Advances in Immunology Volume 76

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ADVANCES IN

Immunology VOLUME 76

This Page Intentionally Left Blank

ADVANCES IN

Immunology EDITED BY FRANK J. DIXON The Scripps Research Institute La Jolla, California ASSOCIATE EDITORS

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

VOLUME 76

San Diego San Francisco New York Boston London Sydney Tokyo

CONTENTS

CONTRIBUTORS

ix

MIC Genes: From Genetics to Biology

SEIAMAK BAHRAM I. II. III. IV. V. VI.

Introduction Genes and Genomics Transcripts and Transcription Biochemistry and Biology Genetics and Immunogenetics Conclusion References

1 5 13 20 30 46 47

CD40-Mediated Regulation of Immune Responses by TRAF-Dependent and TRAF-Independent Signaling Mechanisms

AMRIE C. GRAMMER AND PETER E. LIPSKY I. II. III. IV. V.

Introduction Discovery of CD40 and CD154 Functional Outcomes of CD154–CD40 Signaling Regulation of CD40 and CD154 Expression Role of CD40-Induced Signaling Cascades in Functional Responses of B Cells VI. Conclusion References

61 61 70 78 86 145 146

Cell Death Control in Lymphocytes

KIM NEWTON AND ANDREAS STRASSER I. Introduction II. Apoptosis and the Role of Caspases v

179 179

vi

CONTENTS

III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

Apoptosis Signaling by Death Receptors Apoptosis Signaling through Apaf-1 and Caspase 9 Apoptosis and Its Regulation by Members of the Bcl-2 Family Transcriptional and Posttranslational Control of Bcl-2 Family Members Apoptosis and the Immune System T Cell Development—Apoptosis at the Pre-TCR Checkpoint Positive and Negative Selection at the Pre-T Stage of Development T Cell Apoptosis in Peripheral Lymphoid Organs The TNF-R Family and T Cell Proliferation B Cell Development—Apoptosis at the Pre-BCR Checkpoint Selection of Immature B Cells in the Bone Marrow B Cell Apoptosis in Peripheral Lymphoid Organs Conclusion References

180 184 186 188 189 190 193 195 200 200 202 203 205 206

Systemic Lupus Erythematosus, Complement Deficiency, and Apoptosis

M. C. PICKERING, M. BOTTO, P. R. TAYLOR, P. J. LACHMANN, AND M. J. WALPORT I. Introduction II. Description of the Associations between Complement and SLE in Humans III. Animal Models of Complement Deficiency IV. Complement and Inflammation in SLE V. Lupus Causes Autoantibody Production to C1q VI. Hypotheses for the Association between Complement Deficiency and SLE VII. What Lessons Can Be Learned from Other Murine Models of Autoimmunity? VIII. Conclusions References

227 228 277 279 281 283 297 298 299

Signal Transduction by the High-Affinity Immunoglobulin E Receptor Fc␧RI: Coupling Form to Function

MONICA J. S. NADLER, SHARON A. MATTHEWS, HELEN TURNER, AND JEAN-PIERRE KINET I. II. III. IV. V. VI. VII.

Introduction Signal Initiation: The Central Importance of ITAM Phosphate Transfer and PTKs Phosphate Transfer and PTPs Other Signaling Molecules Integration of Lipid–Protein Interactions and Calcium Flux From Lipid Signaling to Protein Serine–Threonine Protein Kinases

325 327 328 332 335 338 339

CONTENTS

vii

VIII. Lipid Rafts IX. Conclusion References

343 344 345

INDEX CONTENTS OF RECENT VOLUMES

357 367


CONTRIBUTORS

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

Seiamak Bahram (1), Centre de Recherche d’Immunologie et d’He´matologie, Strasbourg 67085, France M. Botto (227), Rheumatology Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London W12 ONN, United Kingdom Amrie C. Grammer (61), Autoimmunity Branch, National Institutes of Health, Bethesda, Maryland 20892 Jean-Pierre Kinet (325), Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 P. J. Lachmann (227), Microbial Immunology Group, Center for Veterinary Science, Cambridge CB3 0ES, United Kingdom Peter E. Lipsky (61), Autoimmunity Branch, National Institutes of Health, Bethesda, Maryland 20892 Sharon A. Matthews (325), Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 Monica J. S. Nadler (325), Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 Kim Newton (179), The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia M. C. Pickering (227), Rheumatology Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London W12 ONN, United Kingdom Andreas Strasser (179), The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia P. R. Taylor (227), Rheumatology Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London W12 ONN, United Kingdom Helen Turner (325), Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 M. J. Walport (227), Rheumatology Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London W12 ONN, United Kingdom ix


ADVANCES IN IMMUNOLOGY, VOL. 76

MIC Genes: From Genetics to Biology SEIAMAK BAHRAM Centre de Recherche d’Immunologie et d’He´matologie, Strasbourg, France

I. Introduction

The specific interaction between the 움/웁 T cell receptor (TCR) and a peptide-bound major histocompatibility complex (MHC) molecule triggers the adaptive immune response (Garboczi and Biddison, 1999; Zinkernagel and Doherty, 1979). MHC class II (MHC-II) molecules present endosomally derived peptides of 12–18 amino acids in length to CD4⫹ helper T lymphocytes (Cresswell, 1994; Germain and Margulies, 1993), whereas MHC-I glycoproteins display endogenously generated nonameric amino acid chains to CD8⫹ cytotoxic T cells (Townsend and Bodmer, 1989; Yewdell and Bennink, 1992). As crucial as the latter reaction is to cellmediated immunity, it uses only a fraction of the available MHC-I molecules. These so-called classical, or class Ia, glycoproteins are encoded by MHC-linked genes, first identified in the mouse model through landmark serological and genetic experiments (for a review, see Snell, 1981; see also Gorer, 1936, 1937; Snell, 1948) and subsequently evidenced in humans, by studying alloimmunized individuals (for a review, see van Rood, 1993; see also Dausset, 1958, 1981). The universality of this system has been verified in almost every vertebrate species examined: In all, a restricted set of ubiquitously expressed, highly polymorphic, 웁2-microglobulin (웁2m)–associated, peptide-loaded class Ia molecules engages a large, almost combinatorial, T cell repertoire (Bjorkman and Parham, 1990; Du Pasquier and Flajnik, 1999; Litman et al., 1999). There are three such classical MHC-I genes in humans: HLA (human leukocyte antigen)–A, –B, and –C, encoded within the MHC on the short arm of the sixth chromosome, band p21.3 (Carroll et al., 1987; Dunham et al., 1987; Malissen et al., 1982). An equivalent number is carried by the mouse MHC on chromosome 17, designated H2-K , -D, and -L (Hood et al., 1983; Weiss et al., 1984).1 Structurally homologous to this first group of molecules are proteins encoded by what is termed nonclassical, or class Ib, MHC genes (Flaherty et al., 1990; Stroynowski, 1990). These are oligomorphic at best, most have an erratic pattern of tissue expression, and they display nonconventional 1 These two species are evidently the best studied with regard to MHC genetics and biology. For a description of MHC genetics in other species, the reader may consult Trowsdale (1995).

1

Copyright 䉷 2001 by Academic Press. All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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SEIAMAK BAHRAM

peptide antigens (mainly hydrophobic polypeptides derived from class Ia signal sequences). The human MHC contains three such genes: HLA-E (Koller et al., 1988), -F (Geraghty et al., 1990), and -G (Ellis et al., 1990; Geraghty et al., 1987; Srivastava et al., 1987), whereas the murine H2 complex is replete with the H2-Q, -T, and -M sequences (with numbers varying from a few to several hundred if not thousands, depending on the haplotype) (Amadou et al., 1999; Delarbre et al., 1992; Teitell et al., 1994).2 The past 20 years have allied the term specialized with MHC-Ib antigens, the murine TL and M3 proteins being the most extensively studied (Lindahl et al., 1997; Shawar et al., 1994; Teitell et al., 1994). However, a unified function has begun to emerge for some of these nonclassical molecules as the interaction with a distinct set of immunoreceptors has been established (Braud et al., 1999; O’Callaghan and Bell, 1998). Indeed, human HLA-E along with mouse Qa-1 interact with the natural killer (NK) cell inhibitory complex CD94/NKG2A-E (Braud et al., 1998; Lee et al., 1998), and human HLA-G is recognized by immunoglobulin (Ig)–like receptors (Allan et al., 1999; Colonna et al., 1997; Ponte et al., 1999) (no function has been ascribed as yet to HLA-F ).3 Hence, ‘‘nonclassical’’ molecules are becoming more and more ‘‘classical’’ (albeit playing a different part), and the enduring usage of this terminology has become more a matter of semantics than as a function-based 2 In general, there seem to be fewer structural constraints on these MHC-Ib genes, and one can confidently state that the actual physical size of the MHC is more or less determined by the topology of these genes along the chromosome. Indeed, molecular genetic analysis of the MHC region in various species clearly reveals a colinearity of the remainder of the complex; that is, most, if not all, class II and III genes chart at regular intervals a segment of roughly 1 Mb each, whereas the class I region varies in length from only a few kilobases in the Syrian hamster to 1.8 Mb in humans. However, this is mainly true in mammals, whereas in the chicken, for example, the MHC seems to be much more compact and to contain only the appropriately designated ‘‘minimal essential genes’’ (Kaufman et al., 1999). 3 Besides the proposed crucial role of HLA-G at the maternofetal interface—that is, protection of the fetus (allograft) (by a still to be experimentally proven mechanism) from rejection exerted by maternal NK or T effector cells—the molecule does not stand the test of time. Indeed, no murine homologue has been reported as of yet; moreover, the HLAG orthologue detected in nonhuman primates, for example, the rhesus monkey (MamuG ), is a pseudogene (Boyson et al., 1996), despite the fact that a functional homologue, Mamu-AG, might perform a similar function (Boyson et al., 1997). Moreover, a rather frequent human HLA-G null allele has been identified (Ober et al., 1998). The same study reported an adult individual homozygous for this allele as well as a first-trimester placenta carrying this allele, with no detectable HLA-G protein. All in all, the physiological relevance of NK–HLA-G interaction awaits further confirmation, and the much stressed unique placental expression of HLA-G might be fortuitous rather than essential.

MIC GENES AND MOLECULES

3

rational nomenclature.4,5 In sum, this first family of MHC class I genes has evolved to interact with two essential effector cell types of the immune systems: T lymphocytes and NK cells. The cloning of the first non–MHC-encoded class I molecule, CD1, was quite unexpected (Calabi and Milstein, 1986), yet more members soon followed (in chronological order): zinc-움2-glycoprotein (Zn움2gp, alternatively designated ZAG ) (Araki et al., 1988), neonatal Fc receptor (FcRn) (Simister and Mostov, 1989), MHC class I–related (MR1) gene (Hashimoto et al., 1995), the endothelial cell protein C/activated protein C receptor, EPCR (Fukudome and Esmon, 1995), and finally the HFE molecule (Feder et al., 1996) (Fig. 1 and Table I). At the outset, most of these molecules were regarded as mere curiosities. However, important functions were quick to emerge for some of them. The most intensely studied member of this group, CD1, is also the only one with a clear immunological mission. Indeed, work in the past decade has clearly established the capacity of this molecule to present mycobacteria-derived lipidic or glycolipidic antigens to the 움/웁 TCR, and thus intervene in host defense (Porcelli, 1995; Porcelli and Modlin, 1999). Other members of this group, however, perform non–defenserelated yet essential functions. FcRn fuels the neonatal bloodstream with much-needed antibodies by transepithelial shuttling of maternal IgG in the gut and the placenta (Simister et al., 1997). The HFE molecule, identified while Feder and colleagues (1996) were investigating the genetic basis for HLA-linked hereditary hemochromatosis, plays a critical role in regulating the body’s iron content. In fact, most patients with primary hemochromatosis carry, at homozygosity, a point mutation within the membrane-proximal 움3 domain of the HFE molecule, rendering association with 웁2m and subsequent surface expression impossible, which leads to gradual iron overload (Bahram et al., 1999; Zhou et al., 1998). ZAG is a soluble 웁2m-independent glycoprotein enriched within various exocrine fluids and might carry a ‘‘fat-depleting factor’’ yet to be identified (Araki et al., 1988; Sanchez et al., 1992; Todorov et al., 1998). The biology and function of MR1 remain to be investigated 4

Indeed, by virtue of their primary sequence (⬎70% sequence identity to class Ia molecules) as well as tridimensional structures so far resolved for HLA-E (O’Callaghan et al., 1998), they are confoundingly similar to class Ia molecules. 5 The imagination of a number of authors has been extremely fertile, as the ‘‘nonclassical’’ genes have been further classified into ‘‘MHC-Ib’’ (HLA-EFG ), ‘‘-Ic’’ (MICA, MICB, and HFE ), or ‘‘-Id’’ (CDI, FcRn, and ZAG ) by Hughes (1999), for some renamed ‘‘class IV’’; this includes both MICA/B and TNF loci (Gruen and Weissman, 1997), or even those termed ‘‘neoclassical’’ (H2–M3) (Wang and Lindahl, 1993). Finally, one is perhaps still better off using the terms classical and nonclassical.


5

(Riegert et al., 1998). Finally, the functional significance of EPCR within the blood coagulation cascade awaits further experiments. Therefore, a unifying view of the cross-genome scattering of MHC-I genes tends to define the MHC-linked members as those performing immunological functions, in contrast to most extra–MHC-encoded loci as engaged in non–defense-oriented roles. The recent identification of a distinct family of intra–MHC-located class I genes fits well with this dichotomy. Members of this MIC (MHC class I chain–related) gene family are stationed along the entire 1.8-Mb MHC class I region (Bahram et al., 1994). Unusual by several criteria (eg, a low degree of homology to other MHC-encoded class I genes, distinct transcriptional control elements, and a peculiar pattern of polymorphism), they appear to interact with T or NK cell receptors. The purpose of this chapter is to describe the short but already rich history of this novel family of histocompatibility antigens. II. Genes and Genomics

A. GENES MIC loci are the latest and the final HLA-encoded class I loci to be identified (Bahram et al., 1994). Their cloning was the culmination of a classic reverse-genetics approach using a 176-kb genomic contig linking HLA-B to BAT-1 in the middle portion of the MHC as a probe. Sequential screening of a fibroblast cDNA library with whole cosmid inserts ultimately led to the isolation of MICA and MICB cDNA clones. Genomic Southern blotting subsequently localized MICC, -D, and -E loci in close proximity to HLA-E, -A, and -F, respectively (Bahram et al., 1994) (Fig. 2). The MICA gene is merely 46.5 kb from HLA-B. MICB is 83 kb centromeric to MICA, and therefore ⬍130 kb from HLA-B. MICC is located 70 kb telomeric to HLA-E; MICD, 28 kb centromeric to HLA-A; MICE and MICG between HLA-G and -F, 18 and 85.5 kb centromeric to the latter, respectively; and finally, MICF, identified 24 kb centromeric to HLA-G (Shiina et al., 1999).

FIG. 1. Genomic dispersion of major histocompatibility complex (MHC) class I molecules. Debuting from an elusive MHC-I protogene, the present mammalian genome harbors class I homologues on at least five or six (human HFE is MHC-linked, unlike the murine counterpart) distinct genomic locations. This schematic representation is not to scale, and within multigene families (HLA, MIC, H2, CD1, as well as RAEs and ULBP), loci are positioned alphabetically. H2-QTM loci depict multiple genes. RAE-1␦ appears within parenthesis as it still needs to be confirmed as an independent locus and not an allele of other RAEs. References are included within the main text.

TABLE I BRIEF CATALOG OF SEMINAL FEATURES OF MHC CLASS I GENES Molecular Weight

웁2m

CD8

HLA-A, -B, -C HLA-E HLA-F HLA-G MICA MICB HFE CD1A, -B, -C

42 42 42 40 60–70 43a 49 43–49

Yes Yes Yes Yes No No (?) Yes Yes

Yes Likely Likely Yes No No (?) No No

CD1D FcRn ZAG MR1 EPCR UL18

37 46 42 39a 46 69

No Yes No

No No

TAP Yes Yes Yes Yes No No (?) No No

No No (?) ? ? ? ? (No) ? ?, Unlikely ?, Unlikely ?, Unlikely Yes ? ?

Expression

Cargo

Ligand

Allele

Ubiquitous Ubiquitous Restricted—B cells Placental trophoblasts Epithelia Epithelia Intestine Myeloid lineage

Cytosolic peptide MHC-I signal peptide ? Cytosolic peptide Nonpeptidic, if any Nonpeptidic, if any None Glycolipids (e.g., GMM and LAM) 움-Glycosylceramide/GPI None Fat-depleting factor ? ? Peptide

움/웁 TCR/KIR2D, -3D CD94/NKG2A ? KIR2DL4/ILT? 웂/␦ TCR/NKG2D 웂/␦ TCR/NKG2D TfR 움/웁 TCR DN/CD8⫹/웂␦-

165/327/88 5 1 14 52 16 ? (mutations) —

Intestine Intestine Exocrine fluids Ubiquitous Endothelium CMV infection

움/웁 TCR DN or CD4 IgG ? ? Protein C LIR-1/ILT2

? ?/— ?/— —/ ? ? —

웁2m, 웁2-Microglobulin; CMV, cytomegalovirus; GMM, glucose monomycolate; GPI, glycosylphosphatidyl inositols; IgG, immunoglobulin G; ILT, immunoglobulin-like transcript; LIR-1, leukocyte immunoglobulin-like receptor-1; LAM, lipoarabinomannan; LM, lipomannan; TAP, transporter associated with antigen processing; TfR, transferrin receptor. a Calculated molecular weight.


7

Both MICA and MICB are unusually large genes of 11,722 and 12,930 bp, respectively, compared to the average 3.5-kb HLA-A to -G genes (Bahram et al., 1996a,b). However, their overall genomic structures parallel those of the canonical MHC-I and more generally members of the Ig superfamily, in which distinct functional domains are encoded by separate exons (Barclay, 1999) (Fig. 2). Peculiarities are restricted to the leading and lagging exons and introns. In both MICA and MICB, large introns of 6840 and 7352 bp, respectively (versus ⬍200 bp in HLA-A to -G ), separate the first two exons and unique exons encode both cytoplasmic tails and 3⬘untranslated segments. Moreover, with respect to MICA, MICB contains an additional (unique) 1-kb 3⬘UT sequence (which incidentally eases transcript identification on Northern blots). The transcriptional–translational significance of these singularities, if any, is presently unknown. MICC, -D, -E, -F, and -G are pseudogenes of 4623, 1928, 7650, 1800, and 2627 bp, respectively (Shiina et al., 1999). Note that the MICG locus is presently available only in GenBank (accession No. AF055066). The truncated MICC gene is devoid of the first exon and intron and carries multiple in-frame termination codons. The crippled MICD gene lacks, in addition to the premier exon and intron, exon 6 and carries insertions or deletions within exon 5. The least problematic MIC pseudogene, MICE, contains all the exons but has multiple deletions both in exons 3–4 and across most introns. Finally, a part of intron 5 as well as exon 6 define the tiniest MIC pseudogene, MICF, as well as MICG. The possibility that some of these loci (especially MICE ) are operational in certain HLA haplotypes, as is the case for other MHC genes, remains to be investigated.6 B. GENOMICS The past 15 years have witnessed the progressive accumulation of a number of related sequences within the MHC, prominent of which are the class I and II multigene families (MHC Sequencing Consortium, 1999).7 Simplistic examination of these two gene families intuitively indicates that they originated from a common ancestor; however, lack of concrete data 6

Indeed, in two circumstances—MHC-II HLA-DRB and MHC-III C4/CYP21 genes— different HLA haplotypes carry a diverse load of genes and pseudogenes (for an overview, see the MHC Sequencing Consortium, 1999). 7 In addition to these, the HLA complex contains the following functional gene clusters: TAP (transporter associated with antigen processing) 1 and 2, LMP (low-molecular-weight polypeptides) 2 and 7 peptide delivery machinery within the class II region, the complement C4A and -B, the 21-hydroxylase (CYP ) A and B, the HSP701, 2, Hom stress protein genes, and finally the cytokines tumor necrosis factor (TNF ), lymphotoxin (LT ) A and B genes within the class III region, as well as the olfactory receptor gene complex on the telomeric side of the class I region. Other multigene families embedded in the class I region are also described within the text.


9

FIG. 2. MIC genes and pseudogenes are stationed along the entire major histocompatibility complex (MHC) class I region. Genomic structures of all loci are highlighted. Data were extracted from the full sequence analysis of the MHC class I region (Shiina et al., 1999). MICA and MICB encode functional glycoproteins. MICC-G are pseudogenes. L, leader; TM, transmembrane; Cyt., Cytoplasmic tail; UT, untranslated.

has precluded definitive conclusions thus far.8 The recent completion of the entire MHC nucleotide sequence provides the much-needed infrastructure to draw an accurate picture of the molecular genealogy of this complex genomic segment. Indeed, this analysis, applied to the class I segment, readily depicts a plausible molecular scenario as to kinetics of the 2-Mb MHC-I region genesis. In brief, shotgun sequencing of a multiplex (YAC, BAC, PAC, and cosmid) contig linking the centromeric MICB gene to the telomeric HLA-F locus allowed Shiina and colleagues (1999) to establish the molecular identity of the entire 1,796,938 bp of this genomic stretch.9 Dot matrix analysis using this entire 1.8 Mb versus itself revealed numerous large-scale duplications. These include the following noticeable homology sections: (i) 35-kb downstream segment of both HLA-B and HLA-C genes as well as 35-kb upstream regions of MICA and MICB genes display 80% and 85% nucleotide identities, respectively; (ii) 앑39 kb upstream of MICD and 35 kb 5⬘ of MICE share significant nucleotide identity not only between themselves but also with (⬎50%) corresponding regions of MICA and MICB loci; and (iii) the 5⬘ segments of MICD and MICF are also homologous to each other. In sum, the upstream segments of all members of the MIC gene family (except MICC ) display sequence homology to each other over distances ⬎15 kb. Interestingly, all these MIC-linked homologous segments share a unique mix of genes, all members of several multigene families. These 8 It is beyond the scope of this chapter to cover the genesis of MHC-II genes, which incidentally might have taken a less ‘‘tormented’’ route than their class I counterparts. Indeed, and as previously alluded to, compiling data from a large number of species reveals a near-perfect colinearity of MHC class II loci, in contrast to an apparent chaotic evolutionary path for the class I region. For example, human HLA-DP, HLA-DO, HLA-DM, HLA-DO, and HLA-DR hold their exact murine counterparts within H2-P, H2-O, H2-M, H2-IA, and H2-IE. Moreover, given the fact that there as been no evidence (so far) of any class II gene outside the MHC, in sharp contrast to the class I situation, it appears safe to state that class II molecules are an ‘‘MHC invention,’’ having most likely appeared later in evolution. 9 Moreover, this research localized a total of 127 genes or potentially coding sequences (one gene every 14.1 kb), as well as a wealth of microsatellites (758 in total). The latter will provide tools for high-resolution mapping of HLA class I–associated disease genes are readily applied to psoriasis vulgaris (Oka et al., 1999). See the work of Shiina and colleagues (1999) for a thorough description.

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SEIAMAK BAHRAM

are HCGIX, 3.8-1, P5, HCGIV, HLA class I, and HCGII (in this order and within the same gene orientation in most cases). These facts strongly imply that successive segmental duplication of this basic unit gave rise to the present human MHC-I system. A model based on these data and supported by dendrograms of various gene families provides a plausible sketch of MHC-I genesis. According to this model, the present telomeric segment of the MHC was most likely the ‘‘ground zero’’ of MHC conception, where HLA-F most possibly served as the proto–MHC-I locus, which in turn gave birth to the MICE and HLA-G genes upon duplication. The basic unit, including MIC, HCGIX, 3.8-1, P5, HCGIV, HLA class I, and HCGII, was therefore created. Two independent subsequent segmental duplications of this elementary unit simultaneously generated both the HLA-A–MICF and MICA–HLA-B segments. The latter gave birth to MICB and HLA-C genes after a single duplicative event. The next partial segmental duplication gave rise to the HLA-80–HCGIV-4 segment from the HLA-A–HCGIV-5 segment. Similarly, four subsequent segmental duplications, including partial ones, led to the present gene organization of the HLA class I region. The order of the generation of each gene predicted by this model is supported by dendrograms of the HLA class I, MIC, HCGIX, P5, 3.8-1, and HCGIV family members (Shiina et al., 1999). Altogether, it is clear that the present MHC was created by a process of gene duplication and selective extinction which may have given the immune system enough leverage to effectively fight the various waves of microbiological aggression encountered through its half-billion years of evolution. C. PHYLOGENY MHC molecules are believed to have appeared concomitantly with other elements of the adaptive immune system (chiefly the TCR and Ig’s) along with early vertebrates, for example, the jawed fishes (Gnathostomata), some 450 million years ago (Du Pasquier and Flajnik, 1999; Litman et al., 1999). All attempts to identify any similar structures in the evolutionary precedent jawless fishes (Agnatha) or nonvertebrates have failed.10 Within the family of MHC-I molecules, there appears to be a clear dichotomy between the evolutionary ‘‘prudent’’ MHC-Ia molecules in comparison to the ‘‘wilder’’ MHC-Ib genes. Indeed, it appears as if the former were passed along from one species to the next (along with their allelic repertoire) 10 Although MHC genes are a vertebrate invention, histocompatibility at a cellular level goes way back to sponges, for example, the colonial tunicate, Botryllus schlosseri (Magor et al., 1999; Weissman et al., 1999), if not to flowering plants (Burnet, 1971).


11

(Figueroa et al., 1988; Lawlor et al., 1988), whereas the latter were largely ‘‘reinvented’’ within the life of each species (Trowsdale, 1995). The evolutionary conservation of MIC genes (with the apparent exception of rodents, as discussed below) clearly breaches this general rule. Indeed, initial zoo-blotting experiments using a human MICA cDNA probe detected homologous sequences in a number of mammalian species. These included nonhuman primates as well as a number of more distant species, including goat, pig, cow, dog, and hamster. Curiously, mouse DNA appeared to be devoid of MIC-related sequences. Further experiments employing degenerate polymerase chain reaction (PCR) using not only congenic laboratory but also outbred mouse DNA as a template failed to reveal any remotely homologous sequences (V. Wanner, unpublished observations). Therefore, one can confidently state that mice (and hence probably rodents in general) are devoid of MIC genes, raising the question (and perhaps providing the answer) as to what extent MIC genes are dispensable (the question becomes even more difficult to evade once the issue of MIC-deficient humans is put forward later in the text). Obviously, from a phylogenetic point of view, mice are only one among thousands of mammalian species and are perhaps not the most interesting, as they appear to be a recent deviance from older rodents.11 To start with, the mouse H2 complex is unique. Besides the previously mentioned expansion–contraction phenomena observed in several congenic H2 haplotypes, one of the premier classical class I genes—indeed the most polymorphic, the H2-K locus—has been inexplicably ejected from the class I region and reinstated, by means of not yet understood phylogenetic gymnastics, to the centromeric segment of the class II segment (Amadou et al., 1999). Pertinent to our interest, the putative location of functional MIC (A and B ) genes in the mouse, the stretch linking H2-D (equivalent to HLA-B ) and BAT1, is substantially shortened as compared to the human MHC: 40 instead of 173 kb (Fig. 3). Hence, one can speculate that MIC genes were lost from the mouse genome during the H2-K translocation. Additional circumstantial evidence suggests that this very segment of the MHC may accommodate some structural frailty, as this is the precise segment where the MICA. gene is deleted in certain Southeast Asian HLA haplotypes (see below for details) (Fig. 3). Certainly, mice appear to be able to live and survive without any MIC genes. Two immediate conclusions, mutually exclusive, could be drawn from this observation: (i) MIC genes are not essential or (ii) they are replaced in this species by ‘‘functional 11 Obviously, this issue will be partially settled once various H2 haplotypes have been sequenced (Amadou et al., 1999) and totally laid to rest upon completion of the nucleotide analysis of the entire mouse genome (http://www.informatics.jax.org/).

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SEIAMAK BAHRAM

FIG. 3. A possible explanation for deletion of MIC genes from the mouse H2 complex. The ‘‘natural’’ BAT1–HLA-B interval is compared to that of some of HLA-B*4801 haplotypes as well as to a syntenic H2 segment. A large (100-kb) deletion eliminates MICA from some B48 haplotype mirrored by a shortened segment in the H2 complex. These events indirectly suggest fragility within this segment of the major histocompatibility complex (MHC). Caution should be instilled, however, as this contention is based solely on structural knowledge of the human and mouse MHCs.

orthologues’’ yet to be identified.12 In favor of the first possibility is that even humans tend to live a ‘‘normal’’ life without MICA and MICB (see the Section V for an explanation). In favor of the latter, there is at least one precedent: HLA-E and Qa-1. Despite not sharing any particular homol12

The use of the term essential could be open to debate. In nonimmunological science, the best example would perhaps be the eukaryotic developmental biology (this is not to get in the prokaryotic world); for a gene to be ‘‘essential’’ means that the natural or targeted removal of the locus from the germline will result in embryonic lethality. However, following this stringent definition, few ‘‘essential’’ genes function specifically within the immune system. Indeed, few ‘‘immune genes’’ are able to make such dramatic statements, and for most, their absence results in more or less serious postnatal complications, with some having no apparent effect at all (Schorle et al., 1991). Examples include that of TAP genes with bronchopulmonary (Donato et al., 1995) and other autoimmune manifestations (MoinsTeisserenc et al., 1999) and those of class I and II genes which result in various degrees of infectious troubles (Frelinger and Serody, 1998; Grusby and Glimcher, 1995; Grusby et al., 1993; Mach et al., 1996).


13

ogy (i.e., they are no more homologous to each other than, say, HLA-E and Qa-2), they both perform similar tasks—presenting MHC-Ia signal peptides to the NK cell inhibitory receptor CD94/NKG2. In addition to humans, the sole species for which high-resolution molecular genetics data are available with respect to MIC genes is the pig (Velten et al., 1999). A sequence-ready contig of the SLA complex has revealed, besides the precise location of a large number of typical class I genes, two MIC-related sequences flanking several class I–related SLA genes on a segment syntenic to the human MHC, within the border between the class I and III segments, close to the pig TNF and BAT1 loci. The pig MIC genes show 앑70% homology to human MIC and no more than 20% to SLA class I sequences (Velten et al., 1999). The initial Southern blot experiment (Bahram et al., 1994) revealed an equivalent number of fragments in nonhuman primates. Efforts by a number of laboratories have culminated in the sequence analysis of these genes (Pellet et al., 1999; Seo et al., 1999; Steinle et al., 1998) (Fig. 4 and Table II). Surprisingly, the degree of sequence identity between human and primate MIC is far lower than that of respective class Ia sequences: 70% for the former versus 앑95% (or higher) for the latter. Moreover, there are several small (3–, 4–, or 5–amino acid) deletions within the 움1 and 움2 distal putative ligand-binding domain of nonhuman primate MIC sequences. No such deletions have been detected within the 움3 domain. Not surprisingly (as is the case for most class I genes), the transmembrane and cytoplasmic domains show the lowest degree of interspecies as well as interlocus conservation. Whether all these divergences reflect functional adaptation to a yet to be identified ligand or instead represent, more generally, a glide toward evolutionary inactivation remains to be appreciated. All in all, the clear conservation of the MIC gene family across mammals defines them as a distinct lineage of MHC-I genes, the eighth, following MHC-Ia/b, CD1, MR1, ZAG, FcRn, HFE, and EPCR. The apparent absence of MIC orthologues/homologues in the mouse genome, mirrored by the reciprocal lack of murine nonclassical loci in other mammals/vertebrates, might best serve as a trace for better understanding the evolutionaly dichotomy of these two well-studied MHC architectures. III. Transcripts and Transcription

A. TRANSCRIPTS The MICA gene encodes an mRNA of 1382 bp harboring a 1149-bp open reading frame that gives rise to a 383–amino acid polypeptide (this length varies based on the number of alanine repeats within the transmem-

14

FIG. 4. Primate and porcine MIC sequences. Domain-by-domain multiple alignment of the presently available primate and pig (only the 움1 exon) MIC sequences. When more than one locus has been identified, they are called MIC1 and MIC2, as the sequences do not show, in general, indisputable MICA- or MIC B-ness. In some cases, allelic variants have also been identified. Data were compiled from Pellet et al. (1999), Seo et al. (1999), and Steinle, et al. (1998).

FIG. 4. (Continued )


17


TABLE II HUMAN, PRIMATE, AND PORCINE MIC SEQUENCES Sequence Human MICA Gene Full-length cDNA Human MICB Gene Full-length cDNA Primate MIC (Genes and cDNAs) Aotus trivirgatus MIC Ateles fusciceps MIC1 Ateles fusciceps MIC2 Callithrix argentata MIC Cercopithecus aethiops MIC Cercopithecus patas MIC Chlorocebus aethiops MIC1 Chlorocebus aethiops MIC2 Gorilla gorilla MIC Hylobates lar MIC1 Hylobates lar MIC2 Macaca mulatta MIC101 Macaca mulatta MIC102 Macaca mulatta MIC201 Macaca mulatta MIC202 Macaca mulatta MIC3 Pan panisus MIC Pan troglodyte MIC Papio hamadryas MIC1 Papio hamadryas MIC2 Papio sp. MIC Pongo pygmaeus MIC1 Pongo pygmaeus MIC2 Porcine MIC (Genes) Sus scrofa MIC1 (exon 2) Sus scrofa MIC2 (exon 2)

Accession

Reference

X92841 L14848

Bahram et al. (1996a) Bahram et al. (1994)

U65416 X91625

Bahram et al. (1996b) Bahram and Spies (1996)

AF055389 AJ242444 AJ242445 AF055390 AF045601 AF045602 AF055385 AF055386 AF045597 AF045596 AF045604 AF055387 AJ242439 AF055388 AJ242440 AJ242441 AF045598 AF055384 AJ242442 AJ242443 AF045603 AF045599 AF045600

Steinle et al. (1998) Seo et al. (1999) Seo et al. (1999) Steinle et al. (1998) Pellet et al. (1999) Pellet et al. (1999) Steinle et al. (1998) Steinle et al. (1998) Pellet et al. (1999) Pellet et al. (1999) Pellet et al. (1999) Steinle et al. (1998) Seo et al. (1999) Steinle et al. (1998) Seo et al. (1999) Seo et al. (1999) Pellet et al. (1999) Steinle et al. (1998) Seo et al. (1999) Seo et al. (1999) Pellet et al. (1999) Pellet et al. (1999) Pellet et al. (1999)

AF083661 AF083662

Velten et al. (1999) Velten et al. (1999)

brane segment; see below) of 43 kDa (Bahram et al., 1994). The MICB transcript of 2376 bp encompasses an open reading frame of equal length bearing 83% amino acid similarity to MICA. MICA and MICB share only an average 21%, 19%, and 34% identity in the 움1, 움2, and 움3 extracellular domains, respectively, with other MHC-I genes; other MHC-I loci, whether human, mouse, classical, or nonclassical, share close to 70% homology with each other (Bahram and Spies, 1996). The MICA and MICB glycoproteins are structured, as are all MHC-I molecules. The mature

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protein contains three extracellular domains (움1–움3) preceding a membrane anchor segment and a relatively short (42–amino acid) cytoplasmic tail. Despite the fact that no canonical targeting motif could be detected within this tail, analysis of an engineered deletion as well as that of a natural mutant lacking this domain has established its role in directing the molecule to the basolateral surface of polarized epithelial cells, the putative physiological emplacement of the MICA glycoprotein (Suemizu et al., unpublished data) (see below). The transmembrane exon does not offer more than the expected hydrophobicity (for length variation, see below). The Ig-like 움3 domain contains no unusual features except for the clear absence of a CD8 coreceptor binding site. The 움1 domain is slightly shorter (85 amino acids instead of 90) and the 움2 domain is barely longer (96 amino acids instead of 92) than MHC-Ia. Moreover, the presence of a large number of N-linked glycosylation sites (eight in MICA and five in MICB) contrasts with an invariant single such site, NX(86)S, within classical MHC-I molecules, incidentally absent in both MICA and MICB. Finally, in addition to the two MHC-I canonical disulfide bridges (i.e., between cysteines 96–164 and 202–259), MICA harbors a third bridge connecting cysteines 35 and 40 (Bahram et al., 1994; Li et al., 1999). The sole notable divergence between the MICA and MICB genes resides within their respective transmembrane exons. Indeed, in contrast to MICB, MICA harbors a short tandem repeat (STR) sequence encoding various numbers of GCT triplets (Mizuki et al., 1997). Based on the number of repeats, these are named (for alanine) A4, A5, A6, A9, A10 (PerezRodriguez et al., 2000), and finally, A5.1. The latter is identical to A5 except for an extra nucleotide insertion (GGCT), leading to a frameshift mutation causing a premature termination codon within the transmembrane exon (the genetics and biological significance of these alleles are discussed below). B. TRANSCRIPTION In contrast to the classical (and most nonclassical) MHC-I genes, MIC genes are not ubiquitously expressed.13 In fact, their transcripts are not 13

As previously mentioned, MHC-Ia genes, as exemplified by HLA-A, -B, and -C in humans show a ubiquitous pattern of expression within all nucleated cells of the organism, with the clear exception of the central nervous system, although the latter changes after viral challenge and the former is not a universal feature of the animal kingdom, as rabbit erythrocytes express MHC molecules (Gorer, 1936). Regarding human MHC-Ib genes, except for HLA-G, which shows a restrictive appearance within the placenta (Ellis et al., 1990)—despite a broader mRNA expression pattern (Ulbrecht et al., 1994)—both HLAE and -F display broader transcription and translation patterns. HLA-F is transcribed in various tissues, although at the cellular level, B cells carry the highest amount of the protein (Wainwright et al., 2000). Finally, HLA-E resembles true class Ia molecules with regard to extent of expression, as it is almost ubiquitously expressed (Geraghty, 1993).


