells responding to LPS (about 30% of normal). Remarkable is the observation that B220'sIgM- cell frequency (fractions A-D, Table 11) appears normal while the frequency of cells responding to IL-7 is reduced to about 60%of normal (as measured in colony forming assays). IL-7 responsiveness normally resides in fraction B and C (Table 11).An interesting follow-up would be to study B220'sIgMcells in the BM of mutants for frequency of fraction B-C cells and IL-7 receptor expression by flow cytometry, as IL-7Ra is a potential target of IRF. The frequencies of cells responding to IL-3 and CSF-1 (normally mediated by multipotential progenitors and macrophage precursors, respectively) are reduced as well, to about 60% of normal. Without further information on erythroid and myeloid differentiation and cellularity in the BM organ these observations are difficult to interpret. In any case, the data indicate an imbalance in B cell genesis as a consequence of inactivating the DNA binding domain of IRF-2. It would be of interest to analyze B cell developmental pathways in IRF-2 mutant mice of various fetal and postnatal age, to see if they exhibit more pronounced defects at any stage. In addition, transplantation experiments may reveal a BM environmental component in the defect. In the spleen of IRF-2 mutant mice, the frequency of B cells appears normal but the response to LPS is somewhat reduced. Serum Ig levels appear normal except for IgG2a which is reduced to about half. Tests of immune defense abilities in uivo show surprising results. Vesicular stomititis virus, a potentially lethal infectious agent, induces a strong humoral response with a switch from IgM to IgG in both normal and IRF-2 mutant mice, indicating that IRF-2 is dispensable for this function. LCMV, however, leads to death of mutant mice but not normal mice by 4 weeks of infection; the cause of death has not been established. Specific CTL activity

tested in uitro at Day 8 appears normal in mutant mice, and by Day 15 the clearance of LCMV from the spleen is complete. Apparently, LCMV triggers iniinunopatliological reactions that are lethal for mice with deficient IHF-2. The observations in ZRF-2 mutant mice, suggesting that IRF-2 is normally active in BM of adult mice, appear coinpatilde with the observed consequences of forced expression of IRF-1 in the B lineage (discussed earlier). In the IHF-1 transgenic animals, IRF-1 may have actually disturbed a iiorinally IRF-l-independent action of IHF-2. IHF-2 may be an actor in cytokine-induced rescue from cell death and proliferation in the B lineage arid possildy other hematopoietic lineages, rather than an initiator of a developmental program. Histologic and kinetic analyses of 13M (see Section 111) may help to clarify this. Hecent i n uitro studies support the notion that IHF-2 has a role beyond acting a s a competitor of IRF-1 for the same cis-elements. IRF-2 inay act independently of I HF-1, by inhibiting nearby transcription activators through its carboxy-terminal transcription repression domain (303). The expression pattern of 1RF genes during normal mouse developiiient has not been described yet. Such a study would help to assess to what extent IHF-1 and IHF-2 coexpress i n uiuo. 8.

Mi$?

The family of myb proteins recognizes a particular consensiis sequence called the Myb motif, found, e.g., in the 5’ flanking region of the huinan CD34 gene (182, 183), in the gene control region of the human and miirine adenosine deaminase gene (CiO), and in CD4. The myb proteins share a characteristic DNA-binding domain, encoded by three imperfect direct repeats, which is widely conserved in evolution (191). They also exhibit a high homology in a domain that can exert negative regulation of transcription. Expression of one of its members, c-myb, in development is evident at E16.5 in liver and thyinus cortex and in epithelium of lung, using ISH (60). C-myb appears high in populations of BM cells from normal mouse enriched for pluripotent progenitors (209). Immature thynocytes and activated T cells also express high levels of c-myb (see (9) and references therein). With regard to the B-lineage, c-myb levels are high in in uitroactivated B cells i n inan (75). The Family member a-myb appears highly expressed in germinal centers of mouse spleen (277), and human tonsil Iymphoblasts (75). These observations suggest that c-myb may inhibit differentiation and induce proliferation during hernopoiesis and lymphocyte activation. C-myb null mutant mice die itz utero at E 15 presumably because of the inability to sustain hepatic erythropoiesis ( 191). Using histology and colony

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forming assays, the frequencies of rnyeloid lineage cells and multipotential progenitors also appear decreased. The null mutation has not yet been further explored for abnormalities in the lymphoid development, e.g., along the lines used for the GATA-2 mutation. A different approach to assess the consequence of lack of functional myb proteins for lymphocyte development has been taken (9). Badiani et al. (9) have constructed dominant interfering myb alleles to block the effects of endogenous myb. One such construct acts as a competitive inhibitor, being a truncated version of c-myb lacking the activator domain but with an intact DNA binding domain. The other is a fusion of the cmyb DNA binding domain to the repressor domain of the Drosophila engrailed transcription factor. Both constructs may interfere not only with c-myb but also with other known myb family members or other as yet unknown proteins capable of occupying myb motifs. The constructs have been put under the control of the 3' flanking region of the human CD2 gene which can direct high levels of expression, in a position-independent manner, to the T lineage in mice. The CD2 control element also directs expression to mature B cells (B precursors have not been analyzed in this respect). Some of the transgenic lines established with either construct are overtly abnormal, with a thymus severely reduced in size at the age of 3-6 weeks. The thymic abnormality is more severe in homozygous than heterozygous mice, indicating that transgene expression levels are important. Among the thymocytes, the CD4-8- population is least affected, possibly because the CD2 regulatory element does not direct significant expression to this stage of T cell development. The number of CD4+8+, CD4+, and CD8' cells is drastically reduced. Interestingly, the spleen shows a reduction in number of T cells as well as sIg+ B cells. The B cell reduction may be a consequence of an intrinsic defect, as transgene expression is detected by Northern analysis of purified splenic B cells. If the transgene is expressed earlier in the B lineage as well, these transgenic mice may be useful for further analysis of B cell development in ontogeny and adult BM to contribute to understanding how myb proteins regulate B cell differentiation and proliferation. Interestingly, in man the factor myeloid zinc finger 1 which is preferentially expressed in primitive hemopoietic cells, appears to regulate transcription of both the CD34 and the c-myb gene (219),while the c-myb also directly regulates the human CD34 gene, The regulation of expression of the murine CD34 gene may therefore be abnormal in the absence of functional myb proteins, and it would be of interest to see the consequences for the murine immune system.

9. Homodomuin Proteins: hlx Homeodomain proteins, a special class of helix-turn-helix proteins, are a group of transcription factors best known for their role in the early

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development of Drosophila and vertebrates (recently reviewed in (145); see also other reviews Cell 78, 1994). Many of the homeodomain proteinencoding genes (hox in mouse, HOX in man) are organized in four different chromosomal complexes, and are all in the same 5’ to 3’ direction for transcription. This organization of the so-called class I genes is highly conserved among vertebrates. Loss-of-function mutations in class I hox genes in mice often lead to changes in cell fate that result in homeotic transformation (a change in “identity”), but may also result in the loss or severe abnormality of particular structures (see (145) for overview). There are several genes encoding homeodomain proteins located outside the four clusters, including hoxll, oct-2, and hlx, which appear relevant for B cell development. The progress in the search for roles of homeodomain proteins in hemopoiesis up to last year has been reviewed by Kehrl(l31) (( 151) is an earlier review). Several lines of evidence, largely involving cell lines and in uitro approaches, suggest a role for certain homeodomain proteins as regulators in blood cell development and an involvement in leukemogenesis. However, few data are currently available that are immediately relevant to the in vivo situation. The attempt recently to describe expression patterns of various homeobox genes in subpopulations of CD34+ cells from normal human BM is an important step forward. The study demonstrates expression of a broad range of class I HOX genes in primitive hemopoietic cells and a regulated expression during their normal development (245).Another area still relatively underexplored involves the target genes for honieodomain proteins involved in hematopoiesis. One of the homeodomain proteins with a potential role in hemopoiesis is encoded by hlx (human homolog: HLX (HB24)),a non-class I gene. Hlx gene products may affect the division of cells and their adhesive properties (for review see (131)). As detected by RNase protection assay (6), hlx is expressed from E8.5, and the level appears to plateau from E10.5 to at least E16.5. Many different tissues dissected from fetuses at E12.5 have significant levels of hlx gene expression. Posterior levels appear higher than anterior levels, suggesting a possible role for hlx in establishing positional information. The liver at E12.5 is rich in hlx mRNA, as is the BM of postnatal mice. In this organ, the Gr-1’ myeloid lineage cells are the highest expressors. B220tsIgM- cells isolated from BM also express hlx, but at a lower level. The thymus contains very low levels of hlx transcripts. Spleen and LN exhibit moderate levels, which could be due to B lymphocyte expression, but this has not been evaluated. Cell lines representing the mature T cell stage, and T cell mitogen-stimulated splenocytes are negative for hlx transcripts. The level in LPS-activated blasts from spleen is rather low.


24 1

A recent study using transgenesis indicates a role for hlx in B cell development in vivo. One line of mice transgenic for hlx under the control of an E, and human CD2 enhancer exhibits defects in both B and T cell development (5). In BM of 7-week-old transgenic mice, the proportion of sIgMt cells is reduced from 12 to 3%, and the proportion of B220+sIgMcells from 15 to 11%. However, the frequency of B220tCD43+ cells is similar to that found in nontransgenic littermates. Thus, the development of B cells in BM appears affected from fraction D (see Table 11) onward. In spleen of 8- to 10-week-old mice, the frequency and number of B220tsIgM+ cells are slightly reduced, but the level of expression of IgM and CD23 in the B220' cell population is normal. However, the proliferative response of spleen cells to LPS is reduced several fold, more than expected on the basis of the somewhat lower frequency of B cells. Serum Ig levels appear normal. Interpretation of the observations is somewhat hindered by the fact that transgene expression has not been analyzed at protein level because of the absence of a specific antibody. In nontransgenic littermates, hlx transcripts are undetectable in Northern analysis of BM and spleen, contrary to what has been found by the (more sensitive) RNase protection assay (6). In transgenic mice, low levels of hlx transcripts are detected but the transcript expression patterns in these organs are unclear. Assuming that the transgene is expressed throughout the B lineage, generating levels of Hlx in the B lineage of transgenic mice that are considerably higher than normal, the observations indicate that Hlx levels in the B lineage are critical for normal control of B cell development from the fraction D stage. A further assessment of the physiological importance of Hlx awaits a loss-of-function mutant mouse. 10. Bmi-1

An exciting area of research concerns the question: who regulates the regulators? Analysis of hox cis-acting elements and their associated factors that govern the specific expression patterns has only just begun. Several genes have been identified which encode proteins that may regulate hox expression (for review see (145)). Some may act by modifymg chromatin structure, e.g., the Polycomb group (Pc-G)of genes which act in Drosophila as negative regulators of homeotic genes by inducing the formation of heterochromatin. Pc-G proteins share a motif called chromodomain, which may play a role in protein-protein interaction (for review see (255)).Genes encoding a homologous domain have been identified in vertebrates, and the bmi-1 gene in mouse is among these. Bmi-1 shows homology with Posterior sex combs (Psc) in Drosophila. Bmi-1 null mutant mice (see below) show a phenotype of posterior transformations which are also observed in mutants of Psc, and mice transgenic for bmi-1 exhibit anterior

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transformation (4), supporting the idea that the fiinction is conserved as well. Whether liox genes are indeed regulated by hmi-1 remains to be investigated. Transcripts of Bmi-l are detected by Northern blotting in mouse enibryos from E10.5. ISH at E12.5 and 14.5 shows that the gene is ubiquitously expressed in the mouse embryo, with relatively high levels in brain, spinal cord, kidney, lung, and gonads anlage (282). h i - 1 null mutant inice show heinopoietic and other abnormalities as well (282). They are born at the expected frequency, but about 50% are cannibalized in the first 3 days after birth. Those surviving the initial days show increasing growth retardation (at 2-3 months of age, their body weight is about half of normal). They also suffer from neurological abnoimalities and waves of sickness, and they die after 3-20 weeks. Pneumonia and opportunistic infections of the intestine, as well as anemia, are often detected in moribund mice. Already at 2 weeks of age, bone cavities are severely hypoplastic and filled with adipocytes. Heinatopoietic cell counts are progressively reduced, to about 30% of wild-type levels at the age of 2-3 months. The B lineage is more affected than the inyeloid lineage. Within the B lineage, stages of B cell development appear differentially affected. In adults, the reduction observed in the B lineage is more severe in the B220’”population (ahout 16% of wild type) than in the B220fCD43+population (about 25% of wild type). Lower body weight and stress because of ill health may contribute to this phenotype, but are unlikely to be the only factors, as BM hypoplasia is already evident so early in life. Similar observations are made for the T lineage. The thymus at birth has about 50% of normal numbers, but by 2-3 months of age it contains less than 1%of the normal number of cells. Those that remain are largely CD4-CD8- and lack TCRaP and CD3. The population size of IL2Rt and HSA’ cells is reduced to the same level as the CD4-CD8- population (roughly 10-15% of wild type), suggesting that the remaining cells are immature T lineage cells. In spleen, the T/B ratio is unchanged; however, the population size is reduced to about 20% of wild type. In heterozygous mutant mice, none of the parameters discussed so fir appear abnormal. A remarkable defect is seen in the response to initogens and growth factors in vitro. Heterozygous mutant mice exhibit an intermediate degree of unresposiveness, indicating that the functions assayed are sensitive to the level of bmi-1 protein. Splenocytes of adult homozygous mutant mice respond poorly to B and T cell antigens, but the LPS resonsiveness in newborn mutants is normal. BM cells of adult homozygous mutant inice do not respond to IL-7 (without or with steel factor) by colony formation in soft agar (at Day 8 after the start of the culture), nor do they respond


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to LPS. The response to M-CSF is also severely reduced. However, colonies are formed in response to IL-3 (an interleukin normally inducing proliferation of multipotential cells) in a frequency similar to wild type. This defect is already observed at 2 weeks of age. Even E14.5 fetal liver cells respond poorly to IL-7 and to M-CSF, but are normal in their response to IL-3. The dramatic unresponsiveness observed implies a functional deficiency in uitro that is less apparent in vivo, and may be due to compartmentalization and the availability of alternative pathways in the latter. There may be an intrinsic defect in hemopoietic cells of Bmi-1 -/- mice, and/or an environmental defect such as an increased level of growth factor inhibitors (secreted by cells that end up in a BM or fetal liver cell suspension) (see, e.g., 295). Some of this may be assessed in vitro by testing the response of wild-type cells in the presence of Bmi-1 -/- cells or Bmi-1 +/- cells. Following the growth of fetal liver or BM cells from wild-type mice in (neonatal) Bmi-1-/- hosts will be equally interesting, and may shed some light on the thymus defect as well. Since serum IgM levels are reduced in adult mutant mice, genesis of the B1 lineage in bmi-1 -/- mice may be affected, an aspect worth investigating.

B. TRANSCRIPTION FACTORS WITH OTHER ROLESI N B CELL DEVELOPMENT A N D FUNCTION 1. Homeodornuin Proteins: oct-2 Oct-1 and Oct-2 are transcription factors that bind to a conserved octamer motif occurring in enhancers of Ig heavy and light chain genes. They are members of the POU family of homeodomain proteins. Their DNA binding domain consists of a POU specific conserved N-terminal segment and a carboxy-terminal homeodomain. Oct-2 shows a predominantly lymphoid-restricted expression pattern, while Oct-1 appears more widely expressed. In man, coactivators of Oct-1 and -2 have been described that may enhance their action in a tissue-specific manner (81).Studies to assess the importance of Oct-2 for B cell development, including an analysis of null mutant mice (41), have been reviewed recently (256). A general conclusion is that Oct-2 is dispensable for Ig gene rearrangement and expression early in development, However, at birth the oct-2 mutant mice do show abnormalities in the spleen and liver that indicate that levels of Oct-2 are critical for the later stages of B cell development. While total nucleated cell numbers in newborn spleen are normal, the ratio of BPl+pcells over BPl-p+ cells is increased in heterozygotes and even more so in homozygous mutant mice. To interpret this as a relative increase in precursor B cell number needs some caution, as BP-1 expression in newborn

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spleen has not been carefully checked for its B-lineage-restricted nature. The frequency of LPS-induced IgM and IgGl secreting clones is reduced in both spleen and liver of newborn mutant mice. The clones that do form produce less Ig in the mutant mice. Again, the reduction is detected in heterozygotes and more severely in homozygous mutant mice. The levels of Oct-2 in B lineage cells of spleen and liver have not been measured and coinpared between newborn wild-type, heterozygous, and homozygous mutant mice, but the phenotype suggests that a single normal allele cannot generate the same levels as two normal alleles. Earlier, at E16-17, the liver of mutant mice contains normal levels of Ig gene rearrangements, and normal frequencies of B220' p', and IL7-clonable cells. Older mutant mice cannot be assessed: the homozygous mutant mice die within a few hours after birth for unknown reason. Transfer of fetal liver cells from Oct-2 -/- mice to adult mice with severe combined immunodeficiency results in reconstitution of the mature B and T lymphocyte pool. Again, however, B cell functions are disturbed , while T cell function appears normal (41, 246). It will also be of interest to follow into adulthood the heterozygous mutant mice, which appear normal in their viability. Such analysis may determine the extent to which B cell development and B cell activation stay dependent on Oct-2 levels.

2. Horneoclomain Proteins: H o d 1 Another homeobox gene located outside the 4 Hox clusters, interesting from the point of view of B cell development, is H o d I . Its counterpart in man was originally isolated from a chromosomal breakpoint in acute lymphoblastic leukemia (ALL) of the T lineage. In this translocation, HOXl1 is brought under the regulation of the T cell receptor a chain gene, resulting in overexpression of HOXll in the T lineage. Mice with forced expression of HOXl1 in the T lineage develop T-ALL, illustrating

the oncogenic potential of HOX11 (see (231) and references therein). In mouse development, ISH shows that Hoxll (almost identical to HOXl1) is first expressed at E8.5, within portions of the developing branchial arches and, from E10.5, in portions of the developing hindbrain and in select cranial ganglia (225,232).From E11.5, Hoxl1 is also expressed at a single site in the abdomen within a portion of the splanchnic mesoderm destined to form the spleen. Its expression follows the developing spleen, histologically recognizable from E12.5, up to E13.5, but is down-regulated thereafter (231). Mice homozygous for a disrupted allele of Horll appear completely healthy with no obvious abnormalities in the tissues deriving from the branchial arches, or in the central nervous system. However, a spleen does not develop in these mice. Possibly as a consequence of this, the relative weight of the stomach (arising from the same embryonic site)

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in hoxll -/- mice is on average higher than normal (232). This is the first example of the genesis of a single organ depending on a single homeodomain protein. The Hoxll -1- mice are ideal to study the role of the spleen in hemopoiesis during mouse development (hemopoietic cells normally enter the spleen from E 15). Preliminary observations postnatally indicate some abnormalities in the blood, with erythrocytes containing nuclear fragments present at 4 days after birth, and (at an unspecified age) a twofold increase in numbers of neutrophils and lymphocytes. Flowcytrometric analyses of T and B lineage cells in thymus, lymph node, and peripheral blood (age of mice again not specified) show normal profiles. Extension of the observations to various (including fetal) ages, and to include BM and peritoneal cavity, is eagerly awaited.

3. Re1 Proteins: p50 The Re1 family are transcription factors residing in the cytoplasm in inactive form, responding to external stimuli by rapid activation and translocation to the nucleus. They are best known for their engagement in the immune response. The Re1 homology domain includes DNA binding and dimerization domains, and a nuclear localization signal. Members of the IKB family, which contain ankynn-like repeats to bind to the nuclear localization signal of Re1 family proteins, mostly serve to prevent entry of Re1 into the nucleus. Stimuli recruiting Re1 to the nucleus act by phophorylating and degrading IKB proteins (for review see (111, 273). Re1 family members are active as homodimers, or heterodimers with other family members. They may also synergize with members of other transcription factor families, e.g., HMG box containing proteins (for review see (10, 254)). Best understood for the role in lymphocyte activation pathways is NF-KB, a dimer of p50 and RelA (p65);both proteins are expressed widely. RelB is a member of the family with a rather restricted expression pattern, and may function as a constitutive rather than inducible binder of KB sites (153,291)). Re1 (c-rel) exhibits a pattern of expression that suggests a role in development of hemopoietic lineages (33).p50 and RelB but not yet Rel, RelA, and p52 (encoded by nfkb2, yet another re1 family member) have been studied for their importance for hemopoietic development and function by generation of null mutant mice. Importance of the p50 member of the Re1 family for hemopoiesis may be indicated by data on expression pattern and potential target genes. Sequential induction of gene expression of re1 family members is apparent when studying cell lines representing different stages in B lymphocyte development (159, 186). P50 is part of DNA-binding heterodimers in preB as well as mature B cell lines, suggesting roles early in B cell genesis. Mice with a null mutation in nfkbl encoding the p105 precursor of p50

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are healthy if housed under specific pathogen-free conditions (251).B and T cell population size and phenotype composition appear normal in spleen, thymus, and BM. The expression of targets of p50 such as the genes encoding I ~ light K chain, MHC class I and I1 appears undisturbed, indicating compensation of p50 function by other transcription factors. An '1bnorinality in immune response is obvious from the susceptibility of p50 mutant mice to infection when housed conventionally. Serum Ig levels are highly abnormal, with IgM slightly elevated but all other isotypes severely reduced. The specific antibody response to a T cell-dependent antigen is also much lower in sera of niutant mice. In light of a possible role in IgH isotype switching for p50, it would be of interest to study the germinal center reactivity in mutant mice. A prominent defect is evident when measuring the activation response in small resting B cells from spleen. In mutant mice, cytoplasmic extracts of these cells exhibit an absence of p50 but also a decrease in other Re1 fainily members. The mutant B cells do not respond to LPS by increasing levels in the nucleus of proteins binding to a KB site, nor by cell proliferation. Using anti-IgM as activating stimulus, the defect is somewhat less pronounced. An abnormality in vitro in T cell stimulation is mentioned as well. The survival rate in response to pathogens is occasionally different between wild-type and mutant mice, with, e.g., increased susceptibility to S. pneumoniue but not H . injuenaae, and increased resistance to EMC virus. I n vitro, embryonic fibroblasts of p50 mutant mice are inducible by Sendai virus to higher levels of transcription of IFNP genes, which may explain the relative resistance to virus observed in vim. The observations in uivo indicate that p50 proteins are crucial for many aspects of regulation of the immune response, including those involving B cells, but are dispensable for the genesis of B lymphocytes.

-

4. Re1 Proteins: Rel-B A discussion of RelB is included because null mutant mice, showing severe dysregulation of hemopoiesis, may become instructive with regard to regulation of B cell genesis. RT-PCR studies on organs of adult normal mice show that thymus, spleen, and intestine express RelB; no transcripts are detectable in BM, liver, kidney, and testis (32). RelB expression has also been studied by ISH on sections of embryos and organs (32, 291). RelB transcripts are undetectable in prenatal development, except for the thymus from E14, expressing RelB in increasing levels, with highest levels in the medulla from the start of its formation. Maximum levels are reached at 1 week after birth, which stay constant up to 6 weeks, the latest age analyzed. Postnatally, RelB transcripts are also detected in splenic white

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pulp (except marginal zone (291))and lymph node, with T cell areas richer than B cell areas (32). Immunohistology shows high levels of RelB in dendritic cells of thymus, and in scattered cells in splenic white pulp. Among dendritic cells isolated from thymus, subcellular distribution of detectable RelB proteins is variable, in both cytoplasm and nucleus, or mainly the nucleus (32). BM has not been studied by ISH and immunohistology. The negative RT-PCR result on cells isolated from this organ still leaves open whether stromal elements of BM are RelB'. Little information exists on the target genes of RelB. RelB would function mainly as heterodimers with p50 or p52 (291). MHC class I1 and IL2R genes, which contain functional KB binding sites in their promoters, are highly expressed in dendritic cells; they may be regulated by RelB. Mice homozygous for a null mutation in RelB (292) develop to birth normally. Binding activity to KB sites is severely reduced in extracts of thymus from homozygous mutant mice at 6 weeks of age, indicating a requirement for RelB in constitutive binding of KB sites in this organ. The reduction observed in extracts from spleen may be due in part to massive increase in red pulp. Between 2 and 6 weeks after birth, mutant mice display increasing signs of illness. Lymphohemopoietic organs exhibit abnormalities. The BM shows myeloid hyperplasia accompanied by a decrease in proportion of erythroid lineage cells. A normal distribution of CD43 and IgM among B220+ cells is mentioned, suggesting no impairment in early B cell development. It is unclear whether B lineage cell numbers in the marrow organ and production rates are changed. Leukocytosis is detected in blood, which appears normal with regard to erythrocytes. The spleen also shows an imbalance in lineages; erythropoiesis and myelopoiesis are increasing with age, reflected by a severe increase in splenic weight. Neutrophils are detected not only in red pulp but also white pulp, a sign of inflammation. At 7 weeks of age, proportions of B220' cells and, more severely, Thy-lt cells, are reduced; it is unclear whether this is accompanied by a reduction in lymphocyte numbers per spleen as well. IgM' to IgD' cell ratio is normal. The thymus exhibits a variable degree of atrophy, with the medulla more affected than the cortex, unexpected if stress alone would be the atrophyinducing factor. From 3 weeks onward, dendritic cells in medulla seem reduced in number per area cross-sectioned, as detected by immunohistology. However, at 7 weeks of age, proportions of CD4+Bt.CD4+,and CD8' cells appear unchanged. Delayed type hypersensitivity reaction is reduced in homozygous mutant mice; the age at which this has been tested is not reported. A defect in dendritic cells of draining lymph nodes caused by the absence of RelB may underly this reduced T cell function.

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Several organs other than spleen become increasingly inflamed, including lymph nodes, lung, and liver. The reaction seems not to be induced by common murine pathogens. Possibly, an excessive response to innocuous antigens is causing the pathology observed, which is also observed in mice with null mutations for various cytokines. On the basis of current knowledge about expression pattern and fuction of RelB, not all abnormalities in mutant mice can easily be understood. It will be very interesting to see whether FL and BM contain RelB' stromal cells, While B cell developmental block is not a consequence of absence of RelB, possible other ways in which the B lineage is affected, for instance in production rates, require a more detailed analysis. A study in mutant mice of germinal center and plasma cell reactions, and serum Ig levels are of interest, because RelB has been found to function as heterodimer with p52 in plasmacytomas and long-term LPS-treated pre-B and B cell lines (159). 5. C-FOS Thefos gene family codes for proteins that share a basic region essential for binding to DNA, and a leucine zipper domain for protein dimerization. Fos proteins typically heterodimerize with proteins encoded by the jun family, and show variable binding affinity to AP-1 sites in the DNA. The AP-1 site (consensus core sequence: TGACTCA) is a cis-element found in a wide variety of genes, e.g., those encoding various metalloproteases, and interleukin-2. Fos/jun binding results most often in activation or (in some cases) repression of gene transcription. Fos proteins may also act in association with members of other families of transactivators, e.g., Re1 (for review see (202)). One of the members, c-fos, is a widely distributed protein and has been studied extensively for its function, by overexpression or inactivation in cell lines and by transgenesis. Such studies indicate an important role for c-fos in the nervous system and in bone formation (for overview see (120)).A role for c-fos in early B cell development is suggested by its expression (mRNA levels and as functional AP1) during culture of initially B220+cell-depleted B M on stroma plus IL-7 (66),showing highest levels at the stage where more than 90% of the cells are B220' sIgM-, and a decrease thereafter, when sIgM+ cells emerge. Trans enic mice generated from a construct ofc-fos under the control of the H-2K promoter (directing expression to mature T and B cells, thymic epithelium and (in low levels) to BM (67))show a decreased ability to respond to antigen by specific IgG production, apparently intrinsic to antigen-specific B cells in spleen (267).In addition, an inhibitory effect of an inducible c-fos transgene on pre-B cell development in vitro has been reported (275). Mice double transgenic for c-fos and v j u n under the control of the H-2Kb promoter

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illustrate that deregulated levels of AP-1 results in early B cell developmental defects (67). Mice have been generated homozygous mutant for c-fos by disruption of exon 2 (289) or exon 1 (120); their phenotypes are quite comparable. In a cell line derived from primary embryonic fibroblasts of c-fos -/mice, some but not all of the stimuli investigated are unable to enhance transcription of some (but not all) of the AP-1 site containing genes, such as metalloprotease genes (106). This illustrates how selective absolute requirement for c-fos may be. When surviving the first day after birth, mutants appear normal in the next 10 days but then rapidly develop growth retardation. Subtle alterations in the nervous system are evident (120). Bone malformation is a very obvious abnormality. The marrow cavity of long bones becomes severely affected by a form of osteopetrosis, reminiscent of that found in young op/op mice which lack functional CSF-1 (see (200) for recent overview). This case of c-fos deficiency appears to have caused abnormal bone resorption as well as directly affected osteogenesis and chondrogenesis. There is severe reduction in space available for hemopoiesis, but also in other microenvironmental elements such as the osteoblastic lining of the cavity (endost). In the initial studies, a reduction has been noted in blood lymphocyte concentration in 4 out of 5 mice at 7-11 weeks of age (120);a reduction of proportion of B220+ cells in spleen has been observed already by 2 weeks after birth (289).The proportion of B220+ cells in liver before birth is normal in mutant mice. A follow-up study with regard to hemopoietic abnormalities has recorded B lymphopenia in about 50% of c-fos -/mice, and the degree of severity increases with age (206). As discussed below, marrow environmental abnormalities appear to account for B cell reduction. Additionally, thymic atrophy correlating with the degree of ill health is seen, and ill health may also contribute to a decrease in hemopoietic activity in marrow. B220+ cell frequency in the peritoneal cavity at 8 weeks of age is normal, suggesting that the abnormality is restricted to the postnatal conventional B cell population (206). The absence of c-fos appears not to cause defects intrinsic to hemopoietic cells. First, although myelopoiesis and erythropoiesis are unusual in c-fos -/- mice in that they reside in spleen rather than marrow, this appears to compensate sufficiently, as the RBC profile of peripheral blood is normal (289) and the frequency of myeloid cells increased rather than decreased (206). Second, also for the B lineage the spleen of mutant mice seems to compensate for marrow in terms of progenitor activity. Using splenocytes for culture of B cell progenitors on stroma plus IL-7, or in methylcellulose in the presence of IL-7, the mutant mice develop B220+sIgMf cells in a frequency similar to (former assay) or higher than (latter assay) wild-type

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mice. Third, when transplanting c-fos -/- splenocytes or c-fos +/+ BM cells to lethally irradiated wild-type mice, long-term (3-6 months) hemopoietic reconstitution of recipients takes place in both cases, including similar levels of B220+ cells in spleen. Yet, while B progenitor activity is abundant in mutant spleen, the peripheral B lymphopenia observed in cfos -/- mice indicates that BM is not efficiently replaced by spleen in its capacity to generate mature B cells. Alternatively, expansion of the peripheral B cell pool is disturbed. Stimulation of primary B cells via sIg or CD40 results in enhanced expression of c-fos (108).Overexpression of c-fos inhibits proliferation of B cells upon stimulation with anti-IgM (138). It will thus be of interest to see the extent to which B cell-dependent immune responses are affected by the lack of c-fos. Proliferative responses of peripheral T cells to T cell receptor stimuli appears impaired in c-fos -1- mice at 4-10 weeks of age (308) but not at 10-14 weeks of age (117). At that older age, no developmental abnormalities in thymus are detected either. 6. Note Added: EBF and Ets-1

Near the completion of this manuscript, publications on mice with a targeted mutation in EBF or ets-1 appeared. EBF is a transcription factor implicated in the regulation of the mb-1 gene . Young adult EBF-deficient mice (157) lack B1 and B2 cells; the BM contains only fraction A type of cells committed to the B lineage (see Table IIIA). Ets-1, implicated in regulation of lymphoid-specific genes, is shown by RAG-2 blastocyst complementation to be unnecessary for genesis of mature T and B cells. However, B and T lineage cell populations derived from ets-1 -/- ES cells exhibit abnormalities, i.e., poor viability in the T lineage, and an unusually large pool of IgM-secreting plasmacells in the spleen (23). V. Concluding Remarks Current informatio,i on the phenotype of mice with mutations in transcription factors indicates that much valuable information may still be retrieved from them. Further identification of genes disturbed in their expression, including those encoding cell cycle and death regulators, will give insight in genetic programs operating in hemopoiesis and B cell development. Novel genetic tools in vivo will allow a follow-up of the effect of mutations in healthier animals. Transcription factors necessary for B cell development may be tested further for ability to drive the differentiation pathways by ectopic expression in suitable cell types, e.g., in hemopoietic stem and progenitor cells, by using the control region of the CD34 gene (172).Together with the growing insight in surface receptor

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engagement and signallingpathways, an integrative vision of B cell development in uiuo is emerging.

ACKNOWLEDGMENTS I express my gratitude to Dr. M. Schilhain for communicating data on sox-4 before they appeared in print; to my graduate students and research assistants, Kwong Yi-hang, Lam Yun-wah, Jeethan Bendoor, Stuart Fraser, Hafejee Essackjee, Lachlan Macdonald, Marjan Veuger, and Peter-Paul Platenburg for contributing their research data and for their moral support; to Mr. Y.S. Ho, Yun-wah, and Lachlan for help with the bibliography: to Teresa Couto for secretarial support; and to Yun-wah, Stuart, and my colleagues Dr. Sham Maihar and Dr. Kathryn Cheah for useful comments on the manuscript. I gratefully acknowledge financial support from the Research Grants Council of Hong Kong (Grants HKU17/90 and HKU374/94M), the Croucher Foundation (Grants 360/032/0918 and 394/032/1236), and the Committee on Research and Conference Grants/ Medical Faculty Research Grants Fund of the University of Hong Kong (Grants 337/032/0009,335/032/0050,337/032/0022, 337/032/0030, 3W032/3948, 361/032/3090, 335/032/0055, and 337/032/7895).

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ADVANCES IN IMMUNOLOGY. VOL. 63

Soluble Cytokine Receptors: Their Roles in Immunoregulation, Disease, and Therapy RAFAEL FERNANDEZ-BOTRAN,' PAULA M. CHILTON,' AND WHE MA+ lXvisian of E x p u i m d lmmundogv and ImmunapalfidoSy,' &patinmnf of Parhahgy, and 'hpatinmnt 0fMicrobidogy and Jmmunahgy, Schod o f Medicim, U n h i t y of h i r v i l k , hisdb, Kmh~ky40292

1. Introduction

One of the hallmarks of the immune system, besides specificity and memory, is its mobility. In contrast to most other organ systems, which occupy defined anatomical spaces, the cells of the immune system are present throughout the body and are able to move from the blood or lymph into practically any tissue or anatomic compartment. A close communication among different cells and cell types is, nevertheless, essential for most immunological processes, including antigen recognition, cell recruitment and migration, and cellular activation, proliferation, and differentiation. The immune system is, therefore, dependent on and regulated by a complex set of interactions that allow cellular communications to take place. These interactions are mediated both by physical cell to cell contact and by soluble factors, the most important of which belong to the group collectively known as cytokines (Vitetta et aZ.,1989). Cytokines are a group of small regulatory glycoproteins, secreted primarily but not exclusively by white blood cells, that mediate many physiologic processes, including inflammation, immunity, and hemopoiesis (Arai et al., 1990; Oppenheim and Saklatvala, 1993; Abbas et aZ., 1994). The effects of cytokines on target cells are exerted by binding to specific membranebound cytokine receptors, which function as ligand-binding and signal transduction moieties, converting a signal (cytokine presence) into a cascade of enzymatic reactions that ultimately result in alterations in phenotypic characteristics and/or gene expression by the target cell (Miyajima et aZ., 1992; Taga and Kishimoto, 1993; Ihle et al., 1995). The activity of a cytokine in uiuo is directly influenced by its concentration in the extracellular compartment and the availability of cytokine receptors on the membrane of responding cells, Therefore, alterations in the extracellular levels of a cytokine and/or in its ability to interact with specific membrane cytokine receptors are two important mechanisms that regulate cytokine activity in uiuo. In the last few years, evidence from numerous laboratories has demonstrated that truncated forms of different cytokine receptors are generated in uivo, either by proteolytic cleavage of the membrane-bound receptor 269 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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or by translation from an alternatively spliced mRNA, separate from that encoding the full-length membrane form. These truncated receptors are released from the cells and appear as soluble cytokine-binding proteins (“soluble cytokine receptors”) in biological fluids or tissue culture supernatants ( Fernandez-Botran, 1991).Very importantly, these soluble cytokine receptors (sCR) appear to have the ability to influence the extracellular levels of their ligand (cytokine) and, at the same time, interfere with its interaction with membrane receptors. These properties, together with their widespread existence, have thus led to the idea that the generation of sCR constitutes a common physiological mechanism for regulating the activity of cytokines ( Fernandez-Botran, 1991; Arend, 1995). Nonetheless, the exact physiological role played by the sCR in the direct regulation of cytokine activity in vivo and their overall contribution to the regulation of immune responses remain unclear. Besides their putative immunoregulatory function, the ability of sCR to specifically alter the biologic activity of their ligands, coupled to a lack of immunogenicity (if used within the same species), has generated considerable interest in potential therapeutic applications, particularly in clinical conditions in which cytokines play a prominent pathophysiological role, such as inflammatory and autoimmune diseases. Moreover, it has become apparent that the levels of different sCR in biological fluids correlate with immune function and/or activation in many pathologic conditions and can be used as helpful “markers” in the diagnosis, management, and prognosis of human diseases. The first part of this review includes discussion of the mechanisms that regulate cytokine activity and the generation and function of sCR as cytokine antagonists and/or cytokine “carriers” in vivo. Then, in the second part we review, in a more specific manner, information about the different sCR, their potential involvement in diseases, and their use as immunotherapeutic agents. II. Regulation of Cytokine Activity

As it is currently used, the term “cytokines” refers to regulatory proteins made by T and B lymphocytes (lymphokines), monocytic cells (monokines), interferons, hematopoietic colony stimulating factors, and connective tissue growth factors. Discussion of the different cytokines and their biologic activities is beyond the scope of this work, and readers are referred to several excellent reviews (Smith, 1988; Van Snick, 1990; Paul, 1991; Vassalli, 1992; Farrar and Schreiber, 1993; Dinarello, 1994). Despite the fact that cytokines are a diverse group of proteins, they share a number of properties, of which pleiotropy (the ability of one

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cytokine to act on many different cell types) and redundancy (the ability of different cytokines to have the same activity on a given cell type) are two of the most biologically significant (Paul, 1989). In contrast to hormones, which are secreted continuously by specialized cells in a defined anatomical location, most cytokines are produced only as a result of cellular activation, and are secreted by many different cell types, practically anywhere in the body. Hormones act exclusively in a systemic or endocrine fashion, whereas cytokine activity is normally exerted locally, in autocrine or paracrine manner, although some cytokines may also act systemically at times. Cytokines are very potent mediators, acting at concentrations well below 100 pM and requiring occupancy of only a fraction of the membrane cytokine receptors on a cell (as low as 10%)in order to exert their biologic effects. Moreover, some cytokines can act as potent inducers of the synthesis of other cytokines, leading to cascades and networks of interacting cytokines that result in amplification of their biological activities (Oppenheim and Saklatvala, 1993). A tight control on cytokine activity in vivo must be, therefore, essential in maintaining homeostasis in the immune system. Consistent with the level of its complexity, the activity of cytokines is simultaneously regulated at multiple points. These include: (1)Cytokine synthesis and secretion; (2) Expression of membrane cytokine receptors on target cells; (3)Interaction of secreted cytokines with membrane receptors; and (4) Events after cytokineheceptor interaction. Table I summarizes the different mechanisms that contribute to the regulation of cytokine activity.

A. CYTOKINE SYNTHESIS AND SECRETION The production of cytokines is under the concurrent control of a variety of transcriptional, translational, and post-translational mechanisms. Most cytokines are not produced constitutively, but are synthesized and secreted in response to a variety of stimuli, depending on the nature of the cytokineproducing cell. In the case of inflammatory cytokines (e.g., TNFa, IL-1, IL-6), in which the major producing cells are monocyte/macrophages, endotoxin and other microbial products, immune complexes, complement components, and contact with activated T cells are the major inducing signals (Oppenheim and Saklatvala, 1993;Vassalli, 1992; Dayer and Burger, 1994; Dinarello, 1994). Antigenic andfor mitogenic stimulation are the major signals inducing cytokine secretion (e.g,. IL-2, IL-4, IFNy) by T lymphocytes (Mosmann et al., 1986; Paul, 1989), and signals mediated by mitogens and FcgR induce cytokine secretion (e.g., IL-3, IL-4, TNFa) by mast cells (Plaut et al., 1989).Cytokines themselves are also potent inducers of the secretion of other cytokines, such as in the case of TNFa and other

MECHANISMSIN Level

I. Cytokine synthesis and secretion

11. Expression of membrane cytokine

receptors

111. Interaction of cytokines with membrane receptors

THE

TABLE I REGULATION OF CYTOKINE Acrnm-i Process

Transcriptional regulation mRNA stability Transcriptiodtranslation Processing and release Increased receptor number and affinity (enhancement) Competition (inhibition) Interference

Enhancement

IV. Events after cytokindreceptor interaction

Signaling ? Sy.iergisdantagonism

Mechanism Transcription factors ' 3'-AU rich motifs Hormones (e.g.,corticosteroids) Drugs (e.g., cyclosporin) Cytokines Proteases (e.g., metalloproteases) Cell activation Cytokines Nonsignahng receptors (e.g., IL-lR type 11) Soluble cytokine receptors Receptor antagonists (e.g., I L l r a ) Nonreceptor cytokine-binding proteins (e.g., uromodulin) Anti-cytokine autoantibodies Soluble cytokine receptors Nonreceptor cytokine-binding proteins (e.g., cu,-macroglobulin) Anti-cytokine autoantibodies Hormones (e.g., corticosteroids) Drugs (e.g., rapamycin) Cytokines


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inflammatory cytokines, including IL-1, IL-6, and IL-8 (Akira et al., 1990; Oppenheim et al., 1991). In other cases, however, cytokines down-regulate the secretion of other cytokines, such as the case of IL-10 inhibiting secretion of IL-2, IFNy, and LT by Thl cells (Fiorentino et al., 1989) and that of IL-4 and IL-10 inhibiting the production of inflammatory cytokines by macrophages (Hart et al., 1989; Standiford et al., 1990; Moore et al., 1993). In general, cytokine secretion is brief and self-limited. Cytokines are not usually stored as preformed molecules, but have to be newly synthesized following cell stimulation. Cytokine-inducing signals normally result in the synthesis or activation of transcriptional factors, which directly, but transiently, activate transcription of cytokine-coding genes (Muegge and Durum, 1990). Moreover, cytokine mRNAs are usually short-lived due to the presence of AU-rich motifs in their 3’ untranslated regions that target them for degradation (Caput et al., 1986). Finally, the release of some cytokines, such as IL-lP and TNFa, is also dependent on posttranscriptional modifications, such as the proteolytic cleavage of precursors into the active mature forms (Black et al., 1988; Gearing et al., 1994).