19

detectable (by means of total cellular RNA Northern blotting) in cells of lymphohematopoietic lineage. MIC are primarily transcribed in fibroblasts and epithelial cell lines as well as various (almost all) tissues harboring these cell types (Bahram et al., 1994, and our unpublished observations; see below). Moreover, at odds with the transcriptional enhancement of MHC-Ia and -II loci following treatment by type I/II interferons, MICA and MICB mRNA levels remain unaffected by these cytokines. In line with this, sequence analysis of upstream genomic sequences of both MICA and MICB loci failed to show any homologies within the well-studied MHCIa promoter sequences; in particular, no sign of the interferon response sequence was evident.14 Intriguing, however, was the presence of a short DNA segment with significant homology to heat shock protein (HSP) gene promoters (Groh et al., 1996). The core of this region is defined by the contiguous and inverse duplications of the pentanucleotide motif 5⬘– nGAAn–3⬘, known as the heat shock response element (HRE), which is common to a number of stress-induced genes, a prototype of which is the MHC-encoded HSP70 gene (⬍400 kb centromeric to MICA and -B ) (Sargent et al., 1989). HRE functions by attracting the stress transcriptional machinery, for which trimerization of the heat shock transcription factor 1 is paramount (for an overview, see Morimoto, 1998).10 The function of the MIC HRE elements has been assessed by shocking epithelial cells for various lengths of time at 42–42.5⬚C and monitoring the level of MICA/B message up-regulation and membrane expression. Transcriptionally, the situation is fairly straightforward. As little as 5– 10 min of heat treatment is enough to induce the transcription of MICA and especially MICB (which is expressed at lower levels in ‘‘healthy’’ cells). The strongest induction encountered so far occurred 1–2 hr poststress. Once the heat shock is removed, the RNA levels return to the basal state after 앑12–16 hr. All of this is somewhat unremarkable, as it faithfully follows the kinetics of HSP mRNA rise and fall, most likely via recruitment of the same transcriptional machinery, although at significantly lesser strength (⬍30 minutes of autoradiography is sufficient to detect changes in HSP transcription as compared to overnight exposures for MIC ). In the first experiments, no obvious correlation between higher MIC mRNA and membrane expression was noted (Groh et al., 1996). However, these early experiments were performed irrespective of the cells’ growth status; when 14 MHC-I transcription is reasonably well characterized (David-Watine et al., 1990; Kimura et al., 1986). In addition to a typical TATA and CAAT box, the critical region seems to reside around position ⫺150 from the transcription start site, where a number of overlapping motifs regulate the transcription. In addition to the above-mentioned interferon response sequence responding to type I (움 or 웁) and II (웂) interferons synergistically with TNF, these include ␬B1 and ␬B2 as well as retinoic acid X receptor 웁 (RXR-웁) binding sites.

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Groh and colleagues (1998) performed similar attempts in confluent (hence, quiescent) cell layers, a marked increase in MIC surface expression was obvious after heat shock. Actually, the physiological relevance of this experimental procedure is doubtful in that the intestinal epithelium is the fastest proliferating tissue in the body, second only to the skin; but it does clearly document, under certain circumstances, enhanced poststress surface deposition of MICA and possibly MICB. Finally, it remains to be seen to what extent MICs are induced by more ‘‘real-life’’ stressful challenges. Obvious examples include epithelial infections with bacteria such as Shigella, Salmonella, Listeria, and Chlamydia: parasites such as Cryptosporidium and Leishmania; and, finally, viruses such as influenza and vesicular stomatitis virus, among many other candidates. Although the presence of an HRE in the putative promoter region of MICA and MICB genes is intriguing, it is not unprecedented. Over a decade ago, during one of the first thorough characterizations of an MHC-I promoter, Kourilsky and co-workers (Kimura et al., 1986) recognized a sequence homologous to HRE in the promoters of the H2-Kb and -Ld mouse class I genes. Although this sequence is probably not functional, as typical MHC-I genes are not stress induced, it may represent vestigial imprints of the universal heat shock defense machinery on the adaptive immune system. IV. Biochemistry and Biology

A. BIOCHEMISTRY A number of anti-MICA/B–specific monoclonal antibodies (mAbs) (Groh et al., 1996, 1998; M. Colonna et al., unpublished observations) and antisera (Suemizu et al., unpublished data) have been obtained.15 These reagents have led to initial biochemical (Groh et al., 1996) and cell biological (Suemizu et al., unpublished data) assessment of MICA function. Indeed, cell surface staining as well as immunoprecipitation experiments using various B cell line transfectants has provided a number of important observations. (i) MICA cDNA stably transfected into 웁2m-deficient Daudi cells allowed detection of a cell surface MICA chain at levels comparable to that in C1R-transfected cells. Moreover, pulse–chase experiments following metabolic labeling revealed identical Endo-H resistance ki15 mAbs 56, 83, and 2C10 were obtained upon immunization with C1R–MICA transfectants (Groh et al., 1996). They are all of the IgG isotype and specific for MICA. mAbs 56 and 2C10 were employed in the biochemical characterization of MICA, whereas mAb 83 was used for immunofluorescent staining. Another mAb, 6D4, was subsequently obtained (Groh et al., 1998) and reported to interact with both MICA and MICB. This, together with 2C10, was used in target recognition experiments (Groh et al., 1998).


21

netics for MICA molecules in both Daudi and C1R transfectants, proving that 웁2m is not required for physiological MICA cell surface expression. (ii) The same set of experiments performed in the TAP-deficient 5.2.4 cells revealed normal surface expression of MICA in the absence of cytosolic peptides. (iii) The native MICA chain is highly glycosylated, with a molecular mass of 65–75 kDa compared to 43 kDa after glycan removal. (iv) Attempts to extract peptides from the putative antigen binding groove were unsuccessful (Groh et al., 1998). This correlates well with the independence of MIC biology from the endogenous antigen presentation pathway as well as with the structure of the putative ligand binding cleft (see below). (v) Finally, staining with this first set of monoclonal antibodies suggested that MICA is specifically expressed in the gastrointestinal epithelium, as revealed by indirect immunofluorescence on tissue sections; the only other site of expression was thymic cortical epithelium (Groh et al., 1996). More recent experiments, however, document a more generalized pattern of expression for MIC genes, extending beyond the intestinal epithelium. This contention is based on two sets of experiments. First, an extensive tissue/ organ Northern blot experiment reveals the presence of both MICA and MICB transcripts in virtually every organ examined, with the clear exception of the central nervous system (unpublished observations). This is most likely due to the presence of epithelial cells within each of these positively examined organs, as initial experiments had documented patent MIC expression in epithelial or fibroblastic cell lines (Bahram et al., 1994; see above). Second, analysis of MIC expression with a novel set of anti-MICA or MICA/B mAbs (M. Colonna et al., unpublished observations) displays a much broader staining pattern; for example, various epithelial components of the kidney and the skin are positive, in contrast to the spleen, which is negative. Therefore, it appears that MIC expression is primarily controlled at the transcriptional level. This was hinted at by the fact that MICA and MICB glycoproteins can be invariably detected in various transfected cells of diverse lineages, for example, lymphoid (C1R, Raji, lymphoblastoid B cells, and so on) (Groh et al., 1996) or epithelial [HeLa (N. Fodil, unpublished observations) and MDCK (Suemizu et al., unpublished data)], strongly implicating that the presence of mRNA suffices for surface expression.16 Nevertheless, why some monoclonal antibodies give restricted staining patterns (our unpublished observations; Groh et al., 1996) remains to be thoroughly investigated. Besides the obvious bigenicity and polyallelism, the fact that both MICA and MICB are heavily glycosylated (perhaps differently in various tissues) should be considered as possible explanations. 16 The opposite is best illustrated by both HLA-E and -F, which do not reach the cell surface despite large levels of transcripts and intracellular deposits in the absence of their physiological ligand. This is known to be true in the case of HLA-E but remains to be proven for HLA-F.

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A mouse antiserum raised against MICA-expressing NS1 transfectants has allowed an initial analysis of MIC cell biology. Using indirect immunofluorescence, immunoelectron as well as laser scanning confocal microscopy, Suemizu and colleagues (unpublished data) were able to localize the MICA glycoprotein at the basolateral surface of human intestinal epithelial cells, one of the putative sites of MICA function. Moreover, subcellular localization studies of an exon 6 (cytoplasmic tail)–less MICA cDNA construct as well as one containing the MICA A5.1 allele establish a function for sequences contained within the MICA (and possibly MICB) cytoplasmic tails for intracellular targeting of the glycoprotein, as following its removal the molecule was diverted to the apical cell membrane. Consequently, MICA A5.1 could be considered de facto a MICA null allele. B. LIGANDS Prior to reviewing MIC ligands, it is perhaps useful to put into perspective the long and often passionate field of MHC-Ib and 웂/␦ and other ‘‘exotic’’ T cell types. Shortly after the discovery of 웂/␦ T cells in humans (Brenner et al., 1986, 1988), Strominger (1989) made a provocative prediction. The thesis postulated that two (then and, in large part, today) enigmatic components of the immune system, that is, 웂/␦ T cells and nonclassical class I molecules (dubbed here MHC-Ib), might interact with each other. What followed [indeed slightly preceded (Matis et al., 1987)] was a constant stream of reports linking these two entities in various systems. The most conclusive efforts were achieved, for obvious experimental reasons, in the murine system.17 A classical example is the description by Tonegawa and 17

The reader is urged, however, to consider fundamental differences in 웂/␦ T cell biology in various species [for detailed information about 웂/␦ T cells, the reader is referred to a number of authoritative review articles which have already fielded the subject (Allison and Havran, 1991; Bluestone et al., 1995; Chien et al., 1996; Kaufmann, 1996)]. First, whereas in humans, 웂/␦ T cells carry a diverse repertoire and are more or less evenly distributed throughout the body (Groh et al., 1989), in the mouse several subpopulations carry a biased TCR (preferential usage of certain V segments, albeit for the fact that they carry a high degree of junctional diversity). The latter are restricted to certain anatomical sites (e.g., the skin, tongue, vagina, and intestinal epithelia), where they represent the majority of T cells and hence are believed to act as the so-called first line of defense. Second, and despite the fact that in both humans and mouse 웂/␦ T cells constitute a rather minute fraction of peripheral T cells (⬍10%), in other species such as cattle, sheep, and chickens, they tend to outnumber 움/웁 T cells within the circulation. Third, in spite of major advances during the last few years, a universal 웂/␦ ligand has yet to be found. In fact, it is becoming increasingly clear that such a ligand may not exist, as these receptors appear to interact with antigen much like Ig. Considering the known ligands, two categories of molecules could be recognized. Despite the formal absence of the paradigmatic 움/웁 MHC restriction (Zinkernagel and Doherty, 1979), MHC and MHC-like structures have been shown to interact with the 웂/␦ TCR. Also, specifically with respect to human 웂/␦ T cells, the situation


23

colleagues of the recognition of H2-T22b (originally named TL 27b) by a 웂/␦ T cell clone (KN6) (Bonneville et al., 1989; Ito et al., 1990). The same molecule was further recognized by another famous 웂/␦ clone, G8 (Schild et al., 1994).18 Besides T22, G8 has also been shown to interact with another murine class Ib chain, T10 (Schild et al., 1994), highly homologous to T22. Another instrumental T cell clone, LBK5, recognizes the class II molecule H2-IEk (Schild et al., 1994), independent of any species or cell type ‘‘barrier,’’ as the recognition is equally potent with human, mouse, or hamster models or in T, B, or fibroblast cell lines. The recognition of nonclassical MHC molecules by the 웂/␦ TCR occurs independently of the wellunderstood class I and II antigen processing pathways, as HLA-DM or TAP mutant cell lines (RMA-S, 721.134, or T2) are equally good stimulators in comparison to wild-type parental lines (Kaliyaperumal et al., 1995).19 Indeed, the sole unequivocal modulator appears to be the level of cell surface expression; even bacterially expressed plastic-bound molecules are able to stimulate a 웂/␦ hybridoma (Crowley et al., 1997). Another murine has been greatly clarified. The human V웂9 (or V웂2, depending on the nomenclature)/V␦2 T cells (these constitute ⬎90% of circulating 웂/␦ cells) respond to a group of phosphatecontaining small molecules, independent of any MHC element (Constant et al., 1994; Morita et al., 1995; Schoel et al., 1994; Tanaka et al., 1994). The fact that several microbiological extracts carry these elements has led to the assumption that this interaction might be in line with the defense role for these cells, which indeed augment numerically during the course of various infectious states (Balbi et al., 1993; Caldwell et al., 1996; Hara et al., 1996; Perera et al., 1994). However, despite the apparent logic in this interaction, it is restricted to human cells, as mouse 웂/␦ T cells do not recognize any of these molecules (M. Bonneville, personal communication). Finally, added to this list of ligands are more ubiquitous molecular entities such as alkylamines derived from a vast spectrum of biological taxa such as microbes, plants, and tea (Bukowski et al., 1999). Hence, we are left with a heteroclitic catalog of targets diverse in both size and structure. 18 A more recent report has evidenced, for the first time, physical interaction between MHC-Ib and a 웂/␦ TCR, that is, binding of H2-T22b to the immobilized G8 protein (Crowley et al., 2000). This, along with the concomitant structural resolution of T22b, allowed the authors to map a putative interaction site with the 웂/␦ TCR on opposite sides of the T22 groove, where the 웁-pleated sheets are directly exposed and on a diametrically opposite acidic patch. This is different from the site proposed by the Strong group (Li et al., 1999) for MICA to interact with the V␦1 receptor: under the 웁-sheet. This situation is very distinct with respect to the MHC-Ia–움/웁 interaction. Thus far, all published crystal structures show a nearly identical picture, that is, an 움/웁 TCR sitting diagonally across from a peptide–MHC complex (Ding et al., 1999b; Garboczi et al., 1996; Garcia et al., 1996, 1998; for a review, see Garcia et al., 1999). 19 This is in contrast to earlier experiments, which incriminated peptide dependency of the 웂/␦ recognition. This was based on site-directed mutagenesis experiments. Once applied to several residues on the floor of the putative antigen-binding cleft of T22, this affected recognition by the KN6 hybridoma (Moriwaki et al., 1993). However, this might have been the indirect consequence of surface expression, as documented by others regarding at least one of these point mutations (Chien et al., 1996).

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MHC-Ib molecule recognized by the 웂/␦ TCR is Qa-1. Vidovic and colleagues (1989) reported the isolation of a CD4⫺CD8⫺ TCR 웂/␦⫹ T cell hybridoma (DGT3) specifically recognizing Qa-1b in conjunction with a peptide ligand, the copolymer poly(Glu50Tyr50). Human MHC-reactive 웂/␦ T cell clones have not been scarce either; almost every HLA locus and a large number of alleles have been recognized as potential targets. Examples include clones stimulated by HLA-A2 (Spits et al., 1990), -A9 (Vandekerckhove et al., 1990), -A24 (Ciccone et al., 1989), -DR2 (Flament et al., 1994), -DR7 ( Jitsukawa et al., 1988), -DRw53 (Holoshitz et al., 1992), and -DQw6 and -DQw7 (Bosnes et al., 1990). Another well-known target for 웂/␦ T cells, including those within the gut, is CD1 (Balk et al., 1991, 1994; Bleicher et al., 1990; Blumberg et al., 1991; Kim et al., 1999; Somnay-Wadgaonkar et al., 1999). Even non-MHC structures can apparently be specifically recognized by 웂/␦ T cells; the most thoroughly studied case is certainly that of the herpes simplex virus glycoprotein, gl. Indeed, a series of elegant experiments by Bluestone and colleagues provides one of the most thorough investigations of the molecular details of 웂/␦ T recognition undertaken to date ( Johnson et al., 1992; Sciammas and Bluestone, 1998; Sciammas et al., 1994). The 웂/␦ T cell clone, Tgl4.4, recognizes gl independently of the class I and II antigen presentation pathways. This recognition is equally strong when a soluble recombinant protein is used. Hence, the same 웂/␦ TCR is able to recognize a large glycoprotein in a native configuration, reminiscent of Ig recognition of target antigen. Maybe the single most important message of research within recent years is that the 웂/␦ mode of recognition resembles Ig rather than 움/웁 TCR. As previously mentioned, and again resembling antibodies, 웂/␦ TCRs are able to recognize a great variety of molecular entities, ranging from small phosphate-bound molecules to large glycoproteins and englobing structures as disparate as alkylamines (Bukowski et al., 1999) and glycolipids. Finally, although the molecular drive behind this promiscuous mode of recognition has yet to be cracked by the crystallographic studies so elegantly applied to both TCR–MHC class I and II ternary complexes (Garboczi et al., 1996; Garcia et al., 1996; Reinherz et al., 1999), sequence analysis of the junctional diversity of 웂/␦ TCRs allows some educated insight into the ‘‘logic card’’ of the receptor.20 This work has shown that the 웂/␦ TCR is more similar to Ig than to the 움/웁 TCR in CDR3 length distribution (Rock et al., 1994). In 움/웁 TCRs, both CDR3s are relatively short and uniform in length, likely governed by coercive forces linked to dual peptide–MHC recognition, whereas in 웂/␦ TCRs, the 웂-chain CDR3 20 The recent resolution of the V␦ chain has indeed shown invaluable structural data and has confirmed previous results indicating a recognition mode similar to those used by Ig’s (Li et al., 1998).


25

chain is short with a limited length distribution, but ␦-chain CDR3 loops are long and quite heterogeneous in length. This is very similar to the situation in Ig, where the light-chain CDR3 loops are short and homogeneous, but the heavy-chain CDR3 loops are longer with a wide length range. Hence, the 웂/␦ TCR could be considered a ‘‘T cell immunoglobulin.’’ The field of NK cells and MHC class I molecules has roots in much older times. The first link between the two entities has been the longstanding experiments of Cudkowicz, Bennett, and Kumar, who in early 1970s, described the so-called ‘‘hybrid resistance’’ phenomenon (Shearer and Cudkowicz, 1975; reviewed in Bennett, 1987; Cudkowicz and Hochman, 1979; Kumar et al., 1997). Briefly, whereas irradiated F1 hybrids between two H2-mismatched strains of inbred mice accept solid organ grafts from either parent, they tend to reject those of bone marrow. The molecular basis for this event was attributed to a hypothetical hematopoietic histocompatibility (Hh) locus which remained mysterious until the recent discovery of the interplay between a growing number of NK receptors and MHC-I molecules (Ka¨rre and Colonna, 1998; Lanier, 1998). Indeed, two sets of NK and T cell subtype–specific receptors interact with a wide range of MHC-I loci and alleles through a site distinct from the well-known footprint of the 움/웁 TCR on MHC 움-helices and peptide. These receptors arise from two distinct gene families: the human chromosome 19q13.4-based Ig-like killer inhibitory receptors (KIRs), including KIR proper as well as the so-called Ig-like transcripts (ILTs) (Colonna, 1997; Lanier, 1998); and the 12p12–13–located CD94 (Lopez-Botet and Bellon, 1999), NKR-P1 (Lanier et al., 1994), and NKG2A-F lectin-type molecules (Houchins et al., 1997; Ryan and Seaman, 1997).21 Among KIRs, KIR2D (nD refers to the number of extracellular domains; for terminology, see Long et al., 1996) binds to HLA-C, and KIR3D binds to HLA-A and -B.16 ILTs may have a broader pattern of interaction with MHC-I glycoproteins; for instance, ILT-2 interacts with HLA-A, -B, and -G (Colonna et al., 1997). Although engagement of cognate MHC ligand by most receptors inhibits effector cell function via recruitment of SHP-1 and SHP-2 phosphatases by the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), certain receptors lack ITIM and associate with signal-transducing modules containing the immunoreceptor tyrosine-based activatory motif (ITAM). Within this context, Groh and colleagues have added MIC to the evergrowing list of 웂/␦ and NK ligands. Based on the intestinal pattern of expression obtained by mAb 83, these authors speculated that MIC might interact with a subset of T lymphocytes bearing the V␦1 웂/␦ receptor, 21 No equivalent to KIR genes have been identified so far in the mouse, where the major inhibitory receptor gene family is the chromosome 6–based lectin-type Ly49 molecule (Yokoyama, 1998), for which a human counterpart has been identified (Westgaard et al., 1998).

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enriched within this same anatomical site. To prove this, gut-derived T cells grown on C1R–MICA– or –MICB-transfected feeder cells were flowsorted using a V␦1-specific mAb. In this manner, two V␦1-bearing 웂/␦ T cell lines and several derived clones were shown to specifically recognize and kill target cells expressing MICA/B molecules (Groh et al., 1998). This recognition was shown to be independent of proteasome-generated, TAP-translocated cytosolic peptides, as lactacystin-treated (proteasomeinhibited) or TAP-deficient transfectants were lysed as well as controls by the same T cell clones. Moreover, attempts to extract peptides using the same conditions that allow peptide isolation from the MHC-Ia groove were unsuccessful. This work clearly establishes the ability of 웂/␦ TCR–bearing T cells to engage MICA- and MICB-expressing target cells, as both antiMIC and V␦1 antibodies were able to inhibit this interaction. However, this inhibition was only partial, and direct physical contact between TCR and MIC remains to be proven by TCR gene transfer experiments. Several other caveats also persist. Chiefly, as previously discussed, MIC expression does not match V␦1 expression and vice versa. Moreover, as the issue of alloreactivity of MIC-interacting T cell clones has not been addressed, the question remains as to their physiological significance. Finally, another recent contender, CD1c, has outcast MICA from the front scene (Spada et al., 2000). Indeed, the comprehensive effort by Spada and colleagues reported very convincing data, including direct TCR involvement in the recognition of CD1c by autoreactive V␦1-bearing 웂/␦ T cells. The situation became more interesting as Bauer and colleagues joined forces with Lanier and co-workers to demonstrate that a previously orphan NK cell receptor, NKG2D, interacts with MICA (Bauer et al., 1999). As previously mentioned, NKG2D is a member of a cluster of genes (NKG2A through F ) encoding type II transmembrane lectin-type molecules on the short arm of human chromosome 12 and murine chromosome 6. Among these, the well-known NKG2A partners with the closely linked CD94 (Lazetic et al., 1996) to create an inhibitory receptor on most NK and CD8⫹ 움/웁 T cells which recognizes the broadly expressed HLA-E molecule.22 By sequence homology, NKG2D is the most divergent member of the NKG2 gene family, as it shares only 20% sequence identify with other members of the family, which bear ⬎90% identity to each other. The gene, transcribed widely in T (CD8⫹ 움/웁 and 웂/␦) and NK cells, encodes a 42-kDa glyco22 NKG2C also associates with CD94, but in this case creates an activatory receptor. In contrast to inhibitory receptors engaging the signal transduction machinery through their cytoplasmic ITIM, the triggering receptors lack such a motif and hence have to recruit appropriate ITAM-bearing modules. For NKG2C/CD94, this is DAP12, a 12-kDa molecule Interacting with NKG2C in part via charged residues in their respective transmembrane segment (Lanier et al., 1998).


27

protein, surface expression of which requires the presence of a signaltransducing unit called DAP10 (Wu et al., 1999) or KAP10 (Chang et al., 1999). DAP10 is both structurally and functionally analogous to as well as physically linked to DAP12, a molecule previously shown to bind CD94/ NKG2C, encoded on chromosome 19q13.1.23 Moreover, the very fact that NKG2D is widely expressed on 움/웁, 웂/␦, and NK cells indirectly questions the physiological relevance of the putative ‘‘exclusive’’ 웂/␦ T cell interaction with MIC (Groh et al., 1998). Finally, the presence of a murine NKG2D orthologue (in contrast to the case for MIC), suggests, at the very least, that MICA/B is not the only ligand for this receptor. This is corroborated by evidence for a promiscuous interaction of NKG2D with MHC-I molecules in general; soluble NKG2D binds to a cocktail of HLA-A, -B, and -C alleles (Ding et al., 1999a). Indeed, a major leap in the field was the identification of a distinct set of ligands for NKG2D. These somewhat remote MHC-I lookalikes are unique in that they lack the proximal 움3 domain. In mouse, these are products of the glycosyl-phosphatidyl inositol (GPI)-linked retinoic acid early transcripts RAE-1움, 웁, 웂, and ␦, as well as the H60 gene product, originally identified as a minor histocompatibility antigen (Cerwenka et al., 2000; Diefenbach et al., 2000). Human equivalents of these murine chromosome 10-encoded loci are located on chromosome 6q25 and were identified during Cosman and colleagues’ search for molecules interacting with the cytomegalovirus UL16 glycoproteins (Cosman et al., 2000) (Fig. 1). Three of these ULBPs (ULBP1-3) were originally reported; at least one more has been identified through in silico genomic screening (B. Cuillerier, O. Cle´ment, S. B., unpublished observations). These molecules do not appear to be induced by cell stress, although some are strongly regulated by retinoic acid during embryonic development. For these transcripts to be absent from most adult tissues, and to be selectively expressed within tumor cells, might indeed represent a powerful signal for NK cell anti-tumor cytotoxicity. The fact that this new set of molecules is conserved between human and mouse, albeit quite remotely, reflects perhaps the redundancy of MIC recognition by NKG2D. In sum, MICA (and likely MICB) have been shown to interact with two distinct immunoreceptors, the 웂/␦ (V␦1) TCR and NKG2D (although effects attributed to V␦1 TCR could indeed be attributable to NKG2D); the physiological significance of these interactions remains a focus for future work. C. STRUCTURE The structure of a soluble MICA glycoprotein (allele 001) expressed in insect cells (Bauer et al., 1998) has been successfully analyzed by x-ray crystal23 An important note must be made, however. In contrast to ITAM-bearing DAP12 capable of transducing signal via interaction with Syk and ZAP70, DAP10 lacks ITAM but bears a YXXM motif binding phosphatidylinositol-3-kinase and Grb-2 molecules.

28

SEIAMAK BAHRAM

lography at 2.8 A˚ (Li et al., 1999). The structure confirms the general configuration of the molecule within an MHC-I–fold (Bjorkman et al., 1987; Madden, 1995), that is, a membrane proximal Ig C-type 움3 domain and a distal putative ligand binding superdomain (움1, 움2) composed of an eightstranded, antiparallel, 웁-pleated sheet, topped by two somewhat parallel 움helices. Although the former does not reveal any surprises, the latter unveils an edifice that seems to have barely stood up to a ‘‘crash test,’’ a structure embodying the extreme sequence divergence from classical HLA molecules. The 움3 structure is unremarkable in that it closely parallels that of other MHC-I molecules as well as 웁2m itself. It is connected to the 움1움2 distal domain by a short minipeptide (L178–T181) which, unlike orthologous linker peptides in other class I sequences, is not in a helical conformation but follows an extended topology, which allows an interdomain flexibility unseen in any other class I structure. Indeed, the most remarkable feature of the MICA structure resides in the fact that the proximal and distal domains make no intimate contact with each other, as indicated by a 113.5-degree deflection (within the studied crystal) in comparison to the prototypic HLA-B27 molecule. However, this alone cannot explain the 웁2m independence of MICA expression, a characteristic shared only with the soluble ZAG. In fact, the crystal structure offers no real structural clues for the absence of 웁2m binding, and the authors (Li et al., 1999) finally attributed this to a catalog of events, including the large number of glycosylation sites (especially N8, although it is not conserved in MICB), lack of well-known 웁2m contact points, and the extreme flexibility of the distal and proximal domains. The 움1움2 cleft is similar to that of other class Ia (Bjorkman et al., 1987; Fremont et al., 1992; Garrett et al., 1989; Madden et al., 1991; Zhang et al., 1992) and Ib molecules (Lebron et al., 1998; Sanchez et al., 1999; Zeng et al., 1997), although an overall comparison reveals major contortions throughout the edifice (Fig. 5). Examples include the loop between the first two, as well as the hairpin connecting the third and fourth, 웁-strands. Deviations also occur within 움-helices. Helix 1 (H1) in the 움1 domain is longer than equivalents in other class I molecules, and hence structures as a well-defined entity.24 This is also the case for H1 and H2b in the 움2 domain, which collectively skew the structure of this domain toward that of the FcRn molecule. The 웁-strands do not deviate from their MHC-Ia counterparts, at least with respect to the six central ones. 24 Based on homology to other class I structures (HLA-B27 has served as a prototype), Li and colleagues divided the 움1 and 움2 domain helices into two and three ‘‘subhelices,’’ respectively. These are helices 1 (residues 45–54) and 2 (60–79) in the 움1 domain and 1 (137–150), 2a (structure not experimentally determined, but deduced), and 2b (164–170) in the 움2 domain.


29

FIG. 5. MICA polymorphism plot. MICA polymorphic residues are highlighted (black) on the ribbon structure of MICA. The figure was generated using RasMol 2.7.1 and Adobe Illustrator with MICA coordinates extracted from Protein Data Bank (http://www.rcsb.org/ pdb/cgi/explore.cgi?pdbld⫽1B3J).

All in all, the MICA putative antigen-binding cleft is less spacious than that of class Ia molecules; the distance between the two helices (C움 trace) is as short as 10 A˚ within the first four 웁-strands and 7 A˚ across the second four as compared to ⬎18 A˚ for the classical H2-Kb. It is even narrower than that of non–peptide-binding CD1 (14.4 A˚) or the empty FcRn (10.2–12.9 A˚) and is only close to that of H2-T22b (Wingren et al., 2000).25 This is certainly in line with the failure of previous attempts to detect any MICA-associated peptides (Groh et al., 1998). Moreover, Li and colleagues (1999) did not encounter any extraelectron density (not accounted for by MICA or solvent), tending to establish that MICA does not enfold any cargo. However, this possibility will only be ruled out once the structure of a 15–amino acid segment (residues 147–161, corresponding to the 움2 H2a) has been established. Indeed, in the reported experimental conditions, residues 147–151 eluded structural resolution and hence had to be modeled assuming a 25 Initial reports suggested that CDId is capable of peptide presentation (Castano et al., 1995); however, the physiological relevance of this interaction has since come into doubt (Brossay and Kronenberg, 1999).

30

SEIAMAK BAHRAM

polyalanine configuration, and amino acids 152–161 failed to reveal their spatial rearrangement. V. Genetics and Immunogenetics

A. DIVERSITY 1. Repertoire Polymorphism has been the driving force behind the discovery of MHC genes, in both mice and humans, and is perhaps the most fascinating feature of these molecules (Parham and Ohta, 1996). MHC-Ia are indeed the most polymorphic genes of the vertebrate genomes. Three hundred twenty-seven alleles have been documented for HLA-B, 165 for HLA-A, and 88 for HLA-C (Bodmer et al., 1999). This extraordinary diversity enables these molecules, at the population level, to present an infinite range of peptide antigens to T cells (Rammensee et al., 1993). It has been argued that this high degree of polymorphism is the result of overdominant selection exerted by infectious agents throughout mammalian evolution (Hill, 1998; Hughes and Nei, 1988).26 In contrast, MHC-Ib genes are oligomorphic at best, for example, 14 alleles are known for HLA-G (Ishitani et al., 1999), 5 for HLA-E (Geraghty et al., 1992), and none for HLA-F (for the latest listing, consult http:// www.anthonynolan.org.uk/HIG/index.html). This lack or low degree of diversity has been variously interpreted. Pamer et al. (1993) postulated that limited polymorphism enables them to present conserved microbial epitopes to the immune system,27 although for others the lack of polymorphism signifies their nonrelevance in an effective immune response (Klein and O’Huigin, 1994). However, in light of the recent discovery of what may be their raison d’eˆtre—interaction with a diverse, yet clonally unchecked NK repertoire—the whole body of literature and hence conclusions regarding these molecules require reappraisal. 26 Despite the fact that the most plausible driving force behind maintenance of MHC diversity is the selective force exerted by infectious agents, it is at the very least ‘‘troublesome’’ to see how relatively little association there is between HLA alleles and infectious threats (Hill, 1998). Indeed, the most notable HLA association is with autoimmune diseases, not infectious ones. Despite this notable degree of association, it is not easy to envision a role for such disorders in shaping the HLA repertoire as, in fact, these disorders affect individuals (at least until early in this third millennium) typically after the reproductive period. 27 Although this has been so far a circumstantial theory, it has been elegantly proven in case of H2–M3. The previous difficulty has been the lack of any biological system allowing an in vivo assessment that differentiates the role of class Ib from Ia molecules. The availability of H2-Db, Kb double-KO mice allowed Seaman and co-workers (1999) to pinpoint a role for H2–M3 for immune response against Listeria monocytogenes.