B. EXPRESSION OF MEMBRANE CYTOKINE RECEPTORS ON TARGET CELLS The activity of a cytokine also depends on the ability of target cells to detect its presence and respond to it, a process that is mediated by specific membrane cytokine receptors. Albeit in low numbers, cell-surface receptors are often constitutively expressed, thus assuring the responsiveness of target cells to regulatory effects of cytokines. Nonetheless, cellular activation normally induces an up-regulation in the number of membrane cytokine receptors and/or an increase in their affinity, thus enhancing the ability of target cells to respond to cytokines (Hemler et al., 1984; Lowenthal et al., 1985; Lowenthal and Greene, 1987; Wang and Smith, 1987; Ohara and Paul, 1988;Loughnan and Nossal, 1989). In some cases, cytokines such as IL-2 or IL-4 induce the increased expression of their own membrane receptors, a process that results in positive feedback regulation (potentiation) of their activity (Bismuth et al., 1984; Smith and Cantrell, 1985; Ohara and Paul, 1988; Renz et al., 1991). The late 1980s and early 1990s saw the systematic cloning of the genes coding for the receptors of most cytokines. Although single chain cytokinebinding proteins were initially referred to as “cytokine receptors,” evidence now indicates that, even though a number of single chain cytokine receptors do exist (e.g., IL-1R and TNF-R), most “functional” cytokine receptors are normally multichain complexes, in which subunits with ligand-binding function associate with signal transducing subunits (Miyajima et al., 1992;

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Taga and Kishimoto, 1993; Ihle et al., 1995).These are often shared among different cytokine receptors, such as the IL-2R-y chain in the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (Kondo et al., 1993; Noguchi et al., 1993; Russellet al., 1993; Grabstein et al., 1994; Kimuraet al., 1995);gp130 in the receptors for IL-6, leukemia-inhibitory factor (LIF), oncostatin-M (OSM), and ciliary neurotropic factor (CNTF) (Gearing et al., 1992): and a common &subunit in the receptors for IL-3, IL-5, and GM-CSF (Tavernier et al., 1991). Although the ligand-binding subunits alone often display relatively low affinities for their ligands (& -1-10 nM), interaction with the signal transducing subunits usually increases their binding affinity, leading to the formation of functional receptors of high affinity (& -10100 pM). Thus, up-regulation of a subunit (by cellular activation and/or cytokines) can lead to a significant enhancement in the affinity of such receptor and an increase in the ability of expressing cells to respond to that particular cytokine. For example, cellular activation results in the transcriptional activation and expression of the IL-2Ra subunit (a low affinity receptor). However, in the presence of the constitutively expressed IL-2RP and -7 chains (Kondo et al., 1993; Russell et al., 1993), a highaffinity IL-2 receptor is formed, allowing the activated cells to respond to very low concentrations of IL-2 (Minami et al., 1993). Information about the subunit composition of different membrane cytokine receptors is summarized in Table 11. Although the expression of membrane cytokine receptors is normally a positive influence on cytokine activity, some receptors may actually have inhibitory effects. Even though most cytokine receptors are capable of signal transduction after binding of their ligands, some receptors, such as the IL-1R type 11, do not appear to generate any signals upon ligandbinding. It has been suggested that such receptors may actually function as “decoys,” preventing binding of IL-1 to the biologically active IL-1R type I (Sims et al., 1993; Colotta et al., 1993). Expression of such a type of receptors would, therefore, result in the negative regulation (inhibition) of IL-1 activity. C. INTERACTION OF SECRETED CYTOKINES WITH MEMBRANE RECEPTORS Once a cytokine is secreted, many factors may influence its ability to interact with its membrane receptors on target cells. Interference with the binding of cytokines to their receptors (and subsequent signal transduction) appears to be a common mechanism in the regulation of the activity of cytokines in vivo. Several different factors may influence the ability of a cytokine to interact with its receptors. Among these are: (1)soluble cytokine receptors, (2) receptor antagonists, and (3) nonreceptor proteins.

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TABLE I1 SUBUNIT COMPOSITION OF CYTOKINE RECEPTORS Cytokine IL-1 family (IL-la, IL-1P, IL-ha) IL-2

IL-3 IL-4 IL-5 IL-6 IL-7 TNF family (TNFa and TNFP) Type I IFN (IFNa, IFNP, IFNL) Type I1 IFN (IFNy) GM-CSF

Subunits Single chain, IL-1R type I Single chain, IL-1R type I1 IL-2Ra ( ~ 5 5 ) IL-2RP ( ~ 7 0 ) IL-2Ry (p64) IL-3Ra IL3RP (130 kDa) IL-4Ra (140 kDa) IL-2Ry ILJRa IL-3RP (130 kDa) IL-6Ra IL-6RP (gp130) IL-7Ra IL-2Ry Single chain, TNF-R type I Single chain, TNF-R type I1 IFNcu/P-Ra IFNdP-RP (IFNAR?)b IFNy-Ra IFNy-rP GM-CSF-Ra IL3RB (130 kDa)

Main functiodaffinitf Binding and signaling (high) Binding, but not signaling (high) Binding (low) Binding and signaling (intermediate) Signaling Binding (low) Signaling Binding (high) Signaling Binding (low) Signaling Binding (low) Signaling Binding (low) Signaling Binding and signaling (high) Binding and signaling (high) Binding and signaling Binding and signaling (?) Binding (low) Signaling Binding (low) Signaling -

1

The relative binding d n i t i e s showri in parentheses are for each subunit alone. Interaction with their signal transducing subunits is usually required for high-affinity binding. IFNAR: Interferon alpha receptor protein.

1 . Soluble Cytokine Receptors Interference with receptor binding is usually cytokine-specificand mediated by cytokine-binding proteins, the majority of which are actually soluble forms of cytokine receptors (sCR) ( Fernandez-Botran, 1991).These sCR function by competing with the cell surface receptors for the binding of free cytokine molecules. In most cases, with the notable exception of soluble IL-6R (Hibi et al., 1990),the soluble receptor/cytokine complexes are unable to interact with their respective membrane-bound signal transducing subunits, and are thus biologically inactive. Hence, sCR prevent cytokines from reaching their membrane receptors and generating a signal, leading to inhibition of cytokine activity. There are situations, however, when the concentration of active cytokines in biological fluids are actually enhanced as a consequence of their interac-

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tion with sCR. Inasmuch as the binding between cytokines and sCR is reversible, cytokine molecules are only temporarily “sequestered” from their membrane receptors. The increased cytokine levels sometimes translate into enhanced biologic responses, particularly when the concentrations of free sCR are not high enough to capture back the dissociating cytokines (Sat0 et al., 1993). The ability of sCR to enhance the concentrations of active cytokines in vim is probably the result of several factors, such as increased cytokine stability, decreased proteolytic degradation, and altered pharmacokinetics (e.g., prolonged in vivo half life, reduced clearance) (Fernandez-Botran and Vitetta, 1991; Aderka et al., 1992). Thus, despite their antagonistic activities, sCR can also enhance the concentrations of cytokines in biological fluids, sometimes even potentiating their effects. Such a function as cytokine “carriers” in vim may help potentiate the systemic or endocrine effects of cytokines. The generation of endogenous sCR is a widespread phenomenon, since soluble receptors for many of the cytokines have been reported in both mice and humans. Table I11 lists the cytokine receptors for which soluble forms have been described. The generation and function of these molecules will be discussed in the next section. TABLE 111 SOLUBLE CYTOKINE RECEPTOHS~ Cytokine

IL-1 family IL-2

I L-4 IL-5 IL-6 IL-7 TNF family

Soluble receptor(s)

SIL-1RI

sIFNcu/P-Ra

Binds IL-la, IL-1/3, and IL-lra (high affinity) Preferential binding of IL-1/3 Low affinity Low affinity High affinity Low affinity Low affinity, can mediate signaling Binds to IL-G/IL-GRa complex Low affinity Preferential binding of TNFa (high affinity) Preferential binding of T N F a (high affinity) Binds type I interferons

sIFNy-Ra sCM-CSF-RCY

Specific for IFNy Low affinity

SIL-1RII sIL-2Ra (p55) sIL-2RB (p70) sIL-4R (murine) SIL-5Ra sIL-6Ra sIL-6Rp (gp130) sIL-7Ra sTNF-RI (p55) sTNF-RII (p75)

Type I IFN (IFNa, IFNP, IFNw) Type I1 IFN (IFNy) GM-CSF

Comments

“ Data are for human sCR, except as indicated.


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2. Receptor Antagonists A different type of mechanism for preventing binding of a cytokine to its membrane receptors are receptor-binding antagonists. These molecules compete with the biologically active cytokine for binding to the same receptor; however, they are unable to generate a signal upon binding and thus effectively block formation of active cytokineheceptor complexes, To date, the only example of this type of inhibitor is the IL-1 receptor antagonist (IL-lra) (Hannum et al., 1990; Eisenberg et al., 1990). Although produced by the same cells that secrete IL-1 (e.g., monocytes), the expression of the IL-lra is regulated by different types of stimuli, particularly immobilized IgG and the cytokines IL-4 and IL-10, both of which interfere with the synthesis of IL-1 (Arend, 1995). Although the IL-lra is likely to function as a negative regulator of IL-1 activity in uiuo, the actual extent of its contribution is unclear, particularly because of the high concentrations required for inhibition. Due to the fact that occupancy of only a few membrane IL-1 receptors is sufficient to generate a biological response, at least 100-fold greater concentrations of IL-lra (as compared to IL-1) are needed to interfere with IL-1 activity (Arend, 1995). Recent evidence has suggested the natural existence of a variant form of human IL-4 that lacks part of its sequence due to an exon-deletion mechanism mediated by alternative RNA-splicing (Sorg et al., 1993).Such a variant may potentially function as an IL-4 receptor antagonist, blocking the binding and activity of native IL-4 while unable to signal by itself. Similar alternative splice variants may also exist for other cytokines, including IL-2, IL-5, and G-CSF (Sorg et al., 1991). This finding suggests that the generation of cytokine variants by alternative RNA splicing might give rise to receptor antagonists and may be an important mechanism for the regulation of cytokine activity. 3. Nonreceptor Proteins In addition to sCR, a number of nonreceptor-related proteins both in serum and urine have the ability to bind certain cytokines, and may also contribute to the regulation of their activity in uiuo. As in the case of sCR, both antagonistic and “carrier” functions have been reported for such proteins. In some cases, these proteins are able to prevent the binding of cytokines to their membrane receptors, inhibiting their biologic activity. For example, Uromodulin, an 85-kDa protein present in the urine of pregnant women, binds both IL-1 and TNFa and acts as their inhibitor (Brown et al., 1986; Hession et al., 1987). In contrast, a2-macroglobulin, a major protein in serum, has been implicated to act as a transport protein rather than as an inhibitor for several cytokines. This function may be

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related to its abilities both to bind cytokines and to act as a protease inhibitor, therefore protecting such cytokines from proteolytic inactivation while transporting them in circulation (Borth and Luger, 1989; Matsuda et al., 1989; James, 1990). The binding properties of az-macroglobulin appear to be relatively nonspecific, since it associates with several cytokines, including IL-1, IL-2, and IL-6, and growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor+ (FGFP). In most cases az-macroglobulin does not appear to affect cytokine activity in uitro; however, inhibition toward certain ligands, such as IL-2 and FGFP, has been reported (James, 1990), suggesting that az-macroglobulin can be a carrier for some cytokines and an inhibitor for others. The roles of these nonreceptor proteins in the regulation of cytokine activity in uiuo are far from clear, but they may act as relatively nonspecific carriers and/or inhibitors of cytokines in the circulation and in organs like the kidney. Some of these proteins, such as a2-macroglobulin,may help retain biologically active cytokines in circulation, whereas others, such as uromodulin, may rather promote their clearance. Such possibilities need to be further investigated. The existence of natural auto-antibodies with specificities for cytokines, including IL-la and IL-6, has been documented (Suzuki et al., 1990; Mae et al., 1991; Takemura et al., 1992; Hansen et al., 1993).Although detected in the sera of approximately 10-25% of normal donors, the titers of such anti-cytokine autoantibodies have been found to be increased in patients with a variety of rheumatic and other autoimmune diseases. The role of these antibodies in the pathology of human disease and as regulators of cytokine activity is not clear. It is possible, however, that similarly to sCR, anti-cytokine antibodies have antagonistic and “carrier” effects on cytokine activity. In fact, neutralizing monoclonal anti-cytokine antibodies have been reported to either neutralize or potentiate cytokine activity in uiuo depending on the relative concentrations of antibodies and cytokine (Finkelman et al., 1993; May et al., 1994). D. EVENTS AFTER CYTOKINEIRECEYTOR INTERACTION Some cytokines can have either synergistic or antagonistic effects on the activities of other cytokines without actually affecting expression of their membrane receptors or their ability to interact with them. Such effects may be mediated through potentiation or interference with the signals generated after cytokineheceptor interaction. A classical example is that of the antagonism between the activities of IFNy and IL-4, particularly on B lymphocytes, where IFNy inhibits IL-4-mediated class I1 MWC and CD23 molecule expression, proliferation and IgG, and IgE secretion, whereas IL-4 interferes with the IFNy-mediated production of IgGzs(Vitettaet al., 1989; Finkelman et a l , 1990).The biochemical bases responsible


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for the synergism or antagonism among cytokines at the intracellular level are poorly understood. Nonetheless, the synergistic and antagonistic relationships among different cytokines appear to be central to the regulation of cytokine networks and the cross-regulation of different CD4+ T cell subsets (e.g., Thl and Th2). Besides cytokines, additional factors may also influence cytokine activity without affecting receptor binding. For example, a 30- to 40-kDa protein present in the urine of febrile humans has been reported to inhibit interleukin-1 activity in thymocyte proliferation assays without interfering with IL1R binding (Rosenstreich et al., 1988). 111. Cytokine Receptors: Membrane and Soluble Forms

Cytokine receptors have been the focus of many studies in recent years. The genes encoding most cytokine receptors have now been cloned and expressed, and many of their structural features and signal transduction mechanisms have been elucidated (Miyajima et al., 1992; Taga and Kishimoto, 1993; Ihle et al., 1995). Based upon the presence of conserved sequence homologies and folding motifs, cytokine receptors have been grouped into five families: ( 1)Immunoglobulin superfamily; (2) Cytokinel hemopoietic growth factor receptor family; (3) Interferon receptor family; (4)TNF receptor family; and (5) Seven transmembrane helix receptor family (Abbas et al., 1994).As discussed previousy, most functionally active receptors are complexes of two or more different subunits, in which ligandbinding chains (a subunits) associate with specialized signal-transducing molecules ( p and y subunits). In many cases, the signal-transducing subunits function as “affinity converters,” increasing the binding affinity of the ligand-binding subunit for its ligand, probably as a consequence of stabilization of the cytokineheceptor complex and/or a reduction in its dissociation rate (LowenthaI and Greene, 1987; Wang and Smith, 1987). Such functional cytokine receptor complexes can have 50- to 1000-fold greater affinities than their ligand-binding subunits alone. Ample evidence now indicates that in addition to their membrane-bound forms, many cytokine receptors also exist naturally in soluble form. The first description of a soluble cytokine receptor was made by Rubin et al. (1985), who discovered the presence of soluble IL-2Ra molecules (the low-affinityIL-2R, or p55), in supernatants from activated peripheral blood T cells. The soluble IL-2 receptor (as it is more commonly known) was later found to be produced as a result of T cell activation both in vitro and in wiwo (Rubin et al., 1985; Osawa et al., 1986; Wagner et al., 1986). Shortly thereafter, it was recognized that the levels of sIL-2R in serum and other biological fluids in a variety of human diseases correlated with

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immunological activation, creating great interest as a potential diagnostic/ prognostic marker (Rubin and Nelson, 1990). However, it was not until the systematic cloning of cytokine receptor genes in the late 1980s and early 199Os, and the discovery of alternatively spliced messages encoding soluble forms, that the widespread existence of soluble cytokine receptors became apparent. Soluble cytokine receptors are truncated versions of their membrane counterparts (in most cases, the ligand-binding a-subunits), lacking the transmembrane and intracytoplasmic domains. Because much of the extracellular domain is conserved in sCR, their ligand (cytokine)-binding ability is retained. Indeed, the affinities of sCR are usually comparable to those of their membrane forms (a-subunits alone). However, inasmuch as the affinities of the membrane-bound ligand-binding subunits are enhanced through association with signal-transducing molecules, the affinities of many soluble receptors are significantly lower than those of functional membrane receptor complexes. In such cases (e.g., the sIL-~R),relatively high concentrations of soluble receptors must be present in order to effectively compete with membrane receptors and inhibit cytokine activity. In contrast, soluble receptors that retain high-affinity binding, such as those derived from single-chain receptors (e.g., sIL-1Rs and sTNF-Rs), are more efficient competitors and are required at lower levels. IV. Production of Soluble Cytokine Receptors

A. MECHANISMS FOR THE GENERATION OF SOLUBLE CYTOKINE RECEPTORS Two major mechanisms appear to be responsible for the generation of endogenous sCR. The first, and more basic, mechanism involves the proteolytic cleavage of the original membrane-bound receptor (a process also known as “receptor shedding”). The second, and more specialized, mechanism relies on the de novo synthesis from alternatively spliced mRNAs specific for the soluble receptor form, and different from those encoding the membrane forms ( Fernandez-Botran, 1991).Soluble cytokine receptors differ in the type of mechanism used for their generation. Some sCR are produced exclusively through “shedding,” others through alternative splicing, and some, like the sIL-GRa, are generated by a combination of both. 1 . Proteolytic Cleavage

Soluble cytokine receptors, such as sIL-2Ra and sTNF-R (type I and 11), must be generated exclusively through the proteolytic cleavage of the membrane forms, since their only receptor-specific mRNAs encode the


28 1

full-length membrane receptors (Rubin et al., 1990; Smith et al., 1994). Proteases acting at the level of the plasma membrane cleave the membrane molecule at or above the transmembrane domain, thus releasing the truncated receptor into the extracellular medium. In this context, membrane cytokine receptors are not the only cell membrane-associated molecules that are released as a result of proteolytic activity. TNFa, for example, is released from a membrane-anchored precursor by proteolytic cleavage (Gearing et al., 1994). In addition, a number of cell surface proteins, such as many cell adhesion molecules (e.g., ICAM-1, E-selectin, VCAM1, CD44) and growth factor receptors [e.g., those for epidermal growth factor (EGF), transforming growth factor-a (TGFa), and nerve growth factor (NGF)] are also released as a consequence of proteolybc cleavage (Gearing and Newman, 1993; Smith et al., 1994). Although the identity of the protease(s) responsible for this activity is not completely clear, recent evidence has indicated that processing of pro-TNFa and release of some membrane molecules are inhibited by synthetic inhibitors of matrix metalloproteinases (Gearing et al., 1994), thus suggesting that this type of protease may also be involved in the generation of soluble cytokine receptors. Even though it is known that cell activation is normally associated with the increased release of different sCR, the mechanisms that regulate the proteolybc process remain to be investigated. Identification of the proteases involved in the generation of sCR may allow the use of synthetic inhibitors that block their production for potential therapeutic purposes. 2. Synthesis from Alternatively Spliced mRNAs The existence of separate mRNAs encoding the membrane and soluble forms of many different cytokine receptors, including IL4R (Mosley et al., 1989), IL-5 (Tavernier et al., 1991), IL-6 (Horiuchi et al., 1994), IL-7 (Goodwin et al., 1990), IFNoJP-R (Novick et al., 1994), and GM-CSF-R (Brown et al., 1995), has been described. Although in all of these cases the transcripts originate from a single gene, alternative RNA splicing gives rise to the two types of messages (Wrighton et al., 1992). Invariably, deletions or termination of translation upstream of the transmembrane domain give rise to truncated receptors in the messages encoding the soluble receptors. Such molecules retain their ligand-binding domain but lack membrane anchoring. The murine interleukin-4 receptor (IL-4R) was the first cytokine receptor for which separate messages encoding its membrane-bound and soluble forms (sIL-4R) were discovered (Mosley et al., 1989). The cDNA encoding the soluble form was found to contain a 114-bp insertion upstream of sequences encoding the transmembrane domain that resulted in addition of six novel amino acids and premature termination, leading to the synthesis of a truncated IL4R lacking the transmembrane and cytoplasmic domains

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of the full-length 140-kDa receptor. It has now been determined that the 114-bp insertion corresponds to exon 8 of the IL-4R gene, and that its inclusion is, in fact, regulated by an alternative splicing mechanism (Wrighton et al., 1992). In the case of cDNA clones encoding the s IL -~R, a deletion involving the sequences responsible for the transmembrane domain was found to alter the translational reading frame, causing the addition of 27 novel amino acids and premature termination (Goodwin et al., 1990).Alternative RNA splicingalso regulates the generation of mRNAs specific for soluble forms of many other cytokine receptors, including both the human and murine IL-5R (Tavernier et al., 1991), the human IL-6R (Horiuchi et al., 1994), the human IFNdP-R (Novick et al., 1994), and the human GM-CSF-R (Brown et al., 1995). The signals and mechanisms regulating the alternative RNA splicing event have not been elucidated; however, it is possible that cellular activation and/or other signals may influence such process, resulting in the preferential transcription of one type of receptor form over the other (e.g., membrane vs soluble). Such a regulatory mechanism could have profound effects on cytokine activity by altering the expression of the membrane or soluble forms of its receptor, thus altering the responsiveness of target cells. Preliminary evidence gathered in our laboratory has shown that antigenic stimulation of murine Th2 clones may result in the preferential up-regulation of soluble over membrane IL-4R transcripts ( Fernandez-Botran et al., 1996),thus suggesting that the alternative splicing event can, indeed, be regulated.

B. REGULATION OF THE PRODUCTION OF SOLUBLE CYTOKINE RECEPTORS Although most of the original descriptions of the different sCR came from in vitro observations and/or cloning studies, the production of sCR in vivo has now been confirmed in both humans and animals. Nonetheless, information regarding the identity of the cells responsible for the production of the different sCR and the factors that regulate their production is, in most cases, incomplete. It is not completely clear, for example, whether sCR are simply released, perhaps as a consequence of membrane protein turnover, or whether their production is actively regulated, particularly during immune responses. In addition, the relationship between the expression of the membrane cytokine receptors and the synthesis and/or release of their soluble forms needs to be better characterized (e.g.,are the membrane and soluble forms coordinately expressed or is there preferential expression of one form over the other?). Consistent with the notion that sCR play a physiologic role as regulators of cytokine activity, information so far available appears to indicate that the generation of sCR is indeed regulated during immune responses.


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Although most sCR are made at low levels under resting conditions, their production is enhanced by cellular activation (Rubin and Nelson, 1990; Honda et al., 1992; Chilton and Fernandez-Botran, 1993; Cope et al., 1995). In agreement with this idea, the serum levels of various sCR, such as sIL-eRa, sTNF-R (types I and 11), and sIL-GRa, have been found to be consistently elevated under clinical conditions associated with active immunological and/or inflammatory responses (Rubin and Nelson, 1990; Arend, 1995).Several factors appear to play a role in regulating the production of sCR during immune or inflammatory responses. As with the membrane receptors, cellular activation and cytokines are two of the most important, and will be discussed next. 1 . Cellular Activation Cellular activation in lymphocytes, monocytes, and other types of cells normally leads to an up-regulation of membrane receptors for different cytokines (Hemler et al., 1984; Lowenthal et al., 1985; Lowenthal and Greene, 1987; Wang and Smith, 1987; Ohara and Paul, 1988). Consequently, it is not surprising that the production of the soluble forms of such cytokine receptors, particularly the ones that are derived by “shedding” of the membrane receptors, is also enhanced by cell activation. The nature of the “activating” signal varies, of course, depending on the identity of the sCR-producing cell (e.g., T lymphocytes and antigens; macrophages and endotoxin). It should be pointed out, however, that the identity of the cells producing the different sCR in uiuo remains, in many cases, poorly characterized, so it is not always easy to predict the type of stimulus that may result in the increased production of a specific sCR in uiuo. Nonetheless, at least in the case of the better-characterized sCR, some generalizations can be made: soluble receptor forms specific for cytokines involved in T cell function and proliferation (e.g., IL-2 and IL-4) are increased following T cell activation (Rubin and Nelson, 1990; Chilton and Fernandez-Botran, 1993),whereas the production of those sCR specific for proinflammatory cytokines (e.g., IL-1, IL-6, and TNFa) is normally up-regulated by monocytic cell activation and/or inflammation (Symonset al., 1990; Leeuwenberg et al., 1994; Kuhns et al., 1995). T cell activation, however, has also been reported to enhance production of sIL-6R and sTNF-R (Honda et al., 1992; Bemelmans et d.,1994; Cope et al., 1995). Inasmuch as a major consequence of cellular activation is the production and release of cytokines, the enhanced production of sCR seen after cell stimulation may be caused, and least in part, by the effects of secreted cytokines. In fact, neutralizing antibodies against IL-4 partially inhibit the production of sIL-4R by murine splenic cells stimulated with the T cell mitogen concanavalin A or with specific antigens (Chilton and Fernandez-

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Botran, 1993;Fernandez-Botran et al., 1995),thus suggesting that cytokines may be important regulators of the production of sCR. 2. Cytokines

Available evidence does indeed indicate that the production of many different sCR is regulated by cytokines. Very importantly, it appears that the production of many sCR, including sIL-2Ra (Lotze et al., 1987; Voss et al., 1989), sIL-4R (Chilton and Fernandez-Botran, 1993), and sTNF-H (Lantz et al., 1990; Cope et al., 1995) is regulated by their own ligands, suggesting a potential “feedback’ regulatory mechanism, whereby the presence of a cytokine induces the production or release of its specific sCR, which in turn, helps down-modulate its activity. Such a process may provide a “buffer,” keeping the actions of cytokines relatively localized. Consistent with such an idea, the production of sCR in vivo is usually up-regulated during responses involving the release of their ligands. Indeed, several lines of evidence from our laboratory have suggested that the production of sIL-4R is tightly linked to those immune responses characterized by the predominant secretion of IL-4 (i.e., Th2 responses), including: (a) enhancement of sIL-4R production by freshly isolated murine T cells, B cells, and macrophages in response to rIL-4 (Chilton and FernandezBotran, 1993); (b) increased expression of sIL-4R-mRNA in splenic cell cultures stimulated with IL-4 (Chilton and Fernandez-Botran, in preparation); (c)correlation between the expression of sIL-4R in uivo and development of Th2 responses in a murine schistosomiasis model (FernandezBotran et al., 1995);and (d) up-regulation of the production of sIL-4R by Th2, but not Thl cell clones after antigenic stimulation ( Fernandez-Botran et al., 1996). Similar relationships exist also for other cytokines and their soluble receptors. In the case of the association of sIL-2R with T cell activation, it is well known that IL-2 itself can up-regulate the membrane expression of the IL-2Ra chain (Bismuth et al., 1984; Smith and Cantrell, 198s). It would seem thus logical that the IL-2 that is produced as a result of T cell activation would also help promote the release of its own sIL-2R. In agreement with such idea, h t z e et al. (1987)have reported that administration of purified human IL-2 to patients with cancer results in the development of IL-2Rt cells and increased levels of sIL-2R in serum. Moreover, increased serum levels of sTNF-R (types I and 11)have also been observed under conditions in which TNFa is secreted, such as in endotoxemia and septic shock (Leeuwenberg et al., 1994; Kuhns et al., 1995), suggesting that TNFa also regulates the production of its own soluble receptors. Indeed, infusion of rTNFa in patients has been reported to result in a rapid increase of sTNF-R levels in their serum (Lantz et al., 1990). The


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mechanisms responsible for the regulation of sCR by their own ligands are not completely clear. Although it is possible that the binding of a cytokine to its membrane receptor may induce its shedding, cytokinemediated increases in the synthesis membrane and/or soluble receptors may be more important mechanisms in mediating the increased production of sCR. In addition to regulation by their own cytokine, many examples also exist for the control of the production of sCR by unrelated cytokines. Loughnan et al. (1989),for example, have reported on the increased expression of membrane IL-2R and release of sIL-2R by murine B cells exposed to IL-5. Both IL-6 and IFNy have been reported to increase production of sIL-4R by a myeloid cell line and bone marrow-derived macrophages (Ruhl et al., 1993), whereas IL-1 has the ability to up-regulate the membrane expression of the TNF-R type I and induces increased sTNF-R levels (both type I and 11)in patients treated with rIL-lP (Bargetzi et al., 1993). Interestingly, some cytokines may cross-regulate each other by inducing the production of each other’s soluble receptors, such as the case of IL-4 and IL-1. For example, rIL-lP induces sIL-4R production by murine Th2 cells ( Fernandez-Botran et al., 1996); whereas IL-4 induces the production of the sIL-1R type I1 and the IL-1 receptor antagonist (IL-lra) (Arend, 1995).

3. Other Factors In addition to direct cellular activation and cytokines, additional signals may also play a role in the regulation of the production or release of sCR. Some of these signals may originate through direct cell to cell contact, and may be putatively mediated by signaling through costimulatory molecules (e.g., CD28/B7B1-2, gp39/CD40). Recent studies from our laboratory have demonstrated that the physical interaction between murine Th2 cells and antigen-presenting cells (irradiated splenic cells) is enough to induce an up-regulation in their production of sIL-4R (FernandezBotran et al., 1996). In other cases, hematologic malignancies or infection by viruses, particularly by the human T lymphotropic virus-1 (HTLV-I), are known to result in the constitutive expression of membrane IL-2R and high levels of production of the sIL-2R (Rubin and Nelson, 1990). V. The lmmunoregulatory Function of Soluble Cytokine RHeptors

The actual role played by sCR in the overall regulation of immune responses is still a matter of speculation and remains as the main question to be answered in the biology of soluble cytokine receptors. The main problem in defining their immunoregulatory function has been the paradox-

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HAFAEL FERNANDEZ-BOTRAN ET AL.

ical observation that even though most sCR act as competitive inhibitors of the binding and activity of their respective cytokines in vitro, they can exert both agonistic and antagonistic effects in vivo. Another significant obstacle has been lack of direct demonstration of their immunoregulatory function in vivo, which is due in part to the difficulty in generating reagents (e.g., monoclonal antibodies) that selectively block cytokine binding by the soluble, but not the membrane, receptors. The antagonistic and “carrier” effects of sCR on cytokine are discussed next. A. ANTAGON~STIC EFFECTS Binding of a molecule of cytokine to its sCR precludes, in most cases, its binding to a functional membrane cytokine receptor, thus preventing subsequent signaling and a biological response. Hence, sCR function as competitive inhibitors of the binding and activity of their respective cytokine in vitro. In fact, all soluble cytokine receptors so far described, even those with considerably lower affinities than their membrane receptors, inhibit the binding and biological activity of their cytokine in in vitro assays. The only exception appears to be the sIL-GRa, which is able to interact with its own signal-transducing subunit, gp130, thus generating a signal (Hibi et al., 1989). Based on their nature as cytokine-binding, competitive inhibitors, the antagonistic effects of sCR on cytokine activity have several important characteristics in common. 1. Their Antagonistic Eflects Are Cytokine-Specijic

Because cytokine-binding is receptor-specific, the inhibition caused by sCR is restricted to the binding and activity of their own ligand. For example, sIL-4R inhibit the IL-4- but not the IL-%mediated proliferation of CTLL or HT-2 indicator cell lines (Mosley et al., 1989; FernandezBotran and Vitetta, 1990). 2. Their Antagonistic Eflects Are Znversely Related to the Concentration of Cytokine Due to its competitive nature, the degree of inhibition caused by a constant amount of a sCR will decrease with increasing concentrations of cytokine (i-e.,the higher the concentration of a cytokine, the more sCR that will be required to prevent its binding to membrane receptors). 3. Their Antagonistic Effects Are lnjuenced by the Binding Afinities of the sCR and the “Functional” Membrane Receptor Complex As discussed previously, association of the ligand-binding chains with signal-transducing subunits at the cell membrane leads to the formation


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of high-affinity membrane receptors. Thus, the affinities of the membrane receptors sometimes can be several orders of magnitude higher than those of the corresponding soluble receptors. For example, the IL-2Rachain (membrane or soluble) binds IL-2 with relatively low affinity (Kd -100 nM), but association with the IL-2Rp and IL-2Ry subunits, results in the formation of the high-affinity IL-2R ( K d -10 pM) (Lowenthal and Greene, 1987; Wang and Smith, 1987; Russell et al., 1993). Hence, sIL-2Ra have an affinity approximately 1000-fold lower than that of the functional membrane IL-2R, and consequently, are not very efficient competitors (Jacques et al., 1987; Baran et al., 1988). Despite the fact that sIL-2R can specifically inhibit IL-2 activity, relatively high concentrations are required (Treiger et al., 1986; Kondo et al., 1988). By contrast, sCR that are able to bind their cytokines with affinities comparable to those of their membrane receptors, such as the sIL-4R and both types of sTNF-R, are much more efficient inhibitors at low concentrations (Femandez-Botran and Vitetta, 1990; Dayer and Burger, 1994). Most sCR, in conclusion, have the potential to function as highly specific “cytokine inhibitors” if present in adequate concentrations. The concentrations of sCR required for inhibition are influenced by the concentration ofcytokine and the difference in affinities between them and the membrane receptors. The antagonistic effects of sCR may play an important role in the down-modulation of immune responses and in preventing “overactivity” and/or toxic effects of some cytokines (Fernandez-Botran, 1991; Debets and Savelkoul, 1994; Maliszewski et al., 1994).The ability of exogenously administered sCR to act as antagonists of cytokine activity in vivo has been confirmed by numerous laboratories (Debets and Savelkoul, 1994; Gessner and Rollinghoff, 1994; Maliszewski et al., 1994; Arend, 1995).

B. “CARRIER” A N D ACONISTICEFFECTS Despite the fact that most sCR have the intrinsic ability to function as competitive inhibitors of the binding and activity of their ligands, several sCR have also been reported to have properties that are consistent with a putative role as “cytokine-carrier proteins”( Femandez-Botran and Vitetta, 1991; Mohler et al., 1993). Furthermore, several sCR have even been shown to potentiate the activity of their own cytokines in vivo (Sat0 et al., 1993; Hale et al., 1995). The mechanisms responsible for these seemingly paradoxical observations may involve several of the following effects on cytokines due to their binding to sCR in vivo. 1 . Increased Cytokine Stability Binding of a cytokine by its soluble receptor may provide increased molecular stability, resulting in reduced activity decay. Aderka and col-

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leagues (1992), for example, have reported that the binding of the bioactive TNF trimer to sTNF-R decreases its breakdown into inactive monomers, resulting in increased biologic activity upon long-term incubation.

2. Protection from Proteolysis Binding of a cytokine to its soluble receptor may protect it from the action of proteolytic enzymes. We intially determined, for example, that the presence of sIL-4R decreases the susceptibility of IL-4 to degradation by trypsin in vitro (Fernandez-Botran and Vitetta, 1991). Consistent with such an observation, we have now recently shown that sIL-4R significantly reduce the proteolytic degradation of radiolabeled IL-4 after intravenous injection in mice (Ma et al., submitted). 3. “Cytokine-Transfer”from sCR to Membrane Receptors Because of the reversible nature of the binding of a ligand to its receptor, binding of a cytokine molecule to a sCR results only in the transient “sequestration” of that cytokine. Upon dissociation, the cytokine molecule then becomes available to membrane (and/or other soluble) cytokine receptors. Thus, the faster the dissociation rate of the soluble receptor, the higher the chance a cytokine will have of dissociating and ultimately binding to a functional membrane receptor. Comparison of the binding properties of soluble and membrane IL-4R disclosed that the soluble forms have much faster dissociation (off-) rate constants than the membrane receptors at 37”C, allowing the dissociation of IL-4 from sIL-4R and subsequent binding to mIL-4R’ cells (Fernandez-Botran and Vitetta, 1991). 4. lncreased in Vivo Half-Lfe and Decreased Clearance The association of a cytokine with its soluble receptors affects its pharmacokinetic behavior in uivo. Recent studies from our laboratory (Ma et al., submitted) in which radiolabeled IL-4 was administered to mice in the , that although IL-4 alone presence and absence of s I L - ~ Rdemonstrated was rapidly removed from circulation, the presence of sIL-4R caused a dose-dependent increase in its in vivo half-life and a concomitant decrease in its blood clearance and elimination in the urine. Binding to sIL-4R also protected IL-4 from proteolytic inactivation in vivo, contributing to higher levels of bioactive IL-4 in circulation. Taken together, these results suggest that sCR may be able to alter the pharmacokinetic properties of their cytokines, prolonging their half-life in circulation and reducing their clearance through diminished renal excretion and/or interference with inactivation in vivo. Such effects are consistent with the reported ability of some sCR to potentiate the activity of their cytokines in viuo.


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The ability of other sCR to modify the pharmacokinetic properties and enhance the activity of their ligands has also been reported. Mohler et al. (1993),for example, found that the half-life of TNFa in uiuo was increased by the administration of recombinant sTNF-R. Moreover, this “carrier” effect is not an exclusive property of sCR, since monoclonal anti-cytokine antibodies, such as anti-IL-4, anti-IL-3, anti-IL-6, and anti-IL-7 ( Finkelman et al., 1993; May et al., 1994),have also been found to potentiate the activity of their respective cytokines in uiuo. In the case of anti-IL-4 antibodies, however, this activity was exhibited by antibodies that blocked binding of IL-4 to membrane IL-4R, but not by an antibody that bound IL-4 but did not prevent its binding (Finkelman et al., 1993). Such an observation suggests that potentiation of cytokine activity may not be simply the result of the passive binding of a cytokine to another molecule, but may involve the active prevention of its uptake by irrelevant cytokine receptor-positive cells and/or a preferential uptake by target cells. In this regard, cells expressing membrane cytokine receptors at high numbers or with increased affinity (i.e., activated cells) will be able to more efficiently compete for cytokine molecules dissociating from sCR, than cells expressing the low numbers of receptors (i.e., resting cells). An exception to the behavior of most sCR is the sIL-6Ra. Complexes between IL-6 and sIL-6Ra can effectively interact with the ligand-transducing, gp130 subunit, and are thus capable of signalling (Hibi et al., 1989).Consistently, sIL-6Ra are not inhibitory and can actually potentiate IL-6 activity both in uitro and in uiuo (Novick et al., 1992b; Suzuki et al., 1993; Tamura et al., 1993; Schobitz et al., 1995). vs AGONISTIC ROLES C. ANTAGONISTIC In conclusion, even though sCR can act as inhibitors of cytokine binding and activity, they can have, at the same time, effects on their ligands that result in increased stability and prolonged half-life in uiuo. The overall effect of sCR on cytokine activity in uiuo must then be dictated by the balance between their antagonistic (competition for binding) and agonistic effects (increased stability and half-life). Such a balance is not influenced exclusivelyby the concentrations of sCR, but also by the local concentration of cytokine and the availability of membrane cytokine receptors on target cells. Consistent with this idea, a recent report by Sat0 et al. (1993)demonstrated that exogenously administered sIL-4R can either potentiate or inhibit the activity of rIL-4 in uiuo (as assessed by the production of IgE antibodies in mice), depending on the relative molar ratios of sIL-4R to IL-4, with relatively low ratios promoting activity and large ratios (a high excess of sIL-4R over IL-4) inhibiting IL-4 function. At the low ratios, the agonistic effects of sIL-4R may have presumably outweighed their

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antagonistic consequences (inhibition of binding); whereas under conditions of a large sIL-4R excess, it was the antagonistic effects that prevailed. Direct demonstration of the actual contribution of endogenously produced sCR to the regulation of inflammatory and immune responses is not yet available. This may have to wait for the availability of reagents that specifically interfere with the function of the soluble, but not the membrane, cytokine receptors, and/or the generation of “knockout” mice in which expression of the soluble receptor form is targeted. Until then, we can only rely on the available information gathered either in vitro or through administration of exogenous recombinant sCR. However, evidence in support of the involvement of sCR in immunoregulatory mechanisms has been provided recently by the discovery of virus-encoded homologs of soluble cytokine receptors, such as those for TNFa, IL-lp, and IFNy (McFadden et ul.,. 1995). These viral proteins behave in much the same manner as sCR do, preventing the binding and activity of their target cytokines, and, although not essential for viral replication, they play a role in their pathogenicity, presumably by protecting the viruses from immunological effector mechanisms. VI. The Properh’es of Soluble Cytokine Receptors and Relationship to Disease

Inasmuch as many details concerning their generation and function are not yet known, it is still difficult to ascertain the actual role of sCR in the pathogenesis and or pathophysiology of human diseases. The fact that sCR are likely to be involved in the regulation of cytokine activity in vivo, however, would predict that they also play an important role in those diseases in which cytokines are directly involved. Although up to date, there are no reports of human diseases caused by “defects” in the generation and/or function of sCR; some, like the sIL-GRa, may contribute to the pathogenesis of certain diseases by potentiating the activity of IL-6. Moreover, there is a considerable amount of information regarding correlations in the serum levels of certain sCR (particularly sIL-~R,sIL-GR, and sTNFR) with many diseases. Indeed, the levels of some of these sCR have shown significant promise as potential diagnostic or prognostic “markers” in a great variety of clinical conditions, including infectious, autoimmune, and malignant diseases. The properties of the different sCR and their associations with diseases will be discussed in this section.

A. SOLUBLE INTERLEUKIN-1 RECEPTORS Interleukin-1 is an extremely pleiotropic cytokine secreted by cells of the monocytic lineage and many other types of cells. IL-1 plays a key role as a mediator of inflammation and has many other important activities


29 1

including the activation and differentiation of T and B lymphocytes (Dinarello, 1994). The regulation of IL-1 activity in vivo is, without doubt, the most complex of all cytokines. It involves: (a) two ligands, IL-la and IL-lp, exhibiting similar activities and synthesized as precursor peptides (Dinarello, 1994); (b) a receptor antagonist, IL-lra, which is structurally related to the other two IL-1 forms but lacks the ability to generate a signal upon binding to the IL-1R (Hannum et al., 1990; Eisenberg et al., 1990); (c) two different types of receptors, IL-1RI and IL-lRII, of which the former is the only one capable of signal transduction whereas the latter may function as a “decoy” receptor (Sims et al., 1993; CoIotta et al., 1993); and (d) soluble cytokine receptors of both types, sIL-1RI and sIL-1RII (Symons et al., 1991; Svenson et al., 1993). Soluble forms of both types of IL-1R are generated by proteolyhc cleavage of the membrane receptors (Arend, 1995). Naturally occurring soluble forms of the type I receptor (sIL-1RI) have been reported in normal human serum (Svensonet al., 1993),with levels increasing during endotoxemia (Arend, 1995). In contrast to the recombinant sIL-1R1, which binds &la, IL-lp, and the IL-lra with similar affinities, the naturally occurring sIL-1RI was found to bind the IL-lra preferentially (-200-fold higher affinity), suggesting not only that truncation at the C-terminal end may affect the binding characteristics of the soluble vs the membrane forms of the receptor, but also that endogenous sIL-1RI may interfere with the inhibition caused by IL-lra (Svenson et al., 1993). Genetically engineered recombinant sIL-1R1, however, have been shown to inhibit binding of both IL-la and IL-lp (Dower et al., 1991) and inhibit the activity of IL-1 in vivo in both animal and human models of inflammation and alloreactivity (Fanslow et al., 1990b; Jacobs et al., 1991a; Mullarkey et al., 1994). Natural forms of the sIL-1RII are also generated, and appear to be the main type of soluble IL-1R in human serum. The presence of sIL-1RII has been reported in normal human plasma (Eastgate et al., 1990), serum, and synovial exudates from patients with rheumatoid arthritis, and in supernatants from activated peripheral blood mononuclear cells (Symons et al., 1990), neutrophils, (Colotta et al., 1993), and human B-lymphoma cell lines (Symons et al., 1991). The soluble form of the type I1 IL-1R is a 47-kDa protein that binds IL-1p with high affinity, but in contrast to the membrane form or the sIL-1R1, its A n i t y for the IL-lra is very low (-2OOO-fold lower than that of the membrane IL-1RII). The sIL-1RII has also been found to block the processing of the inactive IL-1p precursor, thus suggesting that the soluble forms of IL-1RII may inhibit IL-lp activity at two different levels: (a) the proteolytic processing of the pro-IL-lp molecule; and (b) the binding of mature IL-1p to membrane IL-1RI (Symons et al., 1995).In agreement with its low affinity for the IL-lra,

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the sIL-1RII does not interfere with its inhibitory activity on IL-1. Indeed, the combination of both sIL-1RII and IL-lra was reported to have an additive effect in inhibiting the binding of IL-1P to membrane receptors (Symons et al., 1995). Hence, it is possible that sIL-IRII and IL-lra may act in concert to inhibit IL-1 activity in vivo. In fact, the production of both molecules appears to be simultaneously regulated by cytokines with anti-inflammatory properties, such as IL-4 (Colotta et al., 1993). Although not much information is yet available on the generation of sIL-1RII during pathological conditions, the fact that their production is enhanced by the activation of mononuclear cells with agents such as endotoxin, TNFa, and phorbol esters (Symons et al., 1990),predicts that elevated levels of sIL1RII in biologic fluids may occur during both local and systemic inflammatory responses (e.g., synovial fluid in rheumatoid arthritis and serum in endotoxemia). In conclusion, soluble forms of the type I1 receptor may play a significant role as inhibitors of IL-1P activity in vivo, possibly in combination with the IL-lra. The physiologic role of the sIL-1RI is, however, not as clear, since it could potentially have both antagonistic (inhibition of IL-la and IL-1P from binding to membrane IL-1RI) and potentiating effects (interference with the inhibition caused by the IL-lra) on IL-1 activity.