31

The MICA gene was initially sequenced from 92 homozygous typing cell lines collected during the course of the 10th International Histocompatibility Workshop (Bahram et al., 1994; Fodil et al., 1996, 1999). Nucleotide sequence determination of exons 2, 3, and 4 was performed either by sequencing of cloned PCR fragments or by direct sequencing of PCRamplified material. In conjunction with data reported by other investigators (Petersdorf et al., 1999; Visser et al., 1999; Yao et al., 1999), 54 MICA alleles have been identified to date, among which 47 encode distinct putative glycoproteins (Fig. 6 and Tables III and IV) (for the current status, the reader is referred to http://mhc-x.u-strasbg.fr). These are defined by a total of 40 nucleotide substitutions, 30 of which are nonsynonymous: 5 of 8 in 움1, 15 of 16 in 움2, and 10 of 16 in 움3 (Fodil et al., 1999) (Tables III and IV). Resembling classical HLA class I molecules, MICA polymorphic residues are slightly more prevalent in the 움2 domain (Fodil et al., 1996); however, in contrast to classical class I molecules, MICA seem equally diverse within the 움3 domain. MHC-Ia polymorphic residues are concentrated within the antigen-binding cleft in contact with the peptide or the TCR (Bjorkman and Parham, 1990). Moreover, the majority of nucleotide changes in this cleft are nonsynonymous (Hughes and Nei, 1988), creating a strong case for overdominant selection as (mostly likely) exerted by infectious agents in maintaining MHC alleles (Hill, 1998; Klein and O’Huigin, 1994; Parham and Ohta, 1996). In this context, the nature of selective forces maintaining MIC polymorphism is an open question; interestingly, an overwhelming majority of nucleotide variations in the MICA putative antigen-binding cleft give rise to nonconservative amino acid substitutions, whereas the opposite trend is seen in the Ig-like 움3 domain. Among the amino acid substitutions, several are drastic in nature and may have radical effects in putative ligand/ receptor binding. These are predominantly in the 움1 and 움2 domains and include R6P, Q91R, G114R, K125E, H156L, K173E, T181R, W210R, and Q251R. In contrast, six of eight changes in the 움3 domain are conservative, and among these, three are shared by at least one human or murine class I molecule (Bahram et al., 1994; Fodil et al., 1996) (Fig. 6 and Table III). These latter include G206S, T213I, and S215T. It is remarkable that almost none of the MICA variable residues coincide with the polymorphic positions of MHC-I molecules, which are mainly residues in direct contact with the peptide or the TCR, located in the 움1 domain–encoded 움-helices and the 움2 domain–encoded 움-strands (Bjorkman and Parham, 1990; Fodil et al., 1996; Madden, 1995). It is also noteworthy that nine of the MICA variable residues are located precisely at the positions at which MICA and MICB sequences differ, and indeed, eight of these are identical to MICB residues at these positions: T24A, C36Y, K125Q, M129V, G206S, W210R, S215T, and S268G

32

SEIAMAK BAHRAM

(Bahram and Spies, 1996; Fodil et al., 1996). This implicates intergenic conversion events as a mechanism for the establishment of MICA polymorphism, as has been the case for other HLA molecules (Parham and Ohta, 1996). Finally, computation of the allelic variation within the three extracellular domains of 움1–움3 led to an average heterozygosity per nucleotide site (nucleotide diversity) of 0.011. [this analysis is based on the thenknown MICA001–016 alleles (Fodil et al., 1996)] This is remarkably close to that of the antigen-presenting class Ia loci (0.04–0.08) and drastically higher than that (0.0002–0.007) for other nuclear genes (Nei et al., 1997), strongly favoring overdominant selection as the mechanism for maintenance of MIC alleles. Plotting the MICA 움1움2-located variable residues on Li and colleagues’ crystal structure generates a footprint remarkably similar to that of initial simulations (Fig. 5). During the course of genomic sequencing of the MICA locus, a peculiar allelic diversity was uncovered within the transmembrane exon (Mizuki et al., 1997). This exon harbors a multiallelic STR, where a variable number of GCT repeats encode 4, 5, 6, 9, and 10 A residues, respectively, and are therefore called A4, A5, A6, A9 (Mizuki et al., 1997), and A10 (PerezRodriguez et al., 2000). Curiously, no A7 or A8 repeats have been encountered to date, which is surprising given the fact that most STRs have evolved by sequential addition of single repetitive units. Finally, certain MICA alleles carry a nucleotide insertion (GCT 씮 GGCT) causing a frameshift mutation resulting in a premature stop codon within the transmembrane segment (Mizuki et al., 1997). These alleles give rise to a truncated glycoprotein of 35–40 kDa (M. Colonna and S. Bahram, unpublished observations) which eventually reaches the cell surface (albeit at a presumably nonphysiological site; see below for details) and hence might not be secreted as was originally thought. This 5.1 STR defines the transmembrane segment of MICA008 allele (see below), the most frequent MICA allele in several populations examined so far (Fodil et al., 1999). Given the very short distance separating MICA from HLA-B, a vigorous degree of positive linkage disequilibrium is expected. Examples are association of HLA-B*0702 and HLA-B*0801 with MICA008/5.1 (the latter defines the MICA allele within the conserved haplotype HLA-A*0101, B*0801, DRB1*0301), HLA-B*1402 with MICA011/6, HLA-B*27052 with

FIG. 6. MICA alleles. Domain-by-domain multiple alignment of the presently available MICA alleles. Only 47 (of 54) alleles defined by distinct amino acid sequences are shown. For full nucleotide data, see Table IV. For updates, see http://mhc-x.u-strasbg.fr. Dashes define positions identical to those of the MICA001 allele. Transmembrane (TM) sequences report only the short tandem repeat.

34


35


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SEIAMAK BAHRAM

TABLE III MICA NUCLEOTIDE VARIATION Positions Exon 2 6 14 23 24 26 36 56 64 Exon 3 91 105 112 114 122 124 125 129 142 151 156 156 173 175 176 181 Exon 4 191 193 198 205 206 210 213 215 221 247 251 251 255 256 268 271 Exon 5 293 295

Codon

Amino Acid

(␣1) (5 of 8) CGT TGG CTC ACT GTA TGT AAT AGA

씮 씮 씮 씮 씮 씮 씮 씮

CCT GGG CTT GCT GGA TAT AAC AGG

Arg Trp Leu Thr Val Cys Asn Arg

씮 씮 씮 씮 씮 씮 씮 씮

Pro Gly Leu Ala Gly Tyr Asn Arg

CAG AGG TAC GGG CTG ACT AAG ATG GTC ATG CAC CAC AAA GGC GTA ACA

씮 씮 씮 씮 씮 씮 씮 씮 씮 씮 씮 씮 씮 씮 씮 씮

CGG AAG TAT CGG GTG TCT GAG GTG ATC GTG CTC CGC GAA AGC ATA AGA

Gln Arg Tyr Gly Leu Thr Lys Met Val Met His His Lys Gly Val Thr


Arg Lys Tyr Arg Val Ser Glu Val Ile Val Leu Arg Glu Ser Ile Arg

AGC GCC ATT TCT GGC TGG ACA AGC GTA ACC CAA CAA CAG AGG AGC CCT


AGT GCA ATC TCC AGC CGG ATA ACC CTA ACT GAA CGA CAA AGT GGC GCT

Ser Ala Ile Ser Gly Trp Thr Ser Val Thr Gln Gln Gln Arg Ser Pro


Ser Ala Ile Ser Ser Arg Ile Thr Leu Thr Glu Arg Gln Ser Gly Ala

(␣2) (15 of 16)

(␣3) (10 of 16)

(transmembrane) (GCT)4,5,6,9,10 GCT 씮 GGCT

(Ala)4,5,6,9,10 (Ala)2 –stop at 310

Positions refer to amino acid residues. Numbers in parentheses correspond to the ratio of nonsynonymous to total substitutions.

37


TABLE IV AVAILABLE HUMAN MICA ALLELES Allelea MICA001 MICA002 MICA004 MICA005 MICA006 MICA007 MICA008 MICA009 MICA010 MICA011 MICA012 MICA013 MICA014 MICA015 MICA016 MICA017 MICA018 MICA019 MICA020 MICA021 MICA022 MICA023 MICA024 MICA025 MICA026 MICA027 MICA028 MICA029 MICA030 MICA031 MICA032 MICA033 MICA034 MICA035 MICA036 MICA037 MICA038 MICA039 MICA040 MICA041 MICA042 MICA043 MICA044 MICA045

Previous Name

Accession

MUC17 MUC21 MUC22 MUC24 MUC25 MUC26 MUC28 MUC29 MUC30 MUC31 MUC32 MUC33 MUC34 MUC35 MUC36 MICAKWHT MICAAIB MICAAKB MICAALAB MICABCC MICABEA MICABEE MICABHB MICACEA MICACEC

U56940 U56941 U56943 U56944 U56945 U56946 U56947 U56948 U56949 U56950 U56951 U56952 U56953 U56954 U56955 AF097403 AF097404 AF097405 AF097406 Y18110 Y16804 Y16805 Y16807 Y16808 Y16809 Y16811 Y18111 Y18112 Y18113 Y18114 Y18115 Y18116 Y18117 Y18118 AH006333 AH007170 AH007171 AH007172 AH007173 AH007174 AH007182 AH007176 AH007177 AH007178

Reference Bahram et al. (1994) Bahram et al. (1994) Bahram et al. (1994) Bahram et al. (1994) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1996) Fodil et al. (1999) Fodil et al. (1999) Fodil et al. (1999) Fodil et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Yao et al. (1999) Petersdorf et al. (1999) Visser et al. (1999) Visser et al. (1999) Visser et al. (1999) Visser et al. (1999) Visser et al. (1999) Visser et al. (1999) Visser et al. (1999) Visser et al. (1999) Visser et al. (1999) (continues)

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TABLE IV Continued Allelea

Previous Name

Accession

MICA046 MICA047 MICA048 MICA049 MICA050 MICA051 MICA052 MICA053 MICA054

MICACEF MICACIB HSMICGG HSMICGGA HSMICGGB HSMICGGC HSMICGGD HSMICGGE HSMICGGG

AH007179 AH007180 AH007472 AH007473 AH007474 AH007475 AH007476 AH007477 AH007479

Reference Visser et al. (1999) Visser et al. (1999) Y. Mitsuishi et al. (unpublished Y. Mitsuishi et al. (unpublished Y. Mitsuishi et al. (unpublished Y. Mitsuishi et al. (unpublished Y. Mitsuishi et al. (unpublished Y. Mitsuishi et al. (unpublished Y. Mitsuishi et al. (unpublished

observations) observations) observations) observations) observations) observations) observations)

a Alleles are numbered in order of their respective identification. Allele MICA003 has been deleted, as it has not been found in subsequent sequencing efforts.

MICA007/4, HLA-B*4001 with A008/5.1, and finally HLA-B*4402 with MICA008/5.1 (Fodil et al., 1999). Exceptions subsist, however; for example, the most common MICA allele, MICA008/5.1, is variously linked to HLAB*0702, HLA-B*0801, HLA-B*1302, HLA-B*4001, HLA-B*4402, and HLA-B*4701. Several medium-sized population studies allowed an initial assessment of the distribution pattern of MICA alleles within various populations (Fodil et al., 1999; Petersdorf et al., 1999). The gathered information clearly reveals that MICA008, as already mentioned, is the most frequent allele, possibly at the worldwide level. Following are a myriad of other alleles, the most prominent of which are MICA002, -004, or -010, although the distribution of these shows a high degree of interethnic variability. The MICA008 allele is unique in that it carries, almost invariably, the 5.1 transmembrane sequence defined by a shortened transmembrane segment, and consequently has no cytoplasmic tail, giving rise to a diminished 38to 40-kDa glycoprotein (M. Colonna et al., unpublished observations) with an apparent faulty subcellular localization (Suemizu et al., unpublished data). MICB seems to be less diverse than MICA, although this has been less thoroughly explored. Presently, 16 MICB alleles have been documented, among which 13 differ by protein sequence (Fig. 7 and Tables V and VI). MICB alleles are defined by a total of 15 nucleotide substitutions (within

FIG. 7. MICB alleles. Domain-by-domain multiple alignment of the presently available MICB alleles. Only 11 (of 14) alleles with distinct amino acid sequences are shown. For full nucleotide data, see Table VI. For updates, see http://mhc-x.u-strasbg.fr. Dashes define positions identical to those of the MICB001 allele. The MICB010 allele carries a nonsense mutation in the 움2 domain.

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TABLE V MICB NUCLEOTIDE VARIATION Position Exon 2 16 45 52 57 82 Exon 3 98 113 170 Exon 4 189 192 210 243 256 267 268 Exon 5 277

Codon

Amino Acid

(␣1) (5 of 5) GAA CCC GAT AAG GAC

씮 씮 씮 씮 씮

GGA CAC AAT GAG GGC

Glu Pro Asp Lys Asp

씮 씮 씮 씮 씮

Gly His Asn Glu Gly

(␣2) (3 of 3) ATC 씮 ATG GAT 씮 AAT CGA 씮 TGA

Ile 씮 Met Asp 씮 Asn Arg 씮 Stop

ACC 씮 GAG 씮 CGG 씮 ACC 씮 AGG 씮 CAC 씮 GGC 씮

Thr Glu Arg Thr Arg His Gly

(␣3) (4 of 7) ATC AAG CGA ACG AAG CAT AGC

씮 씮 씮 씮 씮 씮 씮

Ile Lys Arg Thr Lys His Ser

(transmembrane) GCG 씮 GTG

Ala 씮 Val

Positions refer to amino acid residues. Numbers in parentheses correspond to the ratio of nonsynonymous to total substitutions.

the extracellular region), 12 of which are nonsynonymous: 5 of 5 (nonsynonymous versus total), 3 of 3, 4 of 7, respectively, in the 움1, 움2, and 움3 domains (Table V) (Ando et al., 1997; Bahram and Spies, 1996; Fischer et al., 2000; Pellet et al., 1997; Visser et al., 1998). Possibly the most interesting aspect of MICB diversity was the report by Ando and colleagues (1997) of a MICB null allele. This allele, MICB010 (also called MICB0107N ), carries a stop codon within the 움2 domain (Table V). Interestingly, this allele is invariably linked to a 100-kb genomic deletion, including the more telomeric MICA gene. This peculiar haplotype is described in detail below. Finally, the presence of a long, 1000-fold TA-repeat microsatellite between MICA and MICB is of interest, as it might represent a potential hot spot for recombination (Ando et al., 1997), although our knowledge of the extent of positive or negative linkage disequilibrium between MICA and MICB alleles is still fragmentary. 2. Disease Susceptibility In this fast-paced genomic era in which few weeks go by without the molecular resolution of a mendelian disorder or a genome-wide scan in

41


TABLE VI AVAILABLE HUMAN MICB ALLELES Allele

Previous Name

Accession

Reference

MICB001 MICB002 MICB003 MICB004 MICB005 MICB006 MICB007 MICB008 MICB009 MICB010 MICB011 MICB012 MICB013 MICB014 MICB015 MICB016

— MICB01021 MICB01022 MICB01023 MICB0103101 MICB0103103 MICB0104 MICB0106 MICB0105 MICB0107N — — — — MICB01022v MICB013101v

X91625 SEG AB003599s SEG AB003600s SEG AB003601s SEG AB003602s SEG AB003604s SEG AB003605s SEG AB003607s SEG AB003606s SEG AB003608s AF021225 AF021226 U95732 U95731 — —

Bahram and Spies (1996) Ando et al. (1997) Ando et al. (1997) Ando et al. (1997) Ando et al. (1997) Ando et al. (1997) Ando et al. (1997) Ando et al. (1997) Ando et al. (1997) Ando et al. (1997) Visser et al. (1998) Visser et al. (1998) Pellet et al. (1997) Pellet et al. (1997) Fischer et al. (2000) Fischer et al. (2000)

search for causative loci behind the so-designated ‘‘complex diseases,’’ it is surprising, if not frustrating, to see how a rather minute segment of the human genome, the HLA complex (3.8 Mb; of the genome), has remained such a formidable genetic puzzle. Nevertheless, the situation is fairly simple. Through the work of a large number of investigators during the past 30 years, we now have a collection of over 100 diseases associated to varying degrees with HLA alleles (Bodmer, 1980; Charron, 1997; Dausset and Svejgaard, 1977). These range from a fairly moderate association of Hodgkin’s lymphoma (which incidentally was the first such disease recognized) with HLA-DPB1*0301 (for a review, see Amiel, 1971) to the near-absolute association of ankylosing spondylitis with HLA-B27 (Brewerton et al., 1973; Schlosstein et al., 1973) as well as narcolepsy with HLADQB1*0602/DQA1*0102 ( Juji et al., 1984; Langdon et al., 1984). Falling between these extremes are most other HLA-linked pathologies. These embrace the linkage of rheumatoid arthritis to HLA-DRB1*0401 (Stastny, 1978), multiple sclerosis with HLA-DR2 (DRB1*1501) (de Moerloose et al., 1979), type I diabetes with DQB1*0302 (Todd et al., 1987; Yunis et al., 1976), systemic lupus erythematosus with HLA-DR2/DR3 alleles (Walport et al., 1982) or complement deficiency (Hauptmann et al., 1974), and IgA deficiency with the conserved HLA-A1, B8, DR3 haplotype (Strothman et al., 1986). Except in two circumstances—association of adrenal hyperplasia with complement genes (Pollack et al., 1981) and hereditary hemochromatosis with HLA-A3, -B14 haplotypes (Simon et al., 1976)

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[which were eventually linked to 21-OH and HFE genes respectively (White et al., 1984; Feder et al., 1996)]—the molecular impetus behind most of these diseases remains unknown.28 The major hurdle hindering the advance in this field is the formidable linkage disequilibrium across the entire MHC, precluding, in most cases, the incrimination of single loci (Ceppellini, 1976). In the array of MHC-associated diseases, several tissue-specific disorders linked specifically to the class I region have unique characteristics. (i) Unlike most MHC-associated diseases, including, for example, type I diabetes, rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus, they are not associated with a MHC haplotype (where MHC-II alleles are usually predominant) but solely with certain MHC-I alleles. (ii) They show a coherent pattern of tissue disturbance, affecting, to various degrees, epithelial/endothelial/conjunctive tissues (i.e., skin, gut, joints, and the eyes), suggesting the existence of an anatomically restricted pathogenic element acting independently or in conjunction with the ubiquitously expressed MHC-I molecules. (iii) They affect young males in their second or third decade, unlike typical MHC-linked autoimmune disorders affecting women in their mid-30s and 40s. These include the well-known associations of seronegative spondyloarthropathies with the HLA-B27 gene (Brewerton et al., 1973; Schlosstein et al., 1973), the association of psoriasis vulgaris with HLA-Cw6 (Oka et al., 1999; Ozawa et al., 1979), and finally the linkage of the Behçet’s disease and HLA-B51 (Ohno et al., 1978). Although it is thought that HLA molecules play a direct role in the etiopathogenesis of these syndromes, most likely via presentation of ‘‘deleterious’’ peptides, to date no direct evidence substantiates this assumption. The high degree of linkage disequilibrium between MICA alleles and those of the closely linked HLA-B gene as well as the unique pattern of MICA tissue expression are sufficient reasons for us to consider MICA as a candidate gene in the disorders mentioned above. A small number of HLA-linked diseases have been examined so far for their potential linkage with MICA alleles, mainly selected on the basis of their known pathophysiology. A preliminary as well as a more thorough investigation of the MICA TM-STR allele did not demonstrate any primary linkage with ankylosing spondylitis (Goto et al., 1997; Yabuki et al., 1999). 28 The most plausible scenario linking HLA alleles to autoimmune disorders is that of ‘‘faulty’’ peptide sampling to T cells, that is, presentation of tissue-specific self antigens not encountered in the course of T cell maturation within the thymus. However, very simple questions remain unanswered. For example, regarding HLA-B27–linked spondylarthropathies, why is it that only a few percent of B27-positive individuals develop the disease (given the fact that B27 seems to be the unique or the most prominent genetic susceptibility element)?


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A satellite disorder of this condition, acute anterior uveitis, did show, however, a more pronounced linkage with the MICA TM-STR A4 allele; however, this requires confirmation on a larger panel as well as investigation of extracellular variants (Goto et al., 1998a,b). Investigation of Behçet’s disease provided the first interesting link between MICA alleles and an HLA-B linked disorder in the Japanese population. Indeed, the MICA TMSTR A6 and MICA009 alleles are significantly linked to Behçet’s disease in several ethnically distinct populations (French, Greek, Iranian, Italian, Japanese, Lebanese, and North African). However, there is a very strong link between HLA-B51 and MICA009 itself, and the strength of this association appears to swing from one locus to the other based on the examined population. It is possible that both of these molecules contribute to the genetics of this complex disorder (Mizuki et al., 1997, 1999; Wallace et al., 1999). Although HLA-Cw6–linked psoriasis does not show any noticeable association with MICA alleles (Oka et al., 1999), psoriatic arthritis apparently displays a specific linkage with MICA A9 STR (linked mainly with the MICA002 allele) independent of HLA-C association (Gonzalez et al., 1999). Finally, two of the MHC-II (or pan-MHC)–linked disorders— multiple sclerosis and celiac disease—do not appear to show any primary linkage to MICA (N. Fodil, M.-P. Roth, and S. Caillat, unpublished observations). 3. Histocompatibility Little (1914) along with Tyzzer (1909), and later Snell (1948), truly initiated the field of transplantation immunogenetics, within the first half of the 1900s.22 A number of seminal experiments, consisting mainly of tissue and tumor grafting between genetically homogenous and, later, inbred strains of mice, led to the identification of the so-designated ‘‘major’’ histocompatibility complex among a large number of histocompatibility loci (H2 was a member of H1–H14).29 To date, the single most important contribution of the MHC in day-to-day clinical practice is to help ensure near-tissue compatibility between donor and recipients of organ and tissue 29

In order to grasp the route to MHC discovery, one must go back to the turn of the 20th century, when Cueńot and Mercier were probably the first scientists to tackle the problem of tumor genetics. Despite their defeat in finding any correlation between coat color and tumor susceptibility, they possibly prepared the way for others’ successful experiments. These came from Loeb (1909), along with Tyzzer in conjunction with his Ph.D. student, Little, who incidentally later, in 1929, founded the Jackson laboratory, where other crucial experiments were about to happen. These were mainly carried out by Snell and in part in collaboration with Gorer, a British scientist on sabbatical leave (Tyzzer, 1909; Little, 1914). This enormous genetic undertaking within the first half of the 20th century clearly paved the way for the understanding of histocompatibility molecules, which were also greatly boosted through the identification of their human counterparts (Dausset, 1958).

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grafts. Although HLA alleles clearly affect the long-term survival rate of kidney transplant recipients, solid organ transplantation in general does not show as critical a reliance on MHC compatibility as does hematopoietic stem cell transplantation [hereafter referred to as bone marrow transplantation (BMT)] (Sasazuki et al., 1998). Several large-scale retrospective analyses have demonstrated an incremental increase in posttransplant complications (mainly the life-threatening graft-versus-host disease) corresponding to the degree of incompatibility between donor and recipients (Sasazuki et al., 1998). Within the framework of allogeneic BMT, clinical results are substantially better when the donor is a haplotype-matched family member rather than an HLA-compatible, unrelated individual (Anasetti et al., 1995; Charron, 1996). At least part of this discordance may reside in yet unidentified polymorphic loci within the MHC. For a number of reasons, MICs are obvious candidates as histocompatibility loci; these include their structure, their unusual degree of polymorphism, and their epithelial pattern of expression (which parallels the complex pathological spectrum of the graft-versus-host disease). The task is complicated, however, given the absence of any murine orthologue or obvious functional equivalent.30 So we are left with the human system, in which retrospective analysis of the increasingly large numbers of unrelated donor BMTs should help decipher the role (if any) of MICA in histocompatibility. Complete sequence analysis of the MICA gene within a large set of complete haplotype-matched unrelated donor BMTs revealed an almost absolute degree of identity between donor and recipients, although a few authentic mismatches existed (B. Cuillerier and M. Ota, unpublished observations). This work may indicate that perhaps MIC typing would not be of additional diagnostic value compared to routinely used HLA typing, but it does not have the power to assess the role of MIC genes as transplantation antigens. To explore this avenue, one may have to analyze an even larger panel of mismatched HLA-B allele grafts and to independently analyze the putative contribution of MICA and eventually MICB by multivariate analysis. B. MICAB⫺/⫺ INDIVIDUALS The study of genetically deficient individuals has taught us a great deal about how the immune system functions in the ‘‘real world.’’ The most notable examples are challenges of immunodeficiency due to the absence of MHC-II or -I molecules (Frelinger and Serody, 1998; Grusby and Glimcher, 1995; Mach et al., 1996). Most of these human disorders have been elegantly paralleled by murine models in which the same genes were 30 Although this issue could be addressed once transgenic mice strains expressing diverse MICA and MICB alleles are investigated as to grafting.


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abrogated by homologous recombination. Obviously, the latter permits direct experimentation and exposure to a well-controlled pathogenic (infectious) environment. As previously mentioned, upon their discovery, it was obvious the rodents were probably the only mammals devoid of MIC genes (Bahram et al., 1994). Hence, we were left in search of elusive human subjects deleted not only for MICA but also for MICB. Watanabe and colleagues (1997) were the first to allude to a large (앑100 kb) deletion within the HLA class I region based on pulsed-field gel electrophoresis experiments. Tokunaga and co-workers were able to further delineate the boundaries of this deletion and made the remarkable discovery that some of these HLA-B*4801– carrying haplotypes have expunged a 100-kb DNA segment including the MICA gene (Komatsu-Wakui et al., 1999). This, combined with the previous report by Ando and co-workers (1997) of a MICB null allele within precisely the same haplotype, made it clear that a fraction of HLA-B*4801 chromosomes are MICA–MICB compound knock-outs (present estimates on a limited number of cases suggest that slightly over half of the HLAB*4801 haplotypes are MIC null). Given the 3.2% frequency of the HLAB*4801 within the Japanese population, a 0.1024% homozygous carriage is expected. So far, and based on a very limited number of cases, 62.5% (5 of 8) of these haplotypes carry the MIC null configuration; hence, given the actual size of the Japanese population, over 80,000 individuals (80,640 based on a population of 126 million) in Japan alone carry this haplotype at homozygosity: MICB*0107N, MICA⫺, HLA-B*4801. The HLA-B*4801 is extremely rare outside of Southeast Asia, with the exception of the Amerindian populations. As the first examined MIC⫺/⫺ individuals were blood donors, ‘‘MIC deficiency’’ apparently does not lead to any manifest clinical symptoms, in clear contrast to the mild to severe immunodeficiencies consequent to the absence of MHC-I or -II molecules. These homozygous individuals (MICAB⫺/⫺) are therefore crucial to dissecting the in vivo role of the MIC genes. These individuals are apparently indistinguishable with respect to other blood donors upon routine clinical examinations. Moreover, an extensive phenotype analysis of various B, T, and NK lymphocyte subpopulations from a limited number of these individuals did not reveal any anomalies. In particular, the number of B (CD19⫹, CD21⫹ ), 움/웁 TCR⫹ (CD4⫹ and CD8⫹ ), 웂/␦ TCR⫹, including V␦1 subpopulations, and, finally NK (CD16⫹, CD56⫹, KIR⫹ ) were present in proportions comparable to those in ‘‘normal’’ (undeleted) HLA-B48–carrying individuals (M. Cella, N. Fodil, M. Ota, H. Inoko, M. Colonna, and S. Bahram, unpublished observations). It might also be worthwhile to consider the fact that even within the HLAB48 haplotypes with an intact MICA gene, most contain the 008 or 010

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allele (Katsuyama et al., 1999). The MICB allele carried by the intact HLAB4801 haplotype is MICB002 (MICB0102, according to Ando et al., 1997) (Ota et al., 2000). MICA008, as previously mentioned, is unique in that it carries a shortened transmembrane segment and displays an aberrant membrane localization; as to allele MICA010, it alone carries a nonconservative amino acid substitution at the beginning of the 움1 domain, R6P (Fodil et al., 1996), which hinders surface expression (Li et al., 2000). Finally, it seems obvious that a more detailed study of the immune system in these individuals, especially with respect to 웂/␦ and NK lymphocyte function, will provide invaluable data with regard to MIC immunobiology. VI. Conclusion

All things considered, MIC genes have mainly added to the confusion dominating the puzzling field of non–peptide-binding MHC-I genes. Present knowledge, especially regarding the absence of these genes in mice as well as their apparent dispensability in humans, suggests that they do not perform functions as fundamental as those ascribed to innate elements of the immune system (Diefenbach and Raulet, 1999). This raises the question as to their true utility to the immune system.31 In this respect, two opposite conclusions could be drawn: either MIC genes have arisen to specifically defend epithelial cells against aggressors, foreign (microbes) or domestic (neoplastic transformation), or they have collected a number of characteristics common to the whole family of histocompatibility molecules (e.g., polymorphism, stress induction, or TCR reactivity). Hence, they may be yet another gene family that has flourished and yet is about to vanish, as this is supposedly the fate of most class I gene families, including the most prominent one, the HLA loci. Although the first hypothesis is in line with today’s conventional immunological school of thoughts, the second is best adapted to a darwinian view of the defense organization, which would stipulate that successive waves of microbiological offensives have constantly remolded the MHC genomics. As often, the truth might reside between the two extremes. This is not an esoteric issue, as it might help resolve outstanding questions that have been stalking the class I loci since their very identification. Among many questions that must be answered regarding MIC genes, perhaps two are most fascinating. First, what is the origin of this second set of class I genes within the MHC itself ?23 Second, what is the driving force behind this peculiar, rather disconcerting pattern of polymorphism? An answer to the first question has been elegantly 31 This question is not limited only to MICs as it applies to almost every MHC-Ib molecule so far identified, even the ones where a function in host defense has been clearly documented (Bouwer et al., 1994; Lo et al., 2000; Pamer et al., 1992; Seaman et al., 1999).


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provided by the genomic work of Shiina et al. (1999), which allows us to trace the origin of MICs to the roots of the human class I region; the answer to the second question is still in the making (Fodil et al., 1996, 1999). The mystery of these genes may be solved once the more general question of why vertebrates utilize a common structure, the MHC-I–fold, in such unrelated functions ranging from iron homeostasis to IgG transport, is answered; in other words, why has the MHC served as a training ground of sorts for nature’s experimentation with such complex structures? In fine, and within the near future, the MIC world should remain focused on the breadth of MICA/B engagement in 웂/␦ and NK biology, the in vivo significance of their stress induction in both anti-infectious and antitumor responses, and the involvement of their diversity in different pathologies as well as solid and marrow organ transplantations. ACKNOWLEDGMENTS I thank Susan Gilfillan, Marco Colonna, and Marc Bonneville for comments on the manuscript; Hidetoshi Inoko for many insightful discussions; as well as Georges Hauptmann and Fritz Melchers for longstanding encouragement and support. Research in our laboratory was performed by Benoıˆt Cuillerier (who compiled the data presented in Figs. 4–6 and 7 as well as in Tables II, IV, and VI), Nassima Fodil, Mirjana Radosavljevic, Vale´rie Wanner, and Sophie Wicker. Support is acknowledged from the Action Concerteé Incitative Jeunes Chercheurs du Ministe`re de l’E´ducation Nationale, de la Recherche et de la Technologie; the Fondation pour la Recherche Me´dicale—Action Recherche Sante´ 2000; the Ligues De´partementales (67–68) Contre le Cancer; and the Association pour la Recherche sur le Cancer.

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CD40-Mediated Regulation of Immune Responses by TRAFDependent and TRAF-Independent Signaling Mechanisms AMRIE C. GRAMMER AND PETER E. LIPSKY Intramural Research Program of National Institute of Arthritis and Musculoskeletal and Skin Diseases, Autoimmunity Branch, National Institutes of Health, Bethesda, Maryland

I. Introduction

CD40 and CD154/CD40 ligand (Fig. 1) are members of the tumor necrosis factor (TNF) superfamily and are known to play a crucial role in normal T cell–dependent and T cell–independent humoral immune responses to both bacteria and viruses. In addition, interactions between these molecules in normal individuals are essential for a panoply of immune responses, including the induction of oral tolerance, the activation of antigen-presenting cells, and the function of tumoricidal natural killer cells, macrophages, and cytotoxic T cells (reviewed in Grewal and Flavell, 1998; Mackey et al., 1998a; Toes et al., 1998; van Kooten and Banchereau, 2000). Moreover, abnormal expression of this pair of TNF superfamily members has been shown to contribute to several pathological conditions (Fig. 2), including a variety of autoimmune conditions, chronic graft-versus-host disease, and survival or proliferation of B cell leukemias and lymphomas as well as the pathogenesis of atherosclerosis (Biancone et al., 1999; Denton et al., 1998; Francisco and Siegall, 1998; Furmann et al., 2000 Grammer et al., 1999b; Laman et al., 1998; Larsen et al., 1997; Mach et al., 1998; Schultze et al., 1998). II. Discovery of CD40 and CD154

Human CD40 was originally described in 1984 as an antigen expressed on the surface of human urinary bladder carcinomas (Koho et al., 1984). In 1985 and 1986, two groups independently described CD40 as a 45- to 50-kDa protein expressed on human B cells with the monoclonal antibodies (mAbs) S2C6 and G28.5 (Clark and Ledbetter, 1986; Paulie et al., 1985). These mAbs were clustered at the Third International Workshop on Leukocyte Antigens in 1986. S2C6 and G28.5 stained B cells freshly isolated from the peripheral blood or tonsil, Burkitt’s lymphoma cells, Epstein–Barr virus (EBV)–transformed lymphoblastoid B cell lines, and freshly isolated B cells from chronic lymphocytic leukemia, CALLA⫹ acute lymphoblastic leukemia, and non-Hodgkin’s lymphoma patients (Law et al., 1990; Paulie 61


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FIG. 1. CD40 and CD154 are members of the tumors necrosis factor (TNF) superfamily. LT, Lymphotoxin; NGF, nerve growth factor; NGFR, NGF receptor; TNFR, TNF receptor.

et al., 1985; Stamenkovic et al., 1989; Uckun et al., 1990, 1991). Examination of the maturational stages at which CD40 is expressed indicated that B cells express CD40 from the pro-B stage through differentiated memory cells. Specifically, CD40 is expressed early in B cell ontogeny before surface expression of CD20, CD21, CD22, CD24, and immunoglobulin M (IgM), but after expression of CD10 and CD19 (Law et al., 1990; Moreau et al., 1993; Saeland et al., 1992; Uckun et al., 1990). By contrast, fully differentiated plasma B cells do not express CD40 (Ling et al., 1987). Although CD40 was originally thought to be a B cell lineage marker, more recent studies have shown that it is also expressed by a wide variety of other cell types (Fig. 3), including T cells (Armitage et al., 1993b; Dunlap et al., 1989; Indzhiia et al., 1992; Wagner et al., 1999; Ware et al., 1991), thymocytes (Brown et al., 1998; Wagner et al., 1999), basophils (Valent et al., 1990), monocytes (Alderson et al., 1993; Brossart et al., 1998; Imaizumi et al., 1999; Kiener et al., 1995; Kornbluth et al., 1998; Kremer et al., 1998; Kuroiwa et al., 1999; Mach et al., 1997; Malik et al., 1996; Nicod and Isler, 1997; Pradier et al., 1996; Shu et al., 1995; Stout et al., 1996; Suttles et al., 1996; Wagner et al., 1994; Zhou et al., 1999), dentritic cells (Bjorck et al., 1997; Buelens et al., 1997; Caux et al., 1994; Cella et al., 1996; Koch et al., 1996; Ludewig et al., 1995; Mackey et al., 1998a,b; McDyer et al., 1999; McLellan et al., 1996; Morse et al., 1998; Mueller et al., 1997; Pinchuk et al., 1994; Ruedl and Hubele, 1997; Van Den Berg et al., 1996), epithelial cells (Cruickshank et al., 1998; Galy and

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FIG. 2. The role of CD154 in disease. ALL, Acute lymphoblastic leukemia; CIDP, chronic demyelinating polyneuropathy; CLL, chronic lymphocytic leukemia; CTLs, cytotoxic T lymphocytes; EAE, experimental allergic encephalomyelitis; GBS, Guillain–Barre´ syndrome; GVHD, graft-versus-host disease; HCL, hairy cell leukemia; Ig, immunoglobulin; KO, knockout; MAIDS, murine acquired immunodeficiency syndrome; NHL, nonHodgkin’s lymphoma; NK, natural killer; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; TD, T cell–dependent; TI, T cell–independent.

Spits, 1992; Gormand et al., 1999; Van Den Berg et al., 1996; Young et al., 1998), epidermal cells (Gaspari et al., 1996; Peguet-Navarro et al., 1995; Salgado et al., 1999; Viac et al., 1997), stromal cells (Banchereau et al., 1991; Fries et al., 1995; Shimaoka et al., 1998), hematopoietic progenitor cells (Flores-Romo et al., 1997), microglia (Aloisi et al., 1999; Nguyen et al., 1998; Wei and Jonakait, 1999), hepatocytes (Afford et al., 1999), endothelial cells (Hollenbaugh et al., 1995; Karmann et al., 1995; Yellin et al., 1995), and smooth muscle cells (Krzesz et al., 1999; Lazaar et al., 1998; Schonbeck et al., 1997a; Suo et al., 1998). Some cells types (eg, T cells, stromal cells, endothelial cells, and smooth muscle cells) do not express CD40 constitutively, but can be induced to express CD40 following exposure to inflammatory mediators.

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FIG. 3. Properties of CD154 and CD40. NK, Natural killer; TNF, tumor necrosis factor.

In 1989, amino acid sequencing (Braesch-Anderson et al., 1989) and expression cloning of human CD40 (Stamenkovic et al., 1989) revealed it to be a 277–amino acid type I membrane glycoprotein related to the nerve growth factor receptor and the TNF receptor. The domains of CD40 include an N-terminal 193–amino acid extracellular portion, a 22–amino acid transmembrane domain, and a 62–amino acid C-terminal cytoplasmic tail. Of note, human CD40 is expressed as a single 1.5-kb mRNA species (Torres and Clark, 1992). The gene for human CD40 was localized to chromosome 20q11–20q13.2 (Lafage-Pochitaloff et al., 1993; Ramesh et al., 1993). Recent experiments have demonstrated that the intracellular portion of CD40 mediates downstream signaling events that lead to nuclear translocation of specific transcription factors following direct association with a family of TNF receptor–associated factor (TRAF) adapter molecules or with JAK3 (of the Janus family of cytoplasmic tyrosine kinases) (Fig. 4). Specifically, distinct sites for TRAFs 1, 2, 3, and 5 are clustered together within amino acids 250–254 of the cytoplasmic tail of CD40 (Pullen et al., 1998), whereas independent binding sites for JAK3 (residues 222–229) (Hanissian and Geha, 1997) and TRAF6 (residues 231–238) (Ishida et al., 1996b) are more membrane proximal. Of note, analysis of CD40-associated proteins revealed that an uncharacterized 23-kDa protein of unknown function is immunoprecipitated with anti-CD40 mAbs from human tonsillar B cells as well as from a variety of B cell lines, including BJAB, Ramos, and Raji (Morio et al., 1995). Finally, initial experiments examining surface


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FIG. 4. The cytoplasmic region of CD40 associates with TRAF adapter molecules and with JAK3.

expression of CD40 demonstrated that it forms multimers on tonsillar B cells and Raji Burkitt’s lymphoma cells but not on cells from the EBVinfected bladder carcinoma cell line (Braesch-Anderson et al., 1989). Early descriptions of functional outcomes following CD40 engagement focused on B lymphocytes. Initial experiments with anti-CD40 mAbs noted that these mAbs stimulated an increase in B cell size (Gordon et al., 1988) and induced homotypic B cell adhesion that was dependent on CD11a/ CD18–CD54 and CD23–CD21 as well as on unknown adhesion pairs that were independent of CD11a/CD18 (Aubry et al., 1992; Barrett et al., 1991; Bjorck et al., 1991, 1993; Kansas and Tedder, 1991). Additionally, early studies emphasized the role of CD40 engagement as a ‘‘progression signal.’’ Whereas nanomolar concentrations of S2C6 and G28.5 induced a small percentage of resting peripheral blood B cells into the G1 phase of the cell cycle (Stamenkovic et al., 1989), the combination of nanomolar concentrations of anti-CD40 mAbs with a ‘‘competence signal’’ such as anti-Ig, anti-CD20 (Bp35), protein kinase C (PKC)–activating phorbol esters, or formalinized Staphylococcus aureus induced progression of a large percentage of resting B cells into the S/G2/M phases of the cell cycle (Gordon et al., 1988; Ledbetter et al., 1987; Stamenkovic et al., 1989). Further studies demonstrated that ligation of CD40 was sufficient for B cell proliferation and differentiation if the level of engagement was above a given threshold. This was achieved by using micromolar concentrations of anti-CD40 mAbs (Gordon et al., 1988; Paulie et al., 1989) or by crosslinking anti-CD40 mAbs with murine fibroblasts expressing Fc웂RII/CD32 (Banchereau et al., 1991). Of interest, anti-CD40 mAbs induced expression of c-myc (Golay et al., 1992), an oncogene critically involved in cellular

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proliferation (reviewed in Schmidt, 1999). Although low-level engagement of CD40 required costimulation with a competence signal such as anti-Ig (Gordon et al., 1988; Paulie et al., 1989; Poudrier and Owens, 1994; Stamenkovic et al., 1989), a higher level of CD40 ligation allowed picomolar amounts of anti-Ig to be stimulatory (Wheeler et al., 1993). Furthermore, addition of interleukin 4 (IL-4) to B cells stimulated with anti-CD40 mAbs under these conditions dramatically increased DNA synthesis (Gordon et al., 1988; Valle et al., 1989) and promoted sustained proliferation of B cell populations that were EBV negative (Banchereau et al., 1991). Of note, IL-13, a cytokine that is homologous to IL-4 (McKenzie et al., 1993; Minty et al., 1993), had a similar effect on CD-40 mediated B cell proliferation (Cocks et al., 1993). Sufficient ligation of CD40 in the presence of cytokines enhanced Ig secretion and induced switching to specific isotypes. For example, the addition of IL-4 or IL-13 to B cells stimulated through CD40 induced switching to and secretion of IgG1 in mice and humans, IgG3 and IgG4 in humans, and IgE in both mice and humans (reviewed in Stavnezer, 2000). The clinical relevance of these findings is emphasized by the fact that IgE is the isotype thought to be involved in allergic responses (Corry and Kheradmand, 1999). In conjunction, these results generated intense interest in characterizing the physiological ligand for CD40. When it was established that CD40 engagement was a potent polyclonal activator of B cells that induced responsiveness to cytokines as well as proliferation and differentiation, the stage was set for cloning of the ligand for CD40. Several model systems of T cell–B cell collaboration were described that induced resting B cells to proliferate and differentiate into Ig-secreting cells in a manner that was contact dependent but not antigen specific or major histocompatibility complex (MHC) restricted (Hirohata et al., 1988; Lohoff et al., 1989; Riedel et al., 1988; Yellin et al., 1991; Zubler et al., 1987). In the human system, T cells activated with immobilized anti-CD3 mAbs induced B cells to proliferate, secrete Ig, and switch to downstream Ig isotypes in a contact-dependent manner (Amoroso and Lipsky, 1990; Hirohata et al., 1988; Nishioka and Lipsky, 1994). Moreover, incubation of T cells with cycloheximide, cyclosporine, or trypsin during anti-CD3 activation abrogated the ability of activated T cells to induce proliferation and differentiation of B cells (Tohma and Lipsky, 1991). Furthermore, stimulation of B cells by activated T cells occurred after the activated T cells were fixed or when B cells were cocultured with membrane preparations prepared from them, indicating that a receptor–ligand interaction rather than a more complex metabolic event was driving T cell– dependent B cell activation. These results were more consistent with the conclusion that activated T cells expressed a cell surface molecule that was critical for polyclonal activation of B cells and required new protein


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synthesis as well as nuclear factor of activated T cells (NF-AT) activation for its expression. Furthermore, the finding that the addition of an antiCD40 mAb (626.1) to a culture of anti-CD3–activated T cells and resting peripheral blood B cells inhibited B cell activation (Blanchard et al., 1993; Splawski et al., 1993) suggested that a novel activity accomplished by engaging CD40 was critical for polyclonal activation of B cells by antiCD3–activated T cells. Independent studies in humans revealed that the D1.1 subclone of the human Jurkat T cell tumor line constitutively expressed a contactdependent helper activity necessary for polyclonal activation of B cells (Yellin et al., 1991). In 1992, a mAb to this subclone (5c8) was isolated that inhibited T cell–B cell collaboration and recognized a cell surface glycoprotein of 앑30 kDa (Lederman et al., 1992a,b). In 1987, a mutant clone of the murine EL4 thymoma cell line had been described that induced proliferation and Ig secretion from a high percentage of murine B cells in a contact-dependent manner that was independent of antigen specificity and MHC restriction (Zubler et al., 1987). Later studies isolated a mouse mAb (MR1) that inhibited polyclonal B cell activation by T helper 1 (Th1)–type T cell clones (Hollenbaugh et al., 1992; Noelle et al., 1992), activated T cells, or membranes from T cells stimulated in the presence of mAbs to the T cell receptor (Brian, 1988; Noelle et al., 1989; Sekita et al., 1988; Swain and Dutton, 1987). Of note, a CD40.Ig construct containing the extracellular domain of human CD40 and the constant region of IgG1 also inhibited T cell–B cell collaboration (Armitage et al., 1992; Fanslow et al., 1992; Hollenbaugh et al., 1992; Noelle et al., 1992). Moreover, both MR1 (Hollenbaugh et al., 1992; Noelle et al., 1992) and CD40.Ig (Armitage et al., 1992; Hollenbaugh et al., 1992) immunoprecipitated a cell surface glycoprotein reported to be 앑33–39 kDa. In 1992, the cDNA encoding murine CD40L was cloned from CD40.Ig binding EL4 cells and found to encode a 33-kDa type II membrane glycoprotein homologous to TNF (Armitage et al., 1992; Hollenbaugh et al., 1992). Human CD40L/CD154 (Fig. 1) was cloned that same year by screening cDNA libraries from activated human T cells with the murine CD40L sequence (Gauchat et al., 1993a; Graf et al., 1992). Similar to murine CD40L, human CD154 was found to be a 33-kDa type II membrane glycoprotein related to TNF. Human CD154 protein consists of a 261–amino acid single-chain polypeptide with a C-terminal extracellular domain of 215 amino acids, a 24–amino acid transmembrane domain, and an N-terminal cytoplasmic tail of 22 amino acids. Molecular modeling and x-ray analysis of CD154 predicted that extracellular domains of CD154 form trimers on the cell surface (Bajorath, 1998; Bajorath et al., 1995a,b; Karpusas et al., 1995; Peitsch and Jongeneel, 1992; Pietraville et al., 1996;

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Singh et al., 1998). Northern blot analysis of activated T cells revealed a major CD154 mRNA species of 1.8–2.1 kb (Gauchat et al., 1993a; Graf et al., 1992; Spriggs et al., 1992; Villa et al., 1994). Subsequent work found that CD154 is not exclusively expressed by activated T cells (Fig. 3), but also is expressed by activated B cells (Blossom et al., 1997; Desai-Mehta et al., 1996; Devi et al., 1998; Furmann et al., 2000; Grammer et al., 1995, 1999a,b; Lipsky et al., 1997; Moses et al., 1997; Pinchuk et al., 1996; Schattner et al., 1998; Trentin et al., 1997; Voorzanger et al., 1996; Wykes et al., 1998), thymocytes (Dunn et al., 1997; Foy et al., 1995; Fuleihan et al., 1995), mast cells (Gauchat et al., 1993b; Pawankar et al., 1997), basophils (Gauchat et al., 1993b), eosinophils (Gauchat et al., 1995), Kupffer cells (Gaweco et al., 1999), monocytes (Mach et al., 1997b), dendritic cells (Pinchuk et al., 1996), natural killer cells (Carbone et al., 1997), smooth muscle cells (Mach et al., 1997b), endothelial cells (Mach et al., 1997b), and platelets (Aukrust et al., 1999; Henn et al., 1998; Lee et al., 2000). Localization of the human CD154 gene to Xq26.3–Xq27.1 (Allen et al., 1993; Aruffo et al., 1993; Graf et al., 1992; Padayachee et al., 1992) led to the identification of humans characterized clinically as having hyperIgM syndrome (HIgMXL) and functionally as having a mutant CD154 with point mutations or deletions in the extracellular domain (reviewed in Garber et al., 1999; Levy et al., 1997; Ramesh et al., 1998; Seyama et al., 1998). Of note, the gene for CD154 consists of five exons and four intervening introns (Villa et al., 1994), of which exon 5 encodes the majority of the extracellular domain, including the binding site for CD40 (Bajorath, 1998; Bajorath et al., 1995a,b; Karpusas et al., 1995; Peitsch and Jongeneel, 1992; Pietraville et al., 1996; Singh et al., 1998). A detailed examination of HIgMXL patients revealed that a subset expresses wild-type CD154 but has defects in signaling through CD40 (Conley et al., 1994; Durandy et al., 1997). Functionally, HIgMXL patients do not undergo IgH chain class switching from IgM to IgG, IgA, or IgE nor do they undergo affinity maturation or germinal center (GC) formation in response to challenge with a T cell–dependent antigen (Aruffo et al., 1993; Facchetti et al., 1995; Ochs et al., 1993). Cloning of CD154 and expression as a soluble construct or as a molecule on the surface of COS cells or Sf9 insect cells allowed direct confirmation of the finding that engagement of CD40 on B cells in vitro induced a panoply of functional responses (Fig. 5). Specifically, in vitro studies with B cells from normal individuals have demonstrated that engagement of CD40 by CD154 induces homotypic adhesion and induction of adhesion molecules [CD23 (Burlinson et al., 1996; Cairns et al., 1998; Lederman et al., 1992b; Maliszewski et al., 1993; Paterson et al., 1996; Saeland et al., 1993; Zupo et al., 1991), CD44H (Guo et al., 1996), CD11a/CD18 and


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FIG. 5. CD154–CD40 interactions mediate T cell–B cell collaboration. GC, Germinal center; GM-CSF, granulocyte/macrophage colony–stimulating factor; Ig, immunoglobulin; IL, interleukin; LT, lymphotoxin; MHC, major histocompatibility complex; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor.

CD54 (Barrett et al., 1991; Kansas and Tedder, 1991; Kennedy et al., 1994; Shinde et al., 1996)]; clonal expansion (Armitage et al., 1993a; Graf et al., 1992; Jumper et al., 1995; Lane, et al., 1993); expression of telomerase (Hu et al., 1997); isotype switching (Stavnezer, 2000); rescue from apoptosis (Holder et al., 1993; Lomo et al., 1997; Schauer et al., 1996, 1998; H. Wang et al., 1996); expression of activation antigens [CD38 (Miyashita et al., 1997), CD80 (Goldstein and Watts, 1996; Kennedy et al., 1994; Yellin et al., 1994a), CD86 (Goldstein and Watts, 1996; Liu et al., 1995; Ranheim and Kipps, 1993; Roy et al., 1995; Wu et al., 1995; Yang and Wilson, 1996), CD95 (Schattner et al., 1995), MHC class II (Hasbold and Klaus, 1994a; Kennedy et al., 1994; Maliszewski et al., 1993), CD154 (Grammer et al., 1999a; Pinchuk et al., 1996), and 4-1BBL/CD137L (DeBenedette et al., 1997)], cytokine and chemokine receptors [CD25 (Burlinson et al., 1996), IL-13R움 (D. Ford et al., 1999), and CXCR4 (Moir et al., 1999)], and the adapter molecules TRAF 1 (Schwenzer et al., 1999), TRAF2 and TRAF4 (Aicher et al., 1999); and secretion of Ig (Nishioka and Lipsky, 1994; Tohma and Lipsky, 1991) and cytokines [TNF-움 (Boussiotis et al., 1994; Goldfeld et al., 1994; Grammer et al., 1998; Tsai et al., 1996), lymphotoxin (LT) (Grammer et al., 1998; Worm and Geha, 1994, 1995; Worm et al., 1998), IL-10 (Grammer et al., 1998), granulocyte/macrophage colony–stimulating factor (GM-CSF) (Grammer et al., 1998), IL-6, and IL-12 (Schultze et al., 1999)].

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III. Functional Outcomes of CD154–CD40 Signaling

A. HUMORAL IMMUNE RESPONSES 1. T Cell–Dependent Responses The importance of CD154–CD40 interactions during T cell–dependent humoral responses as shown from examination of patients with HIgMXL syndrome was emphasized by the study of mice that were treated with an anti-CD154 mAb (MR1) following immunization with a T cell–dependent antigen (Foy et al., 1993, 1994) and mice deficient in expression of CD154 (Oxenius et al., 1996; Renshaw et al., 1994; Xu et al., 1994) or CD40 (Castigli et al., 1994; Kawabe et al., 1994; Oxenius et al., 1996) as a result of gene targeting. Such mice were unable to mount a primary or a secondary antibody response to a T cell–dependent antigen, did not form GCs, and did not generate antigen-specific memory B cells. The GC (Fig. 6) is one of the structures in which maturation of the humoral response to antigen occurs, fostering somatic hypermutation, selection, and isotype switching of activated B cells (reviewed in Choi et al., 1997; Liu and Arpin, 1997; MacLennan et al., 1997). Secondary lymphoid tissues are highly organized structures with distinct T and B cell compartments that collect antigen and antigen-presenting cells expressing antigen in the context of MHC and also recruit lymphocytes. Whereas naive lympho-

FIG. 6. T cell–dependent activation and differentiation in secondary lymphoid organs: the germinal center (GC) reaction. Ab, Antibody; Ag, antigen; C⬘, complement; FDC, follicular dendritic cell; IDC, interdigitating dendritic cell; MHC, major histocompatibility complex; sIg, surface immunoglobulin; TCR, T cell receptor.


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cytes enter lymph nodes and mucosal tissues such as tonsil and Peyer’s patches through high endothelial venules, they enter the spleen directly through the marginal zone. Antigen enters lymph nodes through the afferent lymph; mucosal tissues, across epithelium; and the spleen, through the blood. In all cases, antigen enters the lymphoid tissue and is presented to T cells in the context of MHC by professional antigen-presenting cells such as interdigitating dendritic cells (IDCs). This IDC–T cell interaction occurs in T cell zones such as the deep cortex of lymph nodes and the outer periarteriolar sheaths of the spleen. Stimulation of T cells through the T cell receptor by antigen–MHC complexes results in a signal that has been shown to induce expression of CD154 rapidly (Castle et al., 1993; Covey et al., 1994; Hermann et al., 1993; Lane et al., 1992; Noelle et al., 1992; Nusslein et al., 1996; Roy et al., 1993; Splawski et al., 1996; Spriggs et al., 1992). CD154-expressing T cells have been localized in situ to T cell zones of secondary lymphoid organs by anti-CD40L mAbs MR1 and 5c8 (CasamayorPalleja et al., 1995; Grammer et al., 1999a; Ledbetter et al., 1993; Lederman et al., 1992a; Van den Eertwegh et al., 1993; Vyth-Dreese et al., 1996). Whereas the initial phase of T cell–dependent B cell activation as described above is restricted by antigen and MHC, later phases are antigen and MHC nonspecific and are mediated by contact-dependent signals such as CD154–CD40 interactions and cytokines. It is important to note that the initial phases of T cell activation by antigen-bearing dendritic cells also involve CD154–CD40 interactions (Kato et al., 1997; Kelsall et al., 1996; Ludewig et al., 1995, 1996; MacLennan et al., 1996; Pinchuk et al., 1996; Sousa et al., 1997). In this regard, mature dendritic cells express CD40, and engagement of CD40 on dendritic cells by initially activated T cells expressing CD154 matures the cells by a variety of mechanisms, causing them to be more effective antigen-presenting cells (Fig. 7). This interaction appears to be critical for the full expression of many T cell responses. Ligation of CD40 on B cells has been shown to be sufficient for limited proliferation, Ig secretion (Nishioka and Lipsky, 1994; Tohma and Lipsky, 1991), and induction of sterile transcripts of all Ig isotypes (Fujita et al., 1994; Jumper et al., 1994), but cytokines have been found to amplify and direct the humoral immune response (reviewed in Stavnezer et al., 2000). For example, in humans, IL-2 is the major cytokine driving B cell proliferation and differentiation in an in vitro T cell–B cell collaboration system (Armitage et al., 1993a; Grabstein et al., 1993; Tohma and Lipsky, 1991). In addition, IL-10 promotes B cell proliferation and differentiation (Bonig et al., 1998; Burdin et al., 1995; Inaba et al., 1995; Nonoyama et al., 1993; Rousset et al., 1992), whereas IL-6 promotes differentiation with minimal effects on proliferation (Bonig et al., 1998; Burdin et al., 1995; Splawski et al., 1990). Moreover, cytokines direct which isotypes of Ig are made

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FIG. 7. Functional effects of CD40 engagement on monocytes and dendritic cells. IFN, Interferon; IL, interleukin; MHC, major histocompatibility complex; TF, tissue factor; TNF, tumor necrosis factor.

(reviewed in Stavnezer, 2000). For example, IL-4 or IL-13 promotes secretion of IgG1, IgG3, IgG4, and IgE, whereas transforming growth factor 웁 (TGF-웁) promotes switching to IgA. Finally, interferon 웂 (IFN-웂) inhibits secretion of IgG4 and IgE, and TGF-웁 inhibits secretion of IgE. Despite the importance of cytokines in heavy-chain isotype switching, CD154–CD40 interactions induce initial sterile transcription of many Ig heavy-chain genes, an event that is necessary for subsequent switch recombination ( Jumper et al., 1994). Moreover, CD154–CD40 interactions also provide an important co-signal for production of specific Ig isotypes, as shown by the enhanced IgE production resulting from stimulation with CD154 and IL-4 (reviewed in Stavnezer, 2000). CD154-expressing T cells initiate the GC reaction (Fig. 6) in humans by engaging CD40-expressing B cells in the extrafollicular region of secondary lymphoid organs and inducing the rapid proliferation that results in the formation of the dark zone of the GC ( Jacob and Kelsoe, 1992; Jacob et al., 1991; Kroese et al., 1987; Liu, et al., 1991b). This dark zone B cell population is referred to as centroblasts and is characterized as CD38⫹⫹ (Grammer et al., 1999a), surface Ig (sIg) negative (MacLennan et al., 1991), and myc⫹ (Martinez-Valdez et al., 1996). Of note, the dark zone is devoid of T cells, follicular dendritic cells (FDCs) (Casamayor-Palleja et al., 1995; Hardie et al., 1993; Lederman et al., 1992a; Terashima et al., 1992; Van den Eertwegh et al., 1993; Vyth-Dreese et al., 1996), and antigen/


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antibody/complement immune complexes (Nossal et al., 1968; Tew et al., 1997). After centroblastic B cells have undergone somatic hypermutation in the dark zone, they migrate to the basal light zone, exit the cell cycle by down-regulating myc (Martinez-Valdez et al., 1996), reexpress sIg (MacLennan et al., 1991), and compete for intact antigen held on FDCs in the basal light zone of a GC (Tew et al., 1997). The basal light zone is characterized by a lack of T cells (Vyth-Dreese et al., 1996) and by FDCs that weakly express CD23 (Hardie et al., 1993). Centrocytic B cells (CD38⫹ ) (Grammer et al., 1999a) undergo apoptosis within 7 hr in the basal light zone, unless they express a highly mutated sIg that is able to compete for immune complexes held on FDCs (Y.-J. Liu, et al., 1991b). Those B cells that are able to bind antigen held in immune complexes on FDCs receive a transient survival signal that is mimicked in vitro by anti-Ig (Liu et al., 1989). These high-affinity centrocytes internalize antigen via sIg and migrate to the apical light zone, where they present antigen in the context of MHC to CD4⫹ memory T cells. CD4⫹CD45RO⫹ T cells from the apical light zone of GCs rapidly express CD154 upon T cell receptor engagement in vitro (Casamayor-Palleja et al., 1995). Moreover, CD154⫹ CD4⫹ memory T cells are found in the apical light zone of GCs (Casamayor-Palleja et al., 1995; Grammer et al., 1999a; Hardie et al., 1993; Lederman et al., 1992a; Terashima et al., 1991; Van den Eertwegh et al., 1993; Vyth-Dreese et al., 1996). Finally, isotype switching occurs in B cells activated in the light zone of GCs (Girshick et al., 1999; Hodgkin et al., 1996; Liu et al., 1996; Toellner et al., 1996) as well as during the extrafollicular reaction in the T cell zone (Toellner et al., 1996). Of note, somatic hypermutation and isotype switching have been observed to occur independently, and predominantly in the dark and light zones, respectively (Apel and Berek, 1990; Cumano et al., 1985; Jacob and Kelsoe, 1992; Kaartinen et al., 1983; Klein et al., 1994; Pascual et al., 1994). Although CD154–CD40 interactions are essential for initiation and propagation of the GC reaction, there are stages of the GC reaction that appear to proceed in the absence of CD154⫹ T cells. CD154⫹ T cells are absent from the dark zone, where rapid B cell proliferation and somatic hypermutation occur, and are found infrequently, if at all, in the basal light zone of the GC (Casamayor-Pelleja et al., 1995; Hardie et al., 1993; Lederman et al., 1992a; Van den Eertwegh et al., 1993; Vyth-Dreese et al., 1996), where high-avidity antigen-binding B cells are rescued from apoptosis (Liu et al., 1989; Y.-J. Liu et al., 1991a). Despite the paucity of CD154-expressing T cells, an established GC rapidly disassembles when CD154–CD40 interactions are blocked (Han et al., 1995).

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The finding that human GC B cells express CD154 in situ and immediately ex vivo (Grammer et al., 1999a) suggests that GC B cells may sustain clonal expansion in the absence of T cells by a CD154–CD40– dependent mechanism (Fig. 8). This hypothesis is strengthened by the findings that CD154 expression on B cells leads to homotypic CD154– CD40 interactions and DNA synthesis (Grammer et al., 1995; Lipsky et al., 1997) and that CD40-induced proliferation of B cells is partially mediated by subsequent interactions involving the CD154–CD40 coreceptors (Grammer et al., 1999a). These data confirm earlier in vitro studies that demonstrated rescue of GC B cells from spontaneous apoptosis following treatment with mAbs against CD40 itself (Holder et al., 1993; Liu et al., 1989; Y.-J. Liu et al., 1991). In addition, since ligation of CD40 on B cells induces CD154 expression (Grammer et al., 1999a), homotypic CD154–CD40 interactions between B cells may sustain CD154 expression on B cells in an autocrine or paracrine manner independent of T cells after initial activation. Moreover, the finding that CD154–CD40 interactions between tonsillar B cells propagate GC reactions is further supported by the finding that in vitro differentiation of highly purified GC B cells to those with a memory phenotype was partially blocked by an anti-CD154 mAb (Grammer et al., 1999a). In this regard, earlier in vivo studies demonstrated that inhibiting

FIG. 8. CD154-expressing B cells play a role in initiation and propagation of the germinal center (GC) reaction. Abbreviations are explained in the legend to Fig. 6.


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CD154–CD40–dependent responses following immunization with a T cell–dependent antigen abolished antigen-stimulated clonal expansion of B cells in GCs (Garside et al., 1998; Vora et al., 1998) and interfered with the generation of antigen-specific memory (Gray et al., 1994). Therefore, CD154 expressed by B cells in secondary lymphoid tissues (Grammer et al., 1999a) may be the in vivo source of the signal required for the previously observed CD40-mediated differentiation of GC B cells to memory cells (Arpin et al., 1995). 2. T Cell–Independent Responses The role of homotypic CD154–CD40 interactions between B cells during T cell–independent immune responses to both bacteria and viruses has been elucidated (Fig. 9). Following immunization of mice with Streptococcus pneumoniae, the initial IgM response was shown to be independent of the presence of T cells but dependent on CD154–CD40 interactions (Wu et al., 1999; Hwang et al., 2000). Moreover, the IgG response to polyomavirus infection was shown to be independent of T cells but dependent on CD154-expressing B cells, as severe combined immunodeficiency (SCID) mice reconstituted with CD154⫺/⫺ B cells produced significantly less polyomavirus-specific IgG than SCID mice reconstituted with wildtype B cells (Szomolanyi-Tsuda et al., 2000). The initial polyclonal activation of B cells observed following microbial infection of humans has been demonstrated in vitro to be dependent on homotypic CD154–CD40 interactions between B cells (Grammer et al., 1995). Specifically, engaging sIg induces CD154 expression on the surface of human B cells and polyclonal expansion that is partially dependent on subsequent CD154–CD40 interactions (Grammer et al., 1999a). Moreover, it should be noted that polyclonal activation of B cells has been observed following engagement of sIg with staphylococcal and streptococcal B cell ‘‘superantigens’’ (Domiati-Saad and Lipsky, 1998; Domiati-Saad

FIG. 9. CD154–CD40 interactions between B cells play a role in some T cell– independent (TI) responses. Ag, Antigen; Ig, immunoglobulin; sIg, surface Ig.

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et al., 1996; Vasquez-Kristiansen et al., 1994), pneumococcal polysaccharides (Snapper and Mond, 1996), and DNA in the form of phosphorothioate oligodeoxynucleotides (Liang et al., 1996). Furthermore, the highest density of CD154 in B cell subsets from human secondary lymphoid tissue undergoing a humoral immune response following streptococcal infection was observed in the CD38⫹⫹⫹IgD⫹ plasmablast population hypothesized to be generated by T cell–independent antigen stimulation (Grammer et al., 1999a). Finally, CD154–CD40 interactions between human tonsillar B cells have been shown to be essential for differentiation of activated naive CD38⫺IgD⫹ B cells to CD38⫹⫹⫹IgD⫹ short-lived plasmablasts (our unpublished observations). B. ANTIGEN PRESENTATION, INFLAMMATORY RESPONSES, AND THROMBOSIS Recent experiments have demonstrated that CD154–CD40 interactions not only are important during humoral immune responses but also are involved in the maturation of monocytes to dendritic cells (Fig. 7), activation of dendritic cells from an immature to a mature phenotype, functional responses of fibroblast-like stromal cells during inflammation (Fig. 10), and interaction between activated platelets and endothelial cells (Fig. 11). CD40 engagement on monocytes (Fig. 7) ultimately induces maturation to dendritic cells (reviewed in Banchereau et al., 1998) but also induces adhesion molecules [CD62L (Cella et al., 1999) and CD54 (Kuroiwa et al., 1999)], rescue from apoptosis (Kiener et al., 1995; Suttles et al., 1996,

FIG. 10. Functional effects of CD40 engagement on fibroblast-like stromal cells. IFN, Interferon; TNF, tumor necrosis factor; SDF, stromal cell-derived factor; MMP, matrix metalloproteinase; COX, cyclooxygenase.


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FIG. 11. CD154–CD40 interactions between activated platelets and endothelial cells (ECs). IFN, Interferon; IL, interleukin; TF, tissue factor; TNF, tumor necrosis factor.

1999), expression of cytokines [IL-1움 and IL-1웁 (Wagner et al., 1994), IL-6 (Kuroiwa et al., 1999), IL-12 (Imaizumi et al., 1999; Marriott et al., 1999; Mendez-Samperio et al., 1999), TNF-움 (Imaizumi et al., 1999), and IFN-움 (Cella et al., 1999)], chemokines [MCP1 (monocyte chemotactic protein-1) (Kuroiwa et al., 1999) and IL-8 (Caux et al., 1994)], the chemokine receptor CXCR3 (Cella et al., 1999), the inflammatory mediator nitric oxide (Imaizumi et al., 1999), and the procoagulant tissue factor (Mach et al., 1997). Following CD40-mediated maturation of monocytes to dendritic cells, engagement of CD40 on dendritic cells induces expression of the adhesion molecule CD58 (Caux et al., 1994), activation antigens [CD38 (Miyashita et al., 1997) as well as CD80, CD86, and MHC class II (Caux et al., 1994)], cytokines [IL-12 (Bartholome et al., 1999; Bianchi et al., 1999), TNF-움 (Caux et al., 1994), and IL-15 (Kuniyoshi et al., 1999)], and chemokines [MIP1움 (macrophage inflammatory protein), MIP1웁, IL-8, Rantes, and MCP1 (Sallusto et al., 1999), MPIF-1 (myeloid progenitor inhibitory factor) (Nardelli et al., 1999), and fractalkine (Papadopoulos et al., 1999)]. This CD40 engagement also alters chemokine receptor expression [increases CXCR4 and decreases CCR5 (Chougnet et al., 1999)]. In contrast to the constitutive expression of CD40 on monocytes and dendritic cells, inflammatory mediators are thought to be necessary to induce CD40 on smooth muscle cells (Krzesz et al., 1999; Lazaar et al., 1998; Schonbeck et al., 1997a; Suo et al., 1998), endothelial cells (Hollenbaugh et al., 1995; Karmann et al., 1995; Yellin et al., 1995), and stromal

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cells. Following exposure to IFN-웂, stromal cells express CD40 (Banchereau et al., 1995; Fries et al., 1995; Shimaoka et al., 1998). Engagement of newly expressed CD40 on stromal cells by CD154 results in increased expression of COX-2 (Adawi et al., 1998; Zhang et al., 1998), TNF-움 (Harigai et al., 1999), and stromal-derived factor 1 (Nanki et al., 2000) and decreased expression of MMP1 (matrix metalloproteinase)/interstitial collagenase and MMP3/stromelysin 1 (Wassenaar et al., 1999). Exposure of platelets and endothelial cells to a milieu of inflammatory mediators and stress-induced factors induces CD40 on endothelial cells and CD154 on both platelets and endothelial cells, leading to a cascade of events that favor coagulation (Fig. 11). Whereas IFN-웂, 웁-amyloid, IL-1, TNF-움, and IFN-웁 have been shown to induce CD40 on endothelial cells in vitro (Karmann et al., 1995; Suo et al., 1998), preformed CD154 has been shown to be induced on platelets in situ in fresh thrombi after vessel injury or on atherosclerotic plaques as well as in vitro following exposure to thrombin, collagen, or epinephrine (Aukrust et al., 1999; Henn et al., 1998; Lee et al., 2000). CD40 engagement on endothelial cells induces expression of adhesion molecules [CD54, CD62E, and CD106 (Yellin et al., 1995)]; IL-1–converting enzyme (Schonbeck et al., 1997a), resulting in cleavage of IL-1웁 precursor protein to the active form; tissue factor (Miller et al., 1998; Slupsky et al., 1998), resulting in factor VII– dependent coagulation; stromelysin 3 (Schonbeck et al., 1997b), resulting in the breakdown of atherosclerotic plaques; and MMPs 1, active 2/gelatinase A, 3, and 9/gelatinase B (Mach et al., 1998), resulting in the induction of vascular remodeling and neovascularization. Ligation of CD40 on endothelial cells also decreases expression of thrombomodulin (Miller et al., 1998; Slupsky et al., 1998), resulting in a diminished anticoagulant phenotype. Finally, since studies with lymphocytes have demonstrated that engagement of CD154 itself results in the induction of a variety of signaling cascades and that functional outcomes and endothelial cells express CD154 following exposure to inflammatory mediators, the result of engaging CD154 on endothelial cells must be considered since a variety of cells that come in contact with the endothelium—such as B cells, monocytes, dendritic cells and smooth muscle cells—express CD40 (Fig. 12). IV. Regulation of CD40 and CD154 Expression

Expression of both CD40 and CD154 is regulated at transcriptional, posttranscriptional, and posttranslational levels (Figs. 13–15). Whereas the transcription factors activator protein 1 (AP-1) and nuclear factor kappa beta (NF-␬B) have been demonstrated to regulate transcription of the genes for both receptors [CD40 (Krzesz et al., 1999) and CD154 (Bischof


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FIG. 12. Is there a role for CD154 in modulating the function of endothelial cells (ECs)? DC, Dendritic cell; SM, smooth muscle.

and Melms, 1998; Grammer et al., 1999a; Hodge et al., 1996; Schubert et al., 1995; Splawski et al., 1996; Timmerman et al., 1997; Tsytsykova et al., 1996), calcium signaling leading to nuclear translocation of NF-AT further regulates CD154 transcription (Lobo et al., 1999). In addition, although posttranscriptional mechanisms regulating expression of CD40 and CD154 have not been examined in detail, preliminary mechanisms have been

FIG. 13. Regulation of CD154 and CD40 expression. EC, Endothelial cell; NF-AT, nuclear factor; NF-␬B, nuclear factor kappa beta; TGF, transforming growth factor.