B. SOLUBLE INTERLEUKIN-2 RECEPTORS Interleukin-2 is a cytokine produced by activated CD4+ and some CD8+ T lymphocytes. In addition to being a major T cell growth factor, it also stimulates growth and differentiation of cytotoxic T cell precursors and NK cells, proliferation and differentiation of human B cells, and activation of monocytes (Smith, 1988).The functional IL-2 receptors are composed of at least three different subunits, two of which, a (p55) and P (p75), bind IL-2 with low and intermediate affinities, respectively, and one, y ( p a ) , which functions in signal transduction and as affinity converter for the other subunits (Kondo et al., 1993; Russell et al., 1993). Although the a chain can, by itself, bind IL-2 with low affinity, it is unable to generate a signal in the absence of the other two subunits (Lowenthal and Greene, 1987; Wang and Smith, 1987). Resting T lymphocytes, NK, and other types of cells express low levels of the P and y subunits, allowing them to respond to IL-2, albeit at relatively high concentrations. Normal expression of the (Y subunit, however, is inducible and leads to the formation of highaffinity IL-2R, thus enabling the activated cells to respond to IL-2 at much lower concentrations. Constitutive expression of the IL-2R a chain has been observed in some pathological conditions, such as in some malignant and HTLV-I-infected cells (Rubin and Nelson, 1990).


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Antigenic or mitogenic stimulation of T and B lymphocytes results not only in the induction of the membrane IL-2Ra subunit, but in the generation and release of truncated a-chains as well (Rubin et al., 1985; Osawa et al., 1986; Wagner et al., 1986). These soluble IL-2Ra receptors are approximately 10-15 kDa smaller than the membrane form (p55), but retain their ability to bind IL-2 ( Josimovic-Alasevic et al., 1988b). The binding affinity of the sIL-2Ra for IL-2 is similar to that of the a chain alone ( K d -100 nM), and is thus approximately 1000-times lower than that of the high-affinity IL-2R (Jacques et al., 1987; Baran et al., 1988). It is because of such a large difference in affinities that sIL-2Ra are relatively inefficient in blocking the binding of IL-2 to the biologically active IL-2R on cells, and why their potential biological function as IL-2 antagonists has remained controversial since their discovery. It has been demonstrated, nonetheless, that when used at high concentrations, sIL2Ra can block binding and activity of IL-2 in uitro (Treiger et al., 1986; Kondo et al., 1988).Whether sIL-2Ra could actually reach such inhibitory concentrations in viuo has not yet been unequivocally established. There are, however, several reports pointing to endogenous sIL-2Ra as the molecules responsible for the inhibitory activity toward T cell function found in serum and other biological fluids (e.g., synovial fluid) in pathologic conditions such as rheumatoid arthritis (Symons et al., 1988) and human visceral leishmaniasis (Barral-Netto et aZ., 1991). The generation of the sIL-2Ra is mediated through the proteolytic cleavage of the membrane-bound form. This is based on the existence of only one type of mRNA encoding the full-length membrane IL-2Ra and the absence of alternatively spliced messages potentially encoding soluble forms. In fact, Rubin et al. (1990) demonstrated that transfection of cells with a cDNA encoding the membrane IL-2Ra results not only in cell surface expression, but also in the release of soluble IL-2Ra. Consistently, neoplastic and HTLV-I-infected cells in which the expression of the IL2Ra is constitutive, also produce large amounts of sIL-2Ra (Rubin and Nelson, 1990).The identity of the protease(s) responsible for the generation of sIL-2Ra remains unclear. The existence of endogenously produced soluble forms of the IL-2RP chain (p75) has also been reported (Honda et aZ., 1990). Similarly to the sIL-eRa, generation of the sIL-2RP is also dependent of cellular activation and is mediated through the proteolytic cleavage of the membrane-bound receptor. These soluble IL-2RP are approximately20-25 kDa smaller than the membrane counterpart and bind IL-2 with low affinity. Regardless of their biological function, sIL-2Ra have provided a useful tool for the assessment of immune function in uivo as nonspecific indicators of immune activation (Rubin and Nelson, 1990). Inasmuch as the genera-

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tion of sIL-2Ra correlates with the expression of its membrane form, those conditions resulting in the synthesis of membrane IL-2Ra (i.e., cellular activation, HTLV-I infection) are also associated with increased production of the soluble receptor. Thus, the serum levels of sIL-2Ra appear to be directly related to the degree of T cell activation and/or the number of IL-2Ra' cells in uiuo. Low levels of sIL-2Ra are present in the serum and urine of normal individuals, possibly as a result of a baseline level of immunological activation (Rubin et al., 1985).Increased levels of sIL-2Ra in serum have been observed in a wide variety of clinical conditions, including malignant, autoimmune, and infectious diseases. The serum levels of sIL-2Rm have proved to be valuable indicators of disease activity and/or progression, thus having a direct impact on disease management and prognosis. In the case of hematologic neoplasias, such as in HTLV-I-associated malignancies, hairy cell leukemia, and Hodgkin lymphoma, the serum levels of sIL-2Ra reflect the total tumor burden, with increased levels being associated with disease progression (Richards et al., 1990;Ambrosetti et al., 1993; Kamihira et al., 1994). Moreover, a gradual increase in the mean serum levels of sIL-2Ra, correlating with the natural progression of the disease, has been observed in individuals infected with the human immunodeficiency virus (HIV) (Prince et al., 1988; Honda et al., 1989). In autoimmune diseases, such as rheumatoid arthritis and lupus erythematosus, the levels of sIL2Ra in serum correlate with an active disease and a poor prognosis (Symons et al., 1988; Semenzato et al., 1988; Tokano et al., 1989). Elevated levels of sIL-2Ra have also been reported in many types of viral, bacterial, and parasitic infections, presumably reflecting immunological activation ( Josimovic-Alasevic et al., 1988a; Brown et al., 1989; Muller et al., 1989). Furthermore, high levels of sIL-2Ra have been proposed to be predictive of rejection and/or poor prognosis in transplant recipients (Perkins et al., 1989; Bock et d., 1994). In conclusion, measurement of the levels of sIL-2Ra in serum and/ or other fluids provides a relatively simple and noninvasive tool for the assessment of disease activity, response to treatment, and prognosis in conditions associated with immunological activation. An extensive discussion of the association of different diseases with the levels of sIL-2RdB is outside the scope of this review, however. Table IV provides a summary of clinical conditions in which elevated serum levels of sIL-2Rm and sIL2RP have been reported, as well as selected references and other relevant comments. Readers should be aware that conflictingreports are not uncommon, and therefore are encouraged to consult the original sources.

c. SOLUBLE INTERLEUKIN-4 RECEPTORS

Interleukin-4 is a highly pleiotropic cytokine secreted primarily by the Th2 subset of CD4' T cells, and also by mast cells and some CD8+T cells.

SOLUBLE CYTOKINE RECEITORS

295

IL-4 has many activities on different types of cells, including T and B lymphocytes, macrophages, mast cells, and nonhemopoietic cells. Two of its most important functions are the activation and isotype class switch (IgG,, IgE) of B lymphocytes (Vitetta et al., 1989; Finkelman et al., 1990; Paul, 1991), and the maturation and differentiation of activated precursor CD4+ T lymphocytes (Thp) into the Th2 subset (Swain et al., 1990; Abeshira-Amar et al., 1992; Seder et al., 1992). The effects of IL-4 on target cells are initiated by its binding to membrane-bound IL-4 receptors, composed of a 140-kDa IL-4-binding subunit (Mosley et al., 1989; Harada et al., 1990; Idzerda et al., 1990) in noncovalent association with a signaltransducing subunit, the IL-2R y-chain (Kondo et al., 1993; Russell et al., 1993). Unlike the ligand-binding subunits of the IL-2R (a,fl), the 140-kDa subunit of the IL-4R is able to bind IL-4 with relatively high &nity ( K d -60 pM), even in the absence of the signal transducing y-chain. Membrane IL-4R are present on a wide variety of hemopoietic and nonhemopoietic cell types (Ohara and Paul, 1987; Park et al., 1987; Lowenthal et al., 1988). The murine IL4R was the first cytokine receptor for which an alternatively spliced message encoding its soluble form (sIL-4R) was discovered (Mosley et al., 1989; Wrighton et al., 1992). Although alternatively spliced messages encoding a soluble IL4R have not been found in humans, there is evidence for the production of sIL-4R by human peripheral blood mononuclear cells (Konig et al., 1995). Generation of soluble IL-4R forms in the human may thus occur through a shedding mechanism. Despite the detection of messages encoding the sIL-4R in various murine tissues, it was not completely clear after their discovery, whether natural sIL-4R actually existed in vivo. Shortly after the cloning of the IL-4R, however, a report of the presence of a soluble IL-$-binding protein (IL4BP) in the biological fluids of mice by Fernandez-Botran and Vitetta (1990),suggested that the existence of natural forms of sIL-4R was, indeed, very likely. In fact, using monoclonal antibodies raised against the IL-4R, Fanslow et al. (1990a) later confirmed the existence of sIL-4R in murine biological fluids, while Fernandez-Botran and Vitetta (1991) verified that the protein described by them as IL4BP was identical with the sIL-4R. The natural as well as recombinant sIL-4R were found to bind IL-4 with an affinity similar to that of the mIL-4R (Kd 70-100 pM) (Mosley et al., 1989; Fernandez-Botran and Vitetta, 1990), and, consistent with such high affinity, competed efficiently with membrane IL4R for the binding of IL4, and inhibited its biologic activity in several in uitro assays (Mosley et al., 1989; Fernandez-Botran and Vitetta, 1990; Maliszewski et al., 1990). Moreover, sIL-4R have also been shown to behave as inhibitors of IL-4 activity in vivo. Injection of recombinant sIL-4R into mice was reported to result in lowered cellular responses and prolonged graft survival in

TABLE IV SOLUBLE IL-2 RECEITORS AND HUMAN DISEASE

Hematologic malignancies Adult T-cell leukemia T-cell leukemia T-cell chronic lymphocytic leukemia T-cell acute lymphoblastic leukemia Hairy-cell leukemia Chronic myelogenic leukemia B-chronic lymphocyhc leukemia Malignant lymphoma Multiple myeloma Non-Hodgkin lymphoma Hodgkin lymphoma

Indication of progression, prognosis for survival Elevated Elevated

Kamihira et d.(1994) Motoi et d.(1988) Raziuddin et d.(1994)

Elevated Elevated, marker of disease

Raziuddin et ul. (1994) Chilosi et d.(1987), Richards et al. (1990), Lauria et d.(1992) Motoi et d.(1989)

Elevated, clinical indicator Elevated Elevated in active disease, correlate with disease activity, useful in monitoring Elevated, correlate with disease activity Elevated, monitoring disease, prognostic indicator Elevated, indicator and prognosis

GI tract (esophageal, gastric, colon)

Significantly elevated in patients with metastases Elevated

Pancreatic cancer Breast cancer Lung cancer Primary lung cancer Small cell carcinoma

Elevated Elevated Elevated Elevated Elevated

Solid tumors

Reference

sIL-2Ra levels, comments

Disease

(also in

severe pancreatis)

Semenzato et al. (1987) Hamon et d.(1993),Piccinini et d.(1993), Motokura et d.(1995) Vacca et d.(1991) Chilosi et d.(1989), Stasi et d.(1994a,b) Gause et al. (1991).Ambrosetti et al. (1993) Rove& et d.(1988), Lissoni et al. (199Ob) Berghella d d.(1994),Jablonska et d. (1994), Murakami et d.(1994) P e d et d.(1994) Lissoni et d.(1990a) Buccheri et d.(1991) Poulakis et d.(1991) Yamaguch et al. ( l m ) , Sarandakou et d. (1993)

t o W l

Nasopharyngeal carcinoma Ovarian cancer

Elevated, clinical and prognostic indicators Elevated in advanced, monitor disease course

Cervical and endometrial cancer Melanoma

Elevated Elevated, no correlation with survival

Autoimmune or inflammatory diseases Autoimmune chronic hepatitis Myasthenia gravis Autoimmune thyroid disease Grave’s disease Scleroderma Rheumatoid arthritis

Elevated Elevated Elevated Elevated, useful marker Elevated, internal organ involvement Elevated, indicator of disease activity

Wegener’s granulomatosis Polymyalgia rheumaticdgiant cell arteritis Systemic lupus erythematosus

Elevated, correlate with disease activity Elevated

Subacute cutaneous lupus erythematosus Multiple sclerosis Systemic sclerosis Type I diabetes Glomerulonephritis Inflammatory bowel disease

Elevated

Infectious diseases Bacterial Tuberculosis Lyme disease

Elevated, correlate with disease activity

Lai et d.(1991)

Barton et d.(1993), Gadducci et al. (1994). Hurteau et d.(1994) Ferdeghmi et d.(1993) Ostenstad (1992) Simpson et al. (1995) Confdonieri et al. (1993) Nakanishi et d.(1991) B a l m et al. (1991), Weryha et al. (1991) Uziel et al. (1994), Patrick et al. (1995) Keystone et al. (1988), Semenzato et al. (1988), Symons et al. (1988), Matsumoto et d.(1993) Schmitt et d.(1992) Salvara~et d.(1992) Semenzato et al. (1988). Tokano et al. (1989), Spronk et al. (1994) Neish et al. (1993)

Elevated in some cases, MS in relapse Elevated, correlate with disease activity Elevated Elevated Elevated, sIL-2RP also elevated

Gallo et d.(1989), Sharief et d.(1993) Degiannis et al. (1990) Giordano et al. (1988) Yorioka et d.(1990) Crabtree et al. (1989), Mueller et al. (1990), Nielsen et al. (1995)

Elevated, marker for clinical state

Brown et al. (1989), Takahashi et al (1991), Chan et al. (1995) Nilsson et al. (1994)

Moderately elevated

(continues)

TABLE IV-Continued sIL-2Ra levels, comments

Reference

Elevated, marker for disease progress Elevated in acute and chronic disease Slightly elevated Elevated Elevated Elevated in systemic or long-lasting infections Elevated, vigorous cellular immune response Elevated, disease marker Elevated

Prince et al. (1988), Griffin et d.(1990) Alberti et d.(1989), Muller et al. (1989) Vinante et al. (1994) Marcante et d.(1991) Griffin et al. (1989) Josimovic-Alasevic et al. (1988) Chumpitazi et d. (1990), Riley et d.(1993) Vitale et al. (1992) Iwagaki et d.(1994) Trochu et al. (1992), Bock et al. (1994) Rossi et al. (1994) Perkins et d.(1989), Lalli et al. (1992)

Heart Lung

No correlation Elevated in viral infection Elevated, correlate with the histological grade of rejection, indicator Elevated in severe rejection Marked elevation in rejection, monitoring

Cornea Bone marrow

Elevated in acute rejection Elevated in severe infection and acute GVHD

Gabrielli et d.(1994) Lawrence et d.(1989), Gascoigne et d. (1993) Foster et d.(1993) Siegert et nl. (1990)

Elevated Elevated Elevated

Hashimoto et d.(1993). Lai et al. (1993) Maes et d.(1994) Rapaprt et al. (1994) Bock et al. (1993)

Elevated Elevated Elevated

Parera et al. (1992) Greally et aZ.(1993) Lawrence et al. (1988), Keicho et al. (1990).Pforte et al. (1993)

Disease Viral HIV HAV, HBV EBV (infectious mononucleosis) CMV mononucleosis Measles Parasitic Malaria Visceral leishmaniasis Infectious complications Transplantation Kidney Liver

Other diseases Asthma Schizophrenia Idiopathic nephrotic syndrome of Childhood Idiopathic IgA nephropathy Cystic fibrosis Sarcoidosis


299

two different murine allotransplantation models ( Fanslow et al., 1991). Interestingly, inhibition of IL-4 activity in vivo through the administration of recombinant sIL-~R,was able to shift predominant Th2 into T h l responses, leading to protection of susceptible mice in murine models of cutaneous leishmaniasis (Gessner et al., 1994) and candidasis (Puccetti et al., 1994). Although the high affinity of sIL-4R and their ability to inhibit IL-4 activity are highly suggestive of a potential role as negative regulators of IL-4, sIL-4R may also function as IL-4-“carriers” and/or agonists, as discussed in the previous section. Partly because of the ability of sIL-4R to either potentiate or inhibit IL-4 activity, it has been difficult to predict the type of regulatory function that endogenous sIL-4R may have in uivo. A potential clue toward the elucidation of their contribution to the regulation of IL-4 responses might be obtained from information about how the production of sIL-4R is, itself, regulated; evidence so far available indicates that their production is actively up-regulated during Th2 responses (Chilton and Femandez-Botran, 1993; Femandez-Botran et al., 1995, 1996), thus suggesting that sIL-4R may play an important role, perhaps as negative “feedback” regulators or “buffers”for IL-4, keeping its effects close to the site of production. Whether the levels of sIL-4R in serum may be used as a “marker” for Th2 responses in vivo is a possibility that deserves further investigation. Although the production of sIL-4R by human peripheral blood mononuclear cells stimulated with microbial superantigens has been reported (Konig et al., 1995),very little is known regarding the identity of the cells responsible for their production and the types of signals involved in their regulation. Similarly to its murine counterpart, recombinant human sIL4R are able to inhibit the binding and biologic activity of IL-4 (Garrone et al., 1991).

D. SOLUBLE INTERLEUKIN-5 RECEPTORS Interleukin-5 is a cytokine produced primarily by the Th2 subset of CD4’ T cells. In both mice and humans, IL-5 is the major cytokine responsible for the growth, differentiation, and activation of eosinophils. In the mouse, IL-5 also induces proliferation and differentiation of activated B cells and promotes the differentiation of thymocytes into cytotoxic T lymphocytes (Sanderson et al., 1988). The functional receptors for IL-5 are heterodimers composed of an ILS-binding a-subunit (60 kDa) and a signal-transducing 130-kDa p-subunit (Mita et al., 1989) that is shared with receptors for IL-3 and GM-CSF (Tavernier et al., 1991). Whereas the IL-5Ra subunit alone can bind IL-5 with low affinity ( K d -1 nM),

300


formation of the high-affinity receptor (Kd -40 pM) requires the participation of the IL-5RP subunit (Mita et al., 1989). Attempts to clone the human IL-5Ra resulted in the isolation of a cDNA clone containing a termination codon upstream of the transmembrane domain and encoding a secreted form of the receptor (Tavernier et al., 1991). The presence of putative splice donor sites suggested that this message was generated through an alternative splicing event. After expression, the resulting soluble form of the IL-5Ra was able to interfere with the binding of IL-5 to membrane IL-5Ra molecules expressed on COSl cells and inhibited IL-5 activity in an eosinophil differentiation assay. An alternatively spliced message encoding for a soluble form of the murine IL-5Ra has also been described (Takaki et al.,1990). Whether endogenously produced sIL-5Ra play a role in the regulation of IL-5 activity in duo, perhaps as a protective mechanism against excessive recruitment/activation of eosinophils, remains a matter of speculation. Based on the large difference in affinity between the soluble a-chain ( K d -1 nM) and the high-affinity IL-5R (Kd -40 pM), it could be predicted that relatively high concentrations of the soluble receptor are required in order to achieve significant inhibition of IL-5 activity. However, information regarding the production of sIL-5Ra in uiuo under normal and pathologic conditions is still unavailable. Nonetheless, recombinant sIL-5Ra have been shown to be inhibitory toward IL-5 activity in uiuo, as indicated by the experiments of Yamaguchi et al. (1994), in which administration of sIL-5Ra resulted in suppression of eosinophil accumulation in the bronchoalveolar lavage fluid in a murine model of allergic airway hyperreactivity.

E. SOLUBLE INTERLEUKIN-6 RECEPTORS Interleukin-6 is a multifunctional cytokine that acts on a wide variety of cells. Among its many activities are the induction of immunoglobulin secretion by activated B cells, stimulation of proliferation by hybridomd myelomdplasmacytoma cells, regulation of the acute phase response, hemopoiesis, and T cell mitogenesis (Akira et al., 1990;Van Snick, 1990) . The effects of IL-6 are mediated by a functional IL-6R, which is a heterodimer composed of an IL-6-binding a-chain (60 kDa) and a signal-transducing subunit, gp130 or IL-6RP (Hibi et al., 1990), that is shared with the receptors for oncostatin M (OSM), leukemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) (Gearing et aZ., 1992). The sIL-6Ra alone binds IL-6 with low affinity, but the presence of the signal transducing subunit (gp130)results in the formation of a high-affinity receptor (Hibi et al., 1990; Gearing et al., 1992). In contrast to other sCR, however, the complex between sIL-6Ra and IL-6 is able to interact with

SOLUBLE CITOKINE RECEPTORS

301

the signal transducing subunit on the surface of target cells and is, thus, biologically active. Consistently, sIL-6Ra have been found to potentiate rather than to inhibit the biologic activity of IL-6, both in vitro and in vivo (Novicket al., 1992b;Suzuki et al., 1993;Tamura et al., 1993; Schobitz et al., 1995). Although originally thought to be generated exclusively through the proteolytic cleavage of membrane-bound IL-6Ra chains (Mullberg et al., 1993), alternatively spliced messages encoding a secreted form of the IL6Ra have been described more recently in a variety of normal human cells and cell lines (Horiuchi et al., 1994; Lust et al., 1995).Hence, sIL-6Ra may be generated through both proteolytic and alternative splicing mechanisms. The signals regulating the production of sIL-6Ra are not clear, but cellular activation, particularly by mitogens and activators of protein kinase C (e.g., phorbol myristate acetate), have been reported to greatly enhance their generation in hepatic and lymphoid cells (Mullberg et al., 1993; Korholz et al., 1994). Soluble IL-6Ra are present in normal serum and urine at relatively high levels (Novick et al., 1989; Frieling et al., 1994). The serum levels have been reported to be increased in a variety of clinical conditions, including autoimmune and inflammatory diseases and monoclonal gammopathies (summarized in Table V). The production of sIL-6Ra by promyelocyhc and T cell lines has been reported to be enhanced as a consequence of infection with HTLV-I or HIV, and in fact, increased serum levels of sIL6Ra have also been observed in patients seropositive for HIV (Honda et al., 1992). The elevated levels of sIL-6Ra under these conditions may actually result in the potentiation of IL-6 activity in uivo, with potentially deleterious consequences, such as an enhanced sensitivity of myeloma cells to IL-6 (Gaillard et al., 1993; Greipp, 1994).Interestingly, the serum levels of sIL-6Ra in the mouse have been reported to gradually increase during pregnancy, and decidual cells have been identified as active sources of sIL6Ra (Maeda et al., 1994).Such an observation suggests that potentiation of IL-6 activity in vivo may also play a physiological role during gestation. Other clinical conditions, however, are characterized by decreased serum levels of sIL-6Ra. Patients with severe sepsis have been reported to have elevated serum levels of IL-6, but decreased levels of sIL-6Ra (Frieling et al., 1995). Moreover, decreased serum sIL-6Ra levels have also been reported in patients suffering from schizophrenia (Maes et al., 1994) and juvenile rheumatoid arthritis (De Benedetti et al., 1994). The clinical significance of the reduced levels of sIL-6Ra is not clear, but it may represent an attempt by the immune system to down-regulate IL-6 activity in conditions in which it is overproduced.

TABLE V SOLUBLE IL-6 RECEITORSAND HUMAN DISEASE Disease

sIL-6Ra levels, comments

Reference ~~

Monoclonal gammopathy (benign, early multiple myeloma, established multiple myeloma) Rbeumatoid arthritis Interstitial lung diseases (primary interstitial pneumonia, sarcoidosis) Inflammatory bowel disease (ulcerative colitis, Crohn's disease) Cerebral malaria Severe sepsis Schizophrenia Juvenile rheumatoid arthritis

Elevated, no correlation with tumor m a s

Gaillard et d.(1993)

Elevated in synovial fluid Elevated

Mihara et al. (1995) Yokoyama et al. (1995)

Elevated in active disease

Mitsuyama et d.(1995)

Elevated Decreased (IL-6 is elevated), no correlation with severity and survival Decreased Decreased

Jakobsen et al. (1994) Frieling et al. (1995), Zeni et d.(1995) Maes et nl. (1994) De Benedetti et al. (1994)


303

Naturally produced soluble forms of the signal-transducing gp130 subunit (IL-6RP) have also been reported in human serum (Narazaki et al., 1993).Although the soluble gp130 subunits are unable to bind IL-6 directly, they are able to bind to sIL-6RdIL-6 complexes, preventing their interaction with membrane-bound gp130 on target cells, and thus, inhibiting IL6 activity. In addition, the soluble gp130 is also capable of inhibiting the biologic activity of the other cytokines that utilize the membrane gp130 as part of their receptor (i.e., OSM, LIF, and CNTF). Hence, soluble forms of the gp130 may work as multifunctional antagonists of IL-6 and related cytokines. The mechanisms responsible for the generation of the sIL-GRP and their potential association with human diseases remain unhOWn.

F. SOLUBLE INTERLEUKIN-7 RECEPTORS Interleukin-7 is a cytokine produced by bone marrow and thymic stromal cells that plays a role in the growth and differentiation of B cell and thymic precursor cells (Goodwin et al., 1989).In addition, IL-7 can act as a growth factor for activated T cells (Chazen et al., 1989).Similarly to other cytokine receptors, the functional IL-7R is a heterodimer composed of a ligandbinding a-subunit associated with a common signal transducing molecule, the IL-2Ry chain. Association of the IL-7Ra chain, which binds IL-7 with low affinity, with the signal transducing subunit is required for the generation of the functional high-affinity IL-7 receptor (Noguchi et al., 1993). In the process of cloning the human IL-7 receptor, Goodwin and coworkers (1990) isolated several cDNA clones encoding a putative soluble receptor form, in which a deletion had caused an altered translational reading frame, resulting in the addition of 27 novel amino acids and premature termination upstream of sequences coding for the transmembrane domain. As predicted, when such clones were transfected into COS7 cells, secreted forms of the IL-7R were obtained. The soluble IL-7Ra inhibited binding of IL-7 to cells expressing membrane IL-7R. Based on the difference of affinities between the functional high-affinity IL-7R and sIL-7Ra (low affinity), it appears that relatively high concentrations of the soluble form would be required in order to achieve significant inhibition of IL-7 activity in vivo. The existence of endogenously produced sIL-7Ra in humans has not yet been confirmed. G. SOLUBLETUMOR NECROSIS FACTOR RECEPTORS Tumor necrosis factor-a (TNFa) and tumor necrosis factor+ (TNFP) are two pleiotropic cytokines secreted primarily by monocyte/macrophages and T lymphocytes, respectively. Both forms share biologic activities and

304

RAFAEL FERNANDEL-BOTRAN ET AL

bind to the same receptors on target cells. TNFa is a key mediator of inflammation and septic shock. In addition, T N F d P have many other activities, including activation of monocytes and polymorphonuclear cells, expression of adhesion molecules on endothelial cells, induction of secretion of other proinflammatory cytokines (e.g., IL-1, IL-6, IL-8), and costimulatory effects on T and B lymphocytes (Vassalli, 1992; Dayer and Burger, 1994). The effects of T N F d P are mediated by two distinct, but related, single chain receptors, TNF-RI (55 kDa) and TNF-RII (75 kDa), both of which bind T N F d P with high affinity (Smith et al., 1994). The natural forms of sTNF-R were initially described, in both serum and urine, as proteins with the ability to inhibit the activity of TNFa, and thus were originally refered to as “TNF-inhibitors” (Seckinger et al., 1988; Engelmann et al., 1989; Olsson et al., 1989; Gatanaga et al., 1990). It was not until later that these proteins were identified as soluble TNF receptors based on their ability to bind TNFa and cross-react with anti-TNF-R antibodies (Engelmann et al., 1989; Seckinger et al., 1990). The soluble TNF receptors are considered to represent a natural protection mechanism against the toxic effects of TNFa (Dayer and Burger, 1994). Soluble forms of both types of TNF-R are generated naturally by proteolytic cleavage of the extracellular domains at the cell surface (Nophar et al., 1990). Even though the two membrane TNF receptors bind TNFa and TNFP with similar affinities, the soluble forms bind preferentially to TNFa (Schall et al., 1990; Pennica et al., 1992). Both types of soluble TNF receptors are potent inhibitors of the binding and activity of TNFa, even though the sTNF-RI (p55) has been reported to be 5 to 30 times more potent than the sTNF-RII (p75) in vitro, and a better inhibitor of TNFa activity in vivo (Evans et al., 1994; Hale et al., 1995). Cellular activation appears to be the major stimulus for the production of sTNF-R: lipopolysaccharide, anti-CD3 antibodies, and phorbol esters have been shown to be potent stimulators of sTNF-R production in different types of cells, including monocyte/macrophages (Leeuwenberg et al., 1994), T lymphocytes (Bemelmans et al., 1994; Cope et al., 1995), and granulocytes (Steinshamn et al., 1995). Consistent with the idea that sTNFR function as natural buffers of TNFa activity, their generation is also enhanced by TNFa itself (Cope et al., 1995), suggesting a negative “feedback” regulation pathway. Differences appear to exist in the regulation of production of the two types of sTNF-R, as suggested by the greater sensitivity of the generation of sTNF-RII (p75), in comparison sTNF-RI (p55), by T cells and monocytes in response to stimulation (Cope et al., 1995; Leeuwenberg et al., 1994). However, the production of sTNF-RII, but not that of sTNF-RI, appears to be down-regulated by chronic exposure to TNFa (Cope et al., 1995).These observations suggest that the relative


305

importance of sTNF-RI and sTNF-RII as regulators of TNFa activity may vary under different conditions. It is possible that sTNF-RII may play a major role during acute TNFa secretion, whereas sTNF-RI may be more important during conditions of chronic TNFa release, particularly due to their resistance to down-modulation. Elevated levels of sTNF-R appear to correlate with conditions of acute and chronic inflammation. Even before their identification as soluble TNF receptors, the so called “TNF-inhibitors” were described in the urine of febrile patients (Seckinger et al., 1988), in the serum of patients with malignancies (Gatanaga et al., 1990), and in the serum and other fluids of patients with respiratory distress syndrome (Suter et al., 1992) or arthritis (Cope et al., 1992; Roux-Lombard et al., 1993). Table VI summarizes information about clinical conditions in which alterations in the levels of sTNF-RI and/or sTNF-RII have been reported. The serum levels of sTNFR appear to correlate with disease activity in patients with rheumatoid arthritis (Barrera et al., 1993) and systemic lupus erythematosus (Aderka et al., 1993). In fact, studies by M a n g e and co-workers (1995) suggest that persistently high levels of sTNF-RI are a better indicator of disease activity than measurements of any other cytokines or C-reactive protein in patients with juvenile rheumatoid arthritis. Measurement of serum levels of sTNF-R have also been reported to have management and prognostic value in kidney graft rejection, increasing earlier than biochemical parameters of kidney function, such as creatinine (Keil et al., 1994). Elevated serum levels of sTNF-R also appear to correlate with mortality in cases of bacterial sepsis (Froon et al., 1994)and human falciparum malaria (Kern et al., 1992; Deloron et al., 1994). Moreover, the serum levels of sTNFRII have been reported to be elevated and to increase with the progression of the disease in HIV-infected individuals (Kalinkovich et al., 1992). It even has been suggested that sTNF-R may play a potential protective role during HIV infection, by antagonizing the TNF-mediated transcriptional activation of the virus (Howard et al., 1993). Despite the overwhelming information pointing to the function of sTNFR as inhibitors of TNF activity, as with other sCR, some of their properties are also consistent with a potential role as “carriers.” For example, administration of a dimeric form of the sTNF-RII (a fusion protein with an immunoglobulin Fc fragment), resulted in an increase in the levels of circulating TNFa, albeit in complexed form, in mice challenged with endotoxin (Mohler et al., 1993). Moreover, Hale et al. (1995) reported that combinations of recombinant human sTNF-RI and TNFa at low receptor to cytokine ratios, resulted in a trend toward the enhancement of TNFa activity in vivo. Such observations are reminiscent of the effects of sIL-4R on IL-4, and are consistent with the idea that the effect of

TABLE VI SOLUBLE TNF RECEFTORSAND HUMAN DISEASE Disease

sTNF-R (p55, p75) levels, comments ~~

I. Infectious diseases Bacterial sepsis

p55 and p75 levels correlate with kidney function, significantly higher $5 levels correlate with multiple organ failure and mortality. Subacute bacterial endocarditis Elevated p55 and p75 levels Tuberculosis Elevated Malaria Elevated p55 and p75 levels, higher levels in patients with cerebral malaria HIV Elevated p75, not p55, increased levels correlate with disease progression

CMV

11. Autoimmune diseases Juvenile rheumatoid arthritis

Systemic lupus erythematosus 111. Other

Renal graft rejection

Kidney disease Renal cell carcinoma Myocardial infarction Sarcoidosis

Elevated p55 levels Elevated p55 and p75, persistently high TNF-R levels mark RA activity, increase in p55 seen in response to stress Elevated p55 and p75 levels correlate with disease activity Elevated p55 levels correlate with active rejection, acute and chronic Elevated p55 levels; correlation with kidney function Elevated $5 levels prior to immunotherapy Elevated levels of p55 in severe infarction, no increase in uncomplicated patients Elevated levels of TNFR (type not determined)

____

Reference

Froon et d.(1990), Moldawer (1993), van der Poll et al. (1%3),Ertel et d.(1994) Kern et d.(1993) Rydberg et d.(1995) Kern et d.(1992), Molynew et d.(1993), Wenisch et d.(1994), Hurt et d.(1995) Kalinkovich et d.(1992), W r i e d et d. (1993, 1994) Aukrust et al. (1994), Zangerle et d.(1994a,b) Keil et al. (1994) Heilig et d.(1992), Barrera et al. (1%3), Chikanza et d.(1993),Mangge et d.(1995) Aderka d d.(1993) Bemelman et d.(1994), Keil et d.(1994) Brockhaus et al. (1992), Ward and Gordon (1993), Halwachs et al. (1994), Zemel et d. (1994) Belldegrun et d.(1993) Lantini et al. (1994) Foley et al. (1990)

SOLUBLE CYl'OKINE RECEPTORS

307

cytokine receptors on the activity of their cytokines is dictated by the balance between their agonistic and antagonistic effects.

H. SOLUBLE INTERFERON RECEPTORS Interferons (IFN) are a group of cytokines with the ability to induce an anti-viral state in cells. Interferons have been divided into two main groups: (a) type I or viral interferon, including IFNa (leukocyte-derived), which is actually a family of 23 different polypeptides, IFNP (fibroblast-derived), and IFNw; and (b) type I1 or immune interferon, including IFNy, which is produced mainly by T lymphocytes and NK cells as a result of antigenic or mitogenic stimulation (Pestka et al., 1987; Farrar and Schreiber, 1993). Besides their anti-viral activity, interferons also have important immunomodulatory effects, especially IFNy. Type I interferons (a,P, and o)have similar activities and bind to the same receptor (type I IFN-R), whereas a different membrane receptor is responsible for binding IFNy (type I1 IFN-R) (Branca and Baglioni, 1981; Orchansky et al., 1984). Both types of receptors appear to be heterodimers composed of a-(ligand binding) and @-subunits (mostly responsible for signal transduction) (Farrar and Schreiber, 1993). Soluble forms of the type I IFN-R (IFNd@-R) were initially identified in normal human serum and urine by Western blotting and immunoprecipitation with monoclonal antibodies directed against the IFNdP-R (Novick et al., 1992a). Sequence information obtained from the purified soluble IFNd@-Rwas then used to clone the cDNA encoding the receptor (Novick et al., 1994). Two different cDNA clones were obtained, one encoding a membrane-anchored receptor and another encoding a truncated soluble receptor form, demonstrating that natural sIFNd@-R are generated through an alternative RNA splicing mechanism, rather than by receptor proteolysis. Consistent with the specificity of the type I IFN-R, the s I F N d P-R bound different species of IFNa (aC, a B and a2) and IFNP, but not IFNy. In addition, sIFNd@-Rinhibited the activity of natural human leukocyte interferon (a mixture of many IFNa species), but not that of IFNy, in a viral inhibition assay. The potential involvement of sIFNdPR in the regulation of type I interferons in vivo, particularly during viral diseases, remains to be determined. Although not much information is yet available on natural forms of soluble type I1 ( IFNy) receptors, their existence was demonstrated by Novick and co-workers (1989),who reported the presence of IFNy-binding proteins in normal human urine, and identified them as soluble IFNyR based on their reactivity with monoclonal antibodies directed against the membrane IFNy receptor, Even though the ability of the natural sIFNyR to inhibit binding and activity of IFNy was not tested, experiments

308


performed with genetically engineered recombinant forms of the murine IFNyR have proven their capability to act as IFNy antagonists both in vitro and in vivo (Kurschner d al., 1992; Ozmen et al., 1993a,b).

I. OTHERSOLUBLE CYTOKINE RECEPTORS Natural soluble receptors for human GM-CSF have been reported recently and identified as truncated forms of the a-chain (ligand-binding subunit) of the GM-CSF-R (Brown et al., 1995). These receptors are also generated by translation from an alternatively spliced mRNA, different from that encoding the membrane-bound form. Although the affinity of the soluble receptors is expected to be considerably lower than that of the high-affinity membrane GM-CSF-R complex (aand /3 subunits), recombinant sGM-CSF-R were nonetheless able to block binding of GM-CSF to cells expressing both low- and high-affinity receptors and blocked the biologic activity of GM-CSF in human bone marrow colony forming assays. Additional information regarding the potential regulatory role of sGMCSF-R in vivo and relationship with human disease is not available. Soluble GM-CSF-R have been reported in supernatants from a human choriocarcinoma cell line, but not from several hematopoietic cell lines (Sasaki et al., 1992). The generation of soluble forms is not exclusive of cytokine receptors, and may represent a general regulatory mechanism for the activity of membrane-bound molecules. Evidence also exists for the occurrence of natural forms of several hormone and growth factor receptors, including growth hormone (Leung et al., 1987), epidermal growth factor (Weber et al., 1984), insuline-like growth factors (MacDonald et al., 1989), and transferrin (Ahn and Johnstone, 1993). Soluble forms of other surface molecules playing important roles in immune recognition and cellular interactions, such as MHC class I antigens (Krangel, 1986),CD8 (Fujimoto et al., 1983),CD23 (Guy and Gordon, 1987), and several adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1 (Gearing and Newman, 1993), have also been reported.

J. VIRALSOLUBLECVTOKINE RECEPTORS In the last several years, a growing amount of evidence has pointed to the fact that some viruses, particularly the poxviruses, encode within their genome a number of proteins or “virulence factors” that endow them with increased capacity to propagate within immunocompetent hosts (McFadden et al., 1995). Abrogation of the expression of these “virulence factors” results in attenuation of the pathogenicity of the virus. Although such factors are not essential for replication of the virus in vitro, many of them interfere with different immunological mechanisms, thus allowing the virus


309

to escape destruction by immune effector elements. Interestingly, the genes encoding some of these “virulence factors” were found to have considerable sequence homology with several cellular cytokine receptor genes, including the TNF-R type I1 (Smith et al., 1990; Upton et al., 1991), the IFNyR a-subunit (Upton et al., 1992), and the IL-1R type I1 (Smith and Chan, 1991; Alcami and Smith, 1992; Spriggs et al., 1992). Moreover, these viral receptors were found to behave as soluble cytokine receptors, blocking the binding and inhibiting the activities of their specific cytokines. These proteins are briefly discussed below. The “virulence factors” encoded by the poxviruses are thought to have been acquired from cellular sources, perhaps by recombination through cytoplasmiccDNA intermediates (McFadden et al., 1995).Captured genes that resulted in an enhanced ability to replicate in immunocompetent hosts would thus have been selected. The fact that soluble cytokine receptors are among those factors argues strongly in favor of the idea that endogenous soluble cytokine receptors can, in fact, regulate cytokine activity in vivo and suggests that they play an important role in the regulation of the immune system.

1. Soluble TNF Receptor 11 (T2) The first example of a viral homolog to a cytokine receptor was obtained when the gene encoding the TNF receptor type I1 was found to bear striking sequence homology to a gene, T2, from the Shope fibroma virus (SFV) (Smith et al., 1990, 1991). Similar TNF receptor type II-related genes have been detected in other poxviruses, including myxoma (Upton et al., 1991), cowpox, and variola (Hu et al., 1994). Abrogation of the expression of the T2 gene in the myxoma virus was reported to result in attenuated pathogenicity in susceptible rabbits (Upton et al., 1991). A potential explanation for such observations is that interference with TNFa activity may increase the ability of the virus to propagate in the host, perhaps by inhibiting recruitment and activation of inflammatory cells. 2. Soluble lFNy Receptor (T7) Sequence comparisons between a 37-kDa protein secreted by myxoma virus-infected cells, the T7 protein, and the a-chain of both human and murine IFNy receptors revealed significant homology at the amino acid level, Consistently, the T7 protein proved to have the ability to bind IFNy with an affinity similar to that of the cellular IFNy-Ra and to inhibit its anti-viral activity (Upton et al., 1992; Mossman et al., 1995b). Similar IFNy-R homologs have also been described for other poxviruses, including ectromelia, cowpox, variola and vaccinia (Mossman et al., 1995a).The T7 protein may increase the ability of poxviruses to propagate in immunocom-

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petent hosts by interfering with cellular immune responses and the antiviral effects of IFNy. 3. Soluble ZL-1 Receptor ZZ (B15R)

Database search comparisons using the sequence of the IL-1R type I1 identified several structurally related viral proteins (Smith et al., 1991). It was later demonstrated that, in fact, one of these genes in vaccinia and cowpox viruses (B15R) encoded for a secreted protein with the ability to both bind IL-lP (but not IL-la or IL-lra) and inhibit its biologic activity (Alcami and Smith, 1992; Spriggs et al., 1992). Deletion of the B15R gene from the vaccinia virus correlated with accelerated appearance of symptoms of illness and mortality in intranasally infected mice, suggesting that its expression down-regulates the inflammatory response and modulates the severity of the disease (Alcami and Smith, 1992). VII. Soluble Cytokine Receptors and Therapy

A. INHIBITING THE ACTIVITY OF CYTOKINES IN HUMAN DISEASES There is now overwhelming evidence that cytokines are responsible for the clinical manifestations of an extensive variety of diseases. Most of the manifestations associated with inflammatory responses, including fever, are mediated by the release of cytokines such as TNFa, IL-1, and IL-6. Moreover, the massive production of TNFa and IL-1 is directly responsible for the cardiovascular collapse and metabolic alterations that result in the fatal consequences of the systemic inflammatory response syndrome. The chronic release of proinflammatory cytokines in autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE), is responsible for the infiltration and activation of inflammatory cells that mediate the tissue damage (Akira et al., 1990; Vassalli, 1992; Dinarello, 1994). Allergic reactions are directly dependent on IL-4 (and possibly IL13)for the generation of antibodies of the IgE isotype that sensitize mast cells (Finkelman et al., 1990; Paul, 1991). Furthermore, several premalignant and malignant cells can utilize cytokines as autocrine growth factors, such as in the case of acute myelogenous leukemic cells and IL-1 (Estrov et al., 1992), HTLV-I-infected T cells (Tcell leukemia) and IL-2 (Rubin and Nelson, 1990), and multiple myeloma cells and IL-6 (Van Snick, 1990). Cytokine activity is thus a logical target for therapeutic intervention in many types of diseases. As discussed at the beginning of this chapter, cytokine activity is regulated at many different levels, from its production, expression of specific membrane receptors, interaction of the cytokine with such receptors, and subsequent signalling. Hence, therapeutic approaches for the modulation of


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cytokine activity can be be targeted to one or more of those levels. Moreover, different strategies can be used to accomplish those goals. For example, the production of TNFa may be inhibited by a number of drugs, such as the xanthine derivative pento$line (van Leenen et al., 1993), by interfering with the processing of the TNFa precursor (e.g., inhibitors of metalloproteases) (Gearing et al., 1994), or by the administration of cytokines with anti-inflammatory effects, such as IL-4 and IL-10 (Hart et al., 1989; Standiford et al., 1990; Moore et al., 1993). The interaction of a cytokine with its receptors (e.g., IL-lp), could be interrupted by neutralizing monoclonal anti-IL-lp antibodies, receptor antagonists (&ha), or soluble cytokine receptors (sIL-1RI or sIL-lRII), and signals generated by a cytokine after binding to its receptors could be antagonized by drugs or other cytokines with antagonistic activities (e.g., IFNy-mediated inhibition of IL-4-induced IgE production).

B. SOLUBLE CYTOKINE RECEPTORS AS THERAPEUTIC AGENTS Soluble cytokine receptors have several attractive properties as therapeutic agents for the inhibition of cytokine activity. These include: (a)specificity (sCR target individual cytokines, without affecting the activities of others); (b) affinity (sCR have usually comparable or higher affinities than many available anti-cytokine monoclonal antibodies, making them more potent inhibitors); and (c) lack of immunogenicity (unlike the use of murine mAbs for human therapy, human sCR would not elicit an immune response in humans). Although there are also potential disadvantages to the use of sCR as immunotherapeutic agents for the inhibition of cytokine activity, these are, however, not insurmountable, One potential problem is the relatively short half-life of sCR in uiuo. Jacobs et al. (1991b) found the half-life of the murine sIL-4R to be only 1-3 hr after iv injection, and approximately 6 hr for that of the sIL-1RI (Jacobs et al., 1993). Consistently, Ozmen et al. (199313)reported a half life of the murine IFNy-R of approximately 3 hr (by comparison, monoclonal antibodies usually have half-lives greater than 24 hr). Such a problem has been circumvented for a variety of sCR, by the construction of fusion proteins between the soluble receptors and the Fc portion of an immunoglobulin. Inasmuch as such hybrid receptor molecules have the advantages of increased half-life and, in some cases, of increased affinity due to their dimeric nature, they are usually more effective inhibitors than the monomeric native receptors (Kurschner et al., 1992; Mohler et al., 1993). Another potential problem with the use of sCR as inhibitors is their ability to act as carrier proteins for their ligand, thus leading to an increase in the circulating levels of that cytokine. Although under such conditions the circulating cytokine is complexed with its sCR

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and is, as such, not active, the possibility remains that dissociation of the complexes might result in release of the active cytokine. This problem, however, could be prevented by the administration of a large excess of the inhibitor, so that any cytokine molecule that dissociates from its sCR is again rapidly re-bound and neutralized by free sCR. Alternatively, the ability of sCR to act as agonist or carriers for their ligands could be used to enhance the activity of exogenously administered cytokines. It is well known that most cytokines have toxic side effects when administered in high doses. Unfortunately, since their half-lives in circulation are very short, relatively high concentrations of cytokines must be injected in order to achieve pharmacologic concentrations. The use of sCR as adjuvants to cytokine immunotherapy could allow smaller doses of cytokines to be used, thus reducing toxic side effects. Such treatment would require, however, a careful evaluation of the conditions that result in enhancement, rather than inhibition, of cytokine activity (e.g., sCR to cytokine ratios). The therapeutic potential of several soluble cytokine receptors as cytokine inhibitors has already been demonstrated in experimental animal models and is currently being evaluated in clinical trials for several human diseases. Information regarding the activities of sCR in different disease models is briefly discussed below.

C. SOLUBLECYTOKINE RECEPTORSIN DISEASE MODELS 1. Soluble IL-1 Receptors The ability of sIL-1R type I receptors to inhibit IL-1 activity in vivo has been demonstrated in several disease models. As would be predicted from the inhibition of IL-1 activity, the administration of recombinant sIL-1RI has shown significant ability to decrease inflammation and reduce cellular infiltration in experimental animals. For example, Jacobs et al. (1991a) reported that the administration of rsIL-1RI in rats delayed the onset of symptoms, reduced the severity of paralysis and weight loss, and shortened the duration of the disease in an experimental autoimmune encephalomyelitis model, in which the pathology was exacerbated by injection of rILla. In another model, the administration of recombinant sIL-1R was shown to significantly reduce the accumulation of neutrophils and lower the concentrations of TNFa and IL-6 in the bronchoalveolar lavage fluid of rats challenged intratracheally with lipopolysaccharide (Ulich et al., 1994). Reduced inflammatory responses as a result of sIL-1RI administration have also been reported in a model of IL-la-induced ocular inflammation in rabbits (Rosenbaum and Boney, 1991). The sIL-1RI has also been reported to decrease alloreactivity and prolong the survival of cardiac


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allografts in a murine allogeneic transplantation model ( Fanslow et al., 1990b).In humans, administration of sIL-1RI has been reported to significantly reduce the clinical manifestations of the late-phase allergic reaction (erythema, induration, and itching) following subcutaneous injection of allergen (Mullarkeyet al., 1994). Hence, sIL-1R appear to have considerable potential for the treatment of diseases whose pathogenesis and/or manifestations are directly related to inflammation. 2. Soluble IL4 Receptors The ability of sIL-4R to inhibit the activity of IL-4 in uivo was initially demonstrated by Fanslow and co-workers (1991) in two murine models of allogeneic transplantation. Administration of recombinant sIL-4R was shown to reduce the cellular response of BALB/c mice in response to injection with irradiated C57BV6 splenocytes, and prolonged the survival of heterotopic heart allografts. Consistent with the requirement for IL-4 in the production of IgE antibodies, Sat0 et al. (1993) demonstrated later that administration of sIL-4R in mice challenged with anti-IgD antibodies (a polyclonal B cell activator) resulted in markedly reduced serum levels of IgE. More recently, the ability of recombinant murine sIL-4R to inhibit predominantly Th2 immune responses (which are dependent on IL-4), with a subsequent shift to Thl responses, was demonstrated in murine models of cutaneous leishmaniasis (Gessner et al., 1994) and candidiasis (Pucetti et al., 1994). In both cases, treatment with recombinant sIL-4R rendered naturally susceptible BALB/c mice resistant to infection. The administration of sIL-4R may thus be useful in the treatment of diseases in which the development of predominant Th2 responses is detrimental to the host or those, like allergies, which are dependent on the production of IgE antibodies.

3. Soluble TNF Receptors Because of the central role of TNFa in mediating the cardiovascular and metabolic abnormalities associated with the systemic inflammatory response syndrome, considerable interest has developed in the use of antagonists of TNFa. Recombinant soluble TNF receptors have shown significant potential in this respect. Several studies have shown that the administration of recombinant sTNF-R is able to protect mice and other experimental animals in different models of septic shock (Ashkenaziet al., 1991; Lesslauer et al., 1991). Mohler and collaborators (1993) have also demonstrated that a sTNF-RII-Fc fusion protein conferred protection from lethal endotoxic shock in mice. In a different report, however, Evans et at. (1994)reported that sTNF-RI- but not sTNF-RII-IgG fusion proteins protected baboons from a lethal challenge with Gram-negative bacteria

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(Escherichia coli). The therapeutic value of sTNF-R for the prevention of lethality in humans is curently undergoing clinical trials. Besides their potential in the management of septic shock, soluble TNF receptors may also have significant therapeutic value in other manifestations of TNFa activity, such as in the vascular leak syndrome and in inflammatory diseases such as rheumatoid arthritis. Injection of high doses of IL-2 in both humans and animals results in toxic effects which are probably mediated by TNFa. Thus, sTNF-R may be able to prevent or reduce the toxicity associated with the administration high doses of IL-2. Indeed, sTNF-R have been reported to reduce the pulmonary vascular leak in mice after administration of IL-2 (Dubinett et al., 1994). In regard to their effects on inflammation, soluble TNF-R were reported to arrest the evolution of disease in a murine model of collagen-induced arthritis (Piguet et al., 1992), and a sTNF-RII-Fc fusion protein was shown to significantly reduce the recruitment of neutrophils and eosinophils in the lungs and airways of sensitized mice after intratracheal challenge with antigen (Lukacs et al., 1995). 4. Soluble ZFNy Receptors In agreement with the role of IFNy in mediating a variety of pathologic manifestations, inhibition of IFN y activity in uiuo by administration of recombinant sIFNy-R has shown vely promising results. The ability of sIFNy-R to inhibit IFNy activity in uiuo was initially demonstrated by Ozmen and collaborators (1993a) who reported the neutralization of the protective effect of rIFNy in mice challenged with a lethal dose of the encephalomyocarditis virus. Subsequent reports by the same group also demonstrated the ability of recombinant sIFNy-R to delay the onset of disease in both acute and chronic models of graft-versus host disease (GVHD) (Ozmen et al., 1993b) and to delay the onset of glomerulonephritis and reduce mortality in N Z B N autoimmune mice, a model for SLE (Ozmen et al., 1995). Thus, therapy with sIFNy-R appears to have significant potential for the treatment of autoimmune diseases and in the prevention of GVHD. VIII. Summary and Conclusions

The generation of soluble forms of cytokine receptors appears to be a general mechanism contributing to the regulation of cytokine activity in uiuo. The soluble receptor forms are normally generated by the proteolytw cleavage of the extracellular domain of the membrane-bound receptor (a process also known as “shedding”) or by translation from alternatively spliced mRNAs, different from those encoding the membrane-bound re-


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ceptors. The resulting truncated receptors are released from the cells and accumulate in the serum and biological fluids. Both endogenously produced and genetically engineered sCR retain their ligand-binding ability and exhibit affinities similar to that of the membrane-bound receptor, although in some cases the d n i t y of the functional membrane receptor is significantly higher due to the affinity-conversion effect of the signal transducing subunit. However, most sCR are able to function as competitive inhibitors of the binding and biologic activity of their cytokine, both in vitro and in vivo. One exception is the sIL-GRa, which retains the ability to interact with the membrane-bound signal transducing IL-6RP chain, and is thus capable of signaling. Besides their ability to act as cytokine antagonists, however, some effects of sCR, such as stabilization of cytokine structure, protection from proteolytic degradation, and enhanced in vivo half-life, are consistent with an additional role as cytokine camer proteins, and may, under some conditions, result in potentiation of cytokine activity in viva The overall effect of sCR on the activity of their cytokine, is thus dictated by the balance between their antagonistic and agonistic properties. The exact role of endogenous sCR in the maintenance of homeostasis during immune or inflammatory responses has not yet been unequivocally resolved. The mechanisms regulating the production of different sCR in vivo are not completely understood, but cellular activation and, in many cases, their own ligands enhance their release, suggesting that sCR may act as negative “feedback’ regulators. The levels of some sCR, such as sIL-2Ra and sTNFR, in serum and other biological fluids appear to correlate with cellular activation and/or disease activity in a variety of clinical situations, including malignant, autoimmune, infectious, and inflammatory diseases. Hence, measurements of sCR levels provide simple means of assessing immune function during diseases and may have significant value in their management and prognosis. Moreover, soluble cytokine receptors have proven to be very potent and highly specific “cytokine inhibitors” in experimental animal models, indicating a promising potential as immunotherapeutic agents for human diseases. Elucidation of the relationships of the different sCR with the cytokine networks, both under normal conditions and after immunologic stimulation, is necessary in order to more fully understand their role in the regulation of the immune system. Such knowledge should allow us to more efficiently use sCR to modify cytokine activity in vivo for therapeutic purposes. ACKNOWLEDGMENTS This research was supported in part by grants from the American Heart Association

(93011140)and the National Institutes of Health (AI-34627).We are also indebted to Drs.

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Michael B. Widmer and Tony Troutt (Immunex R & D Corporation, Seattle, WA) for their generous supply of reagents; and to Drs. Ellen S. Vitetta and Jonathan W. Uhr (University of Texas Southwestern Medical Center, Dallas, TX) for both reagents and valuable advice, without which this work would not have been possible.

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This article was accepted for publication on 26 January 1996.

ADVANCES IN IMMUNOLOGY, VOL 63

Cytokine Expression and Cell Activation in Inflammatory Arthritis

1. Inh.oduction

In inflammatory arthritis the synovial tissue lining the joint contains infiltrating immune effector cells and increased numbers of synovial fibroblasts. These cells express an activated phenotype that plays an important role in the pathogenesis of arthritis. Although lymphocytes and monocytes may enter the synovial tissue in a partially activated or preactivated state, these cells, as well as synovial fibroblasts, become fully activated and develop an inflammatory effector phenotype in the synovial microenvironment. Cytokines are important regulators of cell activation and differentiation. An important advance in our understanding of the pathogenesis of inflammatory arthritis has been the characterization of synovial expression of cytokines in several animal models of arthritis and in human diseases such as rheumatoid arthritis (RA) and psoriatic arthritis. Evidence is emerging that many of these cytokines play an important role in cell activation and contribute to the pathogenesis of synovitis. This review will focus upon the expression and role of cytokines in RA, which is one of the most intensively studied and best characterized human inflammatory diseases. The study of cytokines in animal models of arthritis and in seronegative arthritis has revealed both similarities and differences with RA. The role of cytokines in animal models and in seronegative arthritis will be discussed in relation to their role in RA. II. The Inflammatory Synovial Environment

Inflamed synovium consists of a hyperplastic lining layer which overlies a highly vascularized sublining region. The lining layer consists primarily of macrophage-like cells (type A synoviocytes) that are likely bone marrow derived cells which have migrated into the lining layer, and fibroblast-like cells (type B synoviocytes) that appear to have proliferated in situ in the synovium (Edwards, 1987).The lining layer may be many cell layers thick and can develop into pannus, which is hyperplastic inflamed synovial tissue that can invade and destroy cartilage and bone. The phenotype of synovial lining macrophages and fibroblasts can be distinguished from sublining macrophages and fibroblasts on the basis of high expression of certain 337 Copynght 0 1996 by Academic Press, Inc All nghts of reproduction in any form resewed

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LIONEL B. IVASHKIV

effector genes, such as metalloproteases and vascular cell adhesion molecule 1(VCAM-1)(Gravalleseet al., 1991; Firestein et al., 1991; McCachren, 1991; Morales-Ducret et al., 1992). It is not clear if this pattern of gene expression reflects primarily differences in cell phenotype, or exposure to localized regulatory molecules, which may be present in the adjacent synovial fluid (SF) or expressed within the lining layer. The sublining region normally consists of loose connective tissue with occasional blood vessels and scattered macrophages and fibroblasts. During synovitis, this region becomes highly vascularized and infiltrated with activated mononuclear cells, including lymphocytes, plasma cells, monocytel macrophages, and dendritic cells (van Boxel and Paget, 1975; Bankhurst et al., 1976; Konttinen et al., 1981; Broker et al., 1990; Ridley et al., 1990; Thomas et al., 1994). In some patients, these mononuclear infiltrates are present diffusely throughout the sublining region. However, mononuclear cells are often present in perivascular aggregates that are organized into a distinctive architecture which may resemble lymph nodes or Peyer's patches (Ziff, 1974; Kurosawa and Ziff, 1983). The perivenular areas are highly enriched for lymphocytes, which are predominantly small CD4+ T cells. Peripheral to the T cell-enriched areas are transitional zones that contain larger, blast-like CD4' T cells, CD8+ T cells, B cells, macrophages, dendritic cells, and plasma cells. The transitional zone T cells appear to be interacting with interdigitating dendritic cells, which represent a potent antigen presenting cell. Surrounding areas which are highly enriched in plasma cells have also been identified. Up to 25-5096 of synovial T cells, even small cells with a quiescent appearance, can express activation markers such as human leukocyte antigen DR (HLA DR), very late activation antigen 1 (VLA-l), CD69, and B7, and lesser numbers express the a chain of the interleukin 2 (IL-2) receptor (Taq antigen) (Burmesteret al., 1981; Duke et al., 1982; Klareskog et al., 1982; Young et al., 1984; Poulter et al., 1985; Hemler et al., 1986; Pitzalis et al., 1987b; Cush and Lipsky, 1988; Hovedenes et al., 1989; Kidd et al., 1989; Iannone et al., 1994; Verwilghen d al., 1994). A large fraction (in some studies >go%) of synovial T cells express memory markers (Emery et al., 1987; Pitzalis d al., 1987a; Lasky et al., 1988; Hanly et al., 1990). Memory T cells are especially enriched in the transitional and B cell areas, suggesting a local conversion from a nake to a memory phenotype after cell activation (Koch et al., 1988). The activated synovial cells express multiple effector molecules which likely contribute to joint destruction. Activated synovial monocytes express high levels of immune effector molecules such as Fc receptors, HLA class I1 antigens, costimulatory molecules such as B7, and metalloproteases (Broker et al., 1990; Gravallese et al., 1991; Firestein et al., 1991; McCach-

CYTOKINE EXPRESSION IN INFLAMMATORY ARTHRITIS

339

ren, 1991; Ranheim and Kipps, 1994). These molecules can contribute to joint destruction through a variety of mechanisms, including direct destruction of tissues, increased cytotoxicityand release of toxic mediators, and increased (auto)antigen presentation that can drive an (auto)immune response. Synovial monocytes can produce the following cytokines and chemotactic factors (see also Table I): IL-1, IL-6, IL-8, IL-10, tumor necrosis factor a (TNFa), granulocyte macrophage-colony stimulatingfactor (GM-CSF), leukemia inhibitory factor (LIF), transforming factor /3 (TGFP), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), IL-1 receptor antagonist (IL-lra), vascular endothelial growth factor (VEGF), epithelial neutrophil activating peptide-78 (ENATABLE I EXPRESSION OF CYTOKINES IN RHEUMATOID SYNOVIAL FLUIDAND TISSUE Monocytes IL-1 TNFa IL-6 LIF GM-CSF PDGF bFGF sCD23 IL-lraa TGFP IL-10 MCP-1 MIP-la RANTES IL-8 gro ENA-78 VEGF (IFW (IL-2) (IL-4)

+ + + + + + + + + + + + + + + +

Fibroblasts

T cells

+ + + + +

+ + + + + +

+

4-

Note. Cytokines which have been detected in rheumatoid synovial fluid or tissue are shown in the first column. The cytokines enclosed in parentheses have not been detected consistently or only mRNA or low levels of protein have been detected. +, production of the cytokine by this cell type in the synovium has been described. "IL-Ira is also produced by synovial fluid neutrophils. Chondrmytes also contribute to the production of several cytokines, including LIF, TGFP, and MCP-1.

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I,IONEI, 8 . IVASHKIV

78), monocyte chemoattractant protein 1 (MCP-l), and macrophage inflammatory protein la (MIP-la). Activated synovial fibroblasts can proliferate during synovitis and express certain phenotypic characteristics similar to transformed cells, such as large pale nuclei, prominent nucleoli, and high levels of vimentin. Explanted synovial fibroblasts continue to proliferate for multiple passages in tissue culture, grow to high densities, form foci, and are able to grow under anchorage-independent conditions (Labatis et al., 1989a). This abnormal growth control may contribute to synovial hyperplasia, formation of pannus, and invasion and destruction of cartilage and bone. Cultured synovial fibroblastscontinue to secrete prostaglandins and several cytokines through multiple passages in tissue culture (Bucala et a!., 1991). During synovitis, these cells express high levels of proteases and produce these cytokines and chemotactic factors (Table I): IL-6, IL-8, TNFa GM-CSF, G-CSF (CSF-l), LIF, PDGF, bFGF, MCP-1, MIP-la, ENA-78, RANTES, and Gro. An important role for T cells in inflammatory arthritis is suggested by associations of disease with HLA alleles, expression of activation and memory markers by synovial T cells, possible oligoclonal nature of synovial T cells, changes in disease activity in patients who develop immunodeficiency (Winchester et al., 1987; Calabrese et al., 1991; Ornstein et al., 1995), improvement in disease activity after anti-T cell therapy (Moreland et al., 1995; Weinblatt et al., 1995), and direct experimental evidence for the role of T cells in animal models of inflammatory arthritis (Williams et al., 1994). Synovial lymphocytes may contribute to pathogenesis by providing help for antibody responses and driving humoral immunity, or by driving a cellular immune response against persistent antigens or autoantigens. The proximity of T cells, B cells, and antigen presenting cells (APCs) in synovial aggregates, along with the presence of plasma cells that are derived from B cells which appear to have undergone class switching and somatic hypermutation in the synovium, suggests that synovial T cells are capable of providing help for B cells. The activated state of synovial macrophages is highly suggestive of a T cell-activated cellular immune response, and the detection of cytotoxic T cell gene expression, albeit at a low level (Muller-Ladner et al., 1994), supports this conclusion. One of the difficulties in understanding the pathogenic role of synovial T cells has been the absent or low level production of T cell cytokines which are important for helping B cells (IL-4, 1L-5, T cell-derived IL-6) (Miossec et al., 1990; Firestein et al., 1990; Chen et al., 1993; Miyake et al., 1993; Simon et al., 1994) or for activating macrophages ( IFNy) (Husby and Williams, 1982; Firestein and Zvaifler, 1987; Feldmann et al., 1991; Sengupta et al., 1995).


34 1

The contribution of synovial B cells, plasma cells, dendritic cells, mast cells, NK cells, and large granular lymphocyte-like cells to synovial cytokine production has not been established at this time. Neutrophils, which are often the predominant cell type in the synovial fluid but are present at low numbers in the synovial tissue, have been implicated in the production of IL-lra (Malyaket al., 1993).Production of cytokines by chondrocytes and chemoattractant factors by activated endothelial cells is discussed below. 111. Assessment of Cytokine Expression and Activi~yin Synovitis

The activation and phenotype of synovial cells during arthritis will be determined primarily by the cells’ interactions with their environment as they enter and migrate through the synovium. This includes cellular responses to soluble factors such as immune complexes, complement breakdown products, and small inflammatory mediators such as prostaglandins. Equally important are interactions with other cells, which can express cell surface-bound cytokines or stimulatory molecules, and interaction with components of the extracellular matrix (Haskill et al., 1988; Miyake et al., 1993; Ranheim and Kipps, 1994; Thomas et al., 1994; Venvilghen et al., 1994).Because of the importance of soluble cytokines in regulating immune and inflammatory responses, a large effort has been expended in identifylng cytokines that are expressed during synovitis and in trying to determine the contribution of various cytokines to the pathogenesis of synovitis. Cytokines produced during synovitis will accumulate in the synovial fluid, which not only reflects cytokine production, but forms an important component of the inflammatory environment to which cells become exposed when they enter the inflamed joint. Levels of many cytokines in synovial fluid have been measured, and high levels of certain cytokines, for example IL-1 and IL-6 (Fontana et al., 1982; Noun et al., 1984; Miyasaka et al., 1988; Field et al., 1991; Guerne et al., 1989; Westacott et al., 1990), have been consistently detected. Synovial fluid cytokine levels are usually determined using either bioassays or immunoassays. Early problems with lack of specificity of bioassays have been addressed by using specific neutralizing antibodies to individual cytokines to confirm that the measured effect is indeed due to a particular cytokine. Bioassays, however, will not detect cytokines in the presence of inhibitors (such as soluble receptors, anti-cytokine antibodies, and toxic substances) or in the presence of antagonistic cytokines (Cope and Brennan, 1992).Thus, although detection of bioactivity of a given cytokine makes it plausible to infer that the cytokine actually plays a role in the inflammatory process, bioassays do not necessarily accurately reflect cytokine production by synovial cells, or actual cytokine protein levels in the synovial fluid.

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Immunoassays, which depend upon the specific interaction of an antibody with a cytokine, have been used to measure cytokine protein levels. Many immunoassays are sensitive down to the range of 10-50 pg/ml, and thus have sensitivity comparable to that of bioassays. However, there are several problems associated with performing immunoassays using synovial fluids. Falsely low values may be obtained if the cytokine is bound to extracellular matrix, or because the viscosity of synovial fluid interferes with the interactions of antibody with cytokine (Firestein and Zvaifler, 1987). Falsely high values and false positives may be obtained because of rheumatoid factors (antibodies reactive with the Fc region of other antibodies) which are present in RA synovial fluid (Malyak d al., 1991). In addition to the problems mentioned above, measurement of cytokines in synovial fluid will not detect cytokines which have interacted with their receptors and become internalized or “consumed,” and cytokines which are produced locally in the synovium and do not diffuse into the synovial fluid. Paracrine activity, or even direct delivery of cytokines exclusively to adjacent cells (similar to localized release of neurotransmitters into a synapse), has been postulated to be important in regulation of the immune response. Therefore, investigators have assessed cytokine expression, at both the mRNA and protein levels, by cells in synovial tissue. Analysis of mRNA expression using Northern hybridization and the exquisitely sensitive reverse transcription-polymerase chain reaction (RT-PCR) technique has detected mRNA encoding a variety of cytokines, but does not provide any information about the cell type which is expressing the cytokine, and about the localization of expression within the synovium. Cell type and localization of cytokine expression have been investigated using in situ hybridization with tissue sections or dispersed and purified cells of a defined phenotype. mRNA expression of several cytokines has been detected using in situ hybridization, although this technique has limited sensitivity. Cytokine expression can be regulated at the translational level (Haskill d al., 1988),and thus expression of mRNA does not necessarily mean that the cytokine is made and secreted. It is therefore important to confirm that cytokine protein is being produced during synovitis, and this has been accomplished for several cytokines using immunohistochemistry. This technique, however, has limitations in both sensitivity and specificity. Only cytokines which are expressed at relatively high levels will be detected. It is difficult to control for specificity of staining because individual monoclonal antibodies can exhibit differential reactivity independent of antigen specificity. In addition, synovial tissues contain macrophages which express high levels of antibody-binding Fc receptors, and may contain rheumatoid factors, which also bind antibodies. Thus, wherever possible, these studies


343

should be carried out with F(ab)z fragments and with multiple specific and control antibodies. Culture systems have been established to attempt to deal with some of the problems associated with detecting cytokine expression in tissues. In one system, explanted synovial membranes or partially purified fibroblasts or monocytes are placed directly into culture, and cytokine production is measured (Feldmann et al., 1991). Spontaneous production of cytokines can be interpreted as reflecting production in vivo. However, this interpretation needs to be made carefully, since the cultured cells have been removed from the soluble factors in their environment and may begin to express cytokines, such as IFNy, whose expression was repressed in vivo (Feldmann et al., 1991). The great strength of this culture system is that it allows manipulation of levels of endogenous cytokines or addition of exogenous cytokines, thus enabling analysis of the regulation of cytokine production by synovial cells. A different culture system takes the inverse approach by exposing freshly isolated cells from disease-free volunteers to synovial fluids, analyzing cellular activation, and identifylng the triggering cytokine (Sengupta et al., 1995). This approach analyzes responses of relatively homogeneous resting cells to the inflammatory synovial microenvironment, and allows analysis of mechanisms of cell activation. A combination of the experimental approaches described above has been applied to the study of cytokines in RA synovium. Some variability exists in cytokine expression among different patients, suggesting that redundant activities of different cytokines may contribute to the regulation of the activated synovial cell phenotype. Despite variability in expression, some cytokines have been detected in the majority of RA synovia, and are discussed in detail below. In addition to expression in a bioactive form, several additional issues are taken into consideration in assessing which cytokines play an important role in the pathogenesis of synovitis: (i) correspondence between the known biological effects of a cytokine on appropriate target cells and the phenotype of activated synovial cells; (ii)evidence that a cytokine is important in an appropriate animal model of arthritis; and, most importantly, (iii) improvement in synovitis after neutralization or block of cytokine activity.

N. Proinflammatory Cytokines and Growth Factors Multiple inflammatory cytokines have been detected in inflamed synovium and synovial tissue (see above and Table I), and the list is likely to grow as new cytokines are discovered. This discussion will focus upon the cytokines which are present in greatest abundance during synovitis and have been consistently detected in numerous specimens by independent

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groups of investigators, and whose role in synovitis is supported by the majority of available evidence. A. INTERLEUKIN 1 IL-1, which exists in similar a and /3 forms, is a potent proinflammatory cytokine that is derived predominantly from macrophages, and is also produced by fibroblasts, endothelial cells, and lymphocytes. IL-1 has been detected variably in RA synovial fluids, is synthesized by explanted and cultured synovial tissue, and has been detected in dispersed synovial macrophages and in synovial tissue using immunohistochemistry and in situ hybridization. IL-1 is predominantly expressed in perivascular areas in the sublining region, but is also expressed at lower levels in the lining layer, and is expressed at the pannus/cartilage junction (Fontana et al., 1982; Noun et al., 1984; Miyasaka et a!., 1988; Firestein et al., 1990; MacNaul et al., 1990; Westacott et al., 1990; Wood et al., 1992a; Brennan et al., 1991). In tissue culture systems, IL-1 expression can be induced by TNFa and is inhibited by IL-10, suggesting that these cytokines may contribute to the regulation of IL-1 expression during synovitis (Feldmann et al., 1991; Katsikis et al., 1994). IL-1 is a pleiotropic cytokine with multiple effects which may play a role in the pathogenesis of RA. IL-1 stimulates prostaglandin E2 and metalloprotease production by synovial fibroblasts, stimulates fibroblast proliferation through the induction of PDGF expression, activates endothelial cells, and can provide costimulation for T cells. IL-1 stimulates production of additional cytokines, including IL-6, IL-8, and GM-CSF, and stimulates its own synthesis in an autocrine fashion (Dayer et al., 1986; Raines et al., 1989; Dalton et al., 1989; Alvaro-Gracia et al., 1991). IL-1 also can contribute to many systemic symptoms seen in RA, since it can induce fever and promote increased synthesis of acute phase proteins. Injection of IL-1 into joints results in a transient synovitis (Feige et al., 1989) and IL-1 has been implicated in the pathogenesis of arthritis in mice that express a human TNFa transgene in joint tissue (Probert et al., 1995). However, inflamed synovial tissue contains high levels of an IL-1 receptor antagonist (IL-lra) and blockade of IL-1 function has had only modest therapeutic effects in animal models of arthritis. Thus, the extent of the contribution of IL-1 to ongoing, chronic synovitis has not yet been clarified.

B. TUMOR NECROSISFACTOR a TNFa is a macrophage-derived cytokine which is expressed in the lining layer, sublining region, and pannus in inflammatory arthritis. TNFa is produced spontaneously by explanted synovial cells, and its expression is negatively regulated by IL-10 (Di Giovini et al., 1988; S a n e et al., 1988;


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Firestein et al., 1990; MacNaul et al., 1990; Westacott et al., 1990; Chu et al., 1991b; Katsikis et al., 1994). The identity of the stimulus driving TNFa expression in the synovium is not known, although IL-1, IL-6, immune complexes, soluble CD23, and extracellular matrix components that interact with cell surface integrins are reasonable candidates (Armant et al., 1994; Debets et al., 1990; Pope et al., 1994). TNFa stimulates the expression of IL-1 in synovial cells, and has many actions similar to those of IL-1, such as stimulation of PGEz and metalloprotease production, fibroblast proliferation, endothelial cell activation, and production of the cytokines IL-6, IL-8, LIF, TGFP, and MCP-1 (Dayer et al., 1984; AlvaroGracia et al., 1990; Feldmann et al., 1991; Koch et al., 1991; Hamilton et a!., 1993; Villiger et al., 1993a) . Indeed, TNFa and IL-1 reciprocally stimulate production of the other cytokine, and often act synergistically. For example, intraarticular injection of both IL-1 and TNFa results in increased synovitis (Henderson and Pettipher, 1989). Inflamed synovial tissue and serum of active RA patients also contain soluble TNFa receptors, which may function as inhibitors by preventing binding of TNFa to cell surface receptors (Di Gionini et al., 1988; Seckinger et al., 1990; RouxLombard et al., 1993). Recent studies have provided support for an important role for TNFa in animal models of arthritis and in RA. In addition to triggering transient synovitis after intraarticular injection, TNFa accelerates collagen-induced arthritis (Cooper et al., 1992), and causes chronic arthritis in transgenic mice which constitutively express a TNFa transgene in synovial tissue (Keffer et al., 1991).Anti-TNFa antibodies ameliorate the disease process in collagen-induced arthritis, especially when combined with CD4+ T cell depletion or IL-1 receptor blockade (Williamset al., 1994). Exciting studies have characterized the effects of neutralizing anti-TNFa antibodies in the treatment of RA patients who had failed at least one disease-modifymg drug (Elliot et al., 1994a,b).TNFa antibodies significantly diminished joint inflammation in RA patients 4 weeks after treatment (58% of patients had a Paulus 50% response). This response was associated with decreased levels of acute phase proteins and IL-6. However, long term therapy with TNFa antibodies was complicated by diminished efficacy, development of antibodies against the therapeutic monoclonal antibody, development of lupusassociated antibodies, and a high rate of patient dropout. A study using a different anti-TNFa antibody, which has comparable neutralizing ability but a different isotype, demonstrated a more modestly beneficial effect (Rankin et al., 1994). The anti-TNFa studies raise the exciting possibility that therapies directed at cytokines will be important and efficacious in the treatment of RA, and larger scale trials are in progress. However, these studies did not

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demonstrate neutralizing levels of anti-TNFa antibody in the synovium. Since TNFa is expressed as a cell surface molecule prior to proteolytic cleavage and release from the cell (Aversa et al., 1993), the possibility exists that anti-TNF antibodies may work by binding to cell surface TNFa, possibly on cells in the immune system outside the synovium, and targeting these cells for destruction. Thus, a differential effect of neutralizing antibodies of different isotypes may be explained by differential triggering of cytotoxic pathways, as well as by different rates of clearance of immune complexes containing the neutralizing antibody, Future clinical trials with anti-TNFa antibodies should be very helpful in unraveling not only the mechanism of action of these antibodies, but also the role of TNFa in the regulation of cytokine production and in the pathogenesis of RA. C. INTERLEUKIN 6 IL-6 is expressed at high levels in RA synovial fluid, and protein and mRNA expression have been detected in the lining layer, in perivascular cellular aggregates in the sublining region, and in pannus (Houssiau et al., 1988; Bhardwaj et al., 1989; Guerne et al., 1989; Firestein et al., 1990; Field et al., 1991; Wood et al., 1992b; De Benedetti et al., 1994). The source of IL-6 in the lining layer may be either macrophages or synovial fibroblasts, while the predominant source of IL-6 in the sublining region appears to be macrophages (up to 70% of perivascular macrophages express IL-6), and, in one study, up to 10-20% of sublining T cells and antibodyproducing cells expressed IL-6 (Field et al., 1991). IL-6 is constitutively produced by cultured, explanted synovial cells (Feldmann et al., 1991). IL-6 expression is regulated by adherence to extracellular matrix and by several of the soluble factors present during synovitis, including immune complexes, IL-1, and TNFa (Guerne et al., 1989; Haskill et al., 1988; Krutmann et al., 1990; Rosenbaum et al., 1992). However, the identity of the important activating stimulus in vivo during synovitis remains obscure. The IL-6 receptor consists of a gp130 chain ( p subunit) which is important in signal transduction, and an a subunit which facilitates ligand binding (Kishimoto et al., 1994).High levels of soluble a chain of the IL-6 receptor have been detected in synovial fluids. In contrast to soluble cytokine receptors which sequester ligand and function as inhibitors, soluble 1L-6 receptor a chain facilitates the binding of IL-6 to cell surface gp130, and thus functions as a positive inducer of IL-6 signaling. The functions of IL-6 that may be important in synovitis include stimulation of antibody production, coactivation of T cells, and stimulation of production of acute phase proteins (van Snick, 1990). IL-6 can induce expression of immediate early transcription factors of the AP-1 family, TNFa, and Fc receptors. Evidence from experiments with animal models


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of arthritis and other autoimmune diseases support a role for IL-6 in autoantibody production, in the acute phase response, and in inflammatory arthritis (Brozik et al., 1992; van Snick, 1990). Treatment of patients with anti-IL-6 monoclonal antibody in addition to anti-CD4 therapy resulted in additional, but transient, improvement (Wendlinget al., 1993). However, the high levels of synovial IL-6 may be difficult to neutralize, and the efficacy of anti-IL-6 therapy needs to be further evaluated. D. GRANULOCYTE MACROPHAGE-COLONY STIMULATING FACTOR GM-CSF is present in synovial fluids and is expressed by synovial macrophages, although only 2% of macrophages express sufficiently high levels of GM-CSF mRNA to allow detection by in situ hybridization (AlvaroGracia et al., 1989, 1991; Firestein et al., 1990; Campbell et al., 1991). Isolated synovial macrophages constitutively produce GM-CSF in culture, and synovial fibrobasts can be induced to produce GM-CSF by treatment with IL-1 or TNFa (Leizer et al., 1990). Although GM-CSF was originally characterized on the basis of promotion of growth and differentiation of granulocytes and macrophages, it also has several proinflammatory properties which may be important in synovitis. GM-CSF can increase cell surface expression of HLA DR, and experiments with neutralizing antibodies suggest that GM-CSF may play an important role in regulating HLA DR expression in synovitis (Alvaro-Gracia et al., 1989, 1991). GM-CSF maintains macrophage viability (Haskillet al., 1988),may contribute to increased numbers of macrophages in the synovium, and can contribute to the induction of IL-1 and TNFa expression, with resulting increases in macrophage cytotoxicity. GM-CSF also contributes to proliferative activity of media which has been conditioned by synovial fibroblasts (Bucala et al., 1991).

E. PLATELET-DERIVED GROWTH FACTOR AND BASICFIBROBLAST GROWTH FACTOR PDGF and bFGF have been detected in inflammatory synovial fluids and are produced spontaneously by cultured synovial cells and fibroblasts (Remmers et al., 1991; Bucalaet al., 1991).The PDGF B chain is expressed by synovial macrophages, while bFGF is produced by synovial fibroblasts. PDGF is the most potent mitogen for synovial fibroblasts detected to date and can promote anchorage-independent growth of early passage synoviocytes (Kumkumian et al., 1989; Lafyatis et al., 1989). PDGF expression is induced by IL-1, and PDGF may be responsible for the proliferation and for many aspects of the transformed-like phenotype of synovial fibroblasts during synovitis. bFGF can function both as a synovial fibroblast mitogen and as an angiogenesis factor.

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F. OTHERPROINFLAMMATORY CWOKINES LIF and M-CSF have been detected in inflammatory synovial fluids and are produced by cultured synovial cells, and LIF is produced by cultured articular chondrocytes. LIF expression is induced by IL-1, TGFP, PDGF, bFGF, and insulin-like growth factor. M-CSF can stimulate TNFa production, and LIF can induce expression of IL-1, IL-6, and IL-8. LIF can also induce production of its own mRNA (Leizer et al., 1990, Campbell d nl., 1991; Lotz et al., 1992a, Hamilton et al., 1993; Waring et d., 1993). The expression of LIF by chondrocytes suggests a mechanism by which chondrocytes may contribute to synovitis through the secretion of soluble factors. Chondrocytes are capable of producing several proinflammatory mediators (Campbell et al., 1991; Villiger et al., 1992, 1993b), and the role of chondrocyte-derived cytokines in driving synovial inflammation deserves further investigation. Increased expression of soluble CD23 (sCD23) has been detected in RA (Armant et al., 1994). Production of sCD23 is increased by GM-CSF, and sCD23 induces expression of IL-1, IL-6, and TNFa by monocytes/macrophages. sCD23 may play an important role in regulation of TNFa production during synovitis. V. Anti-inflammatory Cytokines and Cytokine Inhibitors: A Double-Edged Sword?

The soluble factors expressed during synovitis also include Factors, such as IL-lra, IL-10, and TGFP that have anti-inflammatory properties and which may be produced as part of an attempt to resolve inflammation. The overall effect of these cytokines is difficult to assess because they may have opposing pro- and anti-inflammatory properties, depending upon the responding cell type, state of cell activation, or interaction with additional cytokines. A. INTEHLEUKIN-1 RECEPTORANTAGONIST IL-lra is a competitive inhibitor of IL-1 at both type I and type I1 receptors, and has no stimulatory activity. Although IL-lra binds to receptors with a similar affinity to IL-1, a large excess of IL-lra is required to block IL-1 activity, likely because IL-1 can elicit a biologic response after binding to only a small fraction of total cellular IL-1 receptors. High levels of IL-lra (up to 50 ng/ml) have been detected in inflammatory synovial fluids, and IL-lra is produced spontaneously by cultured synovial macrophages (Malyak et al., 1993; Firestein et al., 1994; Seitz et al., 1994; Chomarat et al., 1995). IL-lra is expressed in macrophages in perivascular areas of the sublining region, and, to a lesser degree, in lining cells (Firestein et d.,1992). Some IL-lra may also be produced by neutrophils which have entered the synovial fluid (Malyak et nl., 1993). IL-lra production is


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increased by IL-4, IL-10, GM-CSF, and immune complexes (Arend, 1991; Krzesicki et al., 1993; Seitz et al., 1994; Chomarat et al., 1995; Arend and Dayer, 1995) but it is not known which of these stimuli are operative in vivo during synovitis. Trials of IL-lra in animal models of arthritis have demonstrated modest responses.