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FIG. 14. Positive and negative regulation of CD40 expression. GM-CSF, Granulocyte/ macrophage colony–stimulating factor; IFN, interferon; IL, interleukin; NF-␬B, nuclear factor kappa beta; TGF, transforming growth factor; TNF, tumor necrosis factor; BM, bone marrow.

hypothesized from initial experiments. Whereas TGF-웁 has been observed to decrease the stability of CD40 mRNA in epithelial cells and smooth muscle cells (Nguyen et al., 1998), CD154 mRNA has been shown to be stabilized by a CD58-dependent mechanism in endothelial cells (Murakami et al., 1999) or by the addition of ionomycin and phorbol ester to T cells (G. S. Ford et al., 1999; Rigby et al., 1999). Furthermore, both CD40 and CD154 have been shown to be regulated at a posttranslational level. Soluble CD40 and CD154 resulting from a posttranslational cleavage mechanism (Fig. 16) have been observed in culture supernatants from T cells and B cells activated in vitro (van Kooten et al., 1994) as well as at low levels in

FIG. 15. Regulation of CD154 expression. IL, Interleukin; LPS, lipopolysaccharide; NF-␬B, nuclear factor kappa beta; R, receptor; sIg, surface immunoglobulin; PC, phosphatidylcholine; MKK, mitogen-activated protein kinase kinase; SAC, Staphylococcus aureus Cowan I strain; SEB, staphylococcal enterotoxin B; TwHf, tripterygium wilfordii Hookf.


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FIG. 16. Increased soluble (s) CD154 or CD40 in disease. AIDS, acquired immunodeficiency syndrome; CLL, Chronic lymphocytic leukemia; SLE, systemic lupus erythematosus.

the serum and urine of healthy individuals (Aukrust et al., 1999). Of note, increased levels of soluble CD40 and CD154 have been observed in the serum of patients with B cell lymphomas (De Paoli et al., 1997; Younes et al., 1998). In addition, soluble CD40 has been observed in the serum and urine of patients with impaired renal function (Schwabe et al., 1999). Moreover, soluble CD154 has been observed in the serum of systemic lupus erythematosus patients (Vakkalanka et al., 1999) as well as in patients with unstable angina (Aukrust et al., 1999). The functional relevance of the complex regulation of CD40 and CD154 at many levels is highlighted by the observation that expression of these receptors on the surface of cells varies among cell types as well as among varying differentiation stages of the same cell type. For example, activated stromal cells have been observed to express one fifth as many CD40 receptors on their surface compared to normal B cells (Fig. 17). In addition, whereas activated peripheral T cells express 102 to 103 more CD154 than activated peripheral B cells, freshly isolated tonsillar T and B cells express an equivalent level of CD154 that is lower than that expressed by activated peripheral T cells and higher than that expressed by activated peripheral B cells (Grammer et al., 1999a). The lower level of CD154 expressed on tonsillar T cells compared with in vitro activated peripheral T cells may relate to down-regulatory signals present in the inflamed tonsil. Previous studies have observed that B cells, especially activated B cells, have the capacity to down-regulate CD154 expression on T cells activated in vitro (Cantwell et al., 1997; Ludewig et al., 1996; Miyashita et al., 1997; van Kooten et al., 1994; Yellin et al., 1994b). Potential mechanisms have been suggested for this phenomenon, including down-modulation of CD154 mRNA by an undefined signaling pathway as well as direct effects on CD154 protein on the surface of the activated T cell, such as cleavage (Ludewig et al., 1996), capping and endocytosis (Yellin et al., 1994b), or

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FIG. 17. Ramos B cells (䊏) express a higher level of CD40 than stromal cells (䊉). MFI, mean fluorescence intensity. Ramos B cells or stromal cells were incubated for 30 minutes with various concentrations of CD154mCD8 fusion protein and analyzed for amount bound using a PE-labeled mAb to mCD8.

blocking the epitope recognized by the staining mAb by soluble CD40 released from activated B cells (Grammer et al., 1995; van Kooten et al., 1994). IL-10 may be a candidate for direct down-regulation of CD154 mRNA in tonsillar T cells by activated B cells, since IL-10 has been shown to downmodulate CD154 expression on T cells (Splawski et al., 1996), to be secreted in large amounts by activated B cells, and to play a role in GC reactions (Pistoia, 1997). It should be noted that a lower percentage of tonsillar T cells expressed CD154 compared with tonsillar B cells, and, as noted above, the density of tonsillar B cell CD154 is markedly greater than that expressed by peripheral B cells activated in vitro. Therefore, down-regulatory influences on T cell and B cell CD154 in the tonsil in vivo may differ. The relevance of varying receptor expression levels to subsequent signaling cascades and functional outcomes has not been examined in detail, although previous experiments demonstrated that the outcome of signaling through CD40 can be complex, varying from stimulation to apoptosis. This has been studied primarily in B cells, in which the functional outcomes of CD40 ligation appear to depend on the activation and differentiation status of the cell as well as the degree of CD40 engagement (Miyashita et al., 1997). For example, there is a dose–response curve between the degree of CD40 ligation on naive IgD⫹ B cells and the subsequent proliferation that reaches a maximum at 100% engagement. By contrast, postswitch IgD⫺ B cells from the periphery are much more sensitive to CD40 ligation. Specifically, maximal proliferation is reached at 30–40% CD40 engage-


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ment, and higher levels of ligation result in apoptosis. B cell lines such as Ramos are much more sensitive to CD40 engagement in that maximal proliferation is reached at 앑10% CD40 engagement, with higher levels of ligation resulting in programmed cell death. A. TRANSCRIPTIONAL REGULATION OF CD40 Several cytokines have been shown to regulate CD40 expression (Fig. 14). IFN-웂 has been shown to induce CD40 expression in an AP-1–dependent manner (Krzesz et al., 1999) in stromal cells (Shimaoka et al., 1998), epithelial cells (Kooy et al., 1999), smooth muscle cells (Krzesz et al., 1999), and microglia (Gormand et al., 1999; Nguyen et al., 1998; Wei and Jonakait, 1999). By contrast, TNF-움 induces CD40 expression in microglia (Tan et al., 1999), epithelial cells (Kooy et al., 1999), and smooth muscle cells by an NF-␬B– dependent mechanism (Krzesz et al., 1999). The mechanism by which GMCSF induces CD40 expression on microglial cells is unknown (Wei and Jonakait, 1999). Finally, IL-10 has been observed to down-regulate existing CD40 expression on epithelial cells and smooth muscle cells by an unknown mechanism (Kooy et al., 1999). In this regard, it is interesting to note that CD40 expression disappears from activated B cells upon differentiation to plasma cells following exposure to IL-10. Although the effect of IL-10 on B cell CD40 expression has not been tested, these data suggest that IL-10 may contribute to the down-regulation of CD40 on plasma cells. B. TRANSCRIPTIONAL REGULATION OF CD154 T and B lymphocytes have been the primary cell types in which transcriptional regulation of CD154 has been examined (Fig. 15). The pharmacological reagents ionomycin and phorbol ester have been shown to induce CD154 expression by either T cells (Casamayor-Palleja et al., 1995; Covey et al., 1994; Gauchat et al., 1993a; Lane et al., 1992; Nusslein et al., 1996; Splawski et al., 1996; Spriggs et al., 1992) or B cells (Grammer et al., 1995; Lipsky et al., 1997; Pinchuk et al., 1996) by signaling pathways regulated by increasing intracellular calcium and activating PKC. In addition, engagement of the antigen receptor on either T cells (Bischof and Melms, 1998; Hodge et al., 1996; Lobo et al., 1999; Schubert et al., 1995; Splawski et al., 1996; Timmerman et al., 1997; Tsytsykova et al., 1996) or B cells (Grammer et al., 1999a) induces de novo synthesis of CD154 by means of signaling pathways involving calcineurin and therefore likely leading to nuclear translocation of NF-ATc. The relevance of NF-AT to CD154 regulation at the transcriptional level in T cells is emphasized by the finding that deletion of the proximal AP-1/NF-AT site in the CD154 promoter decreases phorbol myristate acetate (PMA)- and concanavalin A (ConA)driven transcription of the CD154 gene in Jurkat T cells. Moreover,

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NF-AT motifs in the 5⬘ promoter region of the CD154 gene in both mice and humans has been shown to bind NF-ATp/c –NF-ATn/AP-1 complexes in nuclear extracts derived from activated T cells and to control transcription of the gene. Although NF-AT has been thought to be a T cell–specific transcription factor, there is a growing body of evidence that B cells can also be induced to activate NF-AT in a cyclosporine-sensitive manner by a variety of stimuli, including engagement of sIg (Choi et al., 1994; Francis et al., 1995; Meyer and Ireland, 1998; Venkataraman et al., 1994; Verweij et al., 1990; Yaseen et al., 1993). Other signal transduction mechanisms that have been shown to be involved in the regulation of anti-CD3–induced CD154, including those revealed by the src kinase inhibitor pyrazol-pyrimidine (Gimsa et al., 1999), the cAMP inducer prostaglandin E2 (Wingett et al., 1999a), glucocorticoids (Bischof and Melms, 1998), and the anti-inflammatory compound TwHF (Ho et al., 1999). Expression of CD154 can be increased over that observed following ligation of CD3 alone by costimulatory signals such as those provided by CD28 (Kehry and Castle, 1994; Klaus et al., 1994b; Pinchuk et al., 1996; Yang and Wilson, 1996; Yin et al., 1999), CD11a/CD18 or CD54 (Roy et al., 1995), CD58 or CD2 (Wingett et al., 1999b), and CD99 (Wingett et al., 1999b) as well as by specific cytokines [IL-2 and IL-4 (Splawski et al., 1996) or IL-12 (Hirohata, 1999; Peng et al., 1998)]; or lyso-PC, an inflammatory mediator produced locally in the plaques of patients with atherosclerosis (Sakata-Kaneko et al., 1998). Finally, the superantigens SEB or TSST-1 (toxic shock syndrome toxin-1), which engage the T cell receptor outside the antigen binding site, have also been shown to induce CD154 expression on T cells ( Jabara and Geha, 1996). Polyclonal activation of B cells with S. aureus Cowan I (Grammer et al., 1995; Lipsky et al., 1997) or lipopolysaccharide (Blossom et al., 1997; Wykes et al., 1998) induces expression of CD154. Moreover, engagement of sIg or CD40 on B cells has been shown to induce CD154 expression by specific pathways of transcriptional regulation. Of importance, both engagement of sIg- and CD40-induced de novo synthesis of CD154 (Grammer et al., 1999a) but not re-expression of preformed protein from intracellular stores have been reported for tonsillar (Casamayor-Palleja et al., 1995) and synovial T cells (MacDonald et al., 1997), anti-Ig–stimulated murine splenic B cells (Wykes et al., 1998), and platelets (Henn et al., 1998). In addition to the apparent role of NF-AT in regulating B cell CD154 expression following engagement of sIg, NF-␬B activation played an important role in up-regulating the expression of CD154 following ligation of either CD40 or sIg. In this regard, examination of the published sequence of the human CD154 promoter (GenBank/EMBL accession No. L47983) (Schubert et al., 1995) revealed the presence of five poten-


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tial NF-␬B binding sites (Gerondakis et al., 1998) at ⫺1200 씮 ⫺1209, ⫺1186 씮 ⫺1195, ⫺812 씮 ⫺821, ⫺656 씮 ⫺665, and ⫺464 씮 ⫺473. At least one of these potential NF-␬B binding sites (⫺464 씮 ⫺473) is within the proximal CD154 promoter (⫺495 씮 ⫹67) necessary for PMAand ConA-driven transcription of the CD154 gene in Jurkat T cells (Schubert et al., 1995). Importantly, ligation of both sIg and CD40 is known to activate NF-␬B in B cells (reviewed in Gerondakis et al., 1998). Moreover, CD40-mediated induction of CD154 mRNA in Daudi B cells was blocked by the src kinase inhibitor herbimycin A (Pinchuk et al., 1996), previously shown to interfere with CD40-induced activation of NF-␬B (Lapointe et al., 1996). Finally, T cell–independent responses of short-lived nonswitched plasmablasts (CD38⫹⫹⫹IgD⫹ ) shown recently to be CD154⫹ (Grammer et al., 1999a) have been demonstrated to be dependent on sIg-mediated signaling leading to activation of BTK (Bruton’s tyrosine kinase) (Satterthwaite et al., 1998) and nuclear translocation of NF-␬B ( J. L. Liu et al., 1991). Together, these results demonstrate that CD154 is transcriptionally regulated following engagement of receptors that induce NF-AT or NF-움B. Receptors that have been demonstrated to induce CD154 transcription include the antigen receptor on either T cells or B cells, receptors that costimulate CD3 signaling on T cells, CD40, as well as receptors for cytokines and inflammatory mediators. The higher level of CD154 expressed by B cells isolated from the tonsil compared to that by peripheral B cells stimulated in vitro suggests that there must be unique and undefined costimulatory features of the tonsil that contribute to the comparatively enhanced expression of CD154 in tonsillar B cell subsets. This could relate to the effects of other signaling molecules that might be present in inflamed secondary lymphoid tissue and costimulate up-regulation of CD154 in tonsillar B cells. These additional signaling molecules may include those previously shown to costimulate anti-CD3–induced CD154 expression by T cells, such as cytokines (IL-2, IL-4, and IL-12), inflammatory mediators (lyso-PC), or interactions between adhesion molecules (CD11a/CD18–CD54). In this regard, IL-12 may be the most relevant cytokine influence during the formation of GCs in primary and secondary follicles in a T cell–dependent response, since B cells express the IL-12 receptor and IL-12 has been shown to be produced by naive and memory cells but not GC B cells following engagement of CD40 but not sIg (Schultze et al., 1999). In addition, IL-12 promotes hyperexpression of CD38 on B cells (Gagro and Gordon, 1999). Moreover, ligation of CD38 has been shown to up-regulate CD154 expression by murine B cells (Wykes et al., 1998), suggesting additional mechanisms by which B cells may be costimulated to express CD154. However, the relevance of this finding to human B cells is questionable, since CD38 marks

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naive, unactivated cells in mice but activated, differentiated B cells in humans (reviewed in Lund et al., 1998; MacLennan et al., 1997). An alternative explanation is that tonsillar B cells have differentiated sufficiently so that signals provided by ligation of sIg or CD40 directly induce exaggerated expression of CD154. Whether enhanced expression of CD154 by tonsillar B cells reflects intrinsic features of the B cells themselves or a facilitating influence of the tonsillar milieu remains to be determined. V. Role of CD40-Induced Signaling Cascades in Functional Responses of B Cells

The crucial role of CD154–CD40 interactions in the normal and abnormal immune responses described above has generated considerable interest in defining the signaling cascades that mediate the functional activities induced by CD40 engagement. Of great interest was the role of CD154– CD40 interactions in the activation of B cells during T cell–dependent and –independent humoral responses. For example, the CD40-mediated signaling pathways that lead to the development and progression of GCs— resulting in IgH class switching, rescue from apoptosis, and B cell memory—are important to delineate. Moreover, understanding the signaling pathways mediating the specific biological outcomes of CD40 engagement that had been observed in vitro—such as adhesion (increases in CD23, CD44H, CD11a/CD18, and CD54), activation (increases in CD38, CD80, CD86, TRAF1, TRAF2, TRAF4, CD95, CD137L, CD154, and MHC class II), responsiveness to cytokines and chemokines (increases in CXCR4, CD25, and IL-13R움), clonal expansion, telomerase expression, cytokine secretion (TNF-움, LT-움, IL-10, GM-CSF, IL-6, and IL-12), IgH class switching, and Ig production—would be an important step in understanding the role of CD40-mediated signaling in B cell biology. Several groups have reported that the functional outcome of CD40 engagement depends on the activation state of the B cell. For example, full CD40 engagement on naive B cells induces proliferation and Ig production. Low-level engagement on postswitch memory B cells induces proliferation and secretion of Ig, but higher levels of engagement induce apoptosis and decreased production of Ig. Moreover, ligation of CD40 on certain B cell lines results in a decrease in both Ig production (Bergman et al., 1996; Grammer et al., 1998) and proliferation that may be related to growth arrest and apoptosis (Bergman et al., 1996; Fluckiger et al., 1992; Funakoshi et al., 1994; Goldstein and Watts, 1996; Grell et al., 1999; Heath et al., 1993; Inui et al., 1990; Murphy et al., 1995). By contrast, engagement of CD40 rescues GC B cells from spontaneous apoptosis (Holder et al., 1993; Y.-J. Liu et al., 1991a,b) and lymphoma B cells from apoptosis induced by sIg cross-linking (Y.-J. Liu et al., 1991a). Therefore, the stage of differentia-


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tion and maturation of the B cell appears to play an important role not only in the functional outcome of CD40 engagement, but presumably also in the nature of the signals generated from CD40 ligation. The biochemical pathways by which CD40 ligation led to diverse functional outcomes that are crucial for the development of humoral immune responses have been examined in detail. Specifically, the question of how CD40 engagement led to proliferation of some B cells, rescue from apoptosis of other B cells, and death by apoptosis in still another B cell subset, even though there is no death domain in the cytoplasmic domain of CD40, is an important issue in understanding the complete role of CD40 in governing B cell responses. Furthermore, since development of pharmaceutical agents that could block specific B cell responses to CD40 engagement would be of potential clinical usefulness, especially in conditions in which there is hyperexpression of CD154 (eg, various autoimmune diseases, graft-versus-host disease, and B cell leukemias and lymphomas) (Fig. 2), great effort has been expended to explore the signaling cascades that are induced following CD40 engagement. A. ACTIVATION OF TRANSCRIPTION FACTORS Engagement of CD40 leads to a variety of biochemical cascades that result in the activation of three major transcription factors [NF-␬B, cAMP response element (CRE), and AP-1] whose binding sites are present in the promoters of genes induced by CD40 ligation (Fig. 18). Specifically, NF-␬B has been shown to be involved in the regulation of TRAF1 (Schwenzer et al., 1999), CD23 (Tinnell et al., 1998), CD54 (H. H. Lee

FIG. 18. Genes that mediate functional responses induced following CD40 engagement on B cells are mediated by specific transcription factors. For abbreviations, see text.

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et al., 1999b; S. J. Lee et al., 1999), CD80 ( Jennings et al., 1999), IL-6 (Vanden Berghe et al., 1999), IL-12 (Gri et al., 1998; Ma et al., 1997; Plevy et al., 1997; Tone et al., 1996; Yoshimoto et al., 1997), GM-CSF (Ghiorzo et al., 1997), LT-움 (Worm et al., 1998), and CD154 (Grammer et al., 1999a). It should be noted that deletion of the NF-␬B sites in the human TNF-움 promoter did not affect its induction in B cells (Tsai et al., 1996). In addition, NF-␬B has been shown to be essential for functional outcomes induced by CD40 ligation, including clonal expansion (Armitage et al., 1993a; Graf et al., 1992; Jumper et al., 1995; Lane et al., 1993) and rescue from apoptosis (Holder et al., 1993; Lomo et al., 1997; Schauer et al., 1996, 1998; H. Wang et al., 1996). CRE sites have been shown to be involved in the regulation of IL-6 (Vanden Berghe et al., 1999), TNF-움 (Brinkman et al., 1999; Tsai et al., 1996), and IL-12 (Lu et al., 1999). AP-1/NF-AT sites have been shown to be involved in the regulation of TNF-움 (Tsai et al., 1996), CD25 (Schuh et al., 1998), and CD154 (Bischof and Melms, 1998; Grammer et al., 1999a; Hodge et al., 1996; Schubert et al., 1995; Splawski et al., 1996; Timmerman et al., 1997; Tsytsykova et al., 1996). Finally, analysis of the IL-10 promoter has revealed potential regulation by the cAmp response element binding protein (CREB) (Eskdale et al., 1997; Mori et al., 1996). 1. AP-1 and NF-AT CD40 engagement on B cells has been shown to induce activation of tyrosine kinases in the src [lyn, fyn, and blk (Faris et al., 1994; Ren et al., 1994)], ZAP-70 [syk (Faris et al., 1994)], and tec [btk (Uckun et al., 1991)] families. Ligation of CD40 also induces activation of the RAS (Gulbins et al., 1996) via the nucleotide guanine exchange factor son of sevenless (SOS) that exchanges GDP for GTP on RAS (Kashiwada et al., 1996, 1998; Li et al., 1996), allowing it to associate with the plasma membrane and be available to recruit effectors such as RAF1 (Gulbins et al., 1996; Kashiwada et al., 1998) and phosphatidylinositol-3-kinase (PI3K) (Aagaard-Tillery and Jelinek, 1996; Fruman et al., 1999; Gulbins et al., 1996; Padmore et al., 1997) via independent mechanisms. CD40 engagement also activated another exchange molecule upstream of PI3K: VAV (Padmore et al., 1997). Association of PI3K with the plasma membrane allows it to phophorylate membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5,trisphosphate (PIP3). CD40-induced activation of phospholipase C웂2 cleaves PIP3 to yield inositol-1,45-trisphosphate (IP3) that increases intracellular calcium levels and diacylglycerol that activates calcium-dependent isoforms of PKC (Gulbins et al., 1996; van Kooten and Banchereau, 1996). Increased intracellular calcium levels following CD40 ligation lead to nuclear translocation of NF-ATc (Klaus et al., 1994). Moreover, CD40


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engagement has been shown to induce signaling cascades that activate the ERK and JNK serine/threonine MAPKs, leading to assembly of the active AP-1 transcription factor (Klaus et al., 1994a). Of note, dimerization of AP-1 and NF-ATc forms another transcription factor, NF-AT (reviewed in Masuda et al., 1998), that has been observed in some circumstances following CD40 ligation on B cells (Choi et al., 1994; Francis et al., 1995). AP-1 is formed following respective phosphorylation of Fos and Jun by dimers of active ERK and JNK that have translocated to the nucleus (reviewed in Kyriakis, 1999a; Wisdom, 1999). In vitro studies have demonstrated that ligation of CD40 on resting murine splenic B cells or the WEHI 231 murine B cell lymphoma cell line activates ERK isoforms within 15 min and 1 min, respectively (Gulbins et al., 1996; Kashiwada et al., 1996; Purkerson and Parker, 1998; Shirakata et al., 1999). In this regard, ERK1 and ERK2 are both activated following CD40 ligation, but ERK2 is consistently activated to a much higher degree than ERK1. Importantly, ERK activation can be observed in WEHI 231 B cells only if the cells have been serum starved before CD40 ligation, implying an important role for costimulation of ERK in this cell line. Additionally, in this situation, CD40 engagement on WEHI 231 B cells induces rapid translocation of phosphorylated ERK from the cytoplasm to the nucleus in a manner that is independent of polymerization of microtubules or actin, since respective pharmacological inhibitors, colchicine and cytochalasin, had no effect on this phenomenon (Shirakata et al., 1999). The ability of CD40 engagement to induce nuclear translocation of ERK implies that it can directly activate the Fos component of AP-1. Experiments examining the contribution of upstream signaling molecules to ERK activation following CD40 engagement have indicated that three independent mechanisms to activate this MAPK are induced (Fig. 19). One CD40-activated signaling cascade leading to ERK activation is independent of calcium-activated isoforms of PKC (Kashiwada et al., 1996; Li et al., 1996) and inhibited by herbimycin A, implying that this pathway utilizes src family kinases (Gulbins et al., 1996) such as lyn, fyn, or blk that have been demonstrated to be stimulated following CD40 ligation (Faris et al., 1994; Ren et al., 1994). Several studies have shown that activation of SOS allows RAS to become activated and recruit RAF1 to the membrane so that it can be phosphorylated and activated by src family kinases (Mason et al., 1999). The last step in this CD40–?–RAS/src kinase–RAF1–dependent ERK activation pathway is mediated by MKK1 (Gulbins et al., 1996; Kashiwada et al., 1996; Li et al., 1996). Dependence of CD40-mediated ERK activation on RAS and RAF1 was demonstrated by inhibition with dominant negative versions of these small G proteins (Gulbins et al., 1996; Kashiwada et al., 1998); dependence upon MKK1 was shown because of inhibition by the

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FIG. 19. CD40-induced signaling pathways activate ERK. For abbreviations, see text.

specific pharmacological inhibitor PD98059 (Li et al., 1996). A second pathway induced by CD40 ligation leading to ERK activation utilizes RAF1–MKK1–ERK in a manner that is independent of the SHC–GRB2– SOS cascade (Purkerson and Parker, 1998), leading to RAS activation (Kashiwada et al., 1998). This pathway is dependent on RAF1 and MKK1, as demonstrated by inhibition by either a dominant negative version of RAF1 or the pharmacological MKK1 inhibitor, PD98059. Of note, CD40-induced ERK activation mediated by TRAF6 appears to utilize this RAS-independent CD40–TRAF6–??–RAF1–MKK1–ERK pathway (Kashiwada et al., 1998). The third signaling cascade that CD40 utilizes to activate ERK may be independent of RAF1. Thus, pharmacological agents that increase cAMP (dibutryl cAMP, cholera toxin, and forskolin) had no effect on this component of CD40-induced activation of ERK (Purkerson and Parker, 1998). Since increased cAMP activates protein kinase A that inhibits the ability of RAF1 to activate MKK1 by phosphorylating RAF1 on S34 to disrupt the RAS–RAF1 interaction and on S261 to allow 14-3-3 to bind and inhibit RAF1 (Cook and McCormick, 1993; Mischak et al., 1996; Wu et al., 1993), it is likely that this CD40-dependent pathway of ERK activation is independent of RAF1. It is possible that this third mechanism of CD40-induced ERK activation may involve calcium-activated isoforms of PKC, since one component of RASdependent ERK activation is inhibited by a pharmacological inhibitor of diglycerides (i.e., diacylglycerol), calphostin (Gulbins et al., 1996). In vitro studies have demonstrated that JNK isoforms are activated rapidly following CD40 engagement on murine splenic B cells (Li et al.,


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1996), WEHI 231 cells (Purkerson and Parker, 1998), M12 murine B cell lymphoma cells (Berberich et al., 1996), activated tonsillar B cells (Berberich et al., 1996; Sakata et al., 1996), or Daudi cells (Berberich et al., 1996) and Ramos (Berberich et al., 1996; Sakata et al., 1996) human B cell lymphoma cells. In this regard, although both JNK1 and JNK2 are activated following CD40 ligation, JNK1 is consistently activated to a much higher degree than JNK2. Of note, CD40 engagement does not activate JNK in resting human B cells unless they are preactivated with reagents such as PMA, anti-IgM, or IL-4 (Sakata et al., 1996); explanations for this observation are uncertain at present. Separate studies have demonstrated that ligation of CD40 on B cells mediates JNK activation via the upstream MAP2Ks, MKK4 (Nishina et al., 1997) or MKK7 (Foltz et al., 1998). Other MAPKs involved in CD40-induced JNK include MEKK1 (Sakata et al., 1996) as well as GCK, GCKR, and GLK (Chin et al., 1999; Katz et al., 1994; Pombo et al., 1995). Of note, in vitro experiments have demonstrated that MEKK1 is a true downstream target of GCK (reviewed in Kyriakis, 1999b). Moreover, MEKK1 has been shown to activate the MKK4 pathway but not the MKK7 pathway (Cuenda and Dorow, 1998), whereas MEKK3 has been shown to activate the MKK7 but not the MKK4 pathway (Deacon and Blank, 1999). Also of interest, the role of MEKK3 in CD40-induced MKK4 activation has not been examined. These results suggest that CD40 ligation activates JNK by a mechanism utilizing MKK7 as well as by one utilizing GCK 씮 MEKK1 씮 MKK4. Other studies have investigated the upstream signaling molecules that may mediate these cascades leading to JNK activation. Both pathways have been demonstrated to be independent of PKC, since depletion with PMA has no effect on CD40-induced activation of JNK in all of the B cells described above (Berberich et al., 1996; Li et al., 1996; Sakata et al., 1999). One study demonstrated that CD40-induced JNK activation in Daudi lymphoma B cells was partially inhibited with a dominant negative version of RAS (RASN17) that also inhibited activation of the RAS-dependent effectors PI3K and RAC (Gulbins et al., 1996). Another study demonstrated that CD40-induced JNK activation in murine splenic B cells or WEHI 231 B cell lymphoma cells was mediated by reactive oxygen intermediates (ROIs) that could be detected within 2 min of receptor ligation (Lee and Koretzky, 1998). In this regard, antioxidants such as N-acetylcysteine, glutathione ethyl ester, or manganese superoxide dismutase partially inhibited CD40-induced JNK activation. The mechanism of ROI activation is unclear but may involve the small G protein RAC (Sulciner et al., 1996) or the ASK1 kinase (Hoeflich et al., 1999; Nishitoh et al., 1998). Together with in vitro studies examining JNK activation in non–B cell lines (reviewed in Davis, 1999), these data suggest that the MEKK1–MKK4

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pathway may be initiated by an unknown CD40-mediated mechanism that activates SOS in a manner that allows RAS to become activated and recruit PI3K to the membrane so that it can phosphorylate PIP2. The phosphorylated form of PIP2, PIP3, binds the pleckstrin homology domain of RAC so that VAV can mediate GDP–GTP exchange, resulting in ROI-dependent activation of RAC and its downstream effectors, PAK, MEKK1, MKK4, and JNK. Finally, preliminary data suggest that the second pathway mediating CD40-induced JNK activation via a MKK7-mediated pathway may be independent of PKC, RAS, and PI3K (Sakata et al., 1999). Details concerning the signaling cascades between CD40 and either GCK or MKK7 remain to be elucidated. 2. CRE The transcription factor that binds CRE is formed by dimerization of Jun and ATF2 which are respectively phosphorylated by the MAPKs JNK and p38 (Andrisani, 1999). Of note, both JNK and p38 have been demonstrated to be involved in posttranscriptional regulation of genes such as TNF-움 that encode mRNAs containing an AU-rich motif in the 3⬘untranslated region (Lee and Young, 1996; Swantek et al., 1997). Engagement of CD40 on tonsillar B cells or Ramos lymphoma B cells activates p38 rapidly (Craxton et al., 1998; Grammer et al., 1998; Sakata et al., 1999; Salmon et al., 1997; Sutherland et al., 1996). In cotransfection experiments, p38 has been shown to be activated specifically by MKK3 (Cuenda et al., 1996; Han et al., 1997) and MKK6 (Han et al., 1996; Moriguchi et al., 1996a,b; Raingeaud et al., 1996; Stein et al., 1996; Zanke et al., 1996), although the particular MAP2Ks that mediate CD40-induced activation of p38 remain to be delineated. In this regard, experiments with pharmacological inhibitors have demonstrated that p38 activation induced by CD40 engagement is independent of PI3K, MKK1, and PKC (Sakata et al., 1999). CD40 ligation leads to phosphorylation of p38 substrates such as MAPKAPK2, ATF2, and p38-dependent transcription of Jun (Craxton et al., 1998; Sutherland et al., 1996). Notably, MAPKAP-K2 has been shown to phosphorylate Ser133 of a CREB, such as ATF2, allowing it to bind a CRE in association with Jun and activate transcription (reviewed in Andrisani, 1999). Therefore, a number of p38-initiated pathways may result in CREdependent gene transcription following CD40 ligation. 3. NF-␬B NF-␬B is regulated primarily by phosphorylation of one of a family of inhibitory proteins, the I␬Bs, which retain NF-␬B dimers of the Rel family of transcription factors (reviewed in Baldwin, 1996) in the cytoplasm of resting cells by a 900-kDa protein kinase complex called the signalosome containing I␬B kinases (IKKs) in association with a scaffolding protein


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(reviewed in Zandi and Karin, 1999). Following stimulation, I␬Bs are phosphorylated on two serine residues by an IKK family member that has been activated by a MAP3K such as NF-␬B–inducing kinase (NIK), MEKK1, or PKC␨. Phosphorylation of I␬B targets it for ubiquitination and degradation by the proteosome, thus releasing NF-␬B dimers so that they can translocate to the nucleus and bind positive regulatory elements in the promoter regions of genes. CD40 engagement has been shown to induce activation of both the 움 and 웁 IKK isoforms (Kosaka et al., 1999), phosphorylation of I␬B at serines 32 and 36, degradation of phophorylated I␬B by the proteosome, and nuclear translocation of released NF-␬B dimers (Hsing et al., 1997; Sutherland et al., 1999). Of note, a pharmacological inhibitor of the proteosome, lactacystin, is often used to determine the role of CD40-induced NF-␬B in primary B cells. B. DISCOVERY OF TRAF ADAPTER MOLECULES Although CD40 was described in 1984 and stimulatory anti-CD40 mAbs were available in 1985, the mechanism by which CD40 mediated the early and late biochemical events observed following receptor ligation was unknown until 1994. Before that time, CD40-mediated activation of signaling cascades was observed, but none of the molecules identified to be involved was directly phosphorylated or interacted with CD40 itself. Moreover, the cytoplasmic tail of CD40 did not contain any known binding motifs for molecules involved in signal transduction, nor did it become phosphorylated upon engagement. A harbinger of the pathways to be delineated, however, was the unexplained observation that a T 씮 A mutation at position 254 in the cytoplasmic tail of CD40 partially interfered with a variety of CD40-induced signaling events and functional outcomes (Inui et al., 1990). In 1994 the yeast two-hybrid system was used to clone cDNAs that coded proteins binding the cytoplasmic domain of CD40 (Fig. 20). It should be noted that a cDNA library from a pre–B cell line was used as ‘‘prey’’ in these experiments. The first cDNA to be identified encoded a 64-kDa protein of 567 amino acids termed the CD40-binding protein (CD40bp) (Hu et al., 1994). The C terminus of CD40bp, later renamed TRAF3, was homologous to two other proteins simultaneously identified by the same method using the cytoplasmic tail of TNF receptor II (TNFRII) as the bait: TRAF1, a 45-kDa protein of 416 amino acids, and TRAF2, a 56-kDa protein of 501 amino acids (Rothe et al., 1994). The CD40 binding capacity of TRAF3 was confirmed within 1 year by two other groups that named the protein CD40 receptor–associated factor 1 (CRAF1) and CD40-associated protein 1 (CAP1) (Cheng et al., 1995; Sato et al., 1995).

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FIG. 20. Characteristics of CD154, CD40, and TRAF adapter molecules. aa, Amino acids; N.D., not determined.

The chromosomal location of TRAF3 was determined to be 14q32.3 in 1998 (van Eyndhoven et al., 1998). In 1995 the yeast two-hybrid system was used to clone cDNAs that expressed proteins binding the cytoplasmic domain of a constitutively active form of CD40, the EBV gene product LMP1. These experiments identified two LMP1-binding proteins: LMP1-associated protein (LAP1), which was identical to TRAF3 (Mosialos et al., 1995), and Epstein–Barr 16 (EB16), which was identical to the TRAF1 family member initially identified because of its ability to associate with TNFRII (Rothe et al., 1994). In 1997, the TRAF1 gene was localized to the q33–q34 region of human chromosome 9 (Siemienski et al., 1997). In 1995, CD40 was shown to associate directly with TRAF2 (Rothe et al., 1995), a family member originally cloned by its ability to bind TNFRII. Whether TRAF1 directly associates with CD40 itself (Pullen et al., 1998, 1999) or does so indirectly (Leo et al., 1999) following heterodimerization with TRAF2 (Rothe et al., 1994) remains unresolved, since there are conflicting reports. In 1995, a new family member, named TRAF4/CART1 (C-rich domain associated with RING and TRAF domains), was identified in breast carcinoma cells and metastatic axillary lymph nodes but was not present in normal human lymphoid tissues (Regnier et al., 1995). The human TRAF4 gene is localized to the q11–q12 region of chromosome 17 (Tomasetto et al., 1995) and encodes a 53-kDa protein of 470 amino acids (Regnier et al., 1995). The issue of whether TRAF4 associates with CD40 in B cells has not been resolved, since there are conflicting reports. Although a study in 1998 demonstrated that TRAF4 does not bind the intracellular tail of CD40 and is not expressed in lymphocytes (Krajewska et al., 1998), two other studies have observed that TRAF4 is expressed in B cells following ligation of CD40 (Aicher et al., 1999; Craxton et al., 1998).