B. TRANSFORMING GROWTH FACTOR /3 High levels of latent and active TGFPl and TGFP2 have been detected in synovial fluids, and explanted synovial cells produce TGFP spontaneously in culture (Fava et al., 1989; Brennan et al., 1990; Miossec et al., 1990; Wahl et al., 1992). TGFP expression by synovial fibroblasts has been detected in the lining layer, the pannus-cartilage junction, and the sublining region. In the sublining region, TGFP is expressed in areas with dense extracellular collagen (Lafyatis et al., 1989b), but expression by occasional cells in lymphoid aggregates has also been detected (Chu et al., 1991). TGFP expression is known to be induced by inflammatory cytokines such as IL-1 and IL-6 (Villiger et al., 1993b), but the regulation of TGFP expression in the inflamed synovium is not currently understood. Despite its name, TGFP exhibits numerous negative effects upon cellular proliferation and immune function. TGFP inhibits proliferation of T cells, B cells, and synovial fibroblasts, and inhibits antibody production (Lotz et al. 1986, 1990). TGFP can skew T cell cytokine production toward a Th2 phenotype (see below) and is a potent deactivator of macrophage functions, including cytotoxicity,phagocytosis, and production of the cytokines TNFa and IL-1 (Wahl et al., 1990;Wahl, 1994).TGFP also stimulates production of extracellular matrix components. A negative role for TGFP in the regulation of inflammatory responses is supported by its ability to suppress inflammatory processes, including collagen-induced arthritis, when administered systemically (Kuruvilla et al., 1991; Racke et al., 1991). TGFP also serves as a chemoattractant for neutrophils and monocytes (Wahl et al., 1987), can promote angiogenesis (Klagsbrun and D’Amore, 1991), and induces expression of inflammatory molecules such as Fc y receptor I11 (Wahl et al., 1992). Indeed, intraarticular injection of TGFP results in recruitment of neutrophils and synovial inflammation and hyperplasia (Fava et al., 1991). Blockade of TGFP activity with neutralizing antibodies suppresses both the acute and the subsequent chronic phase of synovitis in streptococcal cell wall-induced arthritis in the rat (Wahl et al., 1993). Thus, it is difficult to determine whether the role of TGFP in the pathogenesis of RA is primarily proinflammatory or anti-inflammatory, Indeed, TGFP has been proposed to function as a conversion factor that activates resting cells and inhibits activated cells (Wahl, 1994). The role of TGFP may change during the time course of synovitis, with proinflam-

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matory activity in early disease, but predominantly anti-inflammatory activity once synovitis is established. C. INTEHLEUKIN 10 IL-10 has been detected in inflammatory synovial fluids and is expressed by explanted synovial cells in culture (Katsikis et al., 1994; Llorente et nl., 1994).IL-10 expression at the protein level has been detected in monocytes in the synovial lining layer and in T cells in mononuclear cell aggregates in the sublining region. The regulation of IL-10 production by monocytes is not well understood; IL-10 is produced by activated T cells of the Tho, Thl, and Th2 functional phenotypes (see below). IL-10 is a potent inhibitor of Inonocytes that inhibits expression of HLA antigens, production of IL-1, IL-6, IL-8, GM-CSF, and TNFa, and production of nitric oxide, and inhibits cytotoxicity (Howard and O’Garra, 1992).This inhibition of monocyte function results in diminished accessory cell function and subsequent decreased T cell proliferation and cytokine production, especially proliferation of T cells of the T h l phenotype. Neutralization of IL-10 in synovial cell cultures results in increases in IL-1 and TNFa production, and can result in production of detectable levels of IFNy protein (Katsikis et al., 1994). Addition of exogenous IL-10 to synovial cultures results in suppression of IL-1, TNFm, and IL-8 production. IL-10 is responsible, in part, for the synovial fluid-mediated suppression of IFNy production by T cells (Wang et al., 1995). These results suggest that IL-10 functions as a negative regulator of synovitis through the inhibition of cytokine production. Consonant with this interpretation, IL-10 ameliorates inflammation in collagen-induced arthritis, especially when used together with IL-4 (van den Berg et al., 1994). Neutralization of IL-10 activity during the induction of collagen-induced arthritis results in acceleration of onset and increased severity of arthritis (Kasama et al., 1995). IL-10 exerts several immunostimulatory effects upon B cells (Howard and O’Garra, 1992). IL-10 increases B cell expression of HLA class I1 molecules, and augments B cell proliferation and differentiation into antibody-secreting cells. IL-10 may contribute to increased autoantibody production in autoimmune diseases such as systemic lupus (Llorente et al., 1994). IL-10 also has stimulatory effects upon proliferation of thymocytes and T cells which have been treated with IL-4 and are differentiating toward a Th2 phenotype. IL-10 has recently been shown to increase collagenase production by fibroblasts (Reitamo et al., 1994). Thus, similar to TGFP, it is difficult to predict the overall effect of IL-10 upon synovitis. IL-10 likely plays an important role in down-regulating monocyte function and production of the cytokines IL-1, TNFcr, and IFNy. However, in-


35 1

creased IL-10 levels may also lead to stimulation of certain immune and inflammatory functions, especially increased humoral immunity and antibody production, and may contribute to increasing expression of metalloproteases.

D. OTHERANTI-INFLAMMATORY CYTOKINES IL-4 can inhibit monocyte functions (Oswald et al., 1992; Hart et al., 1993) and, together with IL-10, suppresses collagen-induced arthritis (van den Berg et al., 1994). IL-4 mRNA has been variably detected in inflamed synovium, even when RT-PCR has been used (Waalen et al., 1992; Chen et d.,1993; Miyake et d.,1993; Simon et al., 1994), and IL-4 protein levels in synovial fluids are very low or undetectable using an ELISA that can detect 15pg/ml of IL-4 (Miossec et al., 1990).It is possible that low levels or localized expression of IL-4 may contribute to the immune system’s attempt to inhibit synovitis.Since IL-4 stimulates antibody production, it may also contribute to pathogenesis by driving humoral immune responses within the inflamed joint. The chemokines RANTES, MIP-la, and MIP-1P have recently been described to have potent inhibitory activity toward replication of the HIV virus in T cells (Cocchi et al., 1995). Since these cytokines are expressed in inflammatory synovium, it is possible that they may play a role in inhibiting inflammation, in addition to their well established role as chemotaxins and cell activators (see below). Interactions between the immune and neuroendocrine systems may result in either suppression or stimulation of inflammatory responses; these interactions have been reviewed recently (Wilder, 1995). VI. Chemotactic and Angiogenic Factors

Chemotactic and angiogenic factors play an important role in recruiting circulating cells and developing a new vascular supply for inflammatory sites, such as hyperplastic RA synovium. Chemokines are divided into two distinct groups, termed C-C and C-X-C, on the basis of the relative spacing of the first two cysteine residues (Oppenheim et al., 1991). C-C chemokines which have been detected in synovial fluid and are expressed during synovitis include MCP-1, MIP-la, and RANTES (Villiger et al., 1991, 1992; Koch et al., 1992a, 1994b; Akahoshi et al., 1993; Rathaswani et al., 1993). These C-C chemokines are produced by synovial macrophages and fibroblasts, and expression can be augmented by IL-1 and TNFa. These molecules are chemotactic for monocytes, and MIP-la and RANTES are also chemotactic for T cells, especially memory T cells (Schall et al., 1990). MIP-la has been shown to account for 36% of chemotactic activity for monocytes in RA synovial fluids (Koch et al., 1994b). Neutralization of

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MIP-la results in a delay in onset and reduction in severity of collageninduced arthritis (Kasama et al., 1995). C-X-C chemokines expressed during synovitis include IL-8, gro, and ENA-78, which are expressed by macrophages and fibroblasts (Golds et al., 1989; Brennan et al., 1990b; Peichl et al., 1991; Koch et al., 1991). IL8 is also expressed by chondrocytes (Lotz et al., 1992). Expression of these factors can be augmented by IL-1 and TNFa. The C-X-C molecules are chemotactic for neutrophils, and IL-8 is also chemotactic for lymphocytes. IL-8 and ENA-78 have been shown to account for, respectively, 40 and 42% of chemotactic activity for neutrophils in RA synovial fluids (Koch et al., 1991, 1994~). TGFP is another synovial cytokine which is chemotactic for monocytes and neutrophils (Wahl et al., 1987), although it is not a member of the chemokine family. Chemotactic factors can play a role in angiogenesis indirectly, through recruitment of cells which produce angiogenic factors, or may promote angiogenesis directly through chemotactic and mitogenic activity for endothelial cells. IL-8 is a potent mediator of angiogenesis (Koch et al., 1992b) and represents a major fraction of angiogenic activity found in synovial macrophage culture supernatants. Additional angiogenic factors present in synovitis include TGFP, PD-ECGF, and VEGF (Fava et al., 1994; Koch et al., 1994a). VEGF, which is expressed by synovial macrophages, has both mitogenic and chemotactic properties for endothelial cells, and makes a major contribution to chemoattractant activity for endothelial cells in explanted synovial cultures. The adhesion molecules E-selectin and VCAM-1 are expressed on activated vascular endothelium in the synovium, sublining macrophages, and on lining cells. Soluble E-selectin and VCAM-1 are detected in inflammatory synovial fluids, and have been recently been demonstrated to posses angiogenic activity (Koch et al., 1995). Thus, the processes of chemoattraction, adhesion, and angiogenesis appear to be linked. VII. T Cell Cytokines

Many immune responses are characterized by skewing of cytokine production into one of two major patterns, termed T helper 1 (Thl) and Th2 (reviewed in Paul and Seder, 1994; Seder and Paul, 1994). The major T h l cytokine, IFNy, enhances cellular immunity, while the Th2 cytokines, IL-4, IL-5, IL-6, IL-10, and IL-13, drive humoral immune responses. Lymphocytes are considered to be the primary regulators of the balance between Thl and Th2 responses. However, the development of lymphocytes into cells which preferentially produce T h l or Th2 cytokines is determined by factors, often derived from accessory cells, which are present


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during lymphocyte activation. Thl responses are stimulated by the regulatory cytokines IL-12 and IFNy, and Th2 cytokine production is stimulated by IL-4, IL-10, E series prostaglandins (PGEs, which elevate intracellular CAMP levels), and, under certain conditions, TGFP. Interestingly, there is crossregulation, in that agents that promote T h l cytokine production inhibit Th2 cytokine production, and vice versa. The emergence of the appropriate pattern of cytokines is important for the clearance of many pathogens. Unbalanced expression of Thl or Th2 cytokines has been associated with inflammation in different animal disease models, and may play a role in human inflammatory diseases (Romagnani, 1994). Gene targeting experiments have revealed that mice with homozygous deletions in IL-2, IL-10, or a$ T cell receptor (TcR) genes develop inflammatory bowel disease or an autoimmune syndrome associated with autoantibody production (Mombaerts et al., 1993; Kuhn et al., 1993; Sadlack et al., 1993; Wen et al., 1994). The immune systems of these mice appear unable to normally regulate inflammatory responses. In the IL-2 (Sadlack et al., 1993) and one of the Cup TcR gene knockout models (Wen et al., 1994), inflammation and autoantibody production occurred in the setting of a predisposition to develop Th2 immune responses and elevated Th2 cytokine production. In contrast, experimental allergic encephalitis is associated with a predominant Thl pattern of cytokine expression at the sites of inflammation (Chen et al., 1994; Racke et al., 1994; Kuchroo et al., 1995), and can be attenuated by Th2 cytokines. An imbalance or skewing of the production of cytokines may contribute to the pathogenesis of synovitis.The attenuation of collagen-induced arthritis by the Th2 cytokines IL-4 and IL-10 (van den Berg et al., 1994) and the increased severity of arthritis after neutralization of IL-10 (Kasama et al., 1995) suggest that T h l cytokines may contribute to pathogenesis. However, this conclusion may be somewhat oversimplified, since humoral immunity contributes to pathogenesis of collagen-induced arthritis (Taylor et al., 1995), and treatment with the T h l cytokine IFNy may either ameliorate or exacerbate arthritis, depending upon when in the course of disease IFNy is administered (Boissieret al., 1995). In contrast to collagen-induced arthritis, arthritis associated with caprine arthritis-encephalitis virus (CAEV), which is similar to the synovitis of RA, appears to be mediated by a Th2 response and onset of synovitis correlates with suppression of IFNy expression (Cheevers et al., 1994). Thus, it is difficult to make predictions about possible contributions of T hl and Th2 cytokines to the pathogenesis of RA based upon animal models of arthritis. This is especially true since RA synovitis exhibits features of both cellular and humoral

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immunity, as defined by the presence of activated monocytes and antibodyproducing cells. T cell cytokines are often expressed at low levels, and expression is transient in the absence of continuing stimulation. It has proven difficult to detect expression of T cell cytokines, such as IL-2, IFNy, and IL-4, at the protein level, in synovial fluid and synovial tissue using very sensitive imrnunoassays and bioassays (Husby and Williams, 1985; Firestein and Zvaifler, 1987; Miossec et al., 1990; Feldmann et al., 1991; Cope and Brennan, 1992; Ulfgren et al., 1995; Sengupta et al., 1995). When these cytokines are detected, it is only in a small fraction of samples and they are present at very low levels. A low level of expression does not rule out a potentially important regulatory role. However, low levels of expression of IFNy make it unlikely that IFNy is the major monocyte activator and major inducer of monocyte gene expression throughout the synovium. Recent work has shown that different cytokines, such as IL-6 acting in concert with a still-unknown additional factor(s), can substitute for IFNy in the activation of genes that are typically considered IFNy-inducible (Sengupta et al., 1995). Synovial T cell expression of IL-10 in mononuclear cell aggregates in the sublining region has been detected using immunohistochemistry (Katsikis et al., 1994, Cohen et al., 1995). Since monocytes also express IL-10, the quantitative contribution of T cells to synovial IL-10 production is not clear. However, it is likely that the T cell-derived IL-10 can act locally, in the mononuclear cell aggregates in the sublining region. In contrast to analysis of cytokine protein levels, T cell cytokine mRNA has been detected using Northern hybridization, in situ hybridization, and the more sensitive RT-PCR. mRNA encoding IFNy has been consistently detected, and transcripts encoding IL-2 and IL-4 can be detected in some clinical samples, especially when RT-PCR is used (Feldmann et al., 1991; Waalen et al., 1992; Chen et al., 1993; Miyake et al., 1993; Simon et al., 1994; L. Ivashkiv, unpublished data). In one study, 1 in 300 CD3+T cells in RA synovium expressed IFNy mRNA, as assessed by in situ hybridization (Simon et al., 1994). The IFNy expressing cells were present in small clusters. However, it is clear that in this study in situ hybridization did not detect cytokine mRNA in several samples in which cytokine mRNA was detected using RT-PCR. Thus, the 1 in 300 frequency of positivity may reflect only the most highly expressing cells. Interestingly, in the same study the percentage of synovial samples expressing IL-4 mRNA was significantly higher in patients with seronegative arthritis than in patients with RA. This finding is provocative, but its significance is not clear, given the low levels of IL-4 expression and the low frequency of IL-4 mRNA


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positive cells (in the range of 1 in lo00 CD3+ cells), as determined by in situ hybridization. The discordance between expression of IFNy mRNA and protein suggests that IFNy production may be actively inhibited in the synovium (Fig. 1). Agents which are known to inhibit IFNy expression, such as IL-10, TGFP, and PGEs, are present at high levels in RA synovium. Removal of synovial cells from the inflammatory environment and culture in vitro results in increasing levels of IFNy mRNA expression (Feldmann et al., 1991), and neutralization of IL-10 during culture results in production of IFNy protein (Katsikiset al., 1994).Conversely, RA synovial fluids suppress IFNy production by T cells, and suppression is dependent, in part, on IL10 (Wang et al., 1995). Another approach to analyzing the T cell contribution to the synovial cytokine milieu has been to establish T cell clones derived from synovial T cells (Quayle et al., 1993; Cohen et al., 1995). T cell clones typically maintain a relatively stable pattern of cytokine production over time, and segregate into Thl (produce high levels of IFNy), Th2 (IL-4), and Tho (both IFNy and IL-4) phenotypes. In contrast to murine T cell clones, human T cell clones of all three phenotypes can also produce IL-10 (Katsikis et al., 1995), which stimulates humoral immunity, inhibits monocyte function, and has been considered a Th2 cytokine (Howard and O’Garra, 1992). The ratio of production of IL-10 to IFNy may determine whether IFNy-producing Thl cells are, on balance, proinflammatory or antiinflammatory (Katsikis et al., 1995). T cells producing IFNy predominate among T cell clones which are derived from synovial T cells. A high percentage (50-100%)of the RA-derived T h l and Tho clones also produce high levels of IL-10, and may thus correspond to cells which have a predominantly anti-inflammatoryphenotype, despite a capacity for producing IFNy. The low numbers of Th2 clones derived from synovial T cells may reflect a relative paucity of Th2 cells in the synovium, but may also

IL-I0

TGFb PGE FIG. 1. Suppression of synovial T cells by soluble inhibitors. The inflamed synovium contains high levels of several factors, such as IL-10, TGFP, and PGEs, which suppress Thl T cells and can promote Th2 responses. Thl = Thl T cell.

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be explained by bias introduced by the cloning procedure, or difficulties with maintaining these cells in culture. At this point, it is not possible to draw any firm conclusions about the pattern of T cell cytokine production in RA synovium, and about the possible contributions of T cell cytokines to the pathogenesis of synovitis. Although T cell cytokines are expressed at low levels, and likely by a small fraction of synovial T cells, this is similar to the pattern of expression in other infectioushnflammatory processes that are thought to be T celldependent (Cooper et al., 1989). T cell cytokines may function on nearby cells in a paracrine fashion, and affect more distant cells by regulating production of additional factors or by stimulating cells which then migrate through the synovium. It is tempting to try to fit the pattern of synovial cytokine expression into the ThlRh2 paradigm, with Thl T cells being suppressed by soluble Th2 cytokines in the extracellular environment (Fig. 1) (Lotz et al., 1986, 1990; Feldmann et al., 1991; Katsikis et al., 1994; Allen et al., 1995; Wang et al., 1995). It is difficult to predict whether increases in disease activity would correspond to shifts in cytokine balance toward the T h l or Th2 direction. Th2 cytokines may contribute to pathogenesis by driving local antibody (rheumatoid factor) production, or by suppressing a T h l response, with resulting defective clearance of (auto)antigens, and persistence of an immune stimulus. However, this scenario is likely an oversimplification, and much additional research is required to elucidate patterns of T cell cytokine expression in different regions of synovial tissue, particularly in the lymphoid aggregates, patterns of expression in early disease, changes in expression over time, possible production of novel soluble factors or cytokines (Miltenberg et al., 1995), and whether synovial T cells produce fibroblast growth factors such as EGF and bFGF (Blotnick et al., 1994). Recent progress in the development of in situ RT-PCR, which is performed using tissue sections, offers the promise of combining the sensitivity of PCR without sacrificing knowledge about patterns and localization of cytokine expression. VIII. Mechanisms of Synovial Cell Activation by Cytokines

It is difficult to determine which of the many cytokines expressed during synovitis play an important causative role in pathogenesis because cytokines may have synergistic or antagonistic effects and it is impossible to predict which cytokines will predominate. Indeed, the action of certain cytokines such as TNFa can be altered, and even completely reversed by the activity of additional cytokines (Zimmer and Jones, 1990; Lake et al., 1994). Furthermore, the presence of inhibitors such as soluble receptors, receptor


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antagonists, and antibodies against cytokines make it difficult to determine which cytokines are active in causing disease. One approach to determining which cytokines are important is to examine inflammatorycells for evidence of the action of a particular cytokine, or family of related cytokines. This approach requires an ability to detect physiologicallyimportant and specific cellular responses to cytokines. Cytokines exert their effects by activating cellular signal transduction pathways, with production of second messengers and triggering of cascades of activation of kinases, transcription factors, and genes (Kishimoto et al., 1994; Taniguchi, 1995). The ability to analyze mechanisms of signal transduction activated by cytokines during synovitis has been limited because many signaling events are transient, and many signals triggered by cytokines are not specific, in that they can also be activated by cellular interactions with extracellular matrix, small inflammatory mediators, surface stimulatory molecules, and other extracellular ligands. It has become clear that one important component of signal transduction by cytokines involves activation of members of several families of transcription factors, which transmit signals to the nucleus and initiate a cascade of gene activation. The AP-1, reVNF-KB, and STAT transcription factors play an important role in mediating gene activation by cytokines and inflammatory mediators, and are also important in regulating expression of the cytokines themselves (Darnell et al., 1994; Ihle et al., 1994; Ivashkiv, 1995;Thanos and Maniatis, 1995; Hill and Treisman, 1995).For example, AP-1 factors are important in regulating the promoters of genes which encode TNFa, IL-6, and IL-8, and are absolutely required for expression of the collagenase gene; reYNF-KB factors are important for regulation of TNFa and IL-6, and abnormalities in NF-KB expression can lead to widespread inflammatory processes. Interestingly, both AP-1 and NF-KB proteins are inhibited by glucocorticoids, potent anti-inflammatory agents (Scheinman et al., 1995; Auphan et al., 1995). Analysis of transcription factor activity as a measure of cellular responses to extracellular stimuli offers the advantages that transcription factors activity can be sustained and relatively straightfonvard to measure, and that additional information can be gained about regulation of target genes. Increased expression of the AP-1 protein Fos has been detected in inflammatory arthritis, thus implicating AP-1 factors in the activation of collagenase gene expression during synovitis (Trabant et al., 1992; Handel et al., 1995).Both p50 and p65 subunits of NF-KB are located in the nucleus of synovial cells (Handel et al., 1995) and synovial fluids activate NF-KB in resting monocytes (L. Ivashkiv, unpublished data), suggesting that activated NF-KB contributes to the pathogenesis of synovitis. AP-1 and NF-KB proteins are activated by IL-1 and TNFa, but are also activated by a wide

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variety of noncytokine stimuli, including ligation of T and B cell antigen receptors, phorbol esters, and other agents that activate protein kinase C, double stranded RNA, LPS, ultraviolet light, and integrin-mediated interactions with extracellular matrix (Thanos and Maniatis, 1995; Hill and Treisman, 1995). Thus, it is difficult to ascribe activation of these factors specifically to cytokines present in the synovium. An important advance in the understanding of cellular responses to cytokines has been the identification and characterization of the Jak-STAT (signal transducer and activator of transcription) signal transduction pathway (reviewed in Darnel1 et al., 1994; Ihle et al., 1994; Ivashkiv, 1995). Activation of this signahng pathway appears to be relatively specific for cytokines and growth factors. Many cytokines and inflammatory mediators, including IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IFNa, IFNy, LIF, oncostatin M, GM-CSF, G-CSF, PDGF, and EGF activate Jak-STAT pathways. One of the earliest signaling events is the activation of protein tyrosine kinases (PTKs) of the Janus kinase (Jak) family, which are physically associated with the receptor. After activation, receptor associated PTKs phosphorylate several substrates critical for signal transduction, including specific tyrosine residues in the receptors’ cytoplasmic domains. Typically, stimulation by a particular cytokine results in the activation of a distinct pair of two of the four known Jak kinases (Table 11). Jak kinases are required for tyrosine phosphorylation and activation of STATs, TABLE I1 ACTIVATION OF JAK KINASES AND STATs BY CYTOKINES

Cytohne IFNu IFNy IL-2 IL-4 IL-6 IL-10 IL-12

Activated Jaks

Activated STATs

Jakl,Tyk2 Jakl, Jak2 Jakl, Jak3 Jakl,Jak3 Jakl, Jak2, Tyk2 Unknown

Statl, Stat2, Stat3

Jak2, Tyk2

stat1

Stat3,” Stat5

Stat6

Stat1,b Stat3 Statl, Stat3 Stat3, Stat4

Note. The cytokines shown have been selected to emphasize the relative selectivity of STAT activation. The STATs that may be useful in distinguishing between the activity of different cytokines are shown in bold. “IL-2activates Stat3 in preactivated, but not resting, peripheral blood mononuclear cells. bIL-6activates Statl only at high doses and in a celltype-specific manner.


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although it is not yet clear whether Jaks phosphorylate STATs directly. STATs are latent cytoplasmic transcription factors that are rapidly tyrosinephosphorylatedafter stimulation with cytokines,and subsequently dimerize and translocate to the nucleus, where they can activate transcription after binding to a consensus sequence TTCNNNGAA in gene promoters (Darnell et al., 1994; Ihle et al., 1994; Ivashkiv, 1995). Activation of STAT proteins results in complex changes in cell phenotype, and has been implicated in the activation of several genes important in inflammatory responses, including FcyRI, immunoglobulins, interferon regulatory factor1( IRF-l), intercellular adhesion molecule 1( ICAM-l), and acute phase reactants. To date, six distinct but homologous members of the STAT family have been identified and designated Statl through Stat6. Most STATs are widely expressed in a variety of cell types. An individual STAT protein may be activated by multiple ligands, but certain ligands preferentially activate particular STATs (Table I1). For example, IFNy preferentially activates Statl, I L 6 preferentially activates Stat3, and IL-4 preferentially activates Stat6. Cytokines that are produced during synovitisare capable of activating several STATs (Table 111). For example, Stat3 is activated by IL-6, IL-10, IFNa, PDGF, and LIF. Given the relative specificity of activation of STATs by cytokines, detection of specific activated STATs in synovial cells TABLE I11 POTENTIAL ACTIVATION OF STATs BY CVTOKINES PRESENT DURING SYNOVITIS

Cytokine

Activated STATs

IL-6 LIF GM-CSF PDGF IL-10 (IFW (IL-2) (IL-4)

Statl,” Stat3 Statl, Stat3 Stat5 Statl, Stat3 Statl, Stat3 Statl Stat5 Stat6

Note. The cytokines that are known to activate STATs and are present during synovitis are shown. The cytokines enclosed in parentheses have not been detected consistently or only mRNA or low levels of protein have been detected. To date, only active Stat3 (shown in bold),which is likely activated predominantlyby IL-6 (Sengupta d al., 1995;Wang d al., 1995). has been detected in inflammatory synovial cells. “IL-6 activates Statl only at high doses and in a celltype specific manner.

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would suggest that the activated cells have responded to a specific cytokine or group of cytokines. Specific regulation of STAT activity has been described during several inflammatory and immune responses. In delayed type hypersensitivity, a T h l response, Statl is preferentially activated (Jiang et al., 1994). STAT activity during early stages of T cell activation is specifically and differentially regulated by regulatory cytokines and inflammatory mediators, and Jak-STAT signaling can be inhibited by distinct signaling pathways (hashkiv et al., submitted; Sengupta et al., submitted). Development of a Th2 response correlates with loss of T cell activation of Jak-STAT signaling in response to IL-12 (Szabo et al., 1995), and loss of responsiveness to IFNy and lack of activation of Statl have been demonstrated in Thl T cell clones and T cells grown in the presence of IFNy (Pernis et nl., 1994; Bach et al., 1995).Thus, analysis of patterns and regulation of STAT activity during an immune or inflammatory process can yield interesting information about regulation. Constitutively active Stat3 has been detected in freshly isolated synovial cells obtained from patients with inflammatory arthritis ( Wang et al., 1995). Stat3 activity decays over time when cells are removed from the inflammatory environment and placed in tissue culture (L. Ivashkiv, unpublished data). Conversely, inflammatory synovial fluids activate Stat3 in control resting cells, and induce a DNA-binding complex, termed Stat-SF, that contains Stat3 (Sengupta et al., 1995). IL-6 is the major Stat3-activating factor in synovial fluids, and activation of Stat3 is necessary, but not sufficient, for activation of a monocyte effector gene, FcyRI. Synovial fluids do not activate Statl, which is activated by IFNy, and do not activate any other STATs. In activated T cells, synovial fluids suppress Statl, but not Stat3, activity, and the suppression of Statl activity correlates with the suppression of production of the Thl cytokine IFNy (Wang et al., 1995). These findings suggest that during an active phase of synovitis, the balance of cytokine activity favors the activation of Stat3 over Statl, and that Stat2, Stat4, Stat5, and Stat6 are not activated to a comparable level. Thus, cytokines that activate Stat3 participate actively in the inflammatory process. Further analysis of STAT activity during synovitis may provide interesting information about the effects of the balance of cytokine activity upon synovial cells, and may reveal mechanisms of gene regulation during synovial inflammation. IX. Sustained Cytokine Expression and Cytokine Balance

One theme concerning synovial expression of cytokines that emerges from the data discussed above is an interplay between opposing cytokines.


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Cytokine opposition or antagonism may be arranged about several axes, and shifts in balance in one direction may lead to exacerbations of disease. One axis involves balance between proinflammatory cytokines produced as synovitis develops, and anti-inflammatory cytokines which are presumably produced in an effort to control and resolve inflammation (Table IV). It is puzzling why active synovitis continues despite high levels of expression of cytokine inhibitors (such as IL-lra, soluble receptors, TGFP, and IL10) that seem to be sufficient to block proinflammatory cytokines that are often present at lower levels. Perhaps the “anti-inflammatory” cytokines end up “unintentionally” contributing to and sustaining synovitis through their proinflammatory properties. For example, TGFP can function as a chemotaxin (Wahl et al., 1987),and IL-10 can stimulate antibody production (Howard and O’Garra, 1992).Alternatively, the major active cytokines may be novel proteins, possibly T cell products, for which inhibitors are not produced (Blotnick et al., 1994; Miltenberg et al., 1995). Another axis involves balance between Thl and Th2 cytokines. Shifting the balance toward Th2 cytokine production through several different interventions, namely adding exogenous Th2 cytokines, neutralizing Thl cytokines,blocking interactions between costimulatory molecules and their receptors, or inducing oral tolerance, results in prevention or amelioration of EAE, a Thl cytokine-driven process (Chen et al., 1994; Racke et al., 1994; Kuchroo et al., 1995). Treatment with the combination of the Th2 cytokines IL-4 and IL-10 inhibits collagen-induced arthritis (van den Berg et al., 1994), while blocking IL-10 activity results in increased disease severity (Kasama et al., 1995). In the case of collagen-induced arthritis, exogenous IL-4 and IL-10 may act by promoting a Th2 response, or, more TABLE IV THEBALANCE BETWEEN PROINFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES Proinflammatory

Anti-inflammatory

~~

TNFa IL-1 IL-6 (IFW (IL-2) GM-CSF sCD23 Chemokines

TNF-sR, IL-10 IL-lra, IL-10 Anti-IL-6 antibodies IL-10, TGFB, PGEs Soluble IL-2 receptors

? ? ?

Note. The factorswhich inhibit the expression or activity of proinflammatory cytokines are shown in the second column. sR, soluble receptors.

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likely, by directly suppressing monocyte function. In the RA synovium, there appears to be a balance between Thl-like T cells and soluble factors which are immunosuppressive, but also promote Th2 responses. Given possible contributions to pathogenesis of Th2 cytokines, and the possible importance of Thl responses in clearing persistent antigens, it is difficult to predict the long term consequences of shifting this balance in either direction. An important question is why synovial inflammation does not resolve, similar to noninfectious inflammatory processes which eventually end up with wound healing and resolution (Rappollee and Werb, 1992). One obvious potential explanation is that synovitis may be maintained by antigen-specific reactivity of synovial T cells against persistent foreign antigens or autoantigens. In this hypothesis (Fig. ZA), T cells that become activated in the synovium drive the production of cytokines and cell activation throughout the synovium. The challenge to proponents of this hypothesis, given the low levels of T cell expression of known cytokines, is to explain how activating signals can travel from sublining lymphoid aggregates to

A As Auto-Ag activating factors or cell:cell contact

B

r@< -\+

PDGF

LIF

IL-I TNF

IL-6

recruitment of new cells chemokinesfl TGFb

FIG.2. Models for mechanisms of perpetuation of synovid inflammation. (A). Repeated stimulation of T cells, by persistent antigens, auto-antigens, or possibly by cytokines alone. is the driving force which sustains the inflammatory response. (B) During chronic synovitis, T cells no longer play a critical role. Soluble factors produced by monocytes and fibroblasts set up positive regulatory loops that sustain inflammation. T, M, and F = T cells, monocytes, and fibroblasts.

CYTOKINE EXPRESSION I N INFLAMMATORY ARTHRITIS

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distant and distinct regions of the synovium. An alternative view (Fig. 2B) postulates that, at least during later stages of disease, T cells are no longer critical for ongoing synovitis. In this model, the paracrine and autocrine effects of monocyte- and fibroblast-produced cytokines are sufficient to recruit new cells and to perpetuate the inflammatoryprocess. The challenge to proponents of this hypothesis, given the natural course of resolution of most inflammatory processes driven predominantly by monocytes and fibroblast-like cells, is to explain why synovial inflammation is susceptible to being perpetuated and becoming chronic. X. Conclusions

In the past decade, a wealth of information has become available about the expression of cytokines in inflammatory arthritis, particularly in rheumatoid arthritis. Although much of these data is necessarily descriptive in nature, it has already yielded important insights into the pathogenesis of synovitis. Progress is being made in delineating which of the cytokines expressed during synovitis play an important role in pathogenesis, the mechanisms by which they activate cells, and in developing novel therapeutic strategies aimed at blocking cytokine action. Additional information about cytokines in synovitis is needed, particularly about production of cytokines by T cells, signaling pathways and transcriptional responses that are activated, and the patterns of localized expression of cytokines in synovial tissue. Local cytokine expression in defined regions of synovium where cells interact, such as the lining layer or the lymphoid tissue-like mononuclear cell aggregates, likely plays an important role in the regulation of cellular activation and differentiation. Another important issue that needs to be addressed is the temporal pattern of cytokine expression during the course of disease and in response to therapy. Much of today’s knowledge is based upon work with specimens obtained perioperatively from patients with long-standing, chronic disease who have undergone various courses of therapy. Although these data are valid for trying to understand how cytokines may drive ongoing, chronic disease, it does not address the role of cytokines during establishment of synovitis or during early disease. Indeed, studies of collagen-induced arthritis suggest that cytokine regulation of the inflammatory process may change during the course of disease (Boissieret al., 1995).Increased use of synovial biopsies and arthroscopy early in disease will provide an opportunity to address the important question of cytokine expression during early synovitiS.

Experiments with animal models of arthritis and clinical trials with RA patients suggesting that interventions aimed at inhibiting cytokines have

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significant therapeutic potential represent a particularly exciting development (Wendling et nl. 1993; Elliot et al., 1994a,b; Williams et al., 1994). Several potential problems with therapeutic blockade of cytokine activity include: (i) potential lack of long term efficacy; (ii) problems with toxicity; (iii) potential need to inhibit multiple cytokines; (iv) immune responses against biopharmaceuticals, such as antibodies; (v) unexpected consequences from perturbing cytokine networks; and (vi) difficulty in targeting therapy to sites of inflammation and thus avoiding generalized immunosuppression. Some of these problems may be overcome by targeting signaling pathways or transcription factors that are activated by cytokines, and may be more amenable to inhibition by more conventional therapeutic agents. An example of a successful application of this approach is the prevention of LPS-induced lethal toxicity in mice by protein tyrosine kinase inhibitors (Novogrodskyet nl., 1994).A greater understanding of which cytokines are most important in pathogenesis and the mechanisms of cellular responses to these cytokines will be useful in guiding the design of effective and safe therapies. ACKNOWLEDGMENTS I thank my colleagues in the laboratory and the Division of Rheumatic Diseases for many

helpful discussions and for helping to shape my thoughts about cytokines and inflammatory arthritis.

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ADVANCES IN IMMUNOLOCY. VOL. fi3

Prolactin, Growth Hormone, and Insulin-like Growth Factor-I in the Immune System RON KOOUMAN, EUSABETH L HOOGHE-PETERS, AND ROBERT HOOGHE 0epu-t

of Phatmadqy, Msdirol Shod, Fm University of B N S ~ S , B- loo0 B N ~ s ,M i u r n

1. Introduction

The pituitary gland controls many other endocrine glands and is thus indirectly involved in many physiological functions. Its importance for the immune system has recently been recognized. The hypothalamicpituitary-adrenal axis is globally inhibitory to the immune system (reviewed in Wilder, 1995; Chrousos, 1995), yet hypophysectomy in rats results in immunosuppression. Reconstitution experiments showed that the pituitary hormones prolactin (PRL) and growth hormone (GH) exert immunostimulatory properties. Also, dwarf mice with an inborn deficit in PRL, GH, and thyroid-stimulating hormone (TSH) have an immune deficiency due in part to the lack of GH and PRL. These observations have not received much attention in the past and PRL and GH owe their recent entry into textbooks of immunology mainly to the fact that they belong to the cytokinehemopoietin family. This membership is based upon structural characteristics of ligands (hormones, interleukins, and colony-stimulating factors) sharing the four helix-bundle structure and of the corresponding receptors (Boulay and Paul, 1993; Horseman and Yu-Lee, 1994). In addition to PRL and GH, we will also focus on the role of the insulin-like growth factor (1GF)-I, a known growth factor for many cell types, also in the immune system. Its local expression is controlled to a major extent by GH but also by cytokines (see Fig. 1). The main purpose of this review is to examine whether PRL, GH, and IGF-I are indeed full-fledged immunological growth and differentiation factors. There is now ample evidence, summarized in this review, that receptors for PRL, GH, and IGF-I are differentially expressed on several leukocyte populations. Moreover, PRL, GH, and IGF-I are produced by minor populations of leukocytes. The role played by these factors in the development and the function of the immune system, ignored until recently, is now the subject of intense investigation.The relative contribution of systemic (endocrine) and locally produced (paracrine or autocrine) hormones will be considered. Detailed reviews have been published on PRL and GH in the immune system (Gala, 1991), on the role of GH and lactogenic hormones in immunology (Berczi, 1994) and on PRL, GH, and 377 Copyright 0 IW6 hy Academic Press, Inc All rights of reproduction in m y form resrrvrd.

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WON KOOIJMAK ET A l ,

[TI

Other/factors

u leukocyte

IGF-binding proteins

Fic:. I . Relationship between possihle actions of PRL. CH, and IGFs on the ly111phohe111opoietic system. PHL and GH can exert direct actions on ~yrnphoherno~~~~ietic cells via their own receptors, but primate GH can also act via the PHL-R. Some effects of GH are met1i;ittd by ICF-I. However, the rffccts of IGF-I do not always depend on GH, since it can d s o be expressed under the control of other factors. e.g., cytokines. IGF-I1 shares Iroirrology with ICF-I and mostly acts via the ICF-I-R, but can also exert several actions via its own receptor. Furtherinore, actions of IGFs can be inodulatetl by IGF-binding proteins. PKL and CH are mainly produced by the anterior pituitary, and the hulk of serum IGF-I is produced by the liver. However. these factors are also expressed in several leukocyte poplutions.

IGF-I in relation to the hemopoietic and lymphoid system (Hooghe-Peters and Hooghe, 1995). As a framework for critical examination of the available data, we propose to evaluate the value of the following alternative hypotheses: 1. The immune system contains both a PRL- and GH-secreting complex and target cells that express receptors for and respond to PRL and CH. Some effects of GH are mediated through the local production of IGF-I. PRL, GH, and IGF-I are thus paracrine or autocrine immunologic growth and differentiation factors. 2. A “weakened” version of that hypothesis states that PRL, GH, and IGF-I are imrnunoregulatory hormones; the immune system is one of their targets but local production is negligible. 3. An extreme view holds that PRL, GH, and IGF-I play little if any role in the development and the function of the human iininune system. Observations cited above make this hypothesis untenable in rodents.

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II. Hormones

The pituitary gland comprises two parts: the neurohypophysis or posterior lobe is of nervous origin, contains nerve fibers and glial cells, and secretes oxytocin and vasopressin, synthesized in the hypothalamus. The intermediate and anterior lobes form the endocrine pituitary gland, The intermediate part, vestigial in man, secretes melanocyte-stimulating hormone. The anterior part contains six cell types, each producing a single hormone: the gonadotrope cells secrete luteinizing hormone (LH) or follicle-stimulating hormone (FSH) and the corticotrope cell makes ACTH. The mammotrope cell secretes PRL, the somatotrope cell secretes GH, and the thyreotrope cell secretes TSH. Hormone expression in the latter three cell types is controlled to a large extent by the pituitary-specific transcription factor Pit-l/GHF-1 (for a review on the pituitary gland and hormones, see Baulieu and Kelly, 1990; Imura, 1994; Melmed, 1995; for a review on the PRL cell, see Takahashi, 1995; on the role of Pit-1/GHF1, see Rhodes et nl., 1994). Among pituitary hormones, only PRL and GH are members of the cytokine-hemopoietin family. There was at first sight little evidence that the members of the cytokine-hemopoietin family had more in common than the use of receptors with limited but undisputed homology (see Section 111).However, both the gene organization and the spatial structure revealed that cytokine-hemopoietin peptides are related by their four helixbundle structure. They fall into subfamilies (Boulay and Paul, 1993): the large hemopoietins comprising the GH-PRL family, the IL-6, and the leukemia inhibiting factor (LIF) subgroups have wider helices. The small hemopoietins include IL-2, and the IL-4 and IL-7/IL-9 subgroups. PRL and GH themselves belong to a gene family composed of many members, only some of which code for hormones. Of interest to immunology are PRL, GH, and the placental lactogens which share extensive homology (Berczi and Nagy, 1991). In the conceptus, placental lactogens are especially important before the onset of pituitary GH and PRL production. Placental lactogens bind to GH or PRL receptors (there is so far no published evidence for the existence of specific receptors for placental lactogens). It should also be mentioned that the term placental lactogens designates many hormones related more closely to either GH or PRL, depending on the species. For instance in man, there is 40% homology between PRL and GH or placental lactogens (chorionic somatotropins) whereas GH and the placental lactogens share about 90% homology. In rodents, in contrast, placental lactogens are more closely related to PRL. In the past, PRL and GH were purified from pituitary glands. As they have similar molecular weights, preparations of one hormone were often contaminated with the other. Recombinant hormones are now available.

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This review will focus on the effect of hormones on the immune system. Effects of cytokines on the hypothalamus and the pituitary gland will not be reviewed in detail. It should however be mentioned that several cytokines are produced by endocrine cells (TGFP and migration inhibiting factor) and by nonendocrine cells (IL-1, IL-6, LIF) in the pituitary (Bemhagen d al., 1993; Bums and Sarkar, 1993; Ferrara et al., 1992; Spangelo et al., 1994; Tatsuno et al., 1991; Velkeniers et al., 1994; for review, see Asa and Kovacs, 1994; Hooghe-Peters et al., 1991; and McCann et ul., 1994). Some cytokines act as autocrine or paracrine pituitary growth and differentiation factors and modulate hormone secretion.

A. PHOLACTIN 1 . Structure and Physiology of PRL The pituitary hormone PRL is best known for stimulating the development and the activity of the mammary gland. Many other tissues have receptors for PRL. Among important targets are the gonads, the brain, the intestine, the kidney, the liver, and the embryo, in particular the embryonic lung. PRL stimulates differentiation and growth of the mammary gland and other tissues, affects behavior, and influences for instance water and mineral transport and steroid hormone metabolism. Until recently, PRL expression was described in the anterior pituitary only. It has now also been shown in uterus, decidua, mammary gland, brain, and lymphohemopoietic tissue. The fact that serum levels are dramatically reduced after hypophysectomy confirms that the bulk of PRL production originates from the pituitary (or is under pituitary control). In the rat, serum PRL levels, which are very low right after hypophysectomy,gradually increase several weeks thereafter. This was attributed to tissue regrowth after (incomplete) hypophysectomy (Nagy and Berczi, 1991). In women, the highest serum levels are reached during the luteal phase. Increased PRL levels are seen during pregnancy, lactation, and mating in females (Erskine, 1995). Pituitary adenoma (prolactinoma) and treatment with estrogens or anti-dopamine drugs (e.g.,domperidone and haloperidol) also result in hyperprolactinemia. Dopaminergic drugs (bromocriptine and cabergoline) prevent PRL secretion (Bevan and Davis, 1994). In the human, serum PRL levels should remain below 0.6 nM (15 ng/ml) in man and below 1.6 nM (40 ng/ml) in women. Average values, however, do not differ markedly between men and women. After cleavage of a 30 amino acid leader peptide, human PRL is 199 amino acids in length ( M , 23 kDa). Further cleavage, deamidation, phosphorylation, and glycosylation account for numerous variants (Sinha, 1995). There is a site for N-linked glycosylation in human but not in rat PRL,


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which can undergo O-linked glycosylation (Bollengier et al., 1991). Phosphorylation and glycosylation alter activity (Price et al., 1995; Walker, 1994). Also, dimers (big PRL) and oligomers or high molecular complexes containing PRL (big-big PRL) have reduced biological activity. In some cases, big-big PRL consists of PRL bound to anti-PRL IgG (Bonhoff et al., 1995; Cavaco et al., 1995). A 16-kDa N-terminal fragment of PRL in the rat has anti-angiogenic properties, and a novel receptor, binding the 16-kDa variant but not 23-kDa PRL, has been described (Clapp and Weiner, 1992). Some PRL variants result from alternative splicing of the PRL mRNA in animals, but in man there is no convincing evidence so far. The study of the various PRL forms in physiological and pathological situations is far from being completed (analysis of normal human serum forms is provided in Warner et al., 1993). In particular, the variants expressed in extrapituitary tissues have not been characterized. Also, the quantitative importance of extrapituitary PRL production is poorly appreciated. It is generally assumed that extrapituitary production only serves for autocrine or paracrine functions.