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In 1996, two additional family members, TRAFs 5 and 6, were cloned from a cDNA library made from murine C57 Black Kaplan T lymphoma cells by their ability to associate with the cytoplasmic domain of CD40 (Ishida et al., 1996a,b). TRAF5 was independently identified by degenerate oligonucleotide polymerase chain reaction of RNA from the murine B cell lymphoma A20.2J with primers corresponding to highly conserved regions of TRAFs 1–3 (Ishida et al., 1996a). The human TRAF5 gene, localized to the q32.3–q41.1 region of chromosome 1 in human Daudi B lymphoma cells (Mizushima et al., 1998), encodes a 557–amino acid protein of 64.2 kDa. The chromosomal location of TRAF6, a 530–amino acid protein of 60.1 kDa, has not been reported. In 1998 to 1999 two groups demonstrated that there is minimal association of TRAF5 with CD40 in the absence of other TRAFs, whereas there is easily detectable association as a heterodimer with TRAF3 but not with TRAF 2 or 6 (Leo et al., 1999a; Pullen et al., 1998, 1999). Whether the affinity of TRAF5 for CD40 is dependent on conformational features of TRAF5 that may be induced when it heterodimerizes with TRAF3 or whether TRAF5 does not interact directly with CD40 at all remains to be tested. Finally, although five of the six TRAFs identified in mice and humans have been shown to associate with CD40 and mediate downstream signaling pathways, such as those leading to nuclear translocation of NF-␬B and activation of MAPKs, there are also CD40-induced signaling cascades whose mechanism of CD40 association is unknown. These include activation of phospholipase C웂2 (Gulbins et al., 1996; van Kooten and Banchereau, 1996), PI3K (Aagaard-Tillery and Jelinek, 1996; Padmore et al., 1997), and STAT6 (Karras et al., 1997) as well as the proximal src (Faris et al., 1994; Ren et al., 1994), ZAP-70 [syk (Faris et al., 1994)], and tec [btk(Uckun et al., 1991)] family kinases. Moreover, induction of STAT3 activation by CD40 engagement results from the direct interaction of JAK3 with CD40 in a TRAF-independent manner (Hanissian and Geha, 1997; Karras et al., 1997; Jabara et al., 1998; Tortolani et al., 1995). 1. Structure–Function Relationship of TRAFs Following association of specific TRAF adapter molecules with CD40, signaling cascades occur that have been shown to lead to nuclear translocation of specific transcription factors. TRAFs are a family of adapter proteins without intrinsic enzymatic activity that were originally isolated because of their capacity to associate with TNF family members via a C-terminal homologous domain that they all share, termed the TRAF domain (reviewed in Wajant et al., 1999). As described in the preceding section, five of the six TRAFs that have been identified in mice and humans directly or indirectly associate with CD40 (TRAFs 1, 2, 3, 5, and 6) via the C-

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terminal TRAF domain. In turn, a number of molecules have been found to associate with TRAFs. Whereas the MEKK1 kinase (Baud et al., 1999) binds the RING domain of TRAFs, all other known TRAF-associated molecules bind the TRAF domain. These include kinases [NIK (Malinin et al., 1997; Song et al., 1997), RIP (Takeuchi et al., 1996), RIP2/Cardiak/ RICK (Bertin et al., 1999; Inohara et al., 1998; McCarthy et al., 1998), RIP3 (Yu et al., 1999), ASK1 (Hoeflich et al., 1999; Nishitoh et al., 1998), GCK (Yuasa et al., 1998), GCKR (Chin et al., 1999), TAK1 (NinomiyaTsuji et al., 1999; Sakurai et al., 1998), and CDK9 (MacLachlan et al., 1998)], adapter molecules [I-TRAF/TANK (Cheng and Baltimore, 1996; Rothe et al., 1996), TRADD (Hsu et al., 1996), TRIP (Lee and Choi, 1997), A20 (Song et al., 1996), cIAPs (Shu et al., 1996), and ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) (Kopp et al., 1999)], and molecules with unknown function, such as Peg3/Pw1 (Relaix et al., 1998). The evolutionary importance of TRAF adapter molecules in signaling is emphasized by the observation that TRAF-related molecules have been isolated from Drosophila (DTRAFs) (Liu et al., 1999) and Caenorhabditis elegans (CeTRAFs) (Wajant et al., 1998). Sequence analysis of TRAF family members (Fig. 21) reveals that they are proteins of 409–567 amino acids and contain a C-terminal TRAF domain of 앑150 amino acids, a closely spaced cluster of five (TRAFs 1, 3, 5, and 6) to seven (TRAF4) zinc fingers, and—for TRAFs 2–6 but not TRAF1—an N-terminal RING domain. Additionally, TRAFs 3 and 5 but not TRAFs 1, 2, and 6 have an isoleucine zipper motif between the most C-terminal zinc finger and the TRAF domain that can mediate homoand heteromultimerization of these proteins (reviewed in Wajant et al., 1999). By contrast, multimerization of TRAFs 1 and 2 is mediated by the C-terminal portion of the TRAF domain, TRAF-C. Importantly, (TRAF3)x –(TRAF5)y and (TRAF1)x –(TRAF2)y heteromultimers, but not heteromultimers of other TRAF combinations, have been reported. Importantly, with the exception of the most C-terminal zinc finger that forms a unique CX6CX11HX7H zinc-binding unit, all other zinc fingers of

FIG. 21. Representation of the functional domains of TRAF adapter molecules using TRAF2 as a model. aa, Amino acids; CT, carboxy terminus; NT, amino terminus.


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TRAFs 2–6 but not TRAF1 are organized in a typical CX2,3CX11,12HX3C ‘‘CART’’ pattern. The RING domain is organized in a typical B box motif, CX2CX11,12CX1HX2CX2CX9,11CX2C (reviewed in Borden, 1998). Of note, spacing between the sixth and seventh zinc-coordinating residues in the RING domain can vary from four to 48 residues. The potential functional importance of this variability is highlighted by the finding that insertion of seven additional amino acids (RCASILS) between 62L and 63S in wildtype TRAF2 following alternative splicing of the transcript to form TRAF2A alters the ability of this molecule to initiate downstream signaling cascades (Brink and Lodish, 1998; Dadgostar and Cheng, 1998). Cysteines in the RING and zinc finger motifs coordinate zinc ions. Zinc coordination by proteins containing these motifs has been shown to be essential for DNA binding and suggests that TRAFs may be able to influence gene transcription directly. In this regard, a recent study of TRAF2 demonstrated that these motifs were essential for this TRAF to translocate to the nucleus and transactivate E-selectin transcription (Min et al., 1998). Examples of other proteins with RING domains that have been shown to bind DNA in a functional manner include a variety of DNA repair enzymes as well as the recombinase RAG1 (reviewed in Borden, 1998). It should be noted, however, that it remains uncertain whether TRAFs directly activate gene transcription in vivo or, alternatively, interact with downstream signaling proteins that lead to activation of gene transcription, or both. Further experiments have demonstrated that the function of the TRAF domain is not only to associate with signaling molecules but also to mediate homo- and heteromultimerization of various TRAFs. In this regard, with the exception of TRAF3–TRAF5 association, deletion of the TRAF-C domain prevents TRAF multimerization (Baud et al., 1999). The functional importance of multimerization is indicated by the finding that mutants of TRAF 2 or 6 containing the TRAF-N domain fused to repeats of FK-binding protein (FKBP) are not able to induce downstream signaling cascades until this mutant is multimerized in the presence of FK1012, the ligand for FKBP. Finally, reports have observed TRAFs spontaneously forming homotrimers in solution (Pullen et al., 1999) and a trimer of TRAF2 complexed with three copies of a TRAF binding peptide derived from CD40, implying that trimers of TRAF2 interact with a trimer of CD40 (McWhirter et al., 1999). a. MAPK Activation. The role of TRAFs in activation of the MAPKs kinases ERK (Fig. 19), p38 (Fig. 22), and JNK (Fig. 23) has been examined following overexpression in non-B cell lines. First, overexpression of TRAF 6, but not TRAF 2, 3, or 5, induced ERK activity as measured by an in

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FIG. 22. CD40-induced signaling pathways activate p38.

FIG. 23. CD40-induced signaling pathways activate JNK. For abbreviations, see text.


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vitro kinase assay. Second, although activation of the MAPK p38움 was induced following overexpression of either TRAF 2 or 6 (Baud et al., 1999; Carpentier et al., 1998; Yuasa et al., 1998), it should be noted that the effects of overexpressing the other TRAF family members on p38 activation have not been tested. Third, overexpression of TRAFs 1, 2, 3, 5, and 6 induced JNK kinase activity, as measured by an in vitro kinase assay following immunoprecipitation of JNK1 for TRAFs 1, 2, and 6 and JNK2 for TRAF5 (Baud et al., 1999; Chin et al., 1999; Dadgostar and Cheng, 1998; Schwenzer et al., 1999). The ability of these TRAFs to induce phosphorylation of both JNK isoforms was not tested. Of note, the I-TRAF/TANK adapter molecule costimulated JNK activation induced by TRAF 2, 5, or 6, but not by TRAF3 (Chin et al., 1999). Investigation of the intermediate kinases that mediate TRAF-induced JNK and p38 activation has focused on events induced by TRAFs 2 and 6. Activation of p38 induced by TRAF2 has been shown to be mediated by a RIP–MKK6 pathway (Yuasa et al., 1998). Surprsingly, TRAF2–RIP complexes did not activate JNK. By contrast, a kinase-inactive version of the MAP3K, MEKK1 (Baud et al., 1999), or the MAP2K, MKK4 (Reinhard et al., 1997), partially inhibited JNK1 activation induced by TRAF2 overexpression. In addition, a mutant of TRAF2 that cannot associate with MEKK1 because of alterations in its RING domain activated JNK1 to a lesser degree than the wild-type molecule (Baud et al., 1999). Together, these observations indicate that TRAF2 utilizes at least two mechanisms to activate JNK1, one of which is the TRAF2–MEKK1–MKK4–JNK pathway. An additional pathway for TRAF2-induced JNK activity may involve members of the GC kinase family (GCK, GCKR, and GLK) that associate with the TRAF domain of TRAF2 and with MEKK1 to induce JNK activation (Chin et al., 1999; Shi et al., 1999; Yuasa et al., 1998). Of note, GC kinase family members do not activate ERK, p38, or NF-␬B. TRAF2-induced JNK activation was partially inhibited by the antioxidant compound N-acetylcysteine (Natoli et al., 1997). In this regard, activation of JNK induced by overexpressing TRAF2 was partially inhibited by a kinase inactive version of the MAP3K, ASK1, which is activated by ROIs) following oxidation and release of thioredoxin. ASK1 has been shown to associate with the TRAF domain of TRAF2 but to require the RING domain in order for JNK to be activated (Hoeflich et al., 1999; Nishitoh et al., 1998). It should be noted that since ASK1 associates with TRAFs 5 and 6, but not with 1 or 3, this MAP3K may play a role in activation of JNK by these TRAFs as well, although this has not been directly examined. Importantly, dominant negative versions of either RAC1 or cdc42 do not interfere with TRAF2-induced activation of JNK (Chin et al., 1999).

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The alternatively spliced form of TRAF2 with an insertion in the RING domain, TRAF2A, and wild-type TRAF2 activate JNK comparably upon overexpression (Brink and Lodish, 1998). Furthermore, mutational analysis of TRAF2 demonstrated that the RING domain and the most N terminal zinc finger of TRAF2, but not the second or third zinc finger from the N terminus, are required for its ability to activate JNK (Baud et al., 1999). In this regard, it should be noted that alteration of the spacing between the sixth and seventh zinc binding motifs in the RING domain of TRAF2 did not alter its ability to activate JNK (Dadgostar and Cheng, 1998). Similarly, mutational analysis of TRAF5 indicated that the two most Nterminal zinc fingers, but not C45 or C81 in the RING domain that mediate zinc binding, are required to activate JNK (Dadgostar and Cheng, 1998). Of interest, substitution of the RING domain of TRAF3 for this region of TRAF5 did not alter the ability of TRAF5 to activate JNK. These results suggest that the divergent ability of TRAFs 3 and 5 to activate JNK may be related to differences in their zinc fingers, but not the RING domain. However, the ability of TRAFs to activate JNK has been mapped to the RING domain of TRAFs 2 and 5, in combination with the most N-terminal zinc finger of TRAF2 (Baud et al., 1999) and the second zinc finger from the N-terminal of TRAF5 (Dadgostar and Cheng, 1998). The region of TRAF6 that mediates JNK and p38 activation appears to be located in amino acids 1–188, since overexpression of a mutant of TRAF6 (residues 289–522) that does not contain the N-terminal RING and zinc domains did not induce activation of JNK or p38 in 293 HEK cells (Baud et al., 1999). In this regard, TRAF6 has been shown to associate with a complex containing TAK1 and its upstream activator, TAB1 (Ninomiya-Tsuji et al., 1999). Of interest, two mechanisms have been delineated by which TRAF6 activates JNK or p38. First, association of TAB1/TAK1 with TRAF6, but not TRAF2, following receptor ligation mediates activation of MKK4–JNK and MKK3/6–p38 (Ninomiya-Tsuji et al., 1999; Sakurai et al., 1998). In addition, TRAF6 utilizes a second independent mechanism to activate JNK. Association of TRAF6, but not TRAF 2 or 5, with ECSIT can recruit full-length MEKK1 and mediate its processing to the active, 80-kDa form (Kopp et al., 1999). This 80-kDa form of MEKK1 can then activate JNK via MKK4. b. NF-␬B Activation. Overexpression of wild-type TRAF2 in 293 HEK cells (from the human embryonic kidney cell line) induced NF-␬B activity, as measured by a reporter assay or by supershifting (Rothe et al., 1995; Takeuchi et al., 1996). Of interest, overexpression of an alternatively spliced form of TRAF2, TRAF2A, containing seven additional amino acids in its RING domain that lengthened the spacer between the sixth and seventh


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zinc binding motifs, was not able to induce NF-␬B activation in 293 HEK cells (Brink and Lodish, 1998; Dadgostar and Cheng, 1998). Mutational analysis of TRAF2 has demonstrated that the N-terminal region containing the RING and zinc finger domains are required for its ability to activate NF-␬B (Takeuchi et al., 1996). Specifically, experiments demonstrating that deletion of either the RING domain or the most N-terminal zinc finger abrogated the ability of TRAF2 to activate NF-␬B indicate that both of these domains are essential for this signaling function of TRAF2 (Baud et al., 1999). Surprisingly, deletion of the second zinc finger with respect to the N terminus had no effect, and mutation of the third zinc finger doubled the level of NF-␬B induced by overexpressing TRAF2, suggesting that this domain regulates the ability of TRAF2 to activate NF-␬B. By contrast, overexpression of either TRAF1 in HeLa cells (Schwenzer et al., 1999) or TRAF3 in 293 HEK cells (Dadgostar and Cheng, 1998) did not induce nuclear translocation of NF-␬B dimers. Overexpression of TRAF5 in 293 HEK or Jurkat T lymphoma cells induced NF-␬B activity measured by a reporter assay (Ishida et al., 1996a; Mizushima et al., 1998) and supershifting (Nakano et al., 1996). This pattern of NF-␬B activation is identical to that induced following overexpression of TRAF2 in 293 HEK cells (Rothe et al., 1995). In addition, mutational analysis of TRAF5 indicated that the two most N-terminal zinc fingers as well as cysteines in the RING domain that mediate zinc binding are required for TRAF5 to activate NF-␬B (Dadgostar and Cheng, 1998). Together, these results suggest that TRAFs 2 and 5 may utilize similar mechanisms to activate NF-␬B, since they both require the RING domain and the most N-terminal zinc finger to be able to mediate this signaling event. Finally, cotransfection of 293 HEK cells with a fixed amount of TRAF5 and increasing amounts of TRAF3 demonstrated that high levels of TRAF3 costimulate TRAF5-induced NF-␬B activity, as measured by a reporter assay (Leo et al., 1999a). The mechanism for this observation is unclear, although the ability of these TRAFs to form heteromultimers is likely to be involved. Overexpression of wild-type TRAF6 in either the 293 HEK or Jurkat T cell lymphoma cell line induces activation of NF-␬B, as measured by a reporter assay (Baud et al., 1999; Ishida et al., 1996b; Kashiwada et al., 1998). Of note, in vitro overexpression of TRAF3, which has been demonstrated to costimulate TRAF5-induced NF-␬B activity, had no effect on this activity induced by overexpression of TRAF6 (Leo et al., 1999a). In summary, overexpression of TRAFs 2, 5, and 6 has been shown to induce NF-␬B activity (Fig. 24). In addition, transfection with increasing amounts of TRAF3 costimulates NF-␬B activity induced by fixed amounts of TRAF5, but not by TRAF 2 or 6 assay (Leo et al., 1999a).

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FIG. 24. CD40-induced signaling pathways activate IKKs leading to nuclear translocation of NF-␬B. For abbreviations, see text.

TRAFs 2, 5, and 6 have been shown to activate NF-␬B by a variety of mechanisms. TRAF6 may utilize many independent mechanisms to induce NF-␬B activity, although the relative importance of each of these pathways has not been defined. The first mechanism is mediated by association of TRAF6 with the TAK1–TAB1 complex following receptor ligation (Ninomiya-Tsuji et al., 1999; Sakurai et al., 1998). Subsequently, this signaling cascade induces NF-␬B activity by both a NIK-dependent and a NIKindependent mechanism. Second, TRAF6 associates with ECSIT in a manner that cleaves MEKK1 to its active form, allowing it to phosphorylate IKK-웁 and induce nuclear translocation of NF-␬B (Kopp et al., 1999). Third, TRAF6 induces NF-␬B activity in a manner that can be blocked with a dominant negative version of the MAP3K RAF1 (Kashiwada et al., 1998), which has been shown previously to induce NF-␬B activity. The third and fourth mechanisms that TRAF6 utilizes to activate NF-␬B are also used by TRAFs 1, 2, 5, and 6. These mechanisms utilize RIP family members that bind TRAF2 (RIP) (Takeuchi et al., 1996) or TRAFs 1, 5, and 6 (RIP2/CARDIAK/RICK) (McCarthy et al., 1998). In turn, RIP mediates NF-␬B activation via AKT/PKB in a NIK-dependent manner (Ozes et al., 1999). Alternatively, the IKK-웁 kinase, PKC␨ (Lallena et al.,


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1999), associates with RIP via the membrane localization factor, p62 (Sanz et al., 1999). Both RIP-facilitated mechanisms also require PIP3 that costimulates both AKT/PKB (Romashkova and Makarov, 1999; Rommel et al., 1999; Zimmermann and Moelling, 1999) and PKC␨ (Nakanishi et al., 1993). Of note, PIP3 is generated by a RAS-induced PI3K pathway whose dependence on TRAFs is unknown. Finally, a recently described kinase, TANK-binding kinase 1 (TBK1), induces NF-␬B by a mechanism that requires I-TRAF/TANK, the Nterminal region of TRAF2, and a downstream signaling cascade involving NIK and the IKKs (Pomerantz and Baltimore, 1999). Of note, the role of the newly described I␬B kinase, IKKi, which is expressed by B cells and is induced following stimulation with TNF-움, IL-6, or the murine B cell polyclonal activator, lipopolysaccharide, has not been examined in TRAFinduced NF-␬B activation. Together, these results indicate that whereas TRAFs 1, 2, and 5 utilize signaling cascades involving NIK to activate NF-␬B, TRAF6 may utilize NIK, MEKK1, and RAF1 as well as uncharacterized pathways to induce NF-␬B activity. These observations confirm the finding that a kinase-dead version of NIK completely abrogates NF-␬B activation induced by overexpressing TRAF2 or TRAF5, but only partially inhibits activation of this transcription factor induced by overexpression of TRAF6 (Tsukamoto et al., 1999). Moreover, several signaling molecules, including TAK1–TAB1, AKT/PKB, and TBK1, are cofacilitators of NIK-induced NF-␬B activity in that a dominant negative version of NIK blocks signaling via these molecules, and conversely, dominant negative versions of these molecules block NIK-induced NF-␬B activity. This finding is of interest since NIK has been shown to associate with TRAFs 1–6 directly via a WKI motif in the TRAF-N domain (Malinin et al., 1997; Song et al., 1997), but only TRAFs 2, 5, and 6 are able to activate NF-␬B. In addition, mutants of TRAF2 or TRAF5 that associate with NIK via the WKI motif, but are missing the N-terminal RING and zinc finger domains, cannot activate NF-␬B (Baud et al., 1999; Dadgostar and Cheng, 1998). In contrast to the requirement for NIK to function as a cofacilitator with the N-terminal region of TRAFs or MAPKs such as RIP, TAB1–TAK1, AKT/PKB, and TBK1, MEKK1 and PKC␨ do not require cofacilitation. c. Summary. Initial experiments examining the role of specific TRAFs in receptor-induced signaling cascades utilized dominant negative versions of these molecules that lacked these N-terminal RING domains and zinc fingers. By contrast, studies described above have demonstrated that the C-terminal TRAF domain can also be involved in downstream signaling events by virtue of its ability to associate with other kinases and adapter molecules. It should be pointed out that this finding emphasizes the need

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for reexamination of the results of experiments with dominant negative DN TRAFs lacking the N-terminal RING domains and zinc fingers and for future investigation of CD40-mediated signaling with a series of mutants that lack the TRAF-C or TRAF-N domains. 2. Association of TRAFs with CD40 Specific sites on both the TNF family member and the TRAF adapter molecule(s) mediate their association with each other. The domains on TNF family members that have been demonstrated to be essential for specific TRAF binding consist of short, linear amino acid motifs. Several studies have demonstrated that TRAF6 binds the intracellular tail of CD40 at a membrane-proximal location, 231QEPQEINF238 (Ishida et al., 1996b; Pullen et al., 1998), whereas distinct binding sites for TRAFs 1, 2, 3, and 5 have been shown to be clustered together within a more C-terminal region of the cytoplasmic tail containing 250PVQET254 (Fig. 4) (Franken et al., 1996; Pullen et al., 1998). Although the C-terminal portion of the TRAF domain (TRAF-C) has been shown to be required for binding to TNF family member motifs, the TRAF-N region has also been shown to contribute to this association. Crystallographic analysis of a CD40–TRAF complex has been reported (McWhirter et al., 1999). Specifically, a region of TRAF2 (residues 311– 501), TRAF2–311, containing the TRAF-C domain (residues 356–501) and part of the TRAF-N domain (residues 272–355) was complexed with a functionally defined peptide (Ac–Y250PIQET254 –Am) from the cytoplasmic domain of CD40 previously demonstrated to bind TRAFs 1, 2, 3, and 5. The conservative substitution of I251 for V251 increased the stability of the complex while maintaining the presence of the 웁-branched side chain. The complex contained three TRAF2–311 molecules in association with three CD40 peptide fragments. The CD40 fragments did not make contact with each other, but each associated with one TRAF2 fragment that was part of a mushroom-shaped TRAF2 trimer. This mushroom-shaped TRAF2 trimer had a flexible coiled–coiled stem containing the TRAF-N domain pointed toward the cytoplasm and away from the CD40–membrane interface. This structure predicts that the N-terminal RING domain and zinc fingers of TRAF2 that have been shown to associate with the MAP3K MEKK1 would also point away from the membrane and toward the cytoplasm. Hydrogen bonding between a critical R385 and carbonyl groups in the coiled–coiled region of the TRAF-N domain allows the TRAF2 backbone to turn and initiate the beginning of the 웁-sandwich that gives the mushroom-shaped TRAF-C domain its shape and points it toward the CD40–membrane interface. Of note, amino acids capable of hydrogen


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bonding occupy this position in all TRAFs. Multiple nonpolar, hydrophilic, and hydrogen-bonding intersubunit contacts within the TRAF-C domain stabilize the 웁-sandwich structure. Reports have demonstrated that the TRAF-C domain binds a variety of signaling molecules, including NIK. In this regard, the results from the crystallographic analysis predict that the binding site for NIK in TRAF2, 357WKI, is located under the cap of the ‘‘mushroom.’’ Of note, functional association of NIK with TRAFs depends on availability of P379 that is conserved in TRAFs 1, 2, and 6, but not TRAF3 or TRAF5. Finally, association of TRAF2 with the Y250PIQET254 fragment of CD40 is mediated by multiple interactions. TRAF2 forms a binding pocket for CD40. Proline at position 250 of the cytoplasmic sequence of CD40, with its side chain fully buried, points into the TRAF2 binding pocket and orients residues 251–254 of CD40 so that they make optimal contact with TRAF2. I251 (V251 in the wild-type CD40 molecule) makes contact with TRAF2 via a van der Waals interaction with P470 and hydrogen bonding with G468. Q252 of CD40 forms hydrogen bonds with three TRAF2 serines in the pocket, named the ‘‘serine tongs,’’ and E253 of CD40 interacts with TRAF2 via a hydrogen-bound ion pair with R393 and a hydrogen bond with Y395. Importantly, the T254 residue that has been demonstrated to be crucial for binding of TRAFs 1, 2, 3, and 5 to the cytoplasmic domain of CD40 forms a hydrogen bond with D399 in TRAF2. Of note, this residue in conserved in TRAFs 1 and 3 that do not bind CD40 if T254 is mutated to alanine (Leo et al., 1999a; Pullen et al., 1998). Together, these data indicate that individual CD40 molecules in the CD40 trimer associate with the outside of trimerized TRAF2 that is oriented so that the TRAF-N and N-terminal RING and zinc finger domains point toward the cytoplasm, and the TRAF-C domain is pointed toward the CD40–membrane interface. Because crucial residues mediating CD40–TRAF2 contact are conserved in the other TRAFs that bind the 250PVQET254 membrane distal site, this structure provides a model by which TRAFs interact with this domain of CD40. Detailed experiments to delineate the differences in contact between 250PVQET254 and TRAFs 1, 2, 3, and 5 as well as how TRAF6 interacts with 231QEPQEINF238 remain to be performed. A number of studies determined relative affinities of the various TRAFs for CD40 (Leo et al., 1999a; Pullen et al., 1998, 1999). In initial studies, solid-phase binding of TRAFs to the cytoplasmic domain of CD40 was performed using an enzyme-linked immunosorbent assay (ELISA) in which the concentration of TRAF was held constant and the concentration of a GST–CD40cytoplasmic fusion protein was varied. The ELISA approach revealed that 100 pmol of CD40 gave 앑50% binding to 200 애g of either TRAF2 or TRAF3, whereas 1000 pmol of CD40 was required for 앑50%

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binding to the same amount of either TRAF1 or TRAF6. Subsequently, a CD40cytoplasmic fusion protein was attached to a chip, and binding was examined by plasmon resonance using various concentrations of TRAFs. Although binding of TRAFs 2 and 3 was readily detectable by this technique with respective dissociation constants (KD ) of 2.5 and 13 애M, binding of TRAFs 1 and 6 could not be detected even with 10⫺4 to 10⫺5 M of TRAF protein. Binding of TRAF5 to CD40 was not assayed by either of these experimental techniques. These results indicate that TRAFs 2 and 3 bind CD40 with a higher avidity than either TRAF1 or TRAF6. Of note, multimerization of CD40 may account for the 10-fold increase in the KD of intact TRAF2 for CD40 (2.5 애M ) compared to TRAF2 for its CD40 binding site in peptide form, 250PVQET254 (210 애M ). Within the 250PVQET254 domain shown to bind TRAFs 1, 2, 3, and 5, 254 T has been demonstrated to be essential for association of either TRAF2 or TRAF3 with CD40, whereas 250P and 256V have been shown to be involved in binding of TRAF2, but not TRAF3 (Leo et al., 1999a). In this regard, a report demonstrated that despite presaturation of binding sites for TRAF2 with peptide inhibitors, TRAF3 bound CD40 in a concentration-dependent manner and vice versa. These results are consistent with the conclusion that CD40 may have closely approximated, but not identical, binding sites for TRAFs 2 and 3. Notably, the 250PVQET254 site alone may be required, but not sufficient, for association of TRAF2 or TRAF3 with CD40, since one but not the other PXQXT motif in a related TNF family member, LMP1, binds these TRAFs (Devergne et al., 1996). Furthermore, other evidence has demonstrated that independent amino acids C-terminal of 250 PVQET254 in CD40 influence binding of TRAFs 2 and 3 (Pullen et al., 1999). Specifically, 266G and 263E are crucial for binding TRAF2 and TRAF3, respectively (Leo et al., 1999a). In addition, 271ISVQE275 also influences association of TRAFs 2 and 3 with CD40. The recent findings suggesting that TRAFs 2 and 3 interact with distinct amino acids in the 250PVQET254 motif in the intracellular tail of CD40 (Leo et al., 1999a; Pullen et al., 1999) confirm the earlier observation that following stimulation with recombinant CD154, both TRAFs 2 and 3 were immunoprecipitated with an anti-CD40 mAb (Kuhne et al., 1997). These findings are consistent with the conclusion that TRAFs 2 and 3 may be simultaneously recruited to CD40, presumably by binding distinct sites on the cytoplasmic tail. It is also important to note that binding of one TRAF to the cytoplasmic tail of CD40 may influence the binding of another, even when the TRAFs bind to distinct sites and do not heterodimerize. In this regard, binding of TRAF6 to its motif that is C-terminal of 250PVQET254 may alter the conformation of the intracellular tail of CD40 and thereby influence binding of TRAF3 to 250PVQET254. In addition, the findings that point muta-


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tions in the TRAF6 binding site alter the Ka of 250PVQET254 for TRAFs 1, 2 and 3 and that point mutations in 250PVQET254 also alter the Ka of 231 QEPQEINF238 for TRAF6 (Pullen et al., 1999) further emphasize the possibility that association of one TRAF with CD40 may influence the association of another TRAF at an independent binding site. This conclusion remains to be rigorously examined, however. The possibility that TRAFs 2 and 6 may bind CD40 simultaneously and may influence each other’s binding must be considered and rigorously tested. Finally, since TRAFs 2 and 3 can form homodimers or heterodimerize with TRAFs 1 and 5, respectively, the issue of whether signaling through multimerized CD40 molecules can lead to simultaneous signaling pathways mediated by all or a subset of (TRAF6)3, (TRAF3)3, (TRAF2)3, TRAF3x –TRAF5y, or TRAF2x-TRAF1y multimers must be considered. The mechanisms by which TRAFs mediate CD40-induced signaling cascades leading to nuclear translocation of transcription factors are not well understood. Two reports have examined membrane and cytosolic lysates from unstimulated or CD154-stimulated B cells following immunoprecipitation. The first report immunoprecipitated CD40-associated proteins with an anti-CD40 mAb (BE-1 or S2C6) that can bind CD40 in the presence of bound CD154 (Kuhne et al., 1997). In this experiment, TRAFs in unstimulated B cells from the EBV-negative DND39 cell line were localized to the cytosol and were not found in association with CD40. Following ligation of CD40 with soluble recombinant CD154, TRAFs were maximally depleted from the cytosol within 15 min, and some, but not all, of the TRAFs missing from the cytosol were found in association with CD40. The location of the remaining TRAFs was not determined, although the results of subsequent studies suggest that the TRAF fraction that disappeared from the cytosol and did not associate with CD40 may have been degraded (Duckett and Thompson, 1997) or translocated to the nucleus (Min et al., 1998). These findings formed the basis for the hypothesis that TRAFs are predominantly located in the cytoplasm of a quiescent cell and are recruited to the cytoplasmic tail of CD40 upon receptor engagement. Further evidence for this conclusion was provided by the observation by fluorescence microscopy that expression of LMP1 48 hr following transfection of an EBV-negative B cell line caused TRAF2 to relocalize in cells from the cytoplasm to LMP1 in the membrane (Kaye et al., 1996). An apparently contradictory finding (Chaudhuri et al., 1997) used immunoprecipitation to demonstrate that TRAFs associated with endogenous CD40 in B cells from the EBV-positive Raji cell line or with transfected CD40 in 293 HEK cells were released very rapidly (30 sec to 5 min) upon receptor engagement with the EA-5 anti-CD40 mAb that binds CD40 outside its binding site

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for CD154 (Challa et al., 1999). It should be noted that TRAFs associated with CD40 in this experiment represented ⬍1% of available TRAFs in the cell. Additionally, in contrast with the first experiment described above in which recombinant CD154 was used to stimulate the cells and an antiCD40 mAb was used to immunoprecipitate CD40-associated proteins, the latter experiment stimulated the cells with an anti-CD40 mAb and then immunoprecipitated with goat anti-mouse Ig-conjugated Sepharose beads. Moreover, the state of the cells used in the two experiments was very different. Whereas the first experiment used B cells from EBV-negative cell lines, the latter experiment used B cells from an EBV-positive cell line that expresses LMP1, a TNFR superfamily member that activates cells by a mechanism very similar to CD40, and 293 HEK cells transfected with CD40. In this regard, other groups have studied CD40-mediated signaling in 293 HEK cells by overexpressing this receptor; additional engagement of CD40 with CD154 or anti-CD40 mAb increases signaling but is not required for induction of signaling cascades downstream of CD40. Furthermore, it is known that expression of LMP1 activates a cell as though the CD40 on its surface were engaged constitutively (Eliopoulos and Rickinson, 1998). Together, these observations highlight a very important difference between the two experiments that may explain the apparently contradictory results. Whereas the first experiment utilized cells that were not activated by TNFR superfamily members before CD40 engagement, the second experiment utilized cells that had already been activated by LMP1, in the case of the Raji B cell line, and by CD40 itself, in the case of the 293 HEK cells. This activation in the second experiment may have already recruited TRAFs to the cytoplasmic tail of CD40 that were observed at the beginning of the experiment to be in association with CD40 and then rapidly released upon additional activation by an anti-CD40 mAb. One hypothesis generated by the results from these two experiments is that TRAFs may be held in the cytoplasm by an unknown mechanism and be recruited to TNFR superfamily members upon receptor engagement, and then, following receptor-induced activation, released into the cytosol to mediate downstream signaling cascades. Detailed time course experiments designed to examine the validity of this hypothesis remain to be performed. Data supporting the hypothesis that recruited TRAFs are released from CD40 to mediate downstream signaling cascades were provided by the observation that although the IKKs were activated following CD40 engagement, neither the IKKs nor the associated signalosome was recruited to CD40 upon engagement and did not associate with TRAFs either in unstimulated cells or in those that had been stimulated with recombinant CD154 (Kosaka et al., 1999). Importantly, this observation suggests that