2. Regulation of P R L a. Expression. There is only one gene coding for PRL in man, mouse, or rat. Estrogen, angiotensin 11, TSH, triiodothyronine, and the pituitary adenylate cyclase activating peptide (PACAP) stimulate PRL expression, whereas dopaminergic agents inhibit PRL expression. Most of these factors control PRL gene expression via qualitative or quantitative effects on the pituitary transcription factor Pit-1. PRL expression in human decidua and leukocytes relies on the use of an alternative 5’ far-upstream promoter usage (Benvaer et al., 1994; Gellersen et al., 1994). This results in the transcription of an mRNA 150 bp larger than the pituitary form, with no change, however, in the coding sequence for the mature protein. Another salient feature of PRL transcription in extrapituitary tissues is its independence from control by the Pit-1 factor. Recent experiments by Gellersen et al. (1995) indicated, however, that the pituitary-type mRNA (that is the shorter form) can also be produced in the absence of Pit-1. There is little information available on the control of PRL expression and secretion in extrapituitary sites. At the transcription level, the important question is which promoter is used. In many cases, extrapituitary PRL expression uses the far-upstream promoter and initiation site. In the IM-9-P-3 line, PRL expression was negatively regulated by dexamethasone but was not influenced by dopamine or estrogens (Gellersen et al., 1989a). Prolonged exposure of IM-9-P-3 cells to phorbol esters reduced PRL mRNA levels (Gellersen et d., 1989b).

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b. Secretion. Estrogen, galanin, thyrotropin-releasing hormone and

PACAP 27 induce PRL secretion which is inhibited by dopamine, endothelins, and gamma-aminobutync acid (GABA). In contrast to other pituitary hormones, PRL secretion is regulated negatively by the hypothalamus through dopamine (Ben-Jonathan, 1994).Consequently, pituitary stalk section is followed by a rise in plasma PRL. Also, ectopic pituitary grafts (e.g., under the kidney capsule) will secrete PRL continuously following relief from dopaminergic inhibition: such grafts represent a convenient way to produce chronic hyperprolactineinic rodents (Adler, 1986).A rise in plasma PRL levels was observed after the intracerebroventricular injection of IL-1 or IL-2. IL-2 and IL-6 stimulated and IL-1 inhibited the basal release of PRL by cultured pituitary cells (Bernton et al., 1987; Karanth and McCann, 1991; Spangelo et nl., 1989).We failed, however, to detect any direct effect of IL-1 or IL-6 on PRL secretion by purified lactotropes from the rat pituitary in vitro (Hooghe-Peters, unpublished results). The stiniulation observed in other systems could be explained by the presence of cytokineresponsive cells which could in turn affect the secretory activity of lactotropes. There is very little information about the control of PRL release in extrapituitary sites. In human myometrium explant cultures, endothelin-3 increased PRL release whereas epidermal growth factor, vasoactive intestinal peptide, IFNa, and IL-4 were inhibitory. IL-4 was the most potent inhibitor (Bonhoff and Gellersen, 1994). In human decidual cells, IGF-I stimulated PRL release whereas endothelin-I inhibited both basal and IGF-I-stimulated PRL release (Chao et al., 1993). 3. PRL Expression and Regulation in the Lymphohenwpoietic System a. In rodents. In early studies, critically reviewed by Gala (1991), the evidence for PRL expression by leukocytes was indirect. The fact that antiPRL serum blocked the response to B and T cell mitogens was taken as indication that PRL released by cells present in the culture exerts a stimulatory effect. In one report, leukocytes released bovine PRL that had been taken up from the culture medium (Clevenger et al., 1990a). The release of substances promoting the growth of Nb2 lymphoma cells is also taken as evidence for PRL production. Technical problems related to the characterization of lactogenic activity released by cells in culture are discussed by Gala and Rillema (1995). In a recent study, these authors showed that Con A-stimulated splenocytes release both PRL-like bioactivity and a substance that inhibits Nb2 cell proliferation. The PRL-like activity could not be neutralized by anti-PRL, by anti-GH, or by anti-placental lactogen sera and its nature thus remains elusive (Gala and Shevach, 1994; Gala and Rillema, 1995).

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Touraine et al. (1994) found no PRL mRNA in mouse or rat spleen, bone marrow, lymph nodes, or thymus by PCR. In contrast, Delhase et al. (1993) detected PRL mRNA in rat bone marrow, spleen, thymus, and liver by PCR and by in situ hybridization, and PRL protein by immunocytochemistry. In bone marrow, at least 1% of the cells were positive. In the spleen, positive cells were located in the red pulp and in the marginal zone. In the thymus, the rare positive cells were found in the subcapsular area, at the cortico-medullary junction, and in the medullar area. In the liver, some nonhepatocyte cells were also positive. In kidneys, adrenals, thyroid, and pancreas, all cell types were negative with the exception of an occasional leukocyte in the lumen of blood vessels. In recent unpublished studies (Hooghe-Peters et al. ), it was verified by combined in situ hybridization and immunocytochemistry that the cells expressing PRL mRNA also contained the protein. The reverse, however, was not true, as there were more cells containing the protein than the mRNA, suggesting that PRL had been endocytosed. Alternatively, PRL mRNA could be short-lived or present at low but undetectable levels in certain cells. Most PRL mRNAexpressing cells also expressed GH and the Pit-1 transcription factor. Taken together, these observations indicate that a substantial proportion of rat spleen cells expresses PRL and GH. The colocalization of hormones and Pit-1 would suggest that Pit-1 plays a role in PRL and GH expression. This cannot easily be reconciled with the above-mentioned data by Benvaer et al. (1994) and Gellersen et al. (1994) on the control of “extra-pituitary PRL’ expression. Definitive proof of PRL synthesis during short-term cultures requires metabolic labeling and this was done in a few cases only. After labeling of mouse thymocytes, immunoprecipitation of cell lysates and culture supernatants with anti-PRL serum yielded a 22-kDa product (Montgomery et al., 1990).

b. In Man. PRL cDNA has been cloned from the thymus (O’Neal et al., 1992). As in the decidua, the PRL mRNA in leukocytes was 150 bp

longer than in the pituitary. PCR studies showed strong signals in human thymocytes and T cells from peripheral blood mononuclear cells (PBMC), a weaker signal in B cells, and no signal in monocytes (Pellegrini et al., 1992). Using PCR, in situ hybridization and immunocytochemistry, Delhase et al. (1993) obtained the same results with human as with rat material (see above). Observations by Wu et al. (1996) on PRL mRNA expression are in complete agreement. In addition, they detected PRL expression in tonsils. After metabolic labeling of PBMC, immunoprecipitation yielded products of 23 and 36 kDa (Pellegrini et al., 1992), 27 kDa (Montgomery et

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al., 1992), or 60 kDa (Sabhanval et al., 1992). Cell lysates and culture supernatants from Con A-stimulated thymocytes contained 24 kDa immunoprecipitable PRL (Montgomery et al., 1992).Purified NK cells displayed cytoplasmic PRL immunoreactivity but failed to express PRL mRNA. In contrast, a 27-kDa protein could be specifically immunoprecipitated with anti-human PRL serum from lysates and supernatants of IL-%stimulated PBMC (Matera et al., submitted). The identification of PRL expressing cell lines has been of great value. In particular, IM-9-P3 cells, derived from the EBV-transformed IM-9 human B lymphoblastoid line, have been used extensively for molecular studies of extrapituitary PRL expression (DiMattia et al., 1988). Some sublines of the Jurkat T leukemia produce PRL and release it into the medium. In several other lines (derived from B, T, NK, and myelomonocytic cells) PRL mRNA expression has been detected by PCR (Pellegrini et al., 1992). It is not sure however that all these cells are the transformed counterpart of a normal PRL-producing leukocyte. We will discuss later the possibility that PRL is an autocrine growth factor in some forms of leukemia. c. Which Leukocytes Express PRL? According to Pellegrini et al. (1992), the PCR signal from PRL mRNA was strong in human T cells, weak in B cells, and absent in monocytes. Unpublished results from our group indicate that granulocytes in rodents and man also express PRL.

B. GROWTHHORMONE 1 . Structure and Physiology of Growth Hormone The best known function of GH is the promotion of longitudinal growth. Most target tissues respond to GH by producing IGF-I. This factor acts on chondrocytes and thus mediates growth-promoting effects of GH. GH has many other physiological functions, some of which are mediated only by IGF-I. GH has important metabolic functions in kidney, brain, and adipose tissue. The expression of GH is restricted to somatotrope cell in the anterior pituitary gland, to some leukocytes (see below), and to cells in the placenta. Human GH is a 191-amino-acid protein (22 kDa). A large number of variants are found in man as well as in rat. There is heterogeneity in isoelectric points but also in molecular weight. Some shorter forms result from alternative splicing. High-molecular-weight forms represent dimers or oligomers. Post-translational modifications include glycosylation, phosphorylation, acetylation, and proteolytic cleavage (Baumann, 1991).As for PRL, there is a large family of GH variants, the precise activity and function

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of which are not presently known. The major serum form in the hurnan was recently claimed to be 17, not 22 kDa GH (Warner et al., 1993). In the human, there are two genes coding for GH. The GH-N gene codes for the classical pituitary hormone and the GH-V gene codes for the placental form. As GH-V is released from the placenta into the maternal circulation, GH-N production is gradually suppressed.

2. Regulation of Growth Honnone The expression of GH in the pituitary is stimulated by the hypothalamic factor GH-releasing hormone (GHRH) and inhibited by somatostatin. Negative feedback from IGF-I is also important in the control of GH expression. As for PRL, the action of factors affecting GH expression is mediated in many cases through the pituitary transcription factor Pit-1. There is only one known site for the initiation of transcription of GH. This implies that the same factors could regulate transcription in the pituitary gland and in leukocytes. In the pituitary somatotrope cell, GHRH and somatostatin also control the secretion of GH, in the same way that they affect expression (Thakore and Dinan, 1994).Cytokines also modulate GH secretion. IL-1 given either iv or by intracerebroventricular injection inhibited GH secretion in the rat. IL-1 increased the release of somatostatin and decreased the release of GHRH by hypothalamic fragments in culture (Peisen et al., 1995). There were also reports indicating that IL-1 exerted a stimulatory action on GH secretion (Payne et al., 1992; Rettori et al., 1987).Conflicting results were obtained in vitro as well: though IL-1R and IL-1 are coexpressed on pituitary GH cells, we failed to demonstrate a direct action of IL-1 on GH secretion by purified rat pituitary GH cells (Hooghe-Peters, unpublished results). However, Bernton et al. (1987) and Niimi et al. (1994) found that IL-1 had a stirnulatory action, IL-2 is a potent inducer of GH secretion in vivo, probably through its action on the hypothalamus. IL-6 does not seem to affect GH secretion in vivo. IL-6 is expressed by nonendocrine cells in the normal rat and human pituitary and in human GH- and ACTHsecreting pituitary adenomas (Spangelo et al., 1994; Vankelecom et al., 1989; Velkeniers et al., 1994). Spangelo et al. (1989) found that IL-6 stimulated GH secretion in vitro but, again, we could not confirm these data (Hooghe-Peters, unpublished data). IL-6 as well as 1L-2 also has an inhibitory effect on the growth of normal anterior pituitary cells (Artz et al., 1993).TNFa acts directly on the somatotrope cells and stimulates GH secretion (GH in turn controls the production of TNFa by macrophages (Edwards et al., 1991a)). In several systems, GH is secreted in response to stress and it could also participate in the acute phase response. In vivo, large doses of corticosteroids inhibit GH secretion and growth (Giustina

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arid Wehrenberg, 1992; Thakore and Dinan, 1994). It is important to Inention that G H is secreted in pulses and that the secretion pattern may be abnormal although serum levels are within normal limits. GH deficit is responsible for nanism in children but is well tolerated in adults. The hypersecretion of GH in children leads to excessive growth, whereas in adults acromegaly develops. In adults, serum GH should remain below 0.45 I ~ M(10 ng/ml). GH levels decline significantly with age, as do IGF-I levels.

3. GH Expression and Regulation in the Lyrnphoheiiiopoietic Sr~stetii GH expression has been studied in rat and human mononuclear leuko-

cytes and in several cell lines.

a. In Rats. Weigent and Blalock (1991) detected GH transcripts in rat thymocytes and splenocytes using RT-PCR and Southern blot analysis. In addition, GH mRNA was found in rat PBMC by Northern blot analysis (Weigent et nl., 1988). Additionally, Delhase et al. (1993) demonstrated GH expression in bone marrow, thymus, spleen, lymph nodes, and liver by RT-PCR and in situ hybridization (see Section 1I.A). It was shown by radioimmunoassays that cultured rat bone marrow cells, thymocytes, splenocytes, and peripheral blood cells secreted immunoreactive (ir) GH (14-17 pg/lOficells per 24 hr). Affinity-purified leukocytederived GH augmented DNA synthesis in rat splenocytes. This stimulating activity was abrogated in the presence of an anti-rat GH antibody (Weigent and Blalock, 1991). An autocrine or paracrine role for GH in spleriocyte proliferation is also suggested by inhibition studies with antisense deoxyoligonucleotides (Weigent et al., 1991).Inhibition of GH synthesis in cultured splenocytes in the absence of initogens was shown to result in an 87% decrease in thymidine incorporation, which could be reversed by addition of exogenous GI1. Northern blot analysis revealed that both pituitary cells and cultured rat leukocytes from spleen expressed a 1.0-kb mRNA that hybridized with a rat GH probe. Rohn and Weigent (1995) produced four cDNA clones from the mRNA of rat splenocytes. The sequence of all clones was identical to that of pituitary-derived GH inRNA coding for the 22-kDa GH. Using RT-PCR followed by Southern analysis, Binder et nl. (1994) did not detect GH transcripts in bone marrow, thymus, and spleen of adult or fetal rats. GH mRNA was found only in bone marrow and spleen of neonatal rats. Those data contradict the above-mentioned results of Weigent and Blalock (1991). Hypothalamic hormones are present in the periphery at very low concentrations. With the exception of somatostatin and corticotropin-releasing hormone, hypothalamic hormones are rarely expressed outside the central nervous system. However, some hypothalamic hormones have been de-

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tected in lymphoid tissues. GH-releasing hormone (GHRH) and specific receptors for GHRH are expressed in the immune system (Guarcello et al., 1991; Matsubara et al., 1995).Somatostatin, a hypothalamic GH-release inhibiting factor (which, however, has Inany other functions and is expressed in many tissues, such as the digestive tract), is also produced by lymphocytes and monocytes and these cells express receptors, more so after activation. Somatostatin and its analogs bind to specific receptors and affect the function of lymphocytes and macrophages (Hiruma et al., 1990; Karalis et al., 1995; Van Hagen et al., 1994). IGF-I reduced the number of rat splenocytes that expressed irGH (Baxter et al., 1991). A pituitary-type regulation was also suggested by the presence of the pituitary transcription factor Pit-1 in rat leukocytes (Delhase et al., 1993). However, according to recent findings of Weigent and Blalock (1994), GH was expressed in splenocytes from hypopituitary SnellB a g dwarf mice. Since the pituitary of these mice does not produce detectable levels of GH or PRL, due to a mutation in the transcription factor Pit-1, their results point to an alternative, Pit-l-independent regulation mechanism for GH.

b. In Human. Delhase et al. (1993) made similar observations for GH expression in the human and in the rat. All lymphohemopoietic tissues tested were positive in RT-PCR and in situ hybridization (see detailed summary in Section II.A.3). The presence of GH transcripts in human PBMC was suggested by dot blot analysis (Weigent et al., 1988). Human PBMC secreted a lowmolecular-weight (22 kDa) and a high-molecular-weight ( >300 kDa) immunoreactive GH. The 300-kDa complex yielded 22-kDa molecules upon reduction. The human PBMC-derived affinity-purified GH stimulated DNA-synthesis in the Nb2 rat lymphoma cell line. This effect was blocked by specific antibodies to human GH. Varma et al. ( 1993) assessed the number of GH secreting cells in human PBMC by the enzyme-linked immunoplaque assay. It was found that 1% of unstimulated PBMC secreted GH. The GH secretion was increased by at least 100% by stimulation with the T cell mitogens PHA or IL-2, but not by LPS, a B cell mitogen. Additionally, the authors showed that PHA increased the number of GH-secreting cells by about 50%. Hattori et d. (1990) studied the regulation of GH secretion by human PBMC using an enzyme-linked immunoassay. Unstimulated cells secreted 2-6 pg immunoreactive GH per lo6cells during a 7-day culture period. The secretion of irGH was increased by stimulation with PHA or PWM, but not by LPS. Secreted immunoreactive GH eluted at the position of a 22-kDa protein on gel chromatography. GH increased the secretion of GH by PHAstimulated human PBMC, whereas IGF-I had no effect.

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In contrast with the above mentioned findings in rat splenocytes, two regulators of pituitary GI3 secretion, GHRH and somatostatin, did not affect the spontaneous secretion of GH by PBMC (Hattori et al., 1994). These data point to a GH regulation system in the immune system that is different from that in the pituitary.

c. Ce2l Lines. Autocrine GH is secreted by the human Burkitt lymphoma, sfRamos (Lytras et nl., 1993). Secretion of immunoreactive C H has also been found in human T (H9)and B cell lines (IM9) (Kao et al., 1992). C. INSULIN-LIKE GHOWHFACTOH-I 1 . Structure and Physiology of ZGF-I The insulin-like growth factors (IGF-I and IGF-11) are polypeptides stimulating proliferation and differentiation of a wide variety of cell types (Jones and Clemmons, 1995; Lowe, 1991). IGF-I is the growth promoting mediator of GH. It is a 7.6-kDa single-chain polypeptide, consisting of 70 amino acids, which is structurally related to IGF-11, insulin, and its precursor proinsulin. Since IGF-I is produced by many tissues and abundantly present in serum ( 2 2 5 nM ), it has the potential to act via endocrine as well as autocrine and/or paracrine mechanisms. Serum IGF-I originates mainly from the liver where it is produced under the control of GH. In human and rodents, serum IGF-I levels are low in the neonate, rise to peak levels during puberty, and slowly decline with increasing age. In addition to IGF and insulin receptors, IGF-I binds to six IGF-binding proteins (IGFBPs) with 5-50 times greater affinity as compared to the IGF-IR. As a consequence, IGFs in body fluids can be bound to IGFbinding proteins; e.g., circulating IGFs are predominantly (>95%) found in a 150-kDa complex with IGFBP-3 and an 85-kDa acid-labile subunit. The complete primary structures of six forms of human IGFBP have been determined (IGFBP1-6). IGFBP-1 and -2 contain an RGD sequence which can be recognized by integrins. IGFBPs can influence the half-lives and the tissue distribution of IGFs. At the cellular level, IGFBPs can modulate the metabolic and mitogenic activities of IGF-I. For instance, they can regulate IGF-I bioavailability by blocking the binding to the IGFIR. In addition, specific proteases have been reported to increase the bioavailability of IGFs by degradation of IGFBPs (Jones and Clemmons, 1995). IGF-I1 and IGFBPs are further considered in Section VI. The in vitru effects of IGF-I fall into three categories: DNA-synthesis/ proliferation, differentiation, and metabolic effects. IGF-I stimulates DNA synthesis and/or proliferation in many cell types such as endothelial cells, fibroblasts, muscle cells, chondrocytes, and lymphohemopoietic cells. It also has metabolic effects such as stimulation of glucose and amino acid

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uptake and glycogen and protein synthesis, and it promotes cell differentiation in several cell types. In uiuo, IGF-I mediates the growth promoting effects of GH, either as an endocrine or as an autocrine/paracrine factor. IGF-I also plays a role as a fetal growth factor. In addition to its growth promoting effects, IGFI exerts several metabolic, insulin-like effects. Furthermore, IGF-I has been implicated in tissue regeneration and wound healing (Lowe, 1991). 2. Regulation of IGF-1 As a consequence of alternate splicing of mRNA, several different IGFI precursors exist which all contain the 70 amino acid mature peptide, but differ in their amino-tenninal and carboxy-terminal ends. These precursors contain maximally 194 amino acids in human and 158 amino acids in rodents. One of the two carboxy-terminal precursor peptides (E-domains) contains an N-glycosylation site (Lund, 1994). The biological relevance of different precursor types is not known. Precursor peptides can be secreted by several nonhepatic tissues, including leukocytes (“tissue IGF-I ”). IGF-I mRNAs are present in virtually all fetal tissues and postnatal tissues. After birth, IGF-I mRNA levels in liver are markedly increased. The liver is the major source of circulating IGF-I, and serum levels rise progressively during postnatal development, reaching peak levels during puberty. However, IGF-I mRNA in nonhepatic tissues is differentially expressed. GH controls IGF-I gene expression in liver and several other organs such as the gastrointestinal tract, thymus, and spleen. However, IGF-I transcripts in nonhepatic tissues can also be regulated by many other factors such as estrogens, glucocorticoids, neuropeptides, and cytokines (Adamo et al., 1991).

3. IGF-1 Expression and Regulation in the Lyniphohemopoietic System Expression of IGF-I has been described in different lymphoid organs and in several leukocyte subsets such as PBMC, thymic lymphocytes, splenic lymphocytes, monocytes, and macrophages. IGF-I mRNA is expressed strongly in activated macrophages, but only weakly in lymphocytes. a. Monocytes and Macrophages. Cytokine-regulation and activationor developmental-stage-dependent expression of IGF-I has been well studied in human and murine macrophages. In 1988, IGF-I mRNA was detected in murine macrophages from wound tissue by Rappolee et al. (1988), and Rom et al. (1988) reported that human alveolar macrophages contained amounts of IGF-I transcripts comparable to that in human liver, whereas circulating monocytes were negative. In contrast to human circulating monocytes, alveolar macrophages secreted a 26-kDa IGF-I molecule (Rorn et al., 1988). This peptide competed for binding to fibroblasts IGF-I-Rs

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with '"I-IGF-I (serum IGF-I; 7.6 kDa) and stimulated in vitro IGF-I-R tyrosine kinase activity. Using RNase protection assays, Arkins et a1. (1993) showed that thioglycollate-elicited murine macrophages and the macrophage cell line PU5IR expressed amounts of IGF-I transcripts comparable to those from liver. On the other hand, bone marrow cells, thymocytes, splenocytes, and unstimulated macrophages expressed very low levels of IGF-I mRNA. Likewise, low transcript levels were found in lymphocyte cell lines and in a premyeloid cell line. Arkins et al. (1993) also found a 26-kDa peptide in elicited peritoneal macrophages and in the PU5-IR macrophage cell line. The regulation of IGF-I in monocytes and macrophages has been studied by several groups. Hyaluronate induces IGF-I peptide synthesis in murine bone marrow-derived macrophages via the cell surface adhesion molecule CD44 (Noble et al., 1993). Hyaluronate is a glycosaminoglycan present in the extracellular matrix. Other glycosaminoglycans did not induce IGF-I synthesis. Interestingly, binding of hyaluronate to CD44 seems to depend on the activation or maturation stage of CD44' leukocytes. CD44 variants are possibly involved. Whether these phenomena play a role in developmental-dependent expression of IGF-I in cells of the myeloid lineage (Arkins et al., 1995b) remains to be established. Several studies indicate that TNFa is a key cytokine in the regulation of IGF-I in macrophages. Noble at al. (1993)showed that TNFa induced a transient increase in IGF-I transcripts in murine bone marrow-derived macrophages. IGF-I peptide synthesis was quantified by metabolic labeling followed by immunoprecipitation of cell lysates and polyacrylamide gel electrophoresis. TNFa stimulated the synthesis of a 16- to 17-kDa peptide, and anti-TNFa antiserum blocked asbestos- and hyaluronic acid-stimulated IGF-I synthesis. Since the effect of asbestos was independent of CD44, the authors concluded that TNFa mediates the effects of two different cell surface activation mechanisms of IGF-I synthesis. Furthermore, ILlp, which did not exert a direct effect on IGF-I secretion, enhanced the stimulating effect of TNFa. An increase in IGF-I transcripts in rnurine bone marrow-derived macrophages as a response to TNFa was also observed by Fournier et al. (1995).TNFa also increased the secretion of a 15to 16-kDa IGF-I peptide. Another major inflammatory mediator, PGE2, stimulated IGF-I synthesis (15-16 kDa), although it reduced IGF-I mRNA levels. Post-transcriptional processes such as mRNA stability and translation might thus be involved in PGE2 regulation of IGF-I. In human monocytes, IGF-I transcripts could be induced by advanced glycosylation end products (AGES)(Kirstein et al., 1992). Stimulation experiments with AGE-BSA in the presence of antisera against these factors indicate that IL-10 and PDGF, which are expressed prior to IGF-I, can

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mediate induction of IGF-I. Thus, the competence factor PDGF and progression factor IGF-I may stimulate fibroblast proliferation, for instance during tissue repair, according to the dual control model (Stiles et nl., 1979). Endogenous TNFa was not required for IGF-I mRNA induction by AGES.The induction of IGF-I transcripts by AGE-BSA was completely inhibited by IFNy. Arkins et al. (1995a) showed that IGF-I mRNA levels, IGF-I transcription, and the synthesis of a 17-kDa IGF-I precursor in CSF1 differentiated murine bone marrow macrophages were also inhibited by IFNy. Nagaoka et al. (1990) found IGF-I transcripts in resting U937 cells similar in size and amounts to those of human alveolar macrophages. Both the calcium ionophore A23187 and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) stimulated IGF-I gene transcription, and caused a decrease in IGF-I mRNA levels. They also demonstrated that A23187 or PMA caused a rapid release of IGF-I in the presence of cycloheximide, indicating that U937 cells store a releasable pool of immunoreactive IGF-I. This is in marked contrast with hepatocytes which do not store IGF-I peptides.

b. Peripheral Lymphocytes, Thymus and Spleen. Immunohistochemical analyses in rats revealed the presence of immunoreactive IGF-I in lymphocytes and stromal cells in the spleen and thymus (Hansson et al., 1988; Baxter et al., 1991; Geenen et al., 1993). In contrast, all PBMC were negative. Whether positive cells produce IGF-I themselves or whether IGF-I is taken up from serum was not investigated. Baxter et al. (1991) demonstrated the presence of immunoreactive IGFI in a fraction (4%) of rat splenocytes, and the secretion by these cells of a 7.6-kDa immunoreactive IGF-I. Affinity-purified splenocyte-derived IGF-I stimulated DNA synthesis in fibroblasts. The amount of both cytoplasmic and secreted IGF-I was enhanced by incubation with GII. Since irIGF-I and irGH were found in the same splenocyte subpopulations (Weigent et al., 1992) and an anti-GH antiserum decreased the number of IGF-I containing splenocytes (Baxter et al., 1991),the authors suggested that irGH produced in the immune system might be involved in IGF-I regulation. IGF-I transcripts have also been detected in spleen and thymus. In hypophysectomized rats, reduced IGF-I mRNA levels in spleen and thymus were increased after GH treatment, but the GH dependency was less than in liver (Gosteli-Peter et al., 1994). Mathews et al. (1986) demonstrated the presence of IGF-I transcripts in spleen extracts from normal mice and from dwarf mice with a mutation in the GHRH-R (lit)that leads to reduced serum GH levels. Like in most nonhepatic tissues, IGF-I expression in thymus and spleen was not affected and thus seems to be GH independent.

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In human, IGF-I transcripts were found in the capsule and the interlobular septa of the fetal thymus and the capsule and septa of the fetal spleen (Han et al., 1987). IGF-I mRNA has also been demonstrated in PBMC. Using RT-PCR, Nyman et al. (1993) found IGF-I mRNA in PHA-activated human PBMC, but not in unstimulated cells. This is in agreement with the absence of IGF-I in freshly isolated human peripheral blood as determined by RT-PCR (Cluitmans et al., 1995). A PHA-induced increase of IGF-I mRNA in T cells was also found by Kooijman et al. (unpublished data). Freshly isolated T cells expressed no or only very small amounts of IGF-I mRNA which were barely detectable with RT-PCR followed by Southern hybridization. Merimee et al. (1989) demonstrated that GH enhanced the secretion of IGF-I by EBV-transformed human B lymphocytes. However, GH did not influence the IGF-I mRNA expression by another human B cell line, IM-9B (Clayton et al., 1994). c. Bone Marrow. A recent study on the expression of hemopoietic growth factors and cytokines in freshly isolated bone marrow cells from healthy individuals revealed that IGF-I is one of the factors expressed in human bone marrow that stimulate in oitro hemopoiesis. IL-6, IL-7, M CSF, stem cell factor, erythroid differentiating factor, and erythroid potentiating factor were also detected, but mRNAs of other stimulating factors like IL-3, G-CSF, and GM-CSF were undetectable with RT-PCR (Cluitmans et al., 1995). IGF-I transcripts were also found by RT-PCR in murine bone marrow cells (Arkins et al., 1993). In stromal cell lines, Abboud et al. (1991) reported IGF-I secretion, whereas Landreth et al. (1992) detected IGF-I mRNA. Interestingly, the pro-B cell differentiation activity of the S17 stromal cell line was abrogated by pretreatment of this line with antisense oligonucleotide for IGF-I. Thus, next to macrophages (see previous paragraph), stromal cells are likely candidates as IGF-I producing cells in bone marrow (Fig. 3).In addition, IGF-I mRNA is expressed in a murine pro-B cell line, and Arkins et al. (1995b) showed that IGF-I transcripts are expressed in a developmentally dependent manner, being expressed during differentiation of hemopoietic cells into multiple myeloid lineages. Murine bone marrow cells were cultured in the presence of CSF-1, IL-3, G-CSF, or GM-CSF. CSF-1- and IL-3-differentiated adherent cells (90 and 73% macrophages, respectively) contained higher levels of IGF-I transcripts as compared to freshly isolated bone marrow cells, whereas GM-CSFdifferentiated cells (75% macrophages) and G-CSF differentiated cells showed only a moderate increase in IGF-I expression. Their results also suggest that IGF peptides are secreted and are biologically active, because IGFBP3 inhibited the proliferation of CSF-l-committed progenitors.

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In conclusion, stromal cells and activated macrophages express high levels of IGF-I transcripts, whereas unstimulated monocytes and lymphocytes express very little IGF-I mRNA. Macrophages synthesize and secrete precursor forms of IGF-I. In some cases these peptides have been shown to be biologically active, but more studies are needed to reveal their specific properties (receptor binding, interactions with IGFBPs, biological activity) as compared to serum (7.6 kDa) IGF-I. Although effects of GH on IGFI expression have been described, cytokines and hemopoietic growth and differentiation factors are likely important regulators of IGF-I expression. In addition, hyaluronate and AGES, present in inflammatory sites and damaged tissues, also induce IGF-I expression. The data indicate that IGF-I in macrophages or macrophage-like cells can be controlled at the level of gene transcription, mRNA stability, translation, and secretion. 111. Receptors

A. THECYTOKINE-HEMOPOIETIN RECEPTORFAMILY Receptors for GH and PRL were first cloned in the late 1980s. They belong to the large, heterogeneous family of cytokine-hemopoietin receptors. There is a strong homology between GH-R and PRL-R. The primary structure homology between GH-R or PRL-R and other members of this family is, however, restricted to two extracellular domains of 100 amino acids and intracellular motifs called boxes. Conserved elements include two pairs of cysteine disulfide linkages in the first extracellular domain and a WSXWS sequence (YGEKFS in the mammalian GH receptors) in the second domain. The intracellular box 1 is a hydrophobic proline-rich region that strongly resembles a SH3-binding domain and box 2 is hydrophobic and acidic (Bazan, 1990; Thoreau et al., 1991; reviewed in Horseman and Yu-Lee, 1994; Kelly et al., 1993; Budel et al., 1995). The PRL-R and GH-R are located, together with LIF-R and IL-7R, within a cluster of cytokine-hemopoietin receptor loci on mouse chromosome 15 and human chromosome 5 (Gearing et al., 1993). A detailed description of the cytokine-hemopoietin receptors falls outside the scope of this review. We will mention here only two particular features: (1) In the case of the PRL-R, GH-R, and erythropoietin-R, homodimers are formed upon ligand binding, the same molecules being involved in ligand binding and in signaling. For the other members of the family, two or more polypeptide chains form a complete, functional receptor. The classification of these receptors is based upon the nature of receptor-

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associated chains, which are involved mainly in signaling, the chain related to GH-R and PRL-R contributing primarily ligand binding activity. (2) The extracellular portion of several cytokine-hemopoietin receptors can be found in extracellular fluids where it acts as a binding protein (BP). Depending on the tissue or the species, two different mechanisms can lead to the production of BP: (a) shedding after proteolysis of receptor molecules inserted in the membrane or (b) secretion of a protein that lacks transmembrane and intracellular domains as a consequence of alternative processing of the corresponding mRNA. The physiological and pathological significance of BP is still a matter of speculation, certainly for GH-BP and PRL-BP (Heaney and Golde, 1993; Rose-John and Heinrich, 1994). As mentioned before, specific receptors for placental lactogens have not been formally identified. A receptor that binds the 16-kDa N-terminal fragment of PRL but not 23-kDa PRL has been partially characterized (Clapp and Weiner, 1992).

B. THEPROLACTIN RECEPTOR 1, Structure and Tissue Expression In addition to the full-length (long, 85 kDa) receptor and the BP (36 kDa), a short form (40 kDa) of the receptor, generated by alternative splicing of the mRNA, has been characterized in the rat. In addition, the Nb2 rat lymphoma line, used for many PRL-R studies and bioassays of lactogenic hormones, expresses a truncated form of the long receptor. In man, only the long form was known. Recently, however, preliminary evidence for a short isoform in the human was also reported (Clevenger et al., 1995). The PRL-R is strongly expressed in the mammary gland and in the liver. Actually, most rat tissues express PRL-R, both the long and the short form. The function of the short form is unknown since it appears unable to mediate PRL signaling (O'Neal et al., 1994).

2. PRL Receptor Expression in Lymphohenwpoietic System a. Binding Studies. Following pioneer studies by D. Russell, who demonstrated binding of PRL to human lymphocytes, many investigators have found PRL-R on resting and activated leukocytes (reviewed in Gala, 1991). Matera et al. (1988) confirmed binding of PRL to human B and T lymphocytes and found also PRL-R on large granular lymphocytes ( N K cells). NK cells expressed 660 receptors per cell with a & of about 9.10-"' M , whereas T and B cells expressed an average of 320 receptors per cell (& about 1.7.10-9M ). For comparison, Nb2 cells expressed 12,000 receptors per cell (Russell et al., 1985; Matera et al., 1988).

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b. Zmmunocytochemisty. In rat embryonic thymus, monoclonal antibodies to the PRL-R stained cortical lymphocytes and probably also epithelial cells from Day 19.5 (Royster et al., 1995). Using immunolabeling and flow cytometry, Dardenne et al. (1994) and Gagnerault et al. (1993) detected PRL-R on at least 80% of bone marrow cells from man, mouse, or rat. On human PBMC, labeling was strong on B lymphocytes, intermediate on monocytes and low on T cells. The labeling intensity on thymocytes was weak except for some double negative cells (man, mouse) and some CD4'3 CD8- cells (man) or CD4-. CD8+ cells (mouse). In the murine spleen, PRL-R density was lowest on Thy-1 cells, intermediate on B-220' cells, and highest on Mac-lt cells. Gala and Shevach (1993a) found a high density of PRL-R on mouse peritoneal macrophages. c. RNA Studies. (i) In situ hybridization. Both the long and the short form of the receptor are expressed in most tissues examined. In the fetal rat thymus, expression increased significantly between embryonic Day 17.5 and Day 19 in cortical lymphocytes and thymic epithelial cells (Royster et al., 1995). In the adult thymus, expression was considered to be higher in the cortical than in the medullary area. This may, however, be due to a higher cell density. The expression in thymic epithelial cells was verified by complementary investigations including functional assays (Dardenne et al., 1991). In the spleen, receptor expression was higher in the red pulp than in the white pulp (Ouhtit et al., 1993). (ii) PCR. The expression of PRL-R in murine thymocytes and splenocytes after Con A stimulation was first reported by O'Neal et al. (1991). In human thymocytes and PBMC fractions enriched in monocytes, B or T lymphocytes, Pellegrini et al. (1992) found PRL-R mRNA. Expression of both forms of the receptor was confirmed by PCR in thymus, spleen, lymph nodes, and bone marrow of rats and mice (Touraine et al., 1994).

d. Regulation. There are only few data on the regulation of PRL-R. Receptor expression in rat lymphocytes correlated inversely with PRL serum levels (Di Carlo et al., 1995).The proportion of murine lymph node cells expressing PRL-R increased markedly after nonspecific immunological stimulation in vivo (with either Con A or Freunds adjuvant) (Gala and Shevach, 1993b; Gagnerault et al., 1993; Koh and Phillips, 1993). In the human, hyperprolactinemia had little effect on PRL-R expression in PBMC. In patients with macroprolactinoma, there was a significant decrease only in the percentage of CD8' T cells expressing PRL-R and treatment with bromocriptine had no effect on the expression of PRL-R. High serum GH levels had no effect on the expression of PRL-R on PBMC

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(Leite-de-Moraes et al., 1995). As was the case in rodents, the intensity of labeling in T cells increased after T cell activation (Dardenne et al., 1994). e. Expression in Cell Lines. Leukemia and lymphoma lines often express PRL-R (Dardenne d al., 1994; O'Neal et al., 1991). The role of PRL-R in leukemia development and progression will be considered briefly in Section VIII.

C. THEGROWTHHORMONE RECEPTOR 1. Structure and Tissue Expression In contrast with the PRL-R, no short form of the GH-R has been described. A soluble form (GH-BP) does exist, however. Variability in the length of the 5' untranslated region accounts for the heterogeneity in mRNA size (Southard et al., 1995).The fact that the M , of GH-R (130 kDa) exceeds the value computed from the amino acid sequence (70 kDa) is due to extensive glycosylation and ubiquitinylation. Ubiquitinylation is a feature common to several leukocyte receptors such as the T-cell receptor, membrane immunoglobulin, the lymphocyte homing receptor (MEL-14 protein), the TNF-R, and the high-affinity Fce-R. Its significance remains unknown (Jennissen, 1995). Most tissues express GH-R. In addition to the liver, other target cell types have received much attention recently (adipocytes, islet B cells, nerve cells, the mammary epithelium, and leukocytes), but there is less information about GH-R than PRL-R on leukocytes.

2. GH Receptor Expression in the Lymphohemopoietic System The transformed human B cell line IM-9 has been extensively used for GH-R studies. Lesniak et al. (1974) showed that the cells expressed 4000 GH binding sites with a dissociation constant (&) of 0.77 nM. The GHR on IM-9 cells was characterized by Asakawa et al. (1986) by affinity cross-linking studies. Other workers showed that GH induces tyrosine phosphorylation of several substrates in IM-9B cells (Clayton et al., 1994). Binding sites for bovine GH have been detected on bovine and murine thymocytes (Arrenbrecht, 1974). GH receptors on PBMC were characterized by Kiess and Butenandt (1985). They found an average of 6800 binding sites for human GH per cell with a K,, of 0.67 nM on PBMC that were conditioned in Tris buffer at 20°C. Displacement of '=I-GH by PRL was 40 times less effective as compared to human GH, indicating that the GH binding sites were GHRs. Badolato et al. (1994) studied the distribution of GH-R on PBMC subsets by two-color flow cytometry. They reported high levels of GH-R expression on circulating B cells compared to T cells and NK cells using

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both GH-R-specific mAb and fluorescein-labeled GH (Badolato et al., 1994).Furthermore, they showed that B cells expressed more GH-R transcripts than T cells. Human tonsillar lymphoid cells expressed higher GHR levels when activated in culture (0.Thellin, personal communication). Binding sites for human GH have also been shown on rat liver macrophages (Kover and Moore, 1983),and on a murine thymic epithelial cell line (Ban et al., 1991). Competition binding assays indicated that human GH was bound to the GH-R and not to the PRL-R. THROUGH THE PRL-R A N D GH-R A N D D. SIGNALING GENEREGULATION Binding of GH and PRL to their receptors has been reviewed by Wells et al. (1993) and Horseman and Yu-Lee (1994). PRL and GH only bind to their own receptor; primate GH, however, also binds to the PRL-R in the presence of physiological concentrations of Zn2+and induces effects via this receptor. Like PRL, primate GH also binds to PRL-Rs from other species. Thus, to assess whether effects of human GH are mediated by the GH-R, human GH should not be used. Instead, the use of ovine GH ensures that effects are mediated by a bona fide GH-R. For recent studies, recombinant hormones have been available. Site-directed mutagenesis of the receptor has also been performed to examine structural requirements for the receptor or the hormone molecule (Fu et al., 1992) (see Section V.A.2).