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intermediate signaling molecules such as MEKK1 or NIK that have been demonstrated to bind TRAFs and to mediate CD40-induced NF-␬B activation in a TRAF-dependent manner may be initially recruited to TRAFs but then may be released to bind and activate the IKKs and the signalosome. In this regard, the finding that receptor engagement is required for TRAF recruitment to the TNFR as well as association of the MAP3K ASK1 with TRAF2 (Nishitoh et al., 1998) or the MAP3K TAK1 with TRAF6 (Ninomiya-Tsuji et al., 1999) suggests that this hypothesis may be valid. Finally, the issue has been raised as to whether activation signals alter the specific TRAFs that associate with CD40. Specifically, signals that costimulate B cells in addition to CD40 ligation were shown to alter the TRAFs associating with CD40 (Kuhne et al., 1997). For example, IL-4 increased the amount of TRAF2 bound to CD40 compared to TRAF3, whereas signaling through sIg exerted the opposite effect in B cells from the EBV-negative DND39 cell line. The mechanisms responsible for these results have not been examined. 3. I-TRAF/TANK In 1996, two groups used the yeast two-hybrid method to screen cDNAs for encoded proteins that associated with TRAF family members and independently identified the 425–amino acid cytoplasmic I-TRAF/TANK molecule (Chenga and Baltimore, 1996; Rothe et al., 1996). Both groups found that I-TRAF/TANK bound TRAFs 1, 2, and 3, whereas one provided preliminary evidence that TRAFs 1 and 2 bound I-TRAF/TANK with higher affinity than TRAF3. Later examination of the I-TRAF/TANK protein sequence revealed a 178PXQXT182 domain that, in CD40, has been shown to contribute to the association of TRAFs 1, 2, 3, and 5. Direct in vitro binding of TRAFs 4, 5, and 6 to I-TRAF/TANK has not been examined, although TRAFs 5 and 6 have been shown to synergize with I-TRAF/ TANK to activate JNK (Chin et al., 1999). Binding experiments using mutant I-TRAF/TANK and TRAF molecules were performed by both groups and revealed that the N-terminal (residues 1–168), central TIMtk (residues 169–190), and C-terminal (residues 213–425) regions of I-TRAF/TANK all contributed to the I-TRAF/TANK–TRAF interaction. Further experiments demonstrated that the C-terminal domain of the TRAFs (TRAF-C) was essential for this association. The finding by several groups that I-TRAF/TANK and TNFR family members such as CD40 and LMP1 compete for binding in vitro to the TRAF-C domain indicated that receptor-bound TRAFs cannot bind I-TRAF/TANK and that TRAFs bound to I-TRAF/TANK cannot bind TNFR family members. Therefore, a high level of I-TRAF/TANK expression in a cell may prevent ligand-dependent recruitment of TRAFs to

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TNFR superfamily members such as CD40. Alternatively, high levels of I-TRAF/TANK may competitively recruit TRAF molecules that are complexed with TNFR superfamily members. Experiments to examine whether one or both of these possibilities play a role in CD40-induced signaling cascades mediated by TRAF adapter molecules remain to be performed. Furthermore, the finding that I-TRAF/TANK spontaneously interacts with some TRAF2 molecules regardless of the presence of TNFR ligation raises the possibility that the interaction between TRAFs and I-TRAF/TANK is independent of engagement of the TNFR superfamily member. The role of I-TRAF/TANK in CD40 signaling cascades appears to be extremely complicated. Initial experiments examined the impact of I-TRAF/TANK on a common outcome of TRAF-mediated signaling, translocation of NF-␬B dimers to the nucleus. Initial experiments demonstrated that although expression of small amounts of I-TRAF/TANK in 293 HEK cells induced NF-␬B activation (Cheng and Baltimore, 1996; Pomerantz and Baltimore, 1999), expression of larger amounts of a plasmid expressing I-TRAF/TANK did not induce nuclear translocation of NF-␬B (Kaye et al., 1996; Rothe et al.,1996). In these same experiments, expression of large amounts of I-TRAF/TANK inhibited translocation of NF-␬B dimers to the nucleus induced by overexpression of TRAF2. Similar results were observed following expression of LMP1 (Kaye et al.,1996) or simultaneous expression of CD154 and CD40 (Rothe et al.,1996). By contrast, expression of small amounts of I-TRAF/TANK increased TRAF2-induced nuclear translocation of NF-␬B. Similar results were observed when NF-␬B activation induced by overexpression of TRAF 1, 5, or 6 was examined. These results indicate that I-TRAF/TANK exerts a biphasic effect on the signaling cascade, leading to NF-␬B activation with small amounts enhancing and larger amounts inhibiting nuclear translocation. Further studies have attempted to elucidate the mechanism by which low levels of I-TRAF/TANK costimulate TRAF2-induced NF-␬B activation and high levels inhibit TRAF2-induced NF-␬B activation in 293 HEK cells. Initial experiments noted that deletion of the central domain (residues 169–189) of I-TRAF/TANK, containing the 178PXQXT182 binding site for TRAF 1, 2, 3, and 5, decreased costimulation of TRAF2-induced activation of NF-␬B. By contrast, this central domain of I-TRAF/TANK was not necessary for activation of NF-␬B induced by low amounts of I-TRAF/ TANK in the absence of any costimulators. Moreover, the N-terminal (residues 1–168), but not the C-terminal (residues 190–425), region of I-TRAF/TANK induced translocation of NF-␬B to the nucleus in the absence of costimulation. Transfection of TRAF2 in the presence of the Nterminal region of I-TRAF/TANK costimulated NF-␬B activation, albeit to a lesser degree than the wild-type molecule. These results suggest that


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an interaction between TRAF2 and I-TRAF/TANK via the central domain (residues 169–189) as well as the N-terminal region of I-TRAF/TANK is necessary for the synergistic effect of I-TRAF/TANK on TRAF2-induced NF-␬B activation. Additionally, the importance of the N-terminal region of TRAF2 (residues 1–93) in costimulation of nuclear translocation of NF␬B was elucidated by the finding that enhancement of TRAF2-induced NF-␬B activation in the presence of low amounts of I-TRAF/TANK was not observed with a mutant form of TRAF2 (⌬N, residues 94–501) lacking this region. The inhibitory role of I-TRAF/TANK on NF-␬B activation was further elucidated in experiments demonstrating that the C-terminal portion (residues 190–425) of I-TRAF/TANK inhibited activation of NF-␬B induced by a low amount of I-TRAF/TANK itself or in combination with either TRAF2 overexpression or CD40 engagement. By contrast, a truncated N-terminal version of I-TRAF/TANK (residues 1–168) activated NF-␬B in a concentration-dependent manner alone or in combination with TRAF2 and did not inhibit TRAF2-induced NF-␬B activation. Moreover, the biphasic nature of NF-␬B activation that was observed with increasing amounts of full-length I-TRAF/TANK was not observed with the truncated N-terminal I-TRAF/TANK mutant (residues 1–190). Furthermore, no inhibitory effect of the truncated N-terminal I-TRAF/TANK mutant on TRAF2-induced activation of NF-␬B was observed even when large amounts of this N-terminal I-TRAF/TANK mutant were expressed (Pomerantz and Baltimore, 1999). In conjunction with the former experiments, this result suggests that the C terminus of I-TRAF/TANK mediates inhibition of I-TRAF/TANK or TRAF2-induced NF-␬B activation, whereas the N-terminal portion of I-TRAF/TANK is directly involved in NF-␬B activation. The mechanism by which the C-terminal domain exerts its inhibitory effect remains to be elucidated completely. Finally, it should be noted that recent experiments have demonstrated that full-length I-TRAF/TANK has the ability to inhibit NF-␬B induced by overexpression of TRAF2 or TRAF6, but not by TRAFN–FKBP mutants that have been multimerized by the ligand for FKBP, FK1012 (Baud et al.,1999). These results suggest that the inhibitory action of the C-terminal of I-TRAF/TANK may be due to its ability to interfere with TRAF multimerization that has been shown to be essential for activation of signaling cascades such as NF-␬B. Recent experiments, however, have addressed the potential mechanisms by which I-TRAF/TANK affects nuclear translocation of NF-␬B induced following engagement of TNFR superfamily members. Since I-TRAF/ TANK does not contain homologous regions to any known signaling domains, a yeast two-hybrid screen was carried out on a cDNA library from a human pre-B cell line with the N-terminal and central regions (residues

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1–190) of I-TRAF/TANK previously shown to affect the NF-␬B signaling cascade positively. This screen yielded a novel kinase with homology to the IKKs, named TBK1 (Pomerantz and Baltimore, 1999). Overexpression of TBK1 in 293 HEK cells induced activation of NF-␬B, as measured either by a gel supershift assay for the p50/p65 heterodimer of NF-␬B or by an NF-␬B reporter assay. TBK1-induced NF-␬B activation was partially inhibited by a mutant of TRAF2 (residues 87–501) lacking the N-terminal region containing zinc and RING domains as well as by either the kinaseinactive (559T 씮 A) or kinase-dead (429K 씮 A, 430K 씮 A) versions of the MAP3K NIK, demonstrated to be immediately upstream of both the 움 and 웁 versions of IKK. In addition, kinase-dead versions (44K 씮 A) of either the 움 or 웁 versions of IKK completely abrogated TBK1-induced activation of NF-␬B. Moreover, kinase-dead TBK1 had no effect on NF␬B induced by either wild-type NIK or IKK-웁, indicating that TBK1 acts upstream of these signaling molecules. These findings indicated that TBK1induced NF-␬B activation required the N-terminal region of TRAF2 and induced a downstream signaling cascade involving NIK and the IKKs. The role of I-TRAF/TANK and TRAFs in this cascade was elucidated by the finding that a kinase-dead version of TBK1 (38K 씮 A) partially inhibited NF-␬B activation induced by the N-terminal and central regions (residues 1–190) of I-TRAF/TANK alone or in combination with TRAF 2, 5, or 6. This result indicated that I-TRAF/TANK in the presence of endogenous or exogenously expressed TRAFs could induce NF-␬B activation by a TBK1-dependent mechanism. Of note, TBK1-induced NF-␬B was not affected by the full-length version of I-TRAF/TANK containing the inhibitory C-terminal domain (residues 213–425), even at levels that interfered with TRAF-induced activation of NF-␬B. These findings were strengthened by in vitro binding experiments with recombinant proteins clearly showing that I-TRAF/TANK forms a complex with TBK1 and TRAF2. The functionality of this complex in terms of its ability to induce NF-␬B activation is dependent on the ability of I-TRAF/TANK to bind TRAF2, since a mutant of I-TRAF/TANK lacking the central TRAF binding domain (residues 1–168) failed to convey activity to this complex. It should be noted that neither I-TRAF/TANK nor TRAF2 induced the kinase activity of TBK1. The mechanism by which this kinase becomes activated is unknown. One interpretation of these data is that I-TRAF/TANK may act as a scaffolding molecule to allow TRAFs and TBK1 to interact. Finally, the development of a functional complex was not inhibited by the C-terminal domain of I-TRAF/TANK, although subsequent activation of NF-␬B or that portion induced by TRAF association was.


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These results suggest that there are at least two different mechanisms by which I-TRAF/TANK facilitates TRAF-induced NF-␬B activation. Although both mechanisms require the N-terminal and central regions of I-TRAF/TANK as well as the N-terminal region of TRAF2 containing the zinc and RING domains, one mechanism utilizes TBK1 and is insensitive to inhibition by the C-terminal region of I-TRAF/TANK, whereas the second mechanism is sensitive to the inhibitory effect of the C-terminal region of I-TRAF/TANK and is independent of TBK1. In this regard, expression of a naturally alternatively spliced 웂 form of I-TRAF/TANK that lacks the C-terminal inhibitory domain (Rothe et al., 1996) may drive the cascade to maximal activation of NF-␬B by multiple mechanisms. The latter mechanism may also be involved in TRAF2-induced activation of JNK that is enhanced by the N-terminal and central regions of I-TRAF/ TANK (residues 1–190) in a TBK1-independent manner. Moreover, JNK activation induced by TRAF5 or TRAF6, but not TRAF3, is also enhanced by TANK in a manner that is dependent on members of the GC kinase family (Chin et al., 1999). Finally, the relevance of the observation that I-TRAF/TANK has at least two independent mechanisms by which it facilitates TRAF-induced signaling cascades is highlighted by the observation that CD40-induced NF-␬B activation is partially inhibited (앑30%) by a kinase-dead version of TBK1 and is inhibited to a greater degree in the presence of high levels of wild-type I-TRAF/TANK containing the inhibitory C-terminal domain. The role of TBK1 in CD40-induced activation of NF-␬B was confirmed by the finding that overexpression of wild-type TBK1 enhanced activation of NF-␬B following expression of both CD154 and CD40 in 293 HEK cells. Together, these results suggest that one role of I-TRAF/TANK may be as a scaffolding protein that facilitates close contact of TRAFs with molecules crucial for various signaling cascades. The role of I-TRAF/TANK in TRAF-mediated signaling cascades remains to be fully delineated. Initial experiments demonstrated that I-TRAF/TANK bound TRAFs that were not in association with TNFR superfamily members. The issue of whether I-TRAF/TANK holds TRAFs in a quiescent state until they are recruited to TNFR superfamily members upon engagement or whether CD40-associated TRAFs are recruited to I-TRAF/TANK following receptor ligation remains to be resolved. In this regard, the recent data that I-TRAF/TANK acts as a scaffolding protein to bring TRAFs and signaling molecules such as TBK1 into close proximity support the later hypothesis but do not rule out the possibility that I-TRAF/TANK plays dual roles. Specifically, I-TRAF/TANK may act as a holding protein for quiescent TRAFs before receptor engagement in some circumstances and as a scaffolding protein that facilitates TRAF-mediated

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signaling cascades in others. Experiments to determine the scenarios in which these various activities pertain remain to be performed. 4. Regulation of TRAF Expression The issue of whether availability of a particular TRAF at a given point in activation or differentiation of a cell is highlighted by the observation that some TRAF family members are absent in resting B cells, but are induced by a variety of activation signals (our unpublished observations). Specifically, ligation of CD40 on B cells induces expression of TRAFs 1, 2, and 4 (Aicher et al., 1999; Craxton et al., 1998). The signals regulating expression of TRAFs 3, 5, and 6 remains to be examined. TRAF1 mRNA measured by polymerase chain reaction or by RNase protection is induced in B cells by pharmacological activation with ionomycin and phorbol ester, the polyclonal activator lipopolysaccharide, cross-linking sIg, or engaging CD40 (Craxton et al., 1998; Dunn et al., 1999). In addition, expression of the EBV protein LMP1, which acts as a constitutively active version of CD40, induces TRAF1 expression in B cells (Durkop et al., 1999). Supporting evidence for these observations is provided by an examination of TRAF1 regulation in 293 HEK cells in which overexpression of TNFRI, IL-1R, or CD40-induced transcription of TRAF1 as measured by an RNase protection assay or by activation of a TRAF1-dependent luciferase reporter construct (Schwenzer et al., 1999). Additionally, engagement of CD40 on B cells from the human B lymphoma cell line, Daudi, induced TRAF1 transcription as measured by an RNase protection assay (Craxton et al., 1998). Deletion of the most proximal NF-␬B site in the TRAF1 promoter abrogated TNF-움–induced transcription of the gene, suggesting a critical role for NF-␬B in TRAF1 transcription. The signaling events controlling CD40-induced expression of TRAFs 2 and 4 have not been delineated (Craxton et al., 1998). Finally, the signals regulating expression of TRAFs 3, 5, and 6 remain to be examined. Other mechanisms of TRAF regulation have also been reported. For example, a spliced form of TRAF2, TRAF2A, which can mediate JNK activation, but not nuclear translocation of NF-␬B, is highly expressed in splenocytes but not in other B cells that express wild-type TRAF2 (Brink and Lodish, 1998; Dadgostar et al., 1998). Not only does TRAF2A fail to activate NF-␬B, but also expression of TRAF2A can block the ability of wild-type TRAF2 to activate NF-␬B. TRAF2A expression is tightly regulated with a very short half-life (100 min) compared to wild-type TRAF2. Together, these observations suggest that an additional mechanism of the regulation of TRAF2-mediated signal transduction involves the regulated expression of an alternative splice product, although the mechanism controlling the appearance of TRAF2A is unknown.


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Despite the paucity of data regarding regulation of TRAFs in B cells, comparison of TRAF expression in B cells from the peripheral blood versus those from an activated secondary lymphoid tissue (e.g., the tonsil) demonstrated that resting peripheral B cells only expressed TRAFs 5 and 6, whereas activated tonsillar B cells expressed these TRAFs as well TRAFs 1, 2, and 3 (our unpublished observations). These observations emphasize the potential importance of TRAF expression in regulating CD40 signaling in B cells. C. DELINEATION OF TRAF INVOLVEMENT IN CD40-MEDIATED EVENTS Three experimental approaches have been utilized in an attempt to elucidate the role of TRAFs in CD40-mediated events. Most initial reports transiently overexpressed TRAFs alone or in the presence of human CD40 in non-B cell lines that do not normally express CD40. In some experiments, overexpression of CD40 was sufficient to induce CD40-mediated signaling events. In others, CD154 itself was overexpressed simultaneously or CD40 was engaged with recombinant CD154 or with anti-human CD40 mAb. Two early reports examined CD40-mediated signaling events in the EBVnegative Ramos B cell line (R-2G6) that constitutively expresses TRAF3 and had been permanently transfected with a mutant of TRAF3 (residues 324–567), TRAF3⌬N, that lacks the RING domain and zinc fingers (Cheng et al., 1995; Grammer et al., 1998). The second approach involved studying CD40-mediated events following transfection of non-B cell lines or murine B cell lines with wild-type human CD40 or human CD40 molecules that had mutated or deleted the membrane-proximal 231QEPQEINF238 TRAF6 binding site, the 250PVQET254 site necessary for the overlapping binding sites for TRAFs 1, 2, 3, and 5, or both TRAF binding sites. Similar to the first approach, CD40-mediated signaling events were examined following overexpression of CD154 or stimulation with recombinant CD154 or antihuman CD40 mAb. The third approach utilized mice with genetic alterations in TRAF expression. Mice lacking expression of TRAF2 (Yeh et al., 1997), TRAF3 (Xu et al., 1996), TRAF5 (Nakano et al., 1999), or TRAF6 (Lomaga et al., 1999) have been created. In addition, investigators have produced mice overexpressing the TRAF2⌬N (residues 241–501) mutant that lacks RING domains and zinc fingers (Lee et al., 1997) and mice overexpressing wild-type TRAF1 (Speiser et al., 1997). Examination of the role of various TRAFs in CD40-mediated events has focused on their role in functional responses of B cells that have been shown previously to be involved in humoral immunity. In vivo experiments have investigated whether various TRAFs are involved in the humoral response to T cell–dependent or –independent antigens. In vitro experiments have focused on events following engagement of CD40 expressed

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by purified B cells such as signaling via MAPKs or NF-␬B and functional outcomes such as proliferation and cell survival, Ig secretion, cytokine secretion (TNF-움, LT, IL-10, and GM-CSF), and induction of surface antigens involved in adhesion (CD23, CD54, and CD11a/CD18), antigenpresenting cell function (CD80, CD86, and MHC class II), and apoptosis (CD95). The role of TRAFs in CD40-mediated events in other cell types involved in antigen presentation, inflammation, and thrombosis (Figs. 1 and 5–8) have not as yet been examined using any of these approaches. 1. Signaling Cascades Delineation of the role of various TRAFs in CD40-induced signaling cascades using genetically altered mice focused largely on two pathways that have been shown to be involved in B cell function: the activation of JNK and NF-␬B. In vitro transfection experiments also examined the role of TRAFs in the activation of two other MAPKs, p38 and ERK, that have been demonstrated to be induced following CD40 ligation. It should be noted that JNK has been shown to play a role in gene transcription mediated by a number of DNA-binding proteins, including AP-1 when ERK is activated simultaneously and by CREBP when p38 is activated simultaneously. a. NF-␬B. In vitro experiments with purified splenic B cells from TRAF6 knockout mice (Lomaga et al., 1999) demonstrated that expression of TRAF6 is essential for nuclear translocation of NF-␬B induced following CD40 engagement. By contrast, splenic B cells purified from TRAF5 knockout mice (Nakano et al., 1999) or mice overexpressing a mutant of TRAF2, TRAF2⌬N (residues 247–501), containing the C-terminal TRAF-N (residues 272–355) and TRAF-C (residues 356–501) domains but lacking the N-terminal RING domain (residues 26–97) and zinc fingers (residues 100–249) (Lee et al., 1997) exhibited the same level of NF-␬B activation following CD40 ligation as their wild-type littermates. Similarly, splenic B cells purified from mice overexpressing TRAF1 exhibited CD40mediated NF-␬B activation to the same degree as their wild-type littermates (Speiser et al., 1997). Nuclear translocation of NF-␬B following CD40 engagement has not been examined in mice genetically deficient in TRAF2 or TRAF3 (Xu et al., 1996; Yeh et al., 1997), although it should be noted that TNF-움–induced NF-␬B activation was not affected by the absence of TRAF2. Together with the finding that CD40-induced NF-␬B is not affected by overexpression of TRAF2⌬N, this result supports the conclusion that TRAF2 does not play a role in nuclear translocation of NF-␬B induced following ligation of CD40 on resting, splenic B cells.


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Results from mice genetically deficient in TRAF6 (Lomaga et al., 1999) support those obtained from in vitro experiments in 293 HEK cells in which CD40-mediated NF-␬B activity was inhibited by expression of the TRAF-C domain of TRAF6 (residues 372–522) (Ishida et al., 1996b; Kashiwada et al., 1998) or a mutant of TRAF6 lacking the N-terminal RING domain and zinc fingers (residues 289–522) (Baud et al., 1999). By contrast, the results from knockout mice are in apparent disagreement with the findings that following overexpression of CD40 in 293 HEK cells, wild-type TRAF2 costimulated and TRAF2 containing the C-terminal TRAF-N and TRAF-C domains as well as the N-terminal zinc fingers but lacking the N-terminal RING domain (residues 87–501) (Rothe et al., 1995) inhibited NF-␬B activation, as measured using reporter constructs. These findings indicate that CD40 can utilize wild-type TRAF2 to mediate nuclear translocation of NF-␬B when it is present and that a TRAF2 mutant lacking amino acids 1–86 containing the RING domain interferes with CD40-mediated NF-␬B activation when endogenous TRAF2 is present. In this regard, the HEK 293 cells that were used for this in vitro experiment constitutively express TRAF2. By contrast, the majority of splenic B cells used for the experiments with TRAF2-deficient mice are naive cells that may not express TRAF2, since naive B cells from human peripheral blood and a secondary lymphoid tissue, the tonsil, express little or no TRAF2 (our unpublished observations). Therefore, the finding that CD40-induced NF-␬B in TRAF2-deficient mice was comparable to that in wild-type littermates may indicate that CD40 does not utilize TRAF2 normally to mediate nuclear translocation of NF-␬B in naive B cells. A role for TRAF6 in CD40-induced NF-␬B activation was confirmed by comparing the level of CD40-induced NF-␬B activity in 293 HEK cells following overexpression of a wild-type CD40 molecule versus one that could only bind TRAF6 because it lacked the 250PVQET254 site that binds TRAFs 1, 2, 3, and 5 (CD40⌬246) or had the crucial threonine in this site mutated (254T 씮 A). Cells expressing these CD40 mutants manifested CD40-induced NF-␬B activity, as assayed by a reporter assay (Leo et al., 1999a; Pullen et al., 1999; Tsukamoto et al., 1999) or by degradation of the 움 or 웁 isoforms of I␬B (Hsing et al., 1997) that was 40–50% of that exhibited by cells expressing wild-type CD40, implying that TRAF6 contributes approximately half of the CD40-mediated NF-␬B activation. However, in agreement with the in vitro transfection experiments with TRAF2 described above, this result also indicates that CD40 can utilize other mechanisms when they are available to induce nuclear translocation of NF-␬B in 293 HEK cells. Further support for this conclusion was derived by an examination of NF-␬B activation following overexpression of a CD40 molecule that cannot bind TRAF6, but can bind TRAFs 1, 2,

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3, and 5, as a result of mutation in the glutamic acid necessary for binding of TRAF6 (235 E 씮 A). This experiment indicated that in 293 HEK cells, 앑75–80% of CD40-induced NF-␬B activity is mediated by TRAF6, but 20–25% of CD40-induced NF-␬B activity is mediated by TRAF 1, 2, 3, or 5, or a combination of these, that associate with the membrane distal binding site. That this activity is mediated by TRAF2 is suggested by the finding that NF-␬B activity induced in BI-141 murine T lymphoma cells, expressing a mutant CD40 (232Q 씮 A) molecule that does not bind TRAF2, was decreased compared to cells expressing wild-type CD40 (H. H. Lee et al., 1999b). Together with in vitro transfection experiments described above, this experiment suggests that TRAF2 may play a role in CD40induced NF-␬B activation when it is available. A similar situation may exist for TRAF5, although the effect of wild-type or mutant TRAF5 molecules on CD40-induced NF-␬B has not been examined to date. Discrepancies in the degree to which particular TRAFs are involved in CD40-induced NF-␬B activation may relate to the relative expression of these proteins in cell lines. It has been shown that high levels of TRAF3 costimulate TRAF5-induced NF-␬B activity (Leo et al., 1999a) and that TRAF5 associates better with CD40 when it is in a heterodimer with TRAF3. These findings may explain the early enigmatic data that high, but not low, levels of TRAF3⌬N expression decreased CD40-mediated activation of NF-␬B. Together, these results suggest that TRAF3 homodimers may not activate NF-␬B directly, but TRAF3 may costimulate the ability of TRAF5 to mediate CD40induced NF-␬B activity when both TRAFs are available and are heterodimerized. In this regard, NF-␬B activity induced in BI-141 murine T lymphoma cells that were expressed a mutant CD40 (241V 씮 A) molecule that does not bind TRAF3 homodimers was decreased compared to cells expressing wild-type CD40 (H. H. Lee et al., 1999b). Finally, the observation that overexpression of TRAF1 in vivo or in vitro does not increase CD40-induced NF-␬B activation supports the conclusion that TRAF1 does not play a role in CD40-induced activation of NF-␬B. Together, these data suggest that (TRAF2)3, (TRAF5)3, or (TRAF3)x –(TRAF5)y multimers engaging the 250PVQET254 site on the cytoplasmic domain of CD40 may be responsible for the 20–25% of the total CD40-induced NF-␬B activation in 293 HEK cells that express TRAFs 1, 2, 3, 5, and 6. TRAF6 appears to account for the majority of CD40-mediated NF-␬B in this cell line. By contrast, experiments utilizing chimeric CD8–murine CD40 receptors that consist of CD8 with an insert of varying portions of the cytoplasmic domain of murine CD40 demonstrated that the membrane distal TRAF binding site that can associate with TRAFs 1, 2, 3, and 5, but not the membrane proximal site that binds TRAF6, appears to mediate CD40-induced


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NF-␬B activation in B cells from the WEHI 231 murine B lymphoma cell line (Sutherland et al., 1999). These apparently discrepant results emphasize the hypothesis that TRAFs 2, 5, and 6 can mediate CD40-induced NF␬B activation, but that the particular TRAFs that are utilized by a given B cell depend on the activation and differentiation status of the cell, as well as the absolute and relative amounts of the various TRAFs expressed. Finally, comparison of the results obtained from experiments utilizing CD40 molecules that were mutated in either the proximal or distal TRAF binding sites to a CD40 construct lacking both the proximal TRAF6 binding site (235E 씮 A) and the distal 250PVQET254 site that binds TRAFs 1, 2, 3, and 5 (CD40⌬246 or 254T 씮 A) indicated that 앑20–25% of CD40-induced NF-␬B activity in 293 HEK cells is independent of both known TRAF binding sites. In support of this conclusion, NF-␬B activity induced by overexpression of a CD40 molecule lacking the 250PVQET254 binding site for TRAFs 1, 2, 3, and 5, CD40⌬246, was inhibited by 앑75% by overexpression of the TRAF-C domain of TRAF6 (Kashiwada et al., 1998). Together with the results described above, the other 25% of CD40⌬246-induced NF-␬B activity must represent the portion of CD40-induced NF-␬B that is independent of known TRAFs. Therefore, CD40-induced activation of NF-␬B utilizes both TRAF-dependent and TRAF-independent mechanisms. Availability of TRAFs may govern which ones are utilized for the TRAF-dependent portion, but results from genetically altered mice as well as from in vitro transfection experiments indicate that multimers of (TRAF6)3, (TRAF2)3, (TRAF5)3, or (TRAF3)x –(TRAF5)y, but not (TRAF1)3, (TRAF1)x –(TRAF2)y, or (TRAF3)3, all can mediate CD40induced NF-␬B activity (Fig. 24). Recent experiments have examined the signaling cascades that mediate CD40-induced NF-␬B activation (Figs. 24 and 25). In this regard, the finding that a dominant negative version of RAF1 (1–258), but not dominant negative forms of RAS or MKK1, partially inhibited (앑30%) NF-␬B activation induced following engagement of either a wild-type CD40 molecule or a CD40 mutant that only binds TRAF6 (⌬246) expressed in 293 HEK cells suggests that CD40–TRAF6–induced NF-␬B activity may be partially mediated by a signal transduction cascade involving RAF1 (Kashiwada et al., 1998). In addition, experiments comparing NF-␬B activity in splenic B cells from aly/aly mice that have a point mutation in the C terminus of NIK (Shinkura et al., 1999) to heterozygote littermates (aly/ ⫹) demonstrated that NIK mediates 앑75% of CD40-induced NF-␬B activity in resting splenic B cells (Garceau et al., 2000). Of interest, results obtained from overexpressing CD40 molecules that contained only the membrane proximal or distal TRAF binding sites in 293 HEK cells demonstrated different roles for NIK in TRAF6 versus TRAF2- and TRAF5-

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FIG. 25. The state of activation or differentiation of the B cell or the availability of TRAFs may contribute to TRAF utilization in CD40-mediated activation of (A) ERK, (B), JNK and p38, and (C) IKKs leading to nuclear translocation of NF-␬B. For abbreviations, see text.

mediated NF-␬B activation. Whereas a kinase-dead version of NIK completely abrogated NF-␬B activity mediated by the membrane-distal TRAF binding site that can associate with TRAFs 2 and 5, this same kinase-dead NIK construct only inhibited 앑30% of NF-␬B activity induced by the TRAF6 binding site in 293 HEK cells (Leo et al., 1999a; Tsukamoto et al., 1999). By contrast, the EBV-negative B cell line DND39 does not express NIK or another MAP3K that has been shown to mediate TRAF-


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FIG. 25. (Continued)

induced NF-␬B activation, MEKK1, but does activate NF-␬B following CD40 ligation in a IKK-움/웁–dependent manner (Kosaka et al., 1999). In this case, NF-␬B may be activated by PKC␨ that has been demonstrated to associate with TRAFs 2, 5, and 6 via p62 (Sanz et al., 1999) and RIP family members (Bertin et al., 1999; McCarthy et al., 1998) or by an unknown cascade involving the newly described I␬B kinase, IKKi, that is expressed by B cells and is induced following stimulation with TNF-움, IL6, and the murine B cell polyclonal activator, lipopolysaccharide (Shimada et al., 1999). Finally, it should be noted that a recently described kinase, TBK1, may also be involved in CD40-induced NF-␬B activation by a mechanism that requires I-TRAF/TANK, the N-terminal region of TRAF2, and a downstream signaling cascade involving NIK and the IKKs (Pomerantz and Baltimore, 1999). Together, these results indicate that CD40 utilizes different MAP3Ks to activate the IKKs, resulting in I␬B degradation and nuclear translocation of NF-␬B. The mechanism utilized may depend on the MAP3Ks that are available, which in turn may depend on the activation and differentiation state of the cell. Of note, gene expression induced by NIK-independent NF-␬B activation following recruitment of TRAF 2 or TRAF6 to CD40 is inhibited following binding of A20 in association with ABIN (the A20 inhibitor of NF-␬B activation). Suprisingly, A20 does not interfere with nuclear translocation of NF-␬B dimers or binding of these dimers to DNA (Heyninck and Beyaert, 1999; Heyninck et al., 1999).

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b. JNK. JNK activation has been examined in vitro with purified splenic B cells from transgenic mice expressing a mutant TRAF2 molecule, TRAF2⌬N (residues 241–501), lacking the N-terminal RING domain and zinc fingers but with the C-terminal domains intact (Lee et al., 1997). These experiments demonstrated that CD40-induced activation of JNK was decreased by overexpression of TRAF2⌬N compared to that noted in splenic B cells from normal littermates. In this regard, it should be noted that activation of JNK following stimulation with another TNFR superfamily member, TNF-움, was decreased in cells from TRAF2-deficient mice compared to control littermates (Yeh et al., 1997). However, CD40induced JNK activation was not tested in these mice. Notably, CD40mediated JNK activation was abrogated when a mutant TRAF2 (residues 87–501) molecule lacking the RING domain was overexpressed in 293 HEK cells (Rothe et al., 1995). Together, these findings support the conclusion that TRAF2 plays a role in JNK activation mediated by ligation of CD40. By contrast, CD40-stimulated splenic B cells purified from TRAF5 knockout mice (Nakano et al., 1999) or mice overexpressing TRAF1 (Speiser et al., 1997) exhibited the same level of JNK activation as their wild-type littermates. Activation of JNK following ligation of CD40 has not been examined in mice genetically deficient in TRAF3 (Xu et al., 1996) or TRAF6 (Lomaga et al., 1999). A role for TRAF3 in CD40-induced JNK activation was suggested by the finding that Ramos B cells expressing endogenous TRAF3 and permanently transfected so as to express a low level of a TRAF3 mutant (residues 324–567), TRAF3⌬, that lacks the N-terminal RING domain and zinc fingers had a partial defect in CD40-induced JNK activation compared to cells transfected with control vector (Grammer et al., 1998). This finding is consistent with the observation that CD40-induced JNK activation was decreased in BI-141 murine T lymphoma cells expressing a mutant CD40 molecule, 241V 씮 A, that cannot bind TRAF3 homodimers compared to that noted in cells expressing wild-type CD40 (H. H. Lee et al., 1999b). Roles for TRAFs 2 and 3 as well as TRAF6 in CD40-induced JNK activity were also demonstrated by comparing the level of JNK activity in HeLa cervical epithelial cells or the degree of JNK activation in 293 HEK cells following overexpression of a wild-type CD40 molecule or an altered CD40 molecule with the proximal TRAF6 binding site, the distal binding site for TRAFs 1, 2, 3, and 5, or both sites mutated so that they are unable to bind TRAFs (Leo et al., 1999a; Pullen et al., 1999). Whereas mutation of the glutamic acid crucial for TRAF6 binding (235E 씮 A) decreased CD40-induced JNK activity by 50%, mutation of this site as well as the distal TRAF binding site (CD40⌬246) completely abolished the ability of CD40 to induce JNK to phosphorylate its substrate, recombinant jun (Leo


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et al., 1999a). This result confirmed a role for the distal 250PVQET254 binding site in CD40-mediated JNK activation also demonstrated following transfection of murine WEHI 231 B cells with CD40⌬250 (Hara et al., 1997). In 293 HEK cells, JNK activation was assessed following CD40 engagement with antibodies that recognize the phosphorylated forms of two JNK isoforms, JNK1 and JNK2 (Pullen et al., 1999). Of interest, mutation of the distal TRAF binding site, 254T 씮 A, so that TRAF6 was the only TRAF that could bind CD40, reduced activation of JNK1 to 10% and JNK2 to 50% of the levels obtained in wild-type CD40. Additional mutation of the TRAF6 binding site, so that both TRAF binding sites are nonfunctional, completely abrogated the ability of CD40 to activate both JNK1 and JNK2. Moreover, examination of this CD40-induced signaling pathway in cells expressing a CD40 molecule with the TRAF6 binding site mutated, so that only TRAF 1, 2, 3, or 5 could bind CD40, demonstrated reduced activation of both JNK1 and JNK2 to 10% of control levels. These results suggest that TRAF6 plays major and comparable roles in activation of JNK1 and JNK2, whereas TRAFs that bind the membrane distal site play a more important role in activation of JNK1, although they also contribute to JNK2 activation. Of note, experiments examining why CD40 engagement consistently activates JNK1 to a much higher degree than JNK2 or why resting B cells cannot activate JNK following CD40 ligation unless they are preactivated in some manner have not been performed. Together, these results indicate that CD40 utilizes TRAF-dependent, but not TRAF-independent, mechanisms to activate both isoforms of JNK. In addition, whereas the membrane-proximal and -distal TRAF binding sites equivalently contribute to activation of JNK2, the distal binding site that can bind TRAFs 1, 2, 3, and 5 predominantly mediates CD40-induced activation of JNK1. These results suggest that the membrane-proximal and -distal TRAF binding sites differentially activate the upstream kinases responsible for JNK1 versus JNK2 activation. Experiments to delineate the details of the cascades remain to be performed. Finally, results from genetically altered mice as well as from in vitro transfection experiments suggest that multimers of (TRAF6)3, (TRAF2)3, and (TRAF3)3 mediate CD40-induced activation of JNK (Figs. 23 and 25). The role of TRAF1 or TRAF5 in CD40-induced JNK activation has not been delineated. It should be noted that a role for the RING domain and zinc fingers of TRAF6 in CD40-mediated JNK activation in 293 HEK cells was brought into question by the finding that overexpression of a mutant of TRAF6 (289–530) lacking these domains did not inhibit JNK activation following engagement of CD40⌬246 that lacks the 250PVQET254 binding site for TRAFs 1, 2, 3, and 5 (Leo et al., 1999a). Importantly, this finding does not necessarily rule out a role for TRAF6 in CD40-mediated JNK activa-

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tion, since the C-terminal TRAF-N and TRAF-C domains can be involved in downstream signaling events by virtue of their ability to associate with other kinases and adapter molecules when they are available. Of note, experiments utilizing chimeric CD8–murine CD40 receptors that consist of the CD8 with an insert of varying portions of the cytoplasmic domain of murine CD40 demonstrated that the membrane-distal TRAF binding site that can associate with TRAFs 1, 2, 3, and 5, but not the membrane-proximal site that binds TRAF6, appears to mediate CD40induced JNK activation in B cells from the WEHI murine B lymphoma cell line (Sutherland et al., 1999). Together with the results presented above, this finding indicates that multimers of (TRAF2)3 and (TRAF3)3, but not (TRAF6)3, (TRAF5)3, (TRAF5)x –(TRAF3)y, or (TRAF2)x –(TRAF1)y, mediate CD40-induced JNK in WEHI B cells. Again, the state of activation or differentiation of the B cell or the availability of TRAFs may contribute to their utilization in CD40-mediated JNK activation. c. p38. The role of TRAFs in CD40-induced p38 activity was investigated by comparing the degree of p38 activation in 293 HEK cells following overexpression of a wild-type CD40 molecule or an altered CD40 molecule with the proximal TRAF6 binding site, the distal binding site for TRAFs 1, 2, 3, and 5, or both sites mutated so that they are unable to bind TRAFs (Pullen et al., 1999). Mutation of the amino acid crucial for TRAF6 binding, 235 E 씮 A, so that only TRAFs 1, 2, 3, and 5 can bind, decreased CD40induced p38 activation as detected with a phosphospecific anti-p38 antibody by 50%. The importance of the membrane-distal TRAF binding site in CD40-induced p38 activation was emphasized by the finding that Ramos B cells expressing endogenous TRAF3 and permanently transfected so as to express a low level of a TRAF3 mutant (residues 324–567), TRAF3⌬, that lacks the N-terminal RING domain and zinc fingers were defective in CD40-induced p38 activation compared to cells transfected with control vector (Grammer et al., 1998). Evidence of a role for TRAF6 in CD40-mediated p38 activation was provided by the finding that mutation of the distal TRAF binding site so that only TRAF6 can bind CD40 decreased p38 activation by 20% compared to the level obtained with wild-type CD40. Mutation of both the membraneproximal and -distal sites so that no known TRAFs can bind CD40 decreased p38 activation to 10% of the level obtained with wild-type CD40. These results suggest that CD40-induced activation of p38 is mediated by both TRAF-dependent and -independent mechanisms, although the TRAF-dependent component mediates the majority of CD40-induced p38 activation. Furthermore, these data suggest that TRAF6 makes a greater contribution to activation of p38 than the TRAFs that bind the distal


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PVQET254 site when all TRAFs are available in the cell, although it is important to note that either set of TRAFs is capable of inducing p38 activity. Finally, CD40-induced p38 activation has a TRAF component that has been shown to be mediated by either TRAF3 or TRAF6 and a component that is independent of known TRAFs (Figs. 22 and 25). It should be noted that TRAF2 may also mediate CD40-induced p38 activation, since overexpression of TRAF2 induced p38 activation in 293 HEK cells. The role of TRAF1 or TRAF5 in CD40-induced p38 activation has not been delineated. Experiments to examine the signal transduction pathways that mediate activation of p38 following CD40 engagement remain to be performed. d. ERK. The role of TRAFs in CD40-induced ERK activity (Figs. 19 and 25) was investigated by comparing the degree of ERK activation in 293 HEK cells or in murine WEHI 231 B cells following overexpression of a wild-type CD40 molecule or an altered CD40 molecule with the distal binding site for TRAFs 1, 2, 3, and 5 deleted [CD40⌬246 (Kashiwada et al., 1998) and ⌬252 (Hara et al., 1997)]. CD40 was engaged on 293 HEK and WEHI 231 B cells, respectively, with the G28.5 and 5C3 anti-CD40 mAbs. Whereas ERK activity induced following ligation of wild-type CD40 on 293 HEK cells was inhibited by 25% in the presence of a mutant TRAF6 molecule (residues 352–501) containing the C-terminal TRAF-C domain, this mutant completely inhibited ERK activation following ligation of a mutant CD40 molecule (⌬246) that lacks the membrane-distal TRAF binding site and can only bind TRAF6. These data indicate that CD40induced ERK activation is mediated by both TRAF binding sites. Confirmation for a role for the membrane-distal 250PVQET254 binding site for TRAFs 1, 2, 3, and 5 in CD40⌬246-mediated ERK activation was provided by the finding that engagement of this mutant CD40 molecule resulted in decreased ERK2 activation when compared to ligation of a wild-type CD40 molecule in WEHI 231 B cells. Further experiments demonstrated that CD40-induced ERK activation mediated by the membrane-proximal TRAF6 binding site as well as the membrane-distal site that binds TRAFs 1, 2, 3, and 5 uses a signal transduction pathway that is dependent on RAS–RAF1–MKK1. Of interest, a portion of CD40-induced ERK activation that is mediated by TRAF6 uses a RAS-independent mechanism that has not yet been delineated (Kashiwada et al., 1998), but may involve the recently defined TRAF6–RIP–p62–PKC␨ –MKK1–ERK signaling cascade (Berra et al., 1995; Schonwasser et al., 1998; Takeda et al., 1999). Together, these results indicate that activation of ERK following CD40 ligation is partially mediated by TRAF6. Which of the TRAFs can bind the membrane-distal TRAF binding site in CD40-mediated CD40-induced

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ERK activation and whether there is a TRAF-independent component to activation of this MAPK following CD40 engagement remain to be elucidated. 2. Functional Outcomes The role of various TRAFs in the functional outcomes of CD40 signaling has been examined in mice with genetically altered TRAF expression and in B cell lines following in vitro transfection with wild-type or mutated TRAF molecules. Additional experiments have been performed in murine B cell lines that had been transfected with wild-type human CD40 or human CD40 that had mutations or deletions of the membrane-proximal 231 QEPQEINF238 TRAF6 binding site, the 250PVQET254 site necessary for the overlapping binding sites for TRAFs 1, 2, 3, and 5, or both TRAF binding sites. Functional events induced by CD40 signaling events were examined following stimulation with anti-human CD40 mAb. Specifically, TRAF involvement in proliferation or cell survival, adhesion, antigenpresenting cell function, and secretion of cytokines and Ig has been examined in some detail (Fig. 26). Of note, the role of TRAFs in CD40-mediated

FIG. 26. The state of activation or differentiation of the B cell or the availability of TRAFs may contribute to TRAF utilization in CD40-mediated activation of transcription factors leading to functional outcomes. For abbreviations, see text.


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events such as the induction of activation antigens (TRAF1, TRAF2, MHC class II, and CD154) or in the details of the GC reaction, such as rescue from apoptosis and the development of B cell memory, has not yet been examined in detail. a. Proliferation and Cell Survival. Ligation of CD40 on B cells can result in proliferation or apoptosis, depending on the differentiation state of the cell and the degree of receptor engagement (Bergman et al., 1996; Miyashita et al., 1997). In vitro experiments with purified splenic B cells from mice that were genetically altered in TRAF expression examined the role of TRAFs in CD40-induced proliferation. When compared to purified splenic B cells from their wild-type littermates, CD40-mediated proliferation was partially defective in purified splenic B cells from mice that were genetically deficient in TRAF5 (Nakano et al., 1999) or TRAF6 (Lomaga et al., 1999). By contrast, there was no defect in CD40-mediated proliferation of splenic B cells purified from TRAF3 knockout mice when compared to their wild-type littermates (Xu et al., 1996). Suprisingly, splenic B cells purified from a transgenic mouse expressing a mutant of TRAF2, TRAF2⌬N (residues 247–501), containing the C-terminal TRAF domains but lacking the N-terminal RING domain and zinc fingers, exhibited increased proliferation when compared to splenic B cells from their wildtype littermates (Lee et al., 1997). Together with the observation that the C-terminal regions of TRAF2 associate with a variety of signaling molecules, this finding suggests that the C-terminal region of TRAF2 may induce signaling cascades that mediate proliferation induced by CD40 engagement. Alternatively, or in addition, the N-terminal regions of TRAF2 may lead to apoptosis so that deletion of this region results in increased CD40-mediated proliferation. In this regard, MEKK1 (reviewed in Schlesinger et al., 1998) and RIP2/CARDIAK/RICK (McCarthy et al., 1998) that has been shown to associate with TRAF2 has also been shown to lead to apoptosis by a caspase-dependent pathway. Apoptosis induced following CD40 engagement has been observed in a variety of B cell lines as well as in activated primary B cells. Of interest, early experiments demonstrated that engagement of wild-type human CD40 expressed in a murine B lymphoma cell line, M12, induced growth inhibition (Inui et al., 1990). This CD40-induced growth inhbition, later found to result from apoptosis, was not observed when these murine B cells were transfected with a mutant human CD40 molecule that could bind TRAF6, but not TRAFs 1, 2, 3, and 5 because of a mutation (254T 씮 A) in the membrane-distal 250PVQET254 TRAF binding site. The role of TRAF3 in this phenomenon was ruled out by the finding that CD40-mediated apoptosis observed in wild-type Ramos B cells was not

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inhibited in the presence of a mutant TRAF3 molecule (residues 324–567), TRAF3⌬N, lacking the RING domain and zinc fingers (Grammer et al., 1998). Together, these results indicate that TRAFs 3 and 6 do not play a role in CD40-mediated apoptosis. Moreover, a role for the N terminus of TRAF2 was suggested by the finding that overexpression of TRAF2⌬N increased proliferation in splenic B cells following CD40 engagement compared to wild-type littermates. A role for either TRAF1 or TRAF5 in CD40-induced apoptosis has not been examined. These results suggest that multimers of (TRAF2)3 or (TRAF2)x –(TRAF1)y, but not (TRAF6)3, (TRAF3)3, or (TRAF5)x –(TRAF3)y, may mediate CD40-induced apoptosis. Results from genetically altered mice as well as from transfection experiments indicate that multimers of (TRAF6)3 and (TRAF5)3, but not (TRAF3)3 or (TRAF3)x –(TRAF5)y, mediate CD40-induced proliferation. The possibility that (TRAF2)3 multimers induce proliferation following CD40 engagement was suggested by the results described above, but further experiments must be performed to examine this possibility in detail. Furthermore, preliminary experiments have examined the role of signaling cascades involving two MAP3Ks that can activate NF-␬B, NIK, and MEKK1 in CD40-induced proliferation. The physiological relevance of CD40-mediated NF-␬B is highlighted by the finding that mice genetically deficient in the p50 (Sha et al., 1995; Snapper et al., 1996), RelC (Baldwin, 1996), RelB (Kimata et al., 1994), or p52 (Bauerle and Baltimore, 1996) components of NF-␬B are defective in CD40-mediated proliferation. It should be noted that both NIK and MEKK1 associate with TRAFs and are activated following CD40 engagement. One study examined the role of NIK in CD40-induced proliferation and found that the level of proliferation of resting, splenic B cells from aly/aly NIK-deficient mice induced by CD40 and IL-4 was less than that obtained from stimulating splenic B cells from heterozygote (aly/⫹) littermates in the same manner (Garceau et al., 2000). Of note, since previous studies have demonstrated that costimulation with IL-4 favored association of TRAF2 with CD40 in DND39 EBV-negative B cells (Kuhne et al., 1997) in preference to other TRAFs, that IL-4 itself can induce proliferation of B cells and that addition of IL-4 can mask a CD40-mediated signaling defect (Hennet et al., 1998) must be confirmed with experiments that examine the role of NIK in CD40-induced proliferation of B cells in the absence of IL-4. Second, a role for MKK4, a MAP2K activated by MEKK1, in CD40-induced proliferation was ruled out by the finding that CD40-induced B cell proliferation was similar in B cells purified from MKK4 knockout mice and their wildtype littermates (Nishina et al., 1997). Therefore, the downstream pathways that mediate CD40-induced, TRAF-dependent B cell proliferation remain to be delineated in detail.


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b. Adhesion. Engagement of CD40 induces homotypic adhesion between B cells that has been shown to be mediated by CD54 (ICAM1)– CD11a/CD18 (LFA1) as well as by CD23–CD21 interactions. Whereas B cells express CD11a/CD18 and CD21 that can be increased by CD40 engagement, they require CD40 ligation to induce expression of CD54 and CD23. Expression of these molecules can also mediate adhesion with other cell types that play a role in the induction of B cell responses in secondary lymphoid tissue, such as T cells that express both CD54 and CD11a/CD18 as well as FDCs that express CD21. Initial experiments demonstrated that CD40-induced expression of CD54 and CD23 was abrogated in murine B cells expressing a mutant human CD40 molecule that could bind TRAF6, but not TRAFs 1, 2, 3, and 5 because of a mutation (254T 씮 A) in the membrane-distal 250PVQET254 TRAF binding site (Hostager et al., 1996). By contrast, CD40-induced expression of CD11a/CD18 was unaffected by the inability of TRAFs 1, 2, 3, and 5 to associate with CD40, suggesting that TRAF6 may mediate up-regulation of this adhesion molecule. Data have begun to delineate which of the TRAFs that bind the membrane-distal 250PVQET254 site were responsible for CD40-induced upregulation of CD54 and CD23. Although splenic B cells isolated from TRAF5 knockout mice were partially defective in CD40-induced expression of CD54 and CD23 (Nakano et al., 1999), induction of these molecules following CD40 engagement has not been examined in B cells purified from mice deficient in expression of TRAF 1, 2, or 6. In addition, lowlevel expression of a mutant TRAF3 molecule (residues 324–567), TRAF3⌬N, did not affect CD40-induced CD54 expression on Ramos B cells (Grammer et al., 1998). This observation is supported by the finding that overexpression of a mutant CD40 molecule in which the valine crucial for binding of TRAF3 homodimers (241V 씮 A) to the cytoplasmic tail of CD40 did not affect up-regulation of CD54 following CD40 engagement (H. H. Lee et al., 1999b). Furthermore, engagement of a mutant CD40 molecule with the glutamic acid crucial for TRAF2 binding (232Q 씮 A) altered CD40-induced CD54 expression (H. H. Lee et al., 1999b). These results indicate that (TRAF5)3, but not (TRAF)3 or (TRAF5)x –(TRAF3)y multimers, plays a role in CD40-induced expression of CD54 and CD23. Although roles for (TRAF2)3 and (TRAF2)x –(TRAF1)y in the induction of CD23 have not been examined, TRAF2 multimers have been shown to play a role in CD40-induced CD54 expression. Moreover, examination of CD40-induced CD23 and CD54 expression in resting, splenic B cells of aly/aly NIK-deficient mice demonstrated that these adhesion molecules can be induced in a NIK-independent manner following CD40 ligation on resting, splenic B cells (Garceau et al., 2000). In this regard, it should

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be noted that although CD40 induces CD54 expression by a mechanism that is independent of NIK, CD40 engagement induces expression of CD54 by a mechanism that is mediated by the p65 (RelA)/RelC, but not the p50/p65, heterodimer of NF-␬B (Parry and Mackman, 1994), suggesting that TRAF5 homodimers and TRAF2 multimers can mediate translocation of these components of NF-␬B. By contrast, TRAF6 multimers play a role in the up-regulation of CD11a/CD18 following CD40 ligation on B cells. The TRAFs that mediate CD40-induced CD44H expression remain to be elucidated. c. Antigen-Presenting Cell Function. Induction of CD80/B7-1 and CD86/B7-2 following CD40 ligation on resting B cells has been shown to be essential in order for these cells to differentiate from cells that are ineffective antigen-presenting cells to ones that can prime T cells effectively. The specific TRAFs that mediate CD40 signaling cascades leading to induction of CD80/B7-1 and CD86/B7-2 have been examined using genetically altered mice as well as cell lines transfected with mutant CD40 receptors or TRAF adapter molecules. Initial experiments demonstrated that CD40-induced expression of CD80/B7-1 was abrogated in murine B cells expressing a mutant human CD40 molecule that could bind TRAF6, but not TRAFs 1, 2, 3, and 5 because of a mutation (254T 씮 A) in the membrane-distal 250PVQET254 TRAF binding site (Hostager et al., 1996). Genetic deletion of TRAF5 in mice abrogated the ability of CD40 to induce CD80/B7-1 and CD86/B7-2 in purified splenic TRAF5⫺/⫺ B cells compared to B cells purified from wild-type littermates (Nakano et al., 1999). By contrast, engagement of CD40 on purified splenic B cells from TRAF3 knockout mice resulted in the same levels of CD80/B7-1 and CD86/B7-2 as expressed by B cells from wild-type littermates (Xu et al., 1996). Together, these findings suggest that TRAF5 multimers, but not TRAF3 homodimers or TRAF3–TRAF5 heterodimers, mediate CD40induced expression of CD80/B7-1 and CD86/B7-2. Suprisingly, ligation of CD40 on splenic B cells purified from TRAF2⌬N transgenic mice resulted in higher levels of CD80/B7-1 compared to B cells purified from wild-type littermates (Lee et al., 1997). This result suggests that the N-terminal regions of TRAF2 may induce signaling pathways that interfere with CD80/B7-1 expression. Finally, CD40-induced expression of CD80/B7-1 and CD86/B7-2 by B cells has not been tested in mice genetically deficient in TRAF1 or TRAF6, although in vitro transfection experiments demonstrated that TRAF6 does not play a role in CD40-induced expression of CD80/B7-1 (Hostager et al., 1996). In conjunction, these results indicate that multimers of (TRAF5)3, but not (TRAF3)3 or (TRAF3)x –(TRAF5)y, mediate CD40-induced expression of


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CD80/B7-1 and CD86/B7-2. In addition, TRAF2⌬N increased, and lack of TRAF6 binding to CD40 had no effect on, CD40-induced expression of CD80/B7-1. The role of TRAF1 in either of these processes has not yet been examined. Of note, low-level expression of a mutant TRAF3 molecule (residues 324–501), TRAF3⌬N, in Ramos B cells had no effect on up-regulation of MHC class II expression following CD40 engagement (Grammer et al., 1998). Moreover, a role for NIK in CD40-induced CD80, but not CD86 or MHC class II, expression in resting, splenic B cells was demonstrated by examination of aly/aly mice that have a functional point mutation in the C terminus of NIK (Garceau et al., 2000). d. Cytokine Secretion. The role of TRAF3 in CD40-mediated signaling events leading to secretion of GM-CSF, IL-10, TNF-움, and LT-움 was examined in the EBV-negative Ramos B cell line (R-2G6) that constitutively expresses TRAF3 and was permanently transfected with a mutant of TRAF3 (residues 324–567), TRAF3⌬N, that lacks the RING domain and zinc fingers (Grammer et al., 1998). Low-level expression of TRAF3⌬N partially inhibited CD40-induced secretion of IL-10, TNF-움, and LT-움. By contrast, secretion of GM-CSF following CD40 engagement on Ramos cells expressing TRAF3⌬N was equivalent to that secreted by control Ramos cells. In this regard and in agreement with the observation that CD40-induced NF-␬B is independent of TRAF3, CD40-induced expression of GM-CSF has been shown to be mediated by the p65 (RelA)/RelC, but not the p50/p65, heterodimer of NF-␬B (Parry and Mackman, 1994). The potential roles of TRAFs 1, 2, 5, and 6 in secretion of these cytokines following CD40 ligation have not been tested. Importantly, it should be noted that since expression of both CD54 and GM-CSF utilizes p65 (RelA)/ RelC, but not p50/p65, NF-␬B dimers, the finding that TRAF2 or TRAF5 but not TRAF6 mediates CD40-induced CD54 expression suggests that CD40-mediated GM-CSF expression may be regulated by TRAF2 or TRAF5. Moreover, TRAF involvement in CD40-induced secretion of IL-6 and IL-12 or in CD40-mediated induction of receptors (CD25, IL-13R움, and CXCR4) that give the B cell the ability to respond to cytokines or chemokines has not been examined. e. Regulation of Immunoglobulin Production i. Ig Secretion. The role of TRAFs in CD40-induced Ig secretion has been examined following immunization of mice that are genetically deficient in TRAF expression with T cell–dependent or –independent antigens. The T cell–independent humoral response to NP–Ficoll was unaffected by the absence of either TRAF3 (Xu et al., 1996) or TRAF5 (Nakano

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et al., 1999). Roles for TRAFs 1, 2, and 6 in the T cell–independent humoral response have not been examined. GC formation, the hallmark of T cell–dependent responses, induced by immunization with NP–CGG was unaffected by either the absence of TRAF5 (Nakano et al., 1999) or overexpression of TRAF2⌬ (Lee et al., 1997) when these mice were compared to their wild-type littermates. By contrast, production of antigen-specific IgG following immunization with high-dose NP–CGG was abrogated in mice rendered genetically deficient in TRAF3 (Xu et al., 1996), although histological analysis of GCs was not performed in these mice. The independence of T cell–dependent humoral responses on signaling via TRAF5 was confirmed by the finding that production of antigen-specific IgM and IgG was not affected in TRAF5 knockout mice immunized with high-dose NP–CGG (Nakano et al., 1999). Of note, when these mice were immunized with low-dose NP–CGG, the level of antigen-specific IgM was increased, whereas the level of antigen-specific IgG was unchanged. Suprisingly, TRAF5 knockout mice exhibited a defect in affinity maturation at 3 weeks. In light of the finding that TRAF5 is involved in NF-␬B activation and induction of CD80 and CD86 following CD40 engagement on B cells, this result suggests that TRAF5 may be involved indirectly in events that are crucial for the CD40-mediated rescue signal that centrocytes receive upon presentation of internalized antigen to T cells. Finally, results from genetically altered mice indicate that multimers of (TRAF3)3 and (TRAF5)x –(TRAF3)y mediate CD40 signals involved in the development of T cell–dependent humoral responses. Preliminary data suggest that TRAF2 does not play a role in this process, but detailed experiments have not been carried out to validify this claim. The role of either TRAF1 or TRAF6 in the development of T cell–dependent or –independent responses has not been examined to date. Preliminary experiments have been performed to delineate the intermediate signaling molecules that mediate Ig secretion following CD40 engagement. In this regard, IgM and IgG secretion induced in vitro from splenic B cells following CD40 engagement in the presence of IL-4 and IL-5 was completely abrogated by nonfunctional NIK expressed in B cells from aly/ aly mice compared to their heterozygote (aly/⫹) littermates (Garceau et al., 2000). This result must be interpreted with caution, since IL-4 has been shown to favor the association of CD40 with TRAF2 over other TRAFs and since costimulation with cytokines has previously masked a CD40-mediated signaling defect (Hennet et al., 1998). In addition, the role of NIK in Ig secretion from splenic B cells may be to mediate the proliferation that is necessary for splenic plasmablasts to secrete Ig. By contrast, MKK4 that is activated by the MAP3K, MEKK1, does not play


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a role in the T cell–dependent or –independent humoral response to vesicular stomatitis virus (VSV). Specifically, the size and number of GCs containing VSV-specific B cells were not different in MKK4 knockout mice or their wild-type counterparts. Additionally, levels of antigen-specific IgM and IgG were not affected by the absence of MKK4 (Nishina et al., 1997). In summary, the specific TRAFs involved in antibody responses in vivo have not been fully delineated, although the currently available data indicate that TRAFs 3 and 5, but not TRAF2, play a role in T cell–dependent responses, whereas neither TRAF3 nor TRAF5 is required for T cell– independent responses. The role of TRAF1 or TRAF6 in CD40-mediated events during T cell–dependent or –independent responses remains to be delineated. ii. IgH class switching. The role of TRAFs in CD40-induced IgH class switching was investigated in M12 murine B lymphoma cells transiently transfected with reporter constructs for either the Ig–C웂1 or Ig–C␧ germline promoters. Initial experiments were performed following engagement of overexpressed wild-type human CD40 or altered CD40 with the proximal TRAF6 binding site, the distal binding site for TRAFs 1, 2, 3, and 5, or the specific binding site for TRAF3 mutated so that they are unable to bind TRAFs (Leo et al., 1999b). Mutation of the glutamic acid crucial for TRAF6 binding (235E 씮 A) so that only TRAFs 1, 2, 3, and 5 can bind, decreased activity of the Ig–C웂1 promoter to 20% and activity of the Ig–C␧ promoter to 50% of the level obtained with wild-type CD40. Similar results were obtained with CD40 molecules that lacked the distal TRAF binding site (254T 씮 A) so that they could only bind TRAF6 or the distinct binding site for TRAF3 (263Q 씮 A) so that only TRAFs 1, 2, and 6 could bind. Together, these observations indicate that both the proximal and distal TRAF binding sites contribute to activity of the germ-line promoters of either Ig–C웂1 or Ig–C␧. The relevance of the observation that TRAFs binding to the membraneproximal and -distal sites on CD40 play a role in the germ-line transcription of both Ig–C웂1 and Ig–C␧ was confirmed in experiments in which wildtype CD40 and TRAF⌬N mutants that lacked the N-terminal RING and zinc finger domains were overexpressed in M12 lymphoma B cells. Whereas TRAF2⌬N decreased CD40-induced Ig–C웂1 promoter activity by 30%, germ-line transcription was decreased 앑80% by ⌬N mutants of TRAF 3, 5, or 6. By contrast, TRAF6⌬N decreased CD40-induced Ig–C␧ promoter activity by 앑50%, whereas ⌬N mutants of TRAF 2, 3, or 5 decreased this functional outcome by 앑25%. Finally, overexpression of either TRAF2 or TRAF6, but not TRAF3 or TRAF5 alone, induced transcription of both CH promoters that was blocked by a dominant negative version of I␬B. Of note, overexpression of TRAF5 did not induce germ-line promoter activity

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of Ig–C웂1 or Ig–C␧, although it did induce activation of NF-␬B. Since mutation of the TRAF3 binding site or expression of ⌬N mutants of TRAF3 or TRAF5 interfered with CD40-induced CH transcription, it is likely that (TRAF3)x –(TRAF5)y heteromultimers, but not (TRAF3)3 or (TRAF5)3 multimers, mediate CD40-induced Ig–C웂1 or Ig–C␧ promoter activity. Of note, (TRAF3)x –(TRAF5)y heteromultimers may play a larger role in promoter activity of Ig–C웂1 compared to Ig–C␧ in M12 murine B lymphoma cells. In this regard, it should be noted that the p50/p65 and p50/ RelB dimers contribute to transactivation of the Ig–C웂1 promoter following CD40 ligation (Lin and Stavnezer, 1996). By contrast, p50/RelC dimers mediate transcriptional activation of the Ig–C␧ promoter (Iciek et al., 1997). Moreover, TRAFs 6 and 2 play major and minor roles, respectively, in CH transcription of both germ-line promoters in this cell line where all TRAFs are expressed. The role of TRAF1 in IgH class switching has not been examined. It is important to consider these data in the context of the results of experiments examining the role of TRAFs in the development of T cell–dependent humoral responses in genetically altered mice. The findings described above indicate that TRAFs 2, 3, 5, and 6 all play a role the in promoter activity of Ig–C웂1 and Ig–C␧ in M12 lymphoma B cells that express all of these TRAFs. By contrast, production of antigen-specific IgG1 following immunization with a T cell–dependent antigen was not affected in mice genetically deficient in TRAF5 expression, suggesting that activation of the Ig–C웂1 promoter was intact in the absence of TRAF5. Moreover, production of antigen-specific IgG1 following immunization with a T cell–dependent antigen was abrogated in TRAF3 knockout mice. These data indicate that (TRAF3)3 multimers, in the absence of (TRAF3)x – (TRAF5)y multimers, can mediate IgH class switching. These data are in apparent contradiction with the finding that overexpression of TRAF3 did not induce the germ-line promoter activity of Ig–C웂1. Experiments to resolve this apparent conflict remain to be carried out. Examination of the potential role of IgH class switching has not been examined in mice genetically altered in expression of TRAF 1, 2, or 6. Finally, it should be noted that isotype switching to IgG following immunization with the T cell–dependent antigen VSV was intact in mice deficient in an upstream regulator of JNK, MKK4 (Nishina et al., 1997). 3. Integration of TRAF-Mediated Signals Controlling Life or Death Regulation of whether a B cell proliferates or undergoes apoptosis following engagement of CD40 appears to be dependent on a complex network of interacting signaling cascades. For example, recent experiments have demonstrated that CD40 associates with a RIP family member, RIP2/


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Cardiak/RICK (McCarthy et al., 1998), which has the capacity to induce NF-␬B activity that mediates CD40-induced proliferation or caspase activation that can facilitate CD40-induced apoptosis. In this regard, CD40induced apotosis can be blocked by zVAD–fmk (Grammer et al., 1998), a caspase inhibitor. In addition, RIP2/Cardiak/RICK can activate JNK that can contribute to a variety of signaling cascades that can mediate life or death, depending on the other signaling molecules activated. Activation of JNK following association of TRAF2 with the ROI-activated MAP3K ASK1 may mediate CD40-induced apoptosis of B cells, whereas simultaneous activation of ERK and JNK can induce AP-1 that can mediate B cell proliferation. A role of RIP2/Cardiak/RICK in CD40-mediated signaling events was suggested by immunoprecipitation of CD40-associated molecules in 293 HEK cells. RIP2/Cardiak/RICK was found to associate with CD40 via TRAF 5, 6, or 1 alone, respectively, or in association with TRAF2. However, RIP2/Cardiak/RICK apparently does not directly associate with TRAF2 or TRAF3. Overexpression of RIP2/Cardiak/RICK induces nuclear translocation of NF-␬B, JNK activation, and caspase-mediated apoptosis by independent mechanisms. Suprisingly, even though RIP2/Cardiak/RICK is a serine/threonine kinase, abrogation of kinase activity had no effect on these functional outcomes. The mechanisms mediating the signaling cascades downstream of RIP2/Cardiak/RICK are not straightforward. For example, activation of NF-␬B mediated by RIP2/Cardiak/RICK is blocked by expression of TRAF2 or TRAF6 lacking the N-terminal RING and zinc finger domains. In addition, this activity is blocked by a kinase-dead version of NIK. Moreover, NF-␬B activity induced by RIP2/Cardiak/RICK involves the associated CED4/Apaf1 family member, CARD4 (Bertin et al., 1999), in that CARD4-induced nuclear translocation of NF-␬B is blocked by a dominant negative version of RIP2/Cardiak/RICK (residues 455–541) containing its CARD domain but lacking the region that associates with TRAFs. Together, these results indicate that CD40 engagement recruits (TRAF1)3, (TRAF1)x –(TRAF2)y, (TRAF5)3, or (TRAF6)3 multimers. In turn, these TRAF multimers recruit NIK as well as RIP2/Cardiak/RICK in association with CARD4. Importantly, the kinase domain of NIK and the N-terminal RING and zinc fingers of the TRAFs are essential in order for the RIP2/Cardiak/RICK–CARD4 complex to induce NF-␬B activity. By contrast, RIP2/Cardiak/RICK induces JNK activation by a mechanism that is independent of CARD4 and the N-terminal RING and zinc finger domains of the TRAFs. Finally, apoptosis induced by RIP2/Cardiak/RICK is caspase mediated. Of note, apoptosis induced by this molecule can be blocked with the caspase inhibitor zVAD–fmk or by other inhibitors of apoptosis, such as bclxL or cIAP.

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Functionally, any degree of CD40 engagement on naive B cells mediates proliferation, but never apoptosis. By contrast, low levels of CD40 ligation on an activated B cell induce proliferation, but higher degrees of engagement elicit apoptosis (Bergman et al., 1996; Miyashita et al., 1997). This observation suggests that there are fundamental differences between resting naive B cells and activated differentiated B cells that account for this dichotomy in functional outcome. One possibility is availability of signaling molecules. For example, resting naive B cells express TRAFs 5 and 6, whereas activated differentiated B cells also express TRAF 1, 2, or 3 depending on their milieu (our unpublished observations). Some of this might relate to antecedent signaling events such as CD40-induced TRAF1 expression. In addition, undefined signals may have induced alternative splicing of TRAF2 that can down-regulate wild-type TRAF2-induced nuclear translocation of NF-␬B dimers and directly activate JNK. Since TRAF2-induced JNK activation is partially mediated by the ROI-induced MAP3K ASK1, apoptosis may be favored, as ASK1 has been demonstrated to induce apoptosis upon overexpression (Ichijo et al., 1997). One important feature of the signaling cascades leading to life or death is that the final outcome appears to be determined by the suppression of alternative options. For example, nuclear translocation of NF-␬B induces proliferation directly as well as the transcription of proteins with antiapoptotic function, such as bclxL (Choi et al., 1995; Merino et al., 1995; H. Wang et al., 1996), A1/bfl1 (Kuss et al., 1999; H. H. Lee et al., 1999a), and cIAPs (Craxton et al., 1998). By contrast, CD40 ligation induces NF-␬B dependent transcription of the A20 protein that blocks subsequent NF-␬B–induced gene expression mediated by TRAF2 or TRAF6 (Heyninck and Beyaert, 1999; Heyninck et al., 1999 Krikos et al., 1992; Natoli et al., 1997; Sarma et al., 1996; Song et al., 1996). In addition, the functional outcome of CD40 engagement with respect to apoptosis or proliferation may be determined by a balance between caspase activation and NF-␬B/ or AP-1 activation, since pharmacological inhibitors of signaling molecules contributing to activation of these transcription factors interfered with CD40-induced proliferation. Moreover, the findings that the CD40associated RIP2/Cardiak/RICK molecule induces apoptosis by a caspasedependent mechanism (McCarthy et al., 1998) and that zVAD–fmk inhibits CD40-mediated apoptosis (Grammer et al., 1998) suggest that the life-ordeath decision following CD40 engagement may be influenced by caspasedependent cleavage of proteins involved in the signaling cascades leading to NF-␬B– and AP-1–mediated proliferation, such as MEKK1 and ERK (Widmann et al., 1998). Alternatively, or perhaps in concert, CD40mediated apoptosis may involve activation of the caspases that directly lead to apoptosis.


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D. CD40-MEDIATED SIGNALING EVENTS WHOSE TRAF INVOLVEMENT IS UNCLEAR Since the cytoplasmic domain of CD40 lacks intrinsic enzymatic activity, adapter molecules must mediate the early biochemical events that occur following ligation of CD40 (Fig. 27), such as phosphorylation of tyrosine kinases lyn, fyn, blk, syk, btk (Faris et al., 1994; Ren et al., 1994; Uckun et al., 1991), and JAK3 (Hanissian and Geha, 1997; Jabara et al., 1998; Karras et al., 1997; Tortolani et al., 1995); PI3K (Aagaard-Tillery and Jelinek, 1996; Padmore et al., 1997); phospholipase C웂2 (Gulbins et al., 1996; van Kooten and Banchereau, 1996); and formation of IP3 (Uckun et al., 1991) and diacylglycerol following hydrolysis of PIP2. Increases in intracellular levels of calcium (Klaus et al., 1994) and cAMP (Kato et al., 1994; Knox et al., 1993; Pollok et al., 1991) have also been observed