1. Signaling Upon binding of GH to one receptor molecule, a second receptor molecule is recruited. Dimerization is required for signaling. Indeed, GH-R as well as PRL-R can be stimulated by divalent but not by monovalent antibodies. Also, in Laron dwarfism, as a result of a mutation in the GH-R, GH binding, which is suboptimal, is not followed by dimerization or by signaling (Duquesnoy et al., 1994). General principles of signaling through cytokine-hemopoietin receptors, a field of active research, have been reviewed by Horseman and Yu-Lee (1994) and by Ihle (1995). As several receptors share the same JAK or STAT molecules in their signaling pathways (just as different receptors can signal through CAMP), responses may be amplified if two cytokines reinforce each other’s signal. In contrast, competition for limited amounts of signaling molecules may result in blunted responses to one or to both cytokines (see Fig. 2). a. The JAK-STAT-GAS Pathway. The best-documented signaling pathway after stimulation with either PRL or GH starts with the activation

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FIG.2. Coninion features in signal transduction pathways for PRL, GH, IGF-I, and classical cytokines.The model (see Section 1II.D)shows that GH, like PRL, shares signaling inolecules with other members of the cytokine-hemopoietin faniily (e.g..IL-6). For instance, signaling through the PRL-H, GH-R, and IL-6R depends on their association with Janus kinases (JAK). One of these kinases (JAK2)associates with dl these receptors. Subsequently, coinnion STAT transcription factors will be phosphorylated on tyrosine residues (Y-P) and translocated to the nucleus (Jans et al., 1995). Both IGF-I and IL-4 induce the tyrosine phosphorylation of IRS-1 and IRS-2, which function as docking inolecules for other signaling niolecules, such as phospliatidylinositol-3-kinase(P13-kinase), a tyrosine phosphatase (SHPTP), and Grb2-SOS. The use of coiiiinon signaling molecules may lead to redundaiit actions of PRL, GH, IGF-I, and classical cytokines.

of Janus kinases (JAK) (Ihle, 1995; Ihle et al., 1995). The intracellular proline-rich consensus sequence of the receptor (box 1) interacts with JAK-2. JAK-2 is bound, together with JAK-1, to the PRL-€3. In the case of the GH-R, JAK-2 is recruited upon dimerization. Phosporylation events ensue and include the autophosphorylation of JAK-2, the phosphorylation of the receptor, and the phosphorylation of STAT proteins (STAT1 and 5). STAT stands for signal transducer and activator of transcription. Indeed, activated STAT molecules (as a result of phosphorylation) translocate to the niicleus where they interact with other transcription factors to regulate gene expression (Jans, 1995).Alternatively, they can bind directly to DNA elements and activate gene transcription on their own. Such DNA elements often are GAS, so named because they were first identified during responses to IFNy, hence, gamma IFN activated sequences (Wang and Yu-Lee, 1996). It is important to remember that the JAK-STAT pathways are used by many different growth factors. We will discuss later the implications of redundancy when analyzingthe biological activities of GH and PRL. Recent reports also establish the importance of JAK-STAT pathways (in particular

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STAT 3 and STAT S) in leukemic transformation (Danial et al., 1995; Migone et al., 1995).

b. Other Pathways. Even in model systems, the PRL and GH signaling pathways have not been fully characterized. The respective importance and the overlap between the different pathways are known to a limited extent only. For instance, activation through p59 Fyn (a member of the src family) following stimulation of Nb2 cells with PRL has been reported but the downstream signaling pathway is not known (Clevenger and Medaglia, 1994). Especially relevant to the hemopoietic lineage is the requirement for Vav expression for PRL-driven proliferation of a Nb2 cell line (Clevenger et al., 1995) as Vav is expressed selectively in hemopoietic cells. Following activation, Vav is translocated into the nucleus, where it can interact with various proteins and influence RNA transcription. The involvement of Ras in PRL signaling has been studied recently in Nb2 cells and appeared to be mediated by signaling proteins SHC, growth factor receptor bound 2 (Grbe),and the guanine nucleotide exchange factor son of sevenless (SOS), but not by Vav or p120 Ras-GAP (Erwin et al., 1995). A similar mechanism is used by the T lymphocyte antigen receptor to activate Ras (Holsinger et al., 1995). Raf-1 can be activated by Ras and the activation of Raf-1 has indeed been described after stimulation with PRL (Clevenger et al., 1994). This can initiate a cascade of kinases including MAP-kinase (ERK) and MAP-kinase kinase, with Jun and Fos as targets. In Nb2 cells responding to PRL and to IL-2, cell proliferation was associated with the early GTP binding to Ras and tyrosyl phosphorylation, activation and nuclear translocation of MAP kinase (Rao et al., 1995). Several metabolic responses to GH result from stimulation of the protein kinase C pathway and protein kinase C has also been implicated in PRLR signaling (Buckley et al., 1988; Pasqualini et al., 1994, 1994; Rao et al., 1995). Insulin-like effects of GH, however, are mediated through the insulin receptor substrate-1 (IRS-1) which was phosphorylated within 1 min after addition of GH to rat adipocytes. IRS-1 could be a substrate for JAK 2 or for another kinase activated by GH (Souza et d.,1994). Several independent laboratories have observed the transport of PRL into the nucleus (Clevenger et al., 199Ob).This might be a secondary signal transduction pathway, in addition to the stimulation via the membrane receptor. The internalization of PRL is not required for stimulation, as antibodies can trigger the receptor in the absence of PRL. Ligand-mediated internalization of the GH-R results in down-regulation of the receptor but is not a prerequisite for transcriptional signaling (Allevato et al., 1995).

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2. Gene Regulation by GH and PRL a. PRL. Effects of PRL have been studied most extensively in mammary gland explants and cell lines and in the Nb2 lymphoma cells. PRL is only one of the hormones involved in the development and the activity of the mammary gland. PRL participates to the control of milk protein expression. As some leukocytes express milk proteins (casein, lactoferrin), it could be proposed, if these cells bear PRL-R, that PRL controls the expression of milk protein in leukocytes (Grusby et al., 1990; Maher et al., 1993). It was also noticed in the mammary gland that PRL controlled the expression of glycosyl-transferases (Golden and Rillema, 1995) but it remains to be tested if this is also the case in other systems, in particular in leukocytes. Four hours after exposure to PRL in the presence of cycloheximide, the most strongly induced gene in quiescent Nb2 lymphoma cells was the transcription factor IRF-1. PRL also induced the expression of other transcription factors (c-Fos, c-Myc, Gfi-1),the heat shock protein Hsp70, IFNy, p-actin, the serinehhreonine kinase Pim- 1, and the enzyme ornithinedecarboxylase (catalyzing the production of putrescine and other polyamines). Genes induced by PRL in normal splenocytes have also been studied. The fact that there was a good overlap between genes induced by PRL and those induced by Con A can be taken as an argument for the relevance of PRL as potential lymphoid growth or differentiation factor (Yu-Lee et al., 1990; Schwartz et al., 1992; Horseman and Yu-Lee, 1994; Buckley et al., 1995).

b. GH. Many effects of GH are mediated through IGF-I but GH can also exert direct effects on several cell types. Some metabolic responses, for instance, do not require transcriptional activation and are mediated primarily by protein kinase C. The situation may be more complex: in the J774 macrophage cell line, as in mouse peritoneal macrophages, GH stimulates the uptake and degradation of low-density lipoprotein but this effect is mediated by (autocrine) IGF-I as the effect of GH is abrogated by adding anti-IGF-I antibody to the culture (Hochberg et al., 1992). A rapid rise in the transcription of c-Fos, c-Jun, IGF-I and the serine protease inhibitor Spi 2a (also called Spi 2.1) follows the exposure of hepatocytes to GH (Gronowski and Rotwein, 1995). The induction of cytochrome P450 2C12 and of receptors for EGF, PRL, and GH itself proceeds more slowly (reviewed in Horseman and Yu-Lee, 1994; HooghePeters and Hooghe, 1995). c. Summay. PRL and GH can induce metabolic responses and stimulate the expression of transcription factors, growth factors, receptors for

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growth factors, enzymes, enzyme inhibitors, etc. As a result, cells proliferate, differentiate, or increase their functional activity.

E. INSULIN-LIKE GROWTHFACTOR A N D INSULIN RECEPTORS 1 . Structure and Tissue Expression The cellular effects of IGFs are mediated by transmembrane receptors which include the receptors for insulin ( Ins-R), IGF-I (IGF-I-R), and IGF-II/mannose-6-phosphate (IGF-II-R).Like the Ins-R, the IGF-I-R is a member of the tyrosine-kinase receptor heterotetrameric molecule (@aaP) family. These receptors are composed of two identical light chains (90 kDa) which are exclusively extracellular (a), and 2 identical heavy chains ( 125 kDa) having extracellular, transmembrane, and intracytoplasmic domains (P). The a and P chains are disulfide linked. A single gene codes for the a and the chains, which are post-translationally processed from a single precursor molecule that undergoes glycosylation and cleavage. The IGF-II-R shows no homology with the Ins-R and IGF-I-R. IGF-I binds with a high affinity to the IGF-I-R, and with a much lower affinity to the IGF-II-R and the Ins-R. IGF-I1 binds with a high affinity to its own receptor, but it also binds to the IGF-I-R with a 2-10 times lower affinity as compared to IGF-I (Rechler and Nissley, 1985; Nissley et al., 1991). Many, but not all, in uiuo effects of IGF-I1 are mediated by the IGF-I-R. IGF-I1 also binds to the Ins-R, but insulin only binds to its own receptor and to the IGF-I-R. This low-affinity binding of insulin to the IGF-I-R is only relevant for in uitro studies with pharmacological concentrations of insulin. Most tissues express IGF-I-R, with the exception of the liver. Highest expression is found in the brain, kidney, heart, testis, and lung. A biological effect of IGF-I is considered to be mediated through the IGF-I-R if (1) IGF-I is more potent (elicits the effect at a lower concentration) than insulin in mediating biological effects and (2) the effect is prevented by an antibody to the IGF-I-R, e.g., monoclonal antibody aIR3 (reacting only with the human IGF-I-R). 2. Signaling Ligand binding to the Ins-R and IGF-I-R induces activation of the receptor’s intrinsic tyrosine kinases and subsequent autophosphorylation on tyrosine residues of the intracellular P chains. Tyrosine-phosphorylated sites bind to Src homology 2 (SH2) binding domains in downstream signaling proteins. However, the tyrosine phosphorylated Ins-R and IGF-I-R interact less well with these proteins as compared to other tyrosine kinase receptors such as those for epidermal growth factor and PDGF. Instead, the IGF-I-R and Ins-R phosphorylate insulin receptor substrate (IRS)

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proteins (Myers and White, 1995). IRS-1 regulates mitogenesis, and glucose transport probably via activation of p21"" and phosphatidylinositol-3kinase (PIS-K), respectively. This cytoplasmic protein possesses 21 tyrosine phosphorylation sites and can be considered as docking molecules for SH2 proteins, such as the p85 regulatory subunit of PIS-K, the Grb2-SOS complex, and a tyrosine phosphatase (SHPTPJ. Grb2-SOS regulates p21"' which activates downstream kinases (Raf-1, MAP-kinase kinase, and MAPkinase). An alternative p21"'activation pathway is the binding of Grb2-SOS to the IGF-I-R via SHC. IRS-1 has also been implicated in other receptor signaling systems. They mediate signaling for GH, IL-4, IL-9, IL-13, LIF, IFNa, and IFNP (Waters and Pessin, 1990). For instance, the IGF-I- and IL-4-induced mitogenesis in the 32-D premyeloid cell line requires IRS proteins. A second IRS-signaling protein, IRS-2, recently cloned from the FDC-P2 myeloid progenitor cell line, has also been implicated in both insulin and cytokine signaling (Sun et nl., 1995). A function for IRS proteins in cells of the immune system is also indicated by the stimulation of IRS phosphorylation and its association with PIS-K after stimulation of priniary thymocyte cultures with IGF-I (Kooijmanet al., 199%). An IRS-I independent pathway in niyeloid cells is also indicated by Uddin et nl. (1996), who demonstrated the phosphorylation of the Vav protooncogene product. 3. IGF-1-R Expression and Regdation in the Lyniphohemopoietic System Using radioligand binding studies, IGF-I-Rs were detected on human peripheral T cells (Kozak et nl., 1987; Tapson et al., 1988; Johnson et a l , 1992; Kooijman et nl., 1992a), monocytes (Kooijman et al., 1992a), and thymocytes (Verland and Gammeltoft, 1989; Kooijman et al., 1995b). The K,, of these receptors varied from 0.12 to 0.25 nM. The identity of the IGF-I-R on T cells was confirmed by affinity cross-linking experiments (Kozak et ul., 1987; Tapson et al., 1988; Johnson et al., 1992). Furthermore, binding of 12'I-IGF-I to T cells, monocytes, and thymocytes could be blocked by an anti-IGF-I-R mAb, d R 3 (Kozak et al., 1987; Kooijman et al., 1992a; Kooijman et al., 1995b) which proves that it is bound to IGFI-Rs and not to other binding sites such as IGFBPs. Multicolor flowcytoinetry, using aIR3 as a IGF-I-R specific mAb, revealed that IGF-I-Rs are expressed on all major subpopulations of human peripheral blood mononuclear cells: CD4' T cells, CD8+ T cells, monocytes, NK cells, and B cells (Stuart et al., 1991; Kooijman et al., 1992a). In addition, human thymocytes of all developmental stages (CD4-CD8-, CD4+CD8 CD4-CD8', and CD4'CD8-) expressed IGF-I-R (Kooijman et al., 1995b). Interestingly, it has been shown that IGF-I-R expression on mature peripheral T cells can be modulated by T cell activation induced +

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by anti-CD3 and mitogenic lectins. Kozak et al. (1987) showed a transient increase in the amount of '251-IGF-I bound to T cells after activation with PHA. The same effect was observed by Johnson et al. (1992) after stimulation with soluble as well as immobilized anti-CD3. T cell stimulation with anti-CD3 also resulted in an increase in IGF-I transcripts (Hartmann et al., 1992). Although the effects of T cell stimulation on IGF-I-R expression appeared to be dependent on culture conditions ( Johnson et al., 1992), high levels of IGF-I-R expression during the culture period coincided with high rates of DNA synthesis (Kozak et al., 1987; Johnson et al., 1992). The importance of IGF-I-Rs in human peripheral blood T cell proliferation is also indicated by the finding that T cells cannot enter the S-phase when the expression of IGF-I-Rs is suppressed by anti-sense RNA (Reiss et al., 1992). The modulatory effects of anti-CD3 on IGF-I-R expression, and the observation that both CD4t and CD8+ T cell subsets contain subpopulations expressing different levels of IGF-I-Rs (Kooijman et al., 1992a) indicate that T cells at different stages may express different levels of receptors. Indeed, three-color flow cytometry, using aIR3 as a IGF-I-R specific mAb, revealed that IGF-I-Rs are differentially expressed on T cells at different developmental stages (Kooijman et al., 1995c). Both, CD4' and CD8+ peripheral T cells can be divided into two classes: naive cells which have not encountered antigen and memory T cells which have been stimulated by antigen and have returned into a quiescent state. These two fractions can be identified by the expression of different isoforms of CD45. CD45RA is mainly expressed on naive T cells, whereas CD45RO is expressed on memory T cells. It appeared that 87% of the CD4'CD45RAC cells and 66% of the CD8+CD45RAt cells were aIR3+, whereas only 37% of the CD45RO' cells (CD4+and CD8+) bound aIR3. Furthermore, the fraction of aIR3' cells within in vivo- or in uitro-activated (IILA-DRt) T cells was markedly lower than in nonactivated (HLA-DR-) cells. In vitro PHAactivated T cells and CD4'CD45ROt cells activated with recall antigens also contained much less aIR3' cells (1-6%) than nonactivated cells (30-54%) (Kooijman et al., 1995c).The data do not indicate whether PHA and recall antigens preferentially activate aIR3- cells, or whether the IGF-I-Rs are down-regulated. The differential expression of IGF-I binding sites in rat has been studied by two-color flow cytometry using biotin-labeled des( 1-3)IGF-I, which binds poorly to IGFBP but binds to IGF-I-Rs with an affinity comparable to that of IGF-I (Xu et al., 1995). The binding capacity for des(1-3)IGF-I was high on monocytes, intermediate on B cells, and low on T cells. Furthermore, the number of receptors on CD4' T cells was higher than on CD8', and Con A stimulation increased the number of receptors on both

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CD4' and CD8' T cells. Functional IGF-I-Rs have also been described on human erythroid precursors (Cotton et al., 1991), and on lymphoid and myeloid cell lines (Freund et al., 1994; Cross et al., 1995). IV. In Wvo Effects of PRL, GH, and IGF-I on the Lymphohemopoietic System

A. IN Vrvc., EFFECTSIN RODENTS

The in vivo effects of GH and PRL on the lymphohemopoietic system have been studied to a large extent in hypophysectomized rats and hypopituitary dwarf mice. Hypophysectomized rats exhibit reduced serum levels of all pituitary hormones (see Section IX). They have impaired humoral, cellular, and nonspecific immune responses, and injections of either PRL or GH corrected these deficiencies. Hypopituitary Snell-Bagg dwarf mice are deficient in PRL, GH, and TSH due to a mutation in the pituitary transcription factor Pit-1 (Li et al., 1990). However, splenocytes from dwarf mice secreted a residual amount of bioactive PRL-like activity when compared to that of normal littermates (Gala, 1995b). In addition, dwarf mice showed an atrophy of the thymus and the peripheral lymphoid organs, a decreased number of splenic T and B cells, and a cellular depletion of the bone marrow. The immunological defects included humoral and cellular immune responses. Similar immunological findings were observed in the hypopituitary Ames dwarf mouse. Some groups, however, showed that Snell-Bagg dwarf mice displayed normal humoral and cell-mediated responses. Differences in age and animal nursing could be responsible for this discrepancy. For an overview of early studies on GH and PRL effects on immune cell function and development of hypophysectomized rats, dwarf mice, and other animals, we refer to reviews (Kelley, 1989; Gala, 1991; Hooghe-Peters and Hooghe, 1995). Here, we will present more recent work together with the most important findings that point to a role for PRL and GH in the humoral, cellular, and nonspecific immune system. 1. Hemopoiesis and Myelopoiesis Snell dwarfmice, which lack PRL, GH, and TSH, have decreased peripheral blood counts that affect all lineages. All hematological parameters were improved after 2 weeks of treatment with recombinant human GH (20 pg daily per mouse). In Balb/c mice, GH (20 pg every other day) partially prevented the hematological toxicity of azathioprine (Murphy et al., 1992a). Hypophysectomyis rather well tolerated: 5 weeks after hypophysectomy, leukocytosis was higher in operated than in control rats and anemia and thrombopenia were moderate. Hematological protection was afforded by


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PRL, GH, and human placental lactogen (Berczi and Nagy, 1991). After hypophysectomy, PRL levels, that had dropped to 15%of control values after 5 weeks, returned to 50% of control values after 9 weeks. To achieve a more complete PRL depletion, Nagy and Berczi (1991) have injected hypophysectomized rats with anti PRL serum. After 7 weeks of treatment, all rats had died with a moderate pancytopenia. Although additional work is needed to understand the detailed pathological mechanisms, these experiments indicate that bone marrow hemopoietic activity requires PRL in rats. Transgenic mice expressing either bovine GH or human GHRH have splenic hyperplasia with increased numbers of erythroid and megakaryocytic progenitor cells. Multilineage progenitor cells were also increased in GHRH transgenic mice as indicated by the number of day 10 CFU colonies and splenocytes have increased DNA synthesis (Blazar et al., 1995). In the human, pan-hypopituitarism has been linked occasionally with hematological problems. As a rule, however, hypophysectomy is well tolerated and there is no hematological deficit in patients receiving as substitutive treatment thyroid hormone and steroids only, which implies that pituitary PRL and GH are dispensable. 2. Lymphocyte Development The hypopituitary Snell-Bagg dwarf mouse exhibits an age-dependent loss of CD4'CD8' thymocytes and reduced mitogenic response (Cross et al., 1992). The loss of CD4+CD8' thymocytes after weaning coincided with the appearance of CD4'CD8' cells in the lymph nodes, suggesting that immature thymocytes migrate to the periphery (Murphy et al., 1992b). After treatment with human GH, the distribution of CD4'CD8+ cells was normalized, and thymic hypoplasia was partially corrected. Ovine GH, which in contrast to human GH does not bind to the murine PRL-R, also reversed the decrease in CD4'CD8+ cells in the thymus, indicating that GH actedvia the GH-R. Dwarf mice had hypoplastic spleens with a normal proportion of B cells and T cell subsets (Murphyet al., 1992a).Additionally, pre-B cells (B220+IgG-)were not present in bone marrow, although the proportion of mature B cells (B220+IgG+)was not affected. Treatment with human GH increased the total number of B, CD4+,and CD8' cells, but it did not reconstitiute the missing pre-B cell compartment in bone marrow. The data indicate that a deficiency in other factors also contributes to the reduced B cell counts in dwarf mice. Peripheral blood cell counts revealed a reduced number of leukocytes and lymphocytes which could be partially corrected by GH treatment. In a further study, the authors compared the effects of ovine PRL and ovine GH on T cell development and function in dwarf mice (Murphy et al., 1993). GH increased thymic cellularity with a factor 2, but had little

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effect on peripheral T cell responses. PRL, however, decreased thymic cellularity, but increased the number of lymph node cells in immunized mice but not in unprimed mice. Accordingly, PRL augmented antigenspecific induction of DNA-synthesis in lymph node cells, but not the Con A-induced 3H-thymidine uptake. The role of GI-I in T cell development has been further explored in severe combined immunodeficient (SCID) mice which lack mature lynphocytes. In these mice, human GH promoted the engraftment of syngeneic thymocytes and human PBMC (Murphy et al., 1992c; Taub et d., 1994).The cellular basis of this phenomenon is discussed in Section V.C.l. Impaired T cell-mediated responses in dwarf mice can be explained by a reduced T cell activation in uiuo, because the in vivo IL-2R induction in peripheral lymph node cells by Con A was only 50% of that in controls. This response could be completely corrected by treatment with either bovine PRL or bovine GH (Gala and Shevach, 1993b). However, the reduced number of B cells in lymph nodes was not corrected by hormone treatment. Furthermore, when thyroxine-treated Snell dwarf mice were given a combination of bovine GH and PRL, the reduced numbers of B cells (B220t) and macrophages (Mac-1' ) were corrected (Gala, 1995a). This result indicates that reconstitution with several hormones might be necessary to obtain complete correction of the immune system in dwarf mice. Clark et al. (1993) investigated the in uivo effects of IGF-I on hemopoiesis in Balb/c mice using minipumps that injected 100 p g rhIGF-I per day. After a 7-day treatment period, the lymphocyte numbers in thymus, spleen, and lymph nodes were increased. In peripheral blood leukocytes, only the neutrophil count was significantly increased (from 33 to 48%). Subset analysis showed that the number of splenic B cells was increased by 270%, the CD4+ cells by 200%, and the CD8+ cells by 167%. The thymus of IGF-I-treated mice showed an increased fraction of cells stained by peanut agglutinin, a marker of immature thymocytes. In addition, immunoglobulin production in response to dinitrophenyl ovalbumin was augmented by treatment with IGF-I for 14 days. The effect of IGF-I on B and T cell reconstitution in immunocompromised mice was examined by IGF-I treatment of irradiated mice after reconstitution with syngeneic bone marrow (Jardieu et al., 1994). Fourteen days after irradiation and transplantation, a two- to threefold increase in the number of splenic B and T cells was observed. The effect of IGF-I treatment on the number of IgM' cells in spleen coincided with an increase in numbers of B cell progenitors (sIgMtB220-) and mature B cells (sIgM ) in bone marrow. In addition, the frequency of splenic B220' cells in the S-phase of the cell cycle was increased in IGF-I-treated mice. +

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In addition to the above-mentioned effects of IGF-I on the T cell compartment, there are several indications for a role of IGF-I in T cell development. Binz et al. (1990) demonstrated that administration of IGFI to diabetic rats stimulated DNA synthesis in thymocytes, and increased the number of CD4+/CD8+ thymocytes. Administration of IGF-I also partially corrected the histology of the thymus; the thymus became larger, regained a distinct lobular architecture with a more delineated cortex and medulla. Beschorner et al. (1991) showed that human GH and IGF-I enhanced thymic recovery after cyclosporin treatment. GH or IGF-I treatment resulted in an enlargement of the thymus, an increase in CD4+CD8cells, the regeneration of Hassall's corpuscles, and an increased expression of class I1 MHC. 3. Humoral lmmunity and Cellular lmmunity The antibody response to sheep red blood cells in hypophysectomized rats (reduced to 15%of the response in control rats) could be enhanced by treatment with purified rat and bovine PRL. Rat, bovine, and human GH corrected the antibody response to 60-75% of control values. Contact sensitivity was also impaired in hypophysectomized rats. Treatment with rat GH or rat PRL corrected both the humoral and the cellular response (Nagy et al., 1983). B cell function was tested by immunization of irradiated mice with DNP-ovalbumin 2 weeks after bone marrow reconstitution. IGF-I stimulated antigen-specific IgG synthesis by 60-80%. The primary and secondary antibody responses to DNP-OVA in normal mice with implanted minipumps were also increased by IGF-I as compared to excipient-injected mice (Robbins et al., 1994). 4. The Nonspecijc lmmune System

There are several indications that PRL and GH are important factors in the regulation of nonspecific immune responses. In mice treated with bromocriptine to suppress PRL secretion, the production of IFNy and the T cell-dependent macrophage activation were suppressed (Bernton et al., 1988).Hypophysectomized rats showed an increased susceptibility to lethal effects of Salmonella infection (Edwards et al., 1991b). In both intact and hypophysectomized rats, porcine GH was as effective as IFNy in increasing the survival rate (2-4 times). Additionally, macrophages from hypophysectomized rats exhibited a 50%reduced capacity to kill extracellular Salmonella as compared to those derived from intact rats. In vivo treatment with either GH or IFNy increased the ability of macrophages isolated from intact and hypophysectomized rats to kill extracellular bacteria in vitro by 285%. These results indicate that GH can stimulate the

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nonspecific immune response. In addition, rat and porcine GH and IFNy partially corrected the reduction of in uiuo TNFa synthesis by macrophages (Edwards et al., 1991a),and the increased resistance to Salmonella infection after GH or IFNy treatment correlatedwith an increased release of reactive oxygen metabolites (Edwards et al., 1992). In aged rats, implantation of the GH-secreting syngeneic GH3 cell line in combination with IFNy treatment reversed the reduced superoxide anion production and bactericidal activity of neutrophils (Fu et al., 1994).Di Carlo et al. (1993) reported that normal mice were protected against the lethal effects of Salmonella by treatment with ovine PRL. This treatment also resulted in an increased phagocytosis and intracellular killing by peritoneal macrophages as measured in uitro.

B. IN Vrvo EFFECTSOF GH IN MAN In viuo studies on the role of GH in humans have been confined mainly to GH-deficient children. These children do not show an increased number of infections and they are not considered to be immunodeficient (reviewed by Kelley (1989) and Gala (1991)).They generally show normal levels of T cell subsets, B cells, and serum immunoglobulins. The autologous mixed lymphocyte response and the B and T cell mitogenic responses were not impaired. On the other hand, several changes in the immune system of GH-deficient patients have been noted, such as decreased cytotoxic activity of NK cells (Bozzola et al., 1990; Kiess et al., 1988), impaired in vitro IgM production (Bozzola et al., 1989), reduced phagocytotic capacity of monocytes and polymorphonuclear cells (Manfredi et al., 1994), and reduced in uitro production of IL-la and IL-2 (Casanova et al., 1990). Additionally, children with a rare X-linked combination of GH deficiency and hypogammaglobulinemia showed a reduced B cell count and an impaired ability to synthesize antibodies to several clinically important antigens such as tetanus toxoid and pneumococcal polysaccharide, whereas the cellular immunity was not affected (Fleisher et al., 1980). The defect in humoral immunity was not corrected by GH replacement therapy. The humoral defect resembles that in X-linked agammaglobulinemia (Brutontype) (Sitz et al., 1990), and is not found in other GH-deficient patients. Therefore, it is likely that these patients have a defect in a gene or chromosomal region that is critical for both GH production and B cell development. Studies on the effects of GH replacement therapy in GH-deficient children yielded contradicting results with respect to the number of circulating B cells and T cell subsets (reviewed by Wit et al. (1993)). Decreased B cell numbers during GH therapy were observed in some but not all studies. However, the reductions in B cell counts observed in these studies are generally not indicative of immunodeficiency, and in two studies the effects

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were transient. Moreover, reduced in vitro IgM synthesis in GH-deficient children was partially corrected by GH therapy (Bozzola et nl., 1989). In addition, GH therapy normalized the impaired production of IL-1 and of IL-2 by PHA-activated T cells (Casanova et al., 1990), and completely corrected impaired NK cell activity (Bozzola et al., 1990). GH treatment of adult women with an impaired GH secretion also resulted in an increased natural killer cell activity (Crist et al., 1987). The effects of GH therapy have also been studied in girls with Turner's syndrome. It has been established that endogenous GH secretion is normal in quantitative terms, although some small abnormalities in the profile have been reported. Upon GH treatment, the decreased numbers of NK cell returned to normal, and some patients showed a decrease in circulating B cells, without B cell function impairment as demonstrated by a normal in vitro antibody response (Rongen Westerlaken et al., 1991). Some data indicate that supranormal GH levels stimulate some immune functions in adults. Indeed, supplementation of GH in healthy adults with normal GH secretion resulted in a small increase in NK cell activity (Crist et al., 1987). In acromegalic patients pronounced elevation of GH was related to an enhanced phagocytic activity. However, NK cell activity, serum concentrations of immunoglobulins, and B cells and T cell subsets were normal (Kotzmann et al., 1994). The possible contribution of GH therapy to the development or progression of leukemia is discussed in Section VIII. I N HYPERPROLACTINEMIC PATIENTS C. IMMUNOLOGICAL PARAMETEHS The effects of high serum levels of PRL on the immune system have been addressed in a few studies in hyperprolactinemic patients. Serum PRL levels in these patients range from 1.3 to 80 nM, compared to 0.1 to 0.8 nM in control subjects. Serum concentrations of immunoglobulins, and B and T cell subsets in patients were within the normal range or only slightly changed. One study reported a decreased number of NK cells in women with hyperprolactimemia (Gerli et al., 1986, 1987). In another study, it was demonstrated that the bromocriptine therapy corrected the NK cell number and their activity (Gerli et al., 1987). In a group of 33 female patients, the lymphokine-activated killer cell activity was not affected (Matera et aZ., 1992b). Two studies observed a T cell dysregulation in patients with hyperprolactinemia. Mitogen-induced T cell proliferation and IL-2 production were impaired (Vidaller et al., 1986), and an increased proportion of immature (CDl') T cells and transferrin receptor+ (activated) CD4+ T cells was reported by Gerli et aZ. (1987). The abnormalities were completely or partially corrected after treatment with bromocriptine. A reduced percent-

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age and number of CD8+T cells was observed in PBMC from hyperprolactinemic patients, whereas the total number and percentage of CD4' T cells, B cells, and monocytes was not changed (Leite-de-Moraes et al., 1995). In addition, the CD8+ subset expressed increased levels of PRLR. These defects were not corrected after bromocriptine treatment. It should be noted that in uitro studies (see Section V.C) revealed that the effects of supraphysiological PRL concentrations on T and NK cells are different from those induced by physiological concentrations. The relationship between serum PRL levels and autoimmunity will be discussed in Section VII. V. In Wtm Effech of PRL, GH, and IGF-l on Lymphohemopoietic Cells

IGF-I is a growth and differentiation factor at many stages of hemopoietic development. GH often has the same effect as IGF-I. IGF-I is one of the factors produced by stromal cells and supporting the growth of hemopoietic stem cells (Huang and Terstappen, 1994). PRL increased the frequency of CD34' hemopoietic progenitor cells from human BM, peripheral blood, or umbilical cord blood that respond to the combination of IL-3, GMCSF, and erythropoietin. Optimal response was induced at 2.15 nM, but 0.21 nM already exerted significant effects. Limiting dilution experiments indicated that PRL acts directly on hemopoietic progenitor cells, stimulating both proliferation and differentiation. The most striking effect was seen on erythroid progenitors. Granulocytic colony numbers were also increased (Bellone et al., 1995). We will not discuss erythropoiesis here in detail and simply mention that in several experimental systems, GH and IGF-I also stimulated erythropoiesis (reviewed in Hooghe and Hooghe-Peters, 1995). A. THEMYELOIDLINEAGE 1. Myelopoiesis Myelopoiesis is relevant to our topic as it leads to the production of granulocytes and macrophages. In an early work with human bone marrow, Merchavet al. (1988)have demonstrated that IGF-I (8 nM) or supraphysiological concentrations of GH (11nM) increased myeloid colony formation in the presence of GM-CSF. There was a 40% increase in colony numbers due to a twofold increase in granulocytic colonies. The numbers of monocytic and mixed colonies were not affected. GH had no effect in the absence of adherent cells, presumably monocytes. There was no stimulation with either GH or IGF-I in the presence of anti-IGF-I-R aIR3 mAb. In another study, IGF-I (0.08 nM)potentiated the formation of granulocyhc colonies induced by G-CSF, GM-CSF, or IL-3 and again had little


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effect in the absence of these factors (Merchav et aE., 1993). Both GH and IGF-I stimulated the growth of leukemic myeloblasts from bone marrow taken from patients with active acute myeloid leukemia (AML).In contrast, with peripheral blood from AML patients in remission, no increase in granulocytw colonies could be obtained with either GH or IGF-I (Zadik, 1993). The role of GH and IGF-I in leukemia will be considered in Section VIII. The effect of IGF-I on monocyte precursors has also been investigated. When adherent cells from bone marrow cell cultures were supplemented with IGF-I (2.6nM),there was an increase in proliferation and differentiation into macrophages. These data were obtained with both murine and human cells. In the mouse, the effect of IGF-I was obliterated with antiGM-CSF. It is therefore concluded that IGF-I acts by stimulating GMCSF release (Scheven and Hamilton, 1991). Taken together, the data indicate that physiological concentrations of GH and IGF-I have a stimulatory effect on both differentiation and proliferation of granulocytic and monocytic lineages. There is also evidence that they promote survival by preventing apoptosis (Rodriguez-Tarduchi et al., 1992; McCubrey et al., 1991; Minshall et al., 1996). 2. Functional Studies with Monocytes, Macrophages, and Granulocytes The generation of reactive oxygen intermediates by mononuclear and polynuclear phagocytes is an important element of the defense system against bacteria, parasites, and neoplastic cells. Edwards et al. (1988)demonstrated that the in vivo effects of porcine GH on the priming of rat macrophages for superoxide anion production could be reproduced in vitro. Furthermore, supraphysiological concentrations (14-45 nM ) of PRL and human GH primed human monocytes for enhanced hydrogen peroxide production induced by PMA (Wanvick Davies et al., 1995). The effects of GH were not mediated by IGF-I, as IGF-I, given alone or in combination with GH, had no effect. Whether the effects were mediated via the GHR or the PRL-R was not established. Like other cytokines such as IFNy, human GH and IGF-I primed human neutrophils for superoxide anion secretion in response to protein kinase C activator PMA (Fu et al., 1991). The effects of IGF-I but not of GH were abrogated in the presence of a mAb against the IGF-I-R. Effects of GH required protein synthesis and were mediated by the PRL-R (Fu et al., 1992). Priming of human neutrophils by human GH and bovine neutrophils by IGF-I were also demonstrated by Wiedermann et al. (1991) and Zhao et al. (1993), respectively. Furthermore, human GH was apotent chemoattractant for humanperiphera1 blood monocytes (Wiedermann et al., 1993), but it inhibited chemotaxis of human polymorphonuclear cells toward N-formyl-methionyl-l-

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phenylalanine (fMLP) (Wiedermann et al., 1991; Fornari et al., 1994). This is in accordance with the finding that GH administration to healthy volunteers resulted in an increased random migration, whereas the fMLPinduced chemotaxis was inhibited (Wiedermann et al., 1992). The effects of IGF-I on human peripheral leukocytes were further investigated by Bjerknes and Aarskog (1995). IGF-I (2-20 nM) stimulated the phagocytosis of IgG-opsonized microorganisms, and the PMA- or fMLP-induced oxidative burst. Higher concentrations (30-130 nM ) were necessary to potentiate the fMLP-induced degradation and to stimulate membrane expression of receptors for complement on stimulated and unstimulated cells. In human basophils, IGF-I (0.6mM) enhanced IGE-mediated histamine release. IGF-I also potentiated release initiated by TPA or by the calcium ionophore A23187. It had little or no effect on release caused by FMLP or C5a. The effect of IGF-I was mediated through the IGF-I receptor (Hirai et al., 1993). In conclusion, direct effects of GH or PRL on granulocytes and cells of the monocyte/macrophage lineage, resulting in increased production of reactive oxygen metabolites, increased release of histamine, or altered migration, might play a significant role in the control of these non-specific immune responses.

B. THEB CELLLINEAGE 1 . B Cell Development The mechanism by which IGF-I can increase the number of mature B cells in mice has been addressed by Landreth et al. (1992), who studied the effects of IGF-I on the proliferation and differentiation of pro-B cells. Like IL-7, IGF-I induced the formation of pre-B cells (expressing cytoplasmic p-heavy chain), from mouse bone marrow that had been depleted of B220' cells. The pro-B cell differentiation activity associated with the S17 stromal cell line was blocked by treatment with either anti-IGF-I antibodies or IGF-I antisense oligonucleotide (Fig. 3). In contrast to IL-7, IGF-I did not induce the proliferation of nonadherent cells from longterm bone marrow cultures. However, it potentiated the IL-7-dependent proliferation. In another study, Gibson et al. (1993) studied the effects of IGF-I on the proliferation of a mouse pro-B cell line. Like the ligmd for the c-kit receptor (KL), a factor produced by bone marrow stromal cells and implicated in pro-B cell formation, IGF-I did not induce pro-B cell proliferation in the absence of IL-7, but both IGF-I and KL potentiated the IL-7-stimulated proliferation. Moreover, the effects of IGF-I and KL were additive.

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FIG.3. Hypothesized actions of IGF-I in murine B cell development and function. This model is based on the findings of several groups which are mentioned in Sections II.C.3 and V.B. IGF-I stimulates in uitro pre-B cell proliferation and differentiation (Gibson et al., 1993; Landreth et al., 1992). IGF-I also stimulates plasmocytes to secrete antibodies and to switch antibody class (Kimata et al., 1993). Macrophages and hone marrow stromal cells express IGF-I (Arkins et al., 1993; Abboud et al., 1991). and antibodies against IGF-I and IGF-I-R inhibit pro-B cell differentiation stimulating activity of stromal cell lines (Landreth et al., 1992; see *). Studies on IGF-I regulation in nionOcytes and macrophages demonstrate that IGF-I rnRNA expression in oitro can be regulated by TNFa and IL-1 (Fournier et al., 1995; Kirstein et al.,1992). I n oioo, GH stimulated the production of TNFa and IL-1 (Edwards et al., 1991; Casanova et al., 1990). Therefore, the possibility exists that GH regulates IGF-I expression in uioo via TNFa or IL-1. The idea that IGF-I stimulates the development and function of B cells is in agreement with in oiuo data.

2. B Cell Function A role of PRL in B cell proliferation is indicated by the findings of Matera et al. (1992a) who demonstrated that PRL enhanced DNA synthesis in B cells that were stimulated with submitogenic concentrations of Staphylococcus aureus Cowan (SAC) (Matera et al., 1992a). Additionally, PRL synergized with IL-2 to enhance surface IL-2 receptor expression on anti-IgM-stimulated B human cells, and enhanced IgM and IgG secretion by B cells stimulated by IL-2 in combination with anti-IgM (Lahat et al., 1993). The effects of human GH and IGF-I on in vitro B cell function (proliferation and antibody production) have been studied by Kimata and co-workers. Both GH and IGF-I stimulated B cell proliferation and the secretion of

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antibodies by human B cell lines (Kimata and Yoshida, 1994a,b). Furthermore, both GH and IGF-I enhanced proliferation and antibody secretion of SAC-activated tonsillar B cells. (Yoshidaet al., 1992), and they inducedIgG, and IgE secretion by normal human tonsillar mononuclear cells (Kimata and Fujimoto, 1994). In the latter study, the effects of GH and IGF-I on antibody production were also determined in tonsillar mononuclear cells depleted of sIgG4+and sIgE+ cells to rule out effects on the expansion of IgG4- and IgE-secreting B cell populations. The obtained mononuclear cell fraction did not secrete IgG, and IgE. Both GH and IGF-I (4-10 nM) induced IgE and IgG, secretion, whereas the secretion of other IgG subclasses, IgA, and IgM was not affected. Thus, GH and IGF-I had induced class switch. The GH effects were not mediated by IGF-I, and IGF-I1 and insulin were without effect. Furthermore, unlike the effects of IL-4 and IL-13, the effects of GH and IGF-I were not mediated by either IFNa or IFNy. A N D NK CELLS C. THET CELLLINEAGE 1. T Cells Early studies indicated a role for PRL in T cell proliferation. For instance, the rat Nb2 line depends on exogenous lactogenic hormones for proliferation. Indeed, D N A synthesis was markedly increased by 0.4 nM PRL (Russell d al., 1987). Proliferation of the murine T helper clone L2 does not require additional PRL. However, anti-PRL antibodies inhibited DNA synthesis under serum-free conditions. It appeared that PRL was not synthesized, but had been previously internalized and then released. Further studies suggested a function for nuclear PRL in progression into the S phase (Clevenger et al., 1992, 1990b). Antibodies to pituitary PRL inhibit D N A synthesis in Con A-stimulated murine lymphocytes and PHAstimulated human PBMC that were cultured in RPMI/10% fetal calf serum (Hartman et al., 1989). PHA-induced DNA synthesis in human T cells was increased by 1nM PRL in the presence of both optimal and suboptimal concentrations of PHA (Matera d al., 1992a). However, 4 nM PRL was inhibitory when the cells were stimulated with an optimal doses of PHA. An in uitro autocrine or paracrine function of PRL in human PBMC was indicated by the inhibitory effect of an anti-PRL antiserum on mitogeninduced DNA synthesis (Sabharwal et al., 1992). In mice, PRL potentiated Con A-induced DNA synthesis by splenocytes, but PRL alone gave no response (Spangelo d al., 1987). Furthermore, 0.04 nM PRL stimulated DNA synthesis in a one-way mixed lymphocyte reaction (Shen et al., 1992). On the other hand, physiological levels of PRL inhibited DNA synthesis in a mixed lymphocyte reaction (Hiestand d al., 1986).

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Recent studies show that PRL increased the production of IL-2 and the expression of its receptor. Notably, the in vitro responsiveness of lymphocytes to PRL was influenced by the hormonal status. Mukherjee et al. (1990) found that PRL increased the in vitro proliferative response to IL-2 in splenocytes from ovariectomized (OVX) rats. Flow cytometry revealed that PRL increased the IL-2R expression. In addition to splenocytes from OVX rats, splenocytes from rats in diestrus were also responsive, whereas those from rats in estrus and estrogen-treated OVX rats were not. It was further shown that PRL induced DNA synthesis in splenocytes from OVX rats with an optimal response at 43 nM and an EDSoof about 13 nM (Viselli et al., 1991). PRL also stimulated DNA synthesis in thymocytes, and induced IL-2 production by splenocytes and thymocytes. All these effects were found at high concentrations of PRL (43 nM) and in OVX rats but not in male rats. The effects on DNA synthesis were dependent on the presence of adherent cells. In addition to IL-2 production, the IFNy secretion was stimulated by PRL. Regulation of IFNy production may be a mechanism by which PRL influences in vivo macrophage and T cell function (Bernton et al., 1988), and the defense against bacterial infections (Di Carlo et al., 1993).Cesario et al. (1994) found that PRL (210 nM) enhanced the Con A- and PHAstimulated production of IFNy by human PBMC. Accordingly, Schwartz et al. (1992) described that PRL, like Con A and IL-2, induced IFNregulatory factor-1 in the Nb2 cell line. These studies together point to a role for PRL in T cell-mediated immune responses, via modulation of T cell proliferation and cytokine secretion. The in vitro results indicate that the in uivo effects of pathological concentrations, in hyperprolactinemic patients, can be different from the PRL effects at physiological levels. Furthermore, in vitro studies on the effects of PRL on T cell function are complicated by several factors, such as the production of endogenous PRL, the possible mediatory role of adherent cells, and the hormonal status of the cell donor. Generally, GH has a positive effect on DNA synthesis and growth of transformed and normal T cells. However, GH has also been reported to have no effect, or even a negative effect, on DNA synthesis (reviewed by Kelley, 1989). A positive in uitro effect of autocrine GH in lymphocyte proliferation has been demonstrated by inhibition studies using antisense oligonucleotides (Weigent et al., 1991). An autocrine function for IGF-I as a mediator in GH-stimulated T cell growth is suggested by Geffner et al. (1990).These authors showed that the stimulating effect of human GH on the growth of human T-cell leukemia virus (HTLV)-transformed T cells was abrogated by anti-IGF-I and anti-IGF-I-R antibodies. It is thus compulsory, in order to analyze the in vitro effects of GH, to culture the

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cells under IGF-free conditions. Furthermore, human GH stimulated the migration of resting and activated human peripheral T cells, and the adhesion of these cells to immobilized human and murine adhesion molecules (Taubet al., 1994).The authors suggested that this phenomenon is involved in the stimulating effect of GH on the engraftment of human PBMC in SCID mice. Additionally, human GH stimulated the generation of cytotoxic T cells (Snow et al., 1981),and the proliferation of rat thymic epithelial cells (Timsit et al., 1992). Inhibitory actions of anti-IGF-I and anti-IGFI-R antibodies on the proliferation of thymic epithelial cells indicated that the GH effects were mediated by IGF-I. IGF-I is an important potentiating factor in human and bovine serum for mitogen-induced T cell DNA synthesis (Roldan et al., 1989; Kooijman et al., 1992b). In serum-free cultures, IGF-I did not induce proliferation, but it potentiated lectin- or anti-CD3-induced DNA synthesis in T cells (Tapson et al., 1988; Schimpff et al., 1983).The EDs) of the effects (0.12 nM) were in accordance with the K,, for the IGF-I-R (Kooijman et al., 1992b) and antibodies against this receptor inhibited the effects of IGFI (Johnson et al., 1992), indicating the involvement of theIGF-I-R. The stimulating effects of IGF-I on DNA synthesis are possibly mediated by its potentiating effects on IL-2 production (Kooijman et al., 1996). An important function for IGF-I in T cell proliferation is indicated by the inability of T cells to enter the S phase of the cell cycle when IGF-I-R expression was suppressed by antisense oligonucleotides (Reisset al., 1992). In addition to DNA synthesis in T cells, IGF-I also stimulates DNA synthesis in freshly isolated human thymocytes (Kooijman et al., 199513). Spontaneous DNA synthesis as well as PHA- and IL-2-stimulated D N A synthesis was augmented by IGF-I (ED5" of 0.2 nM). Signaling studies revealed that IGF-I induced the phosphorylation of IRS molecules and its association with phosphatidylinositol-3 kinase (Kooijman et al., 1995a). Since IGF-I and phosphatidylinositol-3 kinase activation have been implicated in inhibition of apoptosis in murine bone marrow cells (Minshall et al., 1996; Rodriguez-Tarduchy et al., 1992), it is tempting to speculate that IGF-I also prevents apoptosis in thymocytes. Other in uitro effects of IGF-I are stimulation of amino acid uptake by thymocytes (Verland and Gammeltoft, 1989) and T cell chemotaxis (Tapson et al., 1988). 2. Natural Killer Cells Physiological concentrations of PRL augmented proliferation of human NK cells (Matera et al., 1992a) induced by suboptimal levels of IL-2, but pharmacological levels of PRL were inhibitory. In addition, physiological concentrations of PRL also stimulated the formation of lymphokine-

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TABLE I ESTABLISHED IN vrmo EFFECTS OF PRL. GH. AND IGF-I ON LEUKOCYTES PRL

In uitro modulation of Proliferation of hemopoietic stem cells Myelopoiesis Proliferation of monocyte precursors Differentiation of monocyte precursors Reactive oxygen intermediate production by Monocyte/Ymacrophages Neutrophils Phagocytosis Pro-B cell proliferation Pro-B cell differentiation Ig production B cell DNA synthesis Ig isotype selection Thymocyte DNA synthesis T cell DNA synthesis Cytokine production Proliferation of NK cells NK cell activity Lymphokine-activated killer cell formation Apoptosis Adhesion Chemotaxis

Yes

Yes Yes

GH”

Yes

Yes Yes”

All classes Yes IgG4 and IgE Yes

Yeslno

Yes

IGF-I

+

++ ++

++ c* ++

Yes Yes Yes Yes No Yes

Yes Yes Yes

All classes Yes IgC, and IgE Yes Yes IL-2 Yes

Yes Yes Yes

Yes Yes

Note. References are given in the text of Section IV. All observed effects, inlcated by “Yes,” were positive. “No” means that no effects were found. Arrows between the last two columns indicate whether the effect of GH was found to be mediated by IGF-I (+) or not (*). “The effects of human GH can be mediated by either the GH-R or the PRL-R. In one case (*), it was established that the effect of GH was exerted via the PRL-R.

activated killer cells in the presence of IL-2 concentrations that were by themselves ineffective (Cesano et al., 1994). Kooijman et al. (1992a) showed that 0.1nM IGF-I increased the cytotoxic activity of human NK cells when cultured in IGF-stripped fetal calf serum. This raises the possibility that the in vivo effects of GH on NK cell activity are mediated by IGF-I. In conclusion, PRL, GH, and IGF-I exert in vitro effects on immature myeloid and lymphoid cells, and on mature macrophages, neutrophils, T lymphocytes, B lymphocytes, and natural NK cells. Most effects of GH are not mediated by IGF-I. Table I summarizes these effects, and for comparison with actions of classical cytokines we refer to “The Cytokine Wall Chart” by Burke et al. (1993). It follows that PRL, GH, and IGF-I

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exert a number of in vitro actions which are comparable to those of several cytokines. VI. IGF-I1 and IGF-Binding Proteins

A. POSSIBLE FUNCTIONS FOR IGF-I1 IN THE LYMPHOHEMOPOIETIC SYSTEM The expression and regulation of IGF-I1 in the immune system have not been studied as well as for IGF-I. It has been shown that IGF-I1 transcripts are expressed in the thymus of adult rats (Brown et al., 1986) and that in human peripheral lymphocytes, IGF-I1 mRNA is induced by PHA (Nyman and Pekonen, 1993). The IGF-I1 peptide is expressed by epithelial cells from the subcapsular cortex and the medulla of human and rat thymus (Geenen et al., 1993). On the other hand, Arkins et al. (1993) did not find IGF-I1 mRNA in mouse thymus, spleen, and macrophages. Most effects of IGF-I1 are mediated by the IGF-I-R (Sara and Hall, 1990). As a consequence, IGF-I1 has the potential to mimic effects of IGF-I. In favor of this idea, the majority of IGF-I1 molecules on IM-9 lymphoblasts were bound to the IGF-I-R (Misra et al., 1986), and the effects of IGF-I1 on T cell proliferation could be blocked by an antibody against the IGF-I-R (Kozak et al., 1987). Furthermore, IGF-I is a more potent stimulator of T cell proliferation than IGF-I1 (Kooijman et al., 1992b), which is in accordance with the lower affinity of IGF-I1 for the IGF-I-R (Kozak et al., 1987). Since the IGF-II-R is identical to the cationindependent mannose 6-phosphate receptor, it has been proposed that this receptor is involved in lysosomal enzyme targeting or IGF-I1 clearance rather than in IGF-I1 signaling. However, there are indications that some effects of IGF-I1 are mediated by IGF-II-R (Nissley et al., 1991). IGF-II-R were found on several types of lymphoid cells using radioligand binding assays. These include PHA- and anti-CD3-activated T cells (Kozak et al., 1987;Johnson et al., 1992),alveolar macrophages, and freshly isolated peripheral blood monocytes (Rorn, 1991). Receptor expression on monocytes could be increased by a factor of 4 by incubation with LPS (Rom, 1991).Furthermore, functional IGF-II-R were identified on rat thymocytes and on a murine T lymphoma cell line (Verland and Gammeltoft, 1989). A twofold increase in the number of receptors on rat thymocytes was found after a 2-day culture period in the presence of Con A. The in vivo effects of IGF-I1 were studied in transgenic mice that overexpress human IGF-I1 under the control of the H-2Kbpromoter. Body length and total weight of transgenic mice as a function of age were not different from controls. The thymus was the only organ whose weight was increased in transgenic mice (van Buul-Offers et al., 1995). Overexpression

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of IGF-I1 increased thymic cellularity and stimulated the generation of phenotypically normal T cells with a preference to CD4+ cells, whereas B cell development was not influenced (Kooijman et al., 1995d). The effects of IGF-I1 on lymphopoiesis were further addressed in the absence of IGF-I. To this end, IGF-I1 transgenic dwarf mice were made by intercrossing IGF-I1 transgenic mice with Snell dwarf mice. We observed that, even in the absence of IGF-I, overexpression of IGF-I1 did not lead to an increase in the number of B cells, whereas T cell development was stimulated to the same extent as in “normal” IGF-I1 transgenic mice. Interestingly, IGF-I1 was more effective in stimulating thymocyte proliferation than IGF-I (Kooijman et al., 1995d), whereas for the stimulation of pro-B cell proliferation, IGF-I1 was less potent that IGF-I (Gibson et al., 1993).Further research on the effects of IGFs on lymphopoiesis is necessary to assess whether IGF-I1 specifically stimulates T cell development via the type I1 IGF receptor. In another transgenic model, IGF-I1 was placed under the control of the rat phosphoenolpyruvate carboy kinase promoter (Wolf et al., 1994). Transgene-specific transcripts were found in the liver, kidney, and several parts of the gut, and serum levels of IGF-I1 were increased by 200-300%. In these mice, however, the weight of the thymus was not affected. Possibly, the high levels of IGF-I1 in the thymus of the IGF-I1 transgenic mice (van Buul-Offers et al., 1995) are essential for the effects on T cell development. Autocrine or paracrine effects of IGF-I1 in T cell development in human and rodents are possible, as indicated by the expression of IGF-I1 transcripts (Brown et al., 1986) and peptides (Geenen et al., 1993; Nagaoka et al., 1990). A suitable model system to address the significance of normal IGF-I1 concentrations in the thymus or in serum for T cell development would be IGF-I1 knockout mice (DeChiara et al., 1990).

B. IGF-BINDING PROTEINS IN THE LYMPHOHEMOPOIETIC SYSTEM The expression and regulation of IGFBPs in the immune system has not been studied extensively, but there are several studies that demonstrate the secretion of IGFBPs by several cells of the immune system. Using RT-PCR, Nyman and Pekonen (1993) showed that IGFBP-2 and -3 transcripts are present in freshly isolated human PBMC. PHA-activated PBMC expressed the same level of IGFBP-3, but increased levels of IGFBP-2, and IGFBP-4 and -5. IGFBP-2 and -4 were secreted by several human B and T cell lines (Neely et al., 1991),and by human myeloid (monocyte and macrophage-like) cell lines (Li et al., 1995).Additionally, several murine myeloid cell lines secreted IGFBP-4 but not IGFBP-2 (Li et al., 1995), and the murine bone marrow stromal cell line TC-1 secreted three different IGFBPs (31,38, and 40 kDa) (Abboud et al., 1991). Li et al. (1995) found

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that murine thymic macrophages secrete a single 25-kDa IGFBP (2 ng/ 106cells per hour) which has been identified as IGFBP-4 by sequencing. Interestingly, desIGF-I, a truncated IGF-I with a reduced affinity for IGFBPs, was a more potent stimulator of DNA synthesis in thymic macrophages than IGF-I. Since this effect was observed in serum-free medium, it was concluded that autocrine or paracrine IGFBP-4 inhibits the proliferative effects of IGF-I on thymic macrophages or other cell types. IGFBPs have also been shown to inhibit the stimulatory effects of IGFs on the proliferation of human T cells (Kooijman et al., 1992b) and mouse bone marrow cells (Lopez Karpovitch et al., 1994). VII. Autoimmunity

Autoimmune diseases result from quantitative or qualitative abnormalities in self-reactivity,due to defects in self-tolerance (Steinberg, 1994).The incidence of several autoimmune diseases is higher in females. Therefore, a role for sex hormones in their pathogenesis has been advocated (Wilder, 1995). PRL has not received the same amount of attention as estrogens and it is not sufficiently realized that estrogens and PRL are members of the same network. Indeed, estrogens are major inducers of PRL secretion and PRL in turn modulates the expression of enzymes involved in the metabolism of steroid hormones and reduces gonadotrophin secretion, which contributes to the feedback resulting in reduced estrogen secretion. Much effort has been devoted to define the possible participation of PRL in the development of diabetes, arthritis, and lupus-like conditions in rodents. Women are much more prone to develop autoimmune diseases than men, but the particular hormones responsible have not been clearly identified. In particular, there is circumstantial evidence for a role of PRL in a few conditions only. Despite this lack of information, patients with uveitis or multiple sclerosis have been treated with bromocriptine with the hope that a reduction in serum PRL levels would improve their symptoms. A. SYSTEMIC LUPUS ERYTIIEMATOSUS (SLE) In SLE, high levels of serum autoantibodies lead to multiorgan inflammation and injury.

1 . Animal Models In several strains of mice, a lupus-like disease is transmitted genetically (Wilder, 1995).In some cases such as lpr and gld, the mutations responsible for the disease have been characterized. The disease is more severe in females. Increased ratios of estrogens to androgens tend to accelerate the disease process (Ansar Ahmed et al., 1985). B/W mice (F1hybrids between


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NZB and NZW) spontaneously develop fatal immune complex glomerulonephritis, with an earlier onset in females. When persistent hyperprolactinemia was induced (with ectopic pituitary grafts), disease onset occurred earlier in females as well as in males. In splenocytes from such hyperprolactinemic mice, the expression of IL-4 and IL-6 was higher than in controls. Conversely, bromocriptine treatment reduced PRL levels and this resulted in delayed onset. Treatment with bromocriptine also inhibited IL-4 and IL-6 expression in splenocytes (McMurray et al., 1991a,b). Female mice with physiological hyperprolactinemia resulting from mating, pregnancy, suckling, or pseudo-pregnancy were also investigated. By comparison with virgin controls, all these conditions favored an early onset of the disease in a higher proportion of mice. Only in pseudo-pregnant mice, however, were the severity of the disease and the shortening of life significantly greater than in virgin controls. Pseudo-pregnancy is also the only physiological condition studied with sustained hyperprolactinemia. An interesting observation was the improvement of biological indicators of disease during suckling, which probably had the same effects as plasmapheresis since immunoglobulins, including immune complexes, are cleared through the milk (McMurray et al., 1993). These studies stress the importance of hormones in this model. It is clear, however, that hormones are not responsible for the breakdown of tolerance and only play an aggravating role.

2. Man Lupus erythematosus is more frequent in women of child-bearing age and flares are common during pregnancy (Petri et al., 1991). Moderately elevated PRL levels have repeatedly been found in patients. Recently, four cases of lupus associated with very high levels of PRL have been reported. In three of these cases, lupus developed after a prolonged period of hyperprolactinemia and two patients suffered from lupus exacerbations after withdrawal of bromocriptine (which results in the rise of serum PRL levels) (McMurray et al., 1994). 3. Conclusion Hormones clearly influence the course of SLE and SLE-like murine disease, The importance of estrogens has been advocated, although men with Klinefelter’ssyndrome do not have a higher incidence of SLE (Lahita, 1992). Rather, the data suggest that sustained hyperproplactinemia plays a deleterious role in the development or the progression of the disease. In the primary antiphospholipid syndrome, an autoimmune disease that shares several features with SLE, the hormonal context is also compatible with a precipitating or aggravating role for PRL as disease occurrence is often related to pregnancy or the puerperium. Weidensaul et al. (1994)

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have recently reviewed the problem and reported one case with high levels of PRL and low levels of estrogens. B. RHEUMATOID ARTHRITIS(RA) RA is a chronic, recurrent, systemic inflammatory disease primarily affecting the joints. Three quarters of the cases occur in women.

1. Rats

After injection of complete Freunds adjuvant, intact rats develop a transient arthritis. Hypophysectomized rats are unable to develop arthritis unless given PRL or GH. Also, bromocriptine treatment prevents arthritis, most probably as a result of reducing serum PRL levels (Neidhart, 1989). Indeed, arthritis does develop in rats given bromocriptine plus PRL (Berczi and Nagy, 1982; Berczi et al., 1984). Following the injection of complete Freund’s adjuvant, a vast array of endocrine as well as immunological responses develop. For instance, after 2 days, there was a sixfold increase in plasma PRL and a twofold increase in plasma GH and ACTH levels (Neidhart and Larson, 1990).

2. Mice Collagen arthritis in mice is induced by the injection of collagen in complete Freund’s adjuvant. The latency of the disease is much longer than that of adjuvant arthritis in rats. There are indications suggesting that PRL favors the induction of collagen arthritis, as PRL injections or pregnancy following immunization resulted in a more severe form of the disease. However, bromocriptine given 1 month after immunization, i.e., at the onset of the disease, also resulted in exacerbation of the disease (Mattsson et al., 1992). Could it be that PRL favors the induction of collagen arthritis but protects at later stages? It should however been kept in mind that bromocriptine has many effects in addition to the inhibition of PRL secretion. In the study reported here, there was no control group receiving bromocriptine plus PRL. 3. Man Evidence supporting the involvement of hormones in the pathogenesis or the progression of RA is weak, although this condition has been investigated extensively. GH levels were within normal limits but circadian fluctuations were not normal and the total amount of GH secreted daily in young patients could be reduced. IGF-I levels were low (Allen et al., 1991). Growth retardation is indeed a frequent problem in such patients. Girls with the juvenile form of RA and a positive antinuclear antibody (ANA) test had significantly higher PRL levels than girls with RA and a negative


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ANA test or than age-matched controls (McMurray et al., 1995). Also, PRL from patients had a low bioactivity. This may be due to abnormal ratios of the various isoforms (Nagy et al., 1991; Berczi et al., 1993). Abnormal glycosylation of PRL (or an abnormal ratio of glycosylated PRL) is a possibility, and this would not be unexpected as immunoglobulins are indeed abnormally glycosylated in rheumatoid arthritis patients (Rademacher et al., 1988). More research is needed in this complex field. PRL itself has been shown to modulate protein glycosylation in the mammary gland (Golden and Rillema, 1995). C. INSULIN-DEPENDENT DIABETES MELLITUSOR TYPE1 DIABETES It is now clear that autoimmunity is responsible for the destruction of insulin-producing P cells of the pancreatic islets leading to insulin deficiency. The non-obese diabetic (NOD) mouse is a relevant model for diabetes type I (insulin-dependent diabetes). The contribution of hormones to disease expression has been investigated in detail by Gala’s group. Diabetes incidence in their colony was 73%in females and 24%in males. Neonatal castration doubled the incidence in males whereas neonatal ovariectomy or testosterone injection reduced the incidence in females, to 13 and 26% respectively (Hawkins et al., 1993). Thus, female hormones stimulate the expression of diabetes whereas male hormones have a protective effect. Surprisingly, however, female mice ovariectomized neonatally and given testosterone had a 100% incidence of diabetes. The authors propose explanations based on the diabetogenic role of GH (induced by testosterone) and the anti-diabetogenic role of continuous estrogenic stimulation (induced by testosterone in intact females only). In a second set of experiments, the same group has shown that hyperprolactinemia, induced by anterior pituitary grafts, increased the incidence in males. In ovariectomized female mice given a anterior pituitary graft, the incidence was the same as in control, intact females but the onset of the disease occurred earlier. In intact males, bromocriptine had no effect on the incidence of diabetes. In males given an anterior pituitary graft, bromocriptine prevented the increased incidence seen with pituitary grafts only. In females, bromocriptine reduced the incidence of diabetes. Taken together, the data are in favor of a strong participation of PRL in the expression of diabetes in the NOD mouse (Hawkins et al., 1994). The possible contribution of PRL to human diabetes has not been investigated. GH clearly is a diabetogenic hormone and this has not been related to its action on the immune system. Whether GH or IGF-I, in addition to their metabolic activity, can influence the autoimmune response in diabetic patients is not known. In the NOD mice, diabetes

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onset can be stimulated by adoptive transfer of autoreactive T cells. In this system, IGF-I treatment delayed the development of the disease (Bergerot et al., 1995). Atrophy of the thymus is one of the consequences of severe insulin deficiency. Thymic lesions in diabetic rats could be corrected by insulin treatment but also by IGF-I, despite persistent hyperglycemia (Binz et al.,1990). A protective action of IGF-I was also observed in experimental allergic encephalitis (EAE, see below).

D. THYROIDITIS High titers of antithyroid antibodies and autoimmune thyroid disorders occur far more frequently in hyperprolactinemic women than in the general population (Ferrari et al., 1983).In a group of 82 women with hyperprolactinemia (idiopathic in half of these cases), 20%had antithyroglobulin antibodies and 12% had antibodies against microsomes. Most patients were euthyroid and a variety of diseases accounted for the cases of hypo- or hyperthyroidy. The relationship between pituitary hormones and specific thyroid diseases deserves further investigation. E. AUTOIMMUNE ENCEPHALITIS AND MULTIPLESCLEROSIS The expression of PRL in the brain has been demonstrated (Emanuele et al., 1992;Clapp et al., 1994).Increased expression was found in hypothalamic wounds (DeVito et al., 1995a).It was further shown that PRL administered at the site of the wound increased the expression of glial fibrillary acidic protein, a marker of reactive astrocytes and TNFa in the tissue surrounding the wound. In vitro, PRL (1 nM) induced proliferation of astrocytes and the expression of IL-la, TNFa, and, later, TGFa (DeVito et al., 1992, 1995b). In unpublished work, Dogusan and Hooghe-Peters have shown that TNFar, in combination with IL-1, induced the apoptosis of rat oligodendrocytes. TNFa and lymphotoxin are also cytotoxic for calf oligodendrocytes. Lymphotoxin was more potent than TNFa and induced apoptosis (Selmaj et al., 1991). Taken together, these observations suggest a role for PRL in regulating the response to injury leading to the death of oligodendrocytes. 1. Experimental Allergic Encephalitis (EAE) EAE is an animal model for autoimmune demyelinating diseases. It is induced in rats by immunization with myelin antigens such as the myelin basic protein in Freund’s adjuvant. We have already mentioned that the injection of Freund’s adjuvant was followed by an increase in the levels of PRL and other hormones. As expected, a rise in PRL levels was also seen after induction of EAE. Bromocriptine treatment not only reduced PRL levels to those of control rats but also reduced the incidence and the


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severity of the disease. Lymphocytes from treated animals had a lower response to the immunizing antigen and to Con A. There was no attempt to reverse the effects of bromocriptine with PRL (Riskind et al., 1991; Dijkstra et al., 1992). There has also been much interest recently in the protective role of IGF-I in EAE. Yao et al. (1995) have started IGF-I injections (200 pg or 1 mg/day, for 8 days) 12 days after induction, when inflammatory lesions and clinical signs were present. Clinical, biochemical, and histological criteria indicated clear-cut improvement in treated rats. Roth et al. (1995) came to similar conclusions after in vitro studies.

2. Multiple Sclerosis The hypothesis that PRL plays a role in multiple sclerosis has been tested by measuring PRL levels in patients. There is no consistent increase although some patients have high serum PRL, in particular during exacerbations of the disease (Kira et al., 1991; Reder et al., 1993). The clinical course is not suggestive of a strong involvement of PRL as the frequency of relapses is not increased during pregnancy. There is, however, an increase during the postpartum (Birket al., 1990).The possibility that a rise in serum PRL may result from a hypothalamic lesion should always be considered in patients with multiple sclerosis.

F. UVEITIS Uveitis is a collective term for intraocular inflammatory disease, sometimes associated with autoimmune conditions. The chance observation that recurrent anterior uveitis improved during bromocriptine therapy given for prolactinoma or Parkinson’s disease in four patients (Hedner and Bynke, 1985) prompted investigations on the possible involvement of PRL in the pathogenesis of autoimmune uveitis. There were unconfirmed reports on the protective effect of bromocriptine in autoimmune uveitis in the rat (Palestine et al., 1987). Patients were also treated with the combination of bromocriptine and cyclosporine, with a resultant reduction in the titer of autoantibodies (Blank et al., 1990). Hyperprolactinemia was later reported in some (9/25) patients with Reiter’s syndrome. The incidence of uveitis, conjunctivitis, and urethritis was higher in the hyperprolactinemic patients. In contrast, PRL levels were normal in patients with acute anterior uveitis (Jara et al., 1994). G. CONCLUSIONS The increased incidence of autoimmune diseases and of exacerbations in women, in particular during pregnancy or the postpartum, is strong, but still only circumstantial as evidence for the role of hormones, in particu-

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lar for SLE, the antiphospholipid syndrome, or lymphocytic hypophysitis (Garber and Hedley-Whyte, 1995; Weidensaul et al., 1994). The complexity of the endocrine network makes it difficult to estimate the contribution of different hormones in increasing the severity of the disease. Special attention should be paid to patients with increases in PRL or estrogen only and to animal models, such as hypopituitary dwarfs and knockout mice, where deficits are more limited than after, e.g., hypophysectomy, castration, or ovariectomy. Finally, if the expression of the fas ligand at the cell surface, as recently found on Sertoli cells (Bellgrau et al., 1995), contributes to the induction or the maintenance of the tolerant state by inducing apoptosis of autoreactive T cells, then factors that increase or decrease the expression of the fas ligand may respectively prevent or favor rupture of tolerance. Future work will establish whether the factors that reduce the expression of fas ligand also increase the severity of autoimmune disease. Sex hormones and PRL are obvious candidates. So far, however, hormones were mostly found to have a stimulatory effect in the immune system. In this respect, future research should look at the selective stimulation of particular helper T cell populations, as an imbalance of T helper activities may be of critical importance in autoimmunity (Romagnani, 1994). VIII. Lymphoproliferative Diseases

PRL and GH can stimulate the growth of malignant blood cells, and have been implicated in hematological malignancies. More important, IGF-I is increasingly recognized as a potent growth factor for many normal and neoplastic cell types, including hemopoietic cells. Therefore, the IGFI-IGF-I-R system is becoming a major target for experimental therapy. Hormones and their receptors can be involved in leukemogenesis. Constitutive activation of PRL-R, GH-R, or IGF-I-R might also lead to uncontrolled proliferation and mutations in these receptors may be oncogenic.

A. EXPRESSION OF PRL, GH, OR IGF-I BY LEUKEMIC CELLS PRL or GH expression by leukemic cells is not common. Some Jurkat T leukemia cells produce PRL and the Burkitt lymphoma line sfRamos produces GH (Lytras et al., 1993; Pellegrini et al., 1992) These cells have been kept in culture for many years and there is no evidence that they have always produced hormones. Actually, this property was found only in selected sublines. It is not known at which stage of in uivo or in uitro neoplastic progression they started to express hormones. For the sfRamos cells, GH is an autocrine growth factor.

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In one clinical study, moderate hyperprolactinemia was found in 16 out of 28 patients with myeloid leukemia. In one of these patients, PRL was found in leukemic blast lysates but there was no attempt to demonstrate actual synthesis in cultured leukocytes (Hatfill et al., 1990). Several human and murine leukemic lines secrete IGF-I, and in some cases this is clearly an autocrine growth factor (Gjerset et al., 1995). Expression of IGF-I was found in different types of leukemia: myeloid, B, or T cells (reviewed by Shimon and Shpilberg, 1995), albeit often at very low levels (Arkins et al., 1993). B. EXPRESSION OF RECEPTORS FOR PRL, GH, OR IGF-I Many leukemic cell lines bear receptors for PRL or GH. It is not often ascertained whether such receptors are functional. Functional PRL-R are expressed in rat Nb2 lymphoma cells and the Nb2 cell line provides a sensitive bioassay for lactogenic hormones (Gout et al., 1980; see Section IX). The 2779 rat T-cell lymphoma is especially interesting: in these cells, expression of the PRL-R was activated by promoter insertion of Moloney MuLV (Barker et al., 1992). When activated in T cells, the PRL-R may thus function as an oncogene. The IGF-I-R is expressed in many malignant cell types including polycythemia Vera erythroblasts, some sublines of the K562 erythroleukemia, myeloid lines (HL60), B and T lymphoblastic leukemias, Burkitt lymphomas, and Hodgkin cells (Vetter et al., 1986; Pepe et al., 1987; reviewed in Shimon and Shpilberg, 1995). In the absence of IGF-I-R, IGF-I could also stimulate leukemic cell growth through the IGF II-R and insulin-R. The presence of such receptors has indeed been demonstrated on some leukemic cells (Freund et al., 1994). C. GROWTHPROMOTING EFFECTOF PRL, GH, AND IGF-I

In addition to the rare cell lines that require PRL or GH for survival or for proliferation, there are many examples of leukemic cells that proliferate faster in the presence of GH or IGF-I. PRL received less attention in this respect. In HL60 myeloid leukemia cells, physiologic concentrations of PRL stimulate DNA synthesis (Nishiguchi et al., 1993) and in a murine T cell line the growth-promoting effect of PRL was synergistic to the effect of IL-2 (Clevenger et al., 1990a). In short-term cultures of leukemic lymphoblasts, physiologic concentrations of GH (10-50 ng/ml, 0.45-2.25 nM) were mitogenic in 6 out of 17 cases (Blatt et al. 1987). Both GH (250-300 ng/ml, 11.3-13.5 nM) and IGF-I (0.05-0.5 ng/ml, 7.10-12-7.10-11M) increased blast colony numbers

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when the ALL or the AML blast assay was performed on T cell-depleted BM cells from pediatric ALL or AML patients, respectively, in the presence of conditioned medium (Zadik et nl., 1993). An erythroleukemic K 562 line proliferated faster in the presence of low concentrations GH (0.1 ng/ml, 45"'-4513M) (Gauwerky et d.,1980). Other lines such as the MOLT4 T-leukemic cells and the PER-255 T-leukemic cells responded only to concentrations higher than 50 ng GH/ml, i.e., 2.25 nM (Blatt et a!., 1987; Baker et nl., 1993). IGF-I is a growth factor for many leukemic cell lines and myeloma cells (Oksenberg et ul., 1990, Baier et ul., 1992; Zadik et al., 1993; F r e u d et al., 1994; Shimon and Shpilberg, 1995). As erythroid progenitors in polycythemia Vera are hypersensitive to IGF-I (Correa et al., 1994), it has been proposed that the mutation responsible for this disease is located in the IGF-I-R gene or in genes involved in IGF-I signaling (Prchal and Prchal, 1994).

D. Is GH LEUKEMOGENIC? For several years, there has been a true concern about the safety of CH treatment in children after reports (quoted by Fradkin et ul., 1993) were released in Japan suggesting a link between treatment with GH and the subsequent development of leukemia. This problem has been addressed by Stahnke and Zeisel(1989), Stahnke (1992), Ritzen (1993), and Fradkin et al.(1993).The number of children treated with GH for primary hypopituitarism, GH-deficiency secondary to cranial irradiation, or growth retardation related, e.g., to renal insufficiency or Turner's syndrome is now close to 100,000.The follow-up amounts to 1,000,000 patient-years. In 1987, a Japanese report suggested that the incidence of leukemia was above the expected figure in children treated with GH. The cases of leukemia in GH-treated children fall into 3 groups:

(1) Children who had leukemia before the treatment and were then treated for nanism secondary to cranial irradiation: (2) Children who had conditions predisposing to the development of leukemia, such as Fanconi's anemia, myelodysplasia, or previous treatment with radiation or with immunosuppressive or cytostatic drugs; (3) Children who were not especially at risk for leukemia before the start of GH treatment. If only children in the last group from all over the world are taken into account, then there is no increased risk of developing leukemia. There is, however, an excess of leukemia among Japanese children treated with GH (relative risk 3.6-4.0). There is no satisfactory explanation for this

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observation. Even for children previously treated for cancer, there was no indication that GH increases the risk of recurrence. For many tumor types, however, the numbers of children treated with GH are still very small (Ritzen, 1993; Moshtang, 1995). Leukemias are myeloid or lymphoid, with a predominance of lymphoid leukemias in the children without additional risk factor. Lymphomas are uncommon. An exceptional case of erythroleukemia at the age of 22 has been reported in a patient treated for 8 years with GH for panhypopituitarism (Bosch Benitez et al., 1994). There is limited evidence that GH is clastogenic. van Buul and van Buul-Offers (1984) induced chromosome abnormalities with supraphysiologic levels of GH in vivo (murine BM cells) and in vitro (CHO cells). More recently, Tedeschi et al. ( 1993) investigated chromosome fragility in children before and during GH therapy (children with short stature, without GH or IGF-I deficiency). After 6 months, there was an increase in bleomycin-induced aberrations. At 6 and 12 months, a slight increase in the number of spontaneous chromosome rearrangements (rings and reciprocal translocations) was also observed. The authors propose that GH therapy increases chromosome fragility, possibly via an increase in superoxide anion production by macrophages (see above, Section V.A.2). In unpublished experiments, increase in chromosome fragility during treatment with GH was not found in GH-deficient children (B. Tedeschi, personal communication). There are only few experimental studies on the leukemogenic properties of GH. Zadik et al. (1993) failed to detect any stimulatory effect of GH or IGF-I in the granulocyte macrophage colony forming assay, with peripheral blood cells from chronic myeloid leukemia patients in remission. In conclusion, PRL, GH, and IGF-I are definitively growth factors for a number of tumor cells. In contrast, a favorable effect of these factors on anti tumor immunity is only a possibility. Therefore, caution is required when treating cancer patients with one of them. There is no evidence that GH favors the development of leukemia or the recurrence or brain tumor. IX. Technical Aspects

In this section we present some supplementary information about techniques and models mentioned in the previous sections and not currently used in immunology laboratories. A. HORMONE ASSAYS

Hormone levels are routinely measured by ELISA or RIA. At the singlecell level, sensitive immunoplaque assays identifylng PRL- or GH-secreting

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cells can now be combined with in situ hybridization (Scarbrough et al., 1991; Varma et al., 1993; Niimi et al., 1994). A bioassay for lactogenic hormones relies on the fact that the rat Nb2 lymphoma line requires PRL for proliferation. Its PRL-R binds PRL and placental lactogens from many species and also GH from primates. All these substances are therefore measured in this bioassay. Further identification of the active factor can be achieved by blocking proliferation with specific antibodies. There is usually a good correlation between hormone levels measured by immunoassay and by bioassay. The Nb2 assay has been characterized thorougly and is specific for lactogenic substances, although some Nb2 sublines also respond to IL-2 (Croze et al., 1988; Rayhel et al., 1988a). Caution is required, however, when evaluating the activity of complex mixtures that may contain several PRL variants but also other factors interfering with the Nb2 lymphoma cell proliferation, such as prostaglandins or TGFP (Rayhel et al., 1988b; Gala and Rillema, 1995). Thus, before stating that a given hormone preparation has an abnormal bioactivityhmmunoreactivityratio, the presence of other substances interfering with Nb2 proliferation should be considered.

B. HORMONE PROINCTION Long-term hyperprolactinemia is obtained conveniently with grafts of pituitary glands or pituitary cell lines. Anterior pituitary glands grafted under the kidney capsule produce only PRL (AdlerJ986). This is the case only after a few weeks, when blood supply to the grafts is established. For several days after surgery, degenerating cells release cell types of pituitary hormones. The rat GH3 line produces GH and PRL in uitro but mainly GH in vivo (Day and Day, 1994). C. HORMONE DEPLETION Hypophysectomyresults in deficiencyof all pituitary hormones. Hypophysectomy is a difficult procedure, however, and, after several months, presumably as a result of regrowth of pituitary tissue, hormone levels may return to relatively high levels (Nagy and Berczi, 1991). GH secretion is inhibited with somatostatin analogs (see Melmed, 1995). A rather selective depletion of PRL in serum is achieved with dopaminergic agents such as bromocriptine and cabergoline which inhibit PRL secretion (Ben-Jonathan, 1994; Bevan and Davis, 1994). In contrast, drugs such as haloperidol have been used to increase PRL secretion and elevate PRL serum levels. In all cases, it should be kept in mind that the pituitary gland is not the only target of these drugs. Of particular relevance to our topic is a recent study by Morikawa et al. (1994) showing direct, in vitro immunosuppressive effects of bromocriptine on lymphocytes. Bromocriptine ad-


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ministration in uivo has often been used to evaluate the contribution of PRL to a given phenomenon. This can be accepted only when the effect of bromocriptine is reversed by PRL.

D. MUTANT,TRANSGENIC, AND KNOCKOUT ANIMALS Different kinds of dwarf mice and rats are available. The Ames (do and Snell-Bagg(dw) dwarf mice have a major deficit in pituitary PRL, GH, and TSH (and also IGF-I and thyroid hormone), little (lit/lit) mice lack GHRH-R (and this accounts for a lack of GH), and dwarf rats have either a selective GH deficit or a combined PRL and GH deficit (Cross et al., 1992; Murphy et al., 1992; Murphy et al., 1993; Ono et al., 1994). Transgenic mice overexpressing the genes coding for hypothalamic hormones or the GH gene ( e g , under the control of the metallothionein promoter) have been produced (Washek, 1995). Knockout mice lacking IGF-I and IGF-I-R genes have been generated (Liu et al., 1993). The breeding of double knockouts may be required to evaluate the extent of redundancy in the cytokine network. E. CLINICAL CONDITIONS In the human, much information can be gained from cases of selective, total or partial, GH deficiency and of the very rare combined PRL, GH, and TSH deficiency. The effects of GH can be evaluated in many patients with GH deficiency or with short stature without GH deficiency, now being treated with human GH. In GH resistance syndromes, due to abnormal GH-R or signaling, GH levels are high but IGF-I levels are low. Patients with pituitary tumor (e.g., prolactinoma, somatotropinoma) have high levels of one or several hormones. Much can be learned through clinical investigation before treatment and after tumor removal by surgery or radiotherapy or during medical treatment. A few transformed lymphoid or monocpc cells express PRL, GH, or IGF-I. It should be remembered that these lines are often particular isolates, subcultures, or subclones and that not all samples of these cell lines produce hormones. In contrast, many cell lines express functional receptors for these hormones. Again, care should be taken to assess the presence of the receptor of interest before starting experiments. X. Perspectives

A. PRL, GH, AND IGF-I AS LYMPHOHEMOPOIETIC GROWTHFACTORS OR CYTOKINES In uivo data presented in this review demonstrate that PRL, GH, and IGF-I stimulate the development of lymphohemopoietic cells, and influ-

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ence several immune functions in rodents. Receptor studies and in vitro data indicate that these effects can be exerted directly on cells of the immune system. The presence of functional receptors for PRL and IGFI has been demonstrated in many populations of leukocytes. Binding sites for GH are also present, but only in a few instances was it established that these binding sites were bona fide GH-Rs, as human GH also binds to the PRL-R. In uitro studies also demonstrated that PRL, GH, and IGFI are growth factors for lymphoid and myeloid cells, and that these factors modulate the immune function of granulocytes, monocytes, macrophages, and lymphocytes (Table I). Taken together the results demonstrate that PRL, GH, and IGF-I are lymphohemopoietic growth factors in rodents with immunomodulatory properties. In particular, in uitro experiments revealed that IGF-I acts as a differentiation factor for murine pro-B cells, and that it induces Ig class switch in human B cells. These results indicate that IGF-I act also as a lymphoid differentiation factor. The fact that serum levels of PRL, GH, and IGF-I can be correlated with immunological reactivity indicate that these factors can be involved in neuroendocrine modulation of the immune system. It is known that pituitary GH and PRL secretion are influenced by stress. However, leukocytes are likely to be exposed to locally produced factors, and there are strong arguments for an autocrine or paracrine function of PRL, GI-I, and IGF-I in the lymphohemopoietic system: PRL, GH, and IGF-I are indeed expressed and secreted in leukocytes and stromal cell of lymphoid organs, and both autocrine and paracrine effects have been demonstrated in vitro. For instance, in vitro effects of GH can be obliterated by anti-sense oligonucleotides to GH mRNA, and in several experiments by anti-IGFI and anti-IGF-I-R. The regulation of these hormones in the immune system is at least in part different from their regulation in the endocrine system. IGF-I can be regulated by several cytokines in addition to GH, and PRL expression is regulated via an alternative 5’ upstream promoter. This suggests that these factors are part of the cytokine network. Although the data summarized in this review strongly support the hypothesis that PRL, GH, and IGF-I act as autocrine or paracrine factors in the lymphohemopoietic system, the evidence so far is circumstantial. A major drawback in the research on autocrine and paracrine functions in the lymphohemopoietic system is the presence of these hormones in serum, and the proven effects of circulating hormones. Definitive evidence for an autocrine or paracrine function for PRL, GH, and IGF-I will be obtained from studies on: ( 1 ) cell-specific targeted disruption or deletion of hormones in the immune system, and (2) stimulation and inhibition of the expression of hormones in the immune system by factors that specifically

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interfere with the regulatory mechanism for these hormones in the lymphohemopoietic system. An important gap in our understanding of the function of hormones in the immune system remains their target cells. In most experimental systems, hormones have a positive action on proliferation and functional activity. Stimulation of B and T cell function has been demonstrated, but it is for instance not known whether the THIand TH2compartments are selectively activated.

B. GH DEFICIENCY (GHD) A N D GH TREATMENT I N HUMANS Isolated GHD on one hand and GH treatment of children with reduced or normal GH levels on the other hand lead to subtle and variable changes in some laboratory parameters of immune function. No clinical symptoms associated with immune dysfunction have been reported in GHD children or in children receiving GH therapy. Patients with a total TSH, PRL, and GH deficiency due to a mutation in the Pit-1 gene also lack clinical symptoms associated with immune dysfunction. Moreover, Laron dwarfs which have a mutation in the GH-R are not immunodeficient (Z. Laron, personal communication). This is in contrast with the effects of hypophysectomy or hypopituitary dwarfism in rodents, and with the numerous effects of PRL, GH, and IGF-I in vitro. With respect to this enigma, we would like to stress that the effects of GH and its mediator IGF-I might be redundant. Figures 1 and 2 give some hints about the levels of redundancy in the cytokine network. For instance, two cytokines bind to the same receptor, e.g., IGF-I and IGF-I1 bind to the IGF-I-R, and both PRL and primate GH bind to the PRL-R. In addition, a number of cytokines use the same signaling machinery. Therefore, PRL, GH, and IGF-I may be dispensable for the human immune system in the presence of other factors which can take over their functions, such as IL-4, IL-6, and IGF-11. Notably, high levels of circulating IGF-I1 are present in human adults, but not in adult rodents. Whether redundancy in human is higher than that in rodents remains to be established, It would be interesting to investigate redundant effects in gene knockout mice. It is expected that mice lacking several factors, such as IGF-I and IL-4, may display major immune deficiencies compared to mice with only one disrupted gene.

C. CLINICAL IMPLICATIONS The increased incidence of autoimmune diseases and of exacerbations

in women, in particular during pregnancy or postpartum, is high but is still only circumstantial evidence, for the role of hormones, in particular in SLE, in the antiphospholipid syndrome, or in lymphocytic hypophysitis. The complexity of the endocrine network makes it difficult to estimate the

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contribution of different hormones in modulating the severity of the disease. Special attention should be paid to patients with increase in PRL or estrogen only, and to animal models, such as hypopituitary dwarfs and knockout mice. More data are necessary before opening new avenues for treatment of patients with, e.g., bromocriptine. To conclude, PRL, GH, and IGF-I hold promise as therapeutic agents in various forms of hemopoietic failure and immune deficiency. Also, dopaminergic agents and somatotropin analogs may be useful in the treatment of autoimmune diseases. Studies of the specific regulation of these hormones in the lymphohemopoietic system can lead to the development of specific clinical tools that interfere with local production of these hormones. However, these factors should be used with discrimination in man and animals. ACKNOWLEDGMENTS We thank our colleagues at the Pharmacology Department and Lina Matera for kind collaboration, and J.R.E. Davis, E. Heinen, R.R. Gala, K. W. Kelley, and J.R.L. Pink for critically reading the manuscript and for their helpful suggestions. We are also indebted to I. Berczi, R.R. Gala, and K.W. Kelley for sharing their recent findings. This work was supported by grants from the European Community (ERB CII-CT930025), the Belgian National Research Foundation (S21/5-AV-E 173), and the VUB-02R.

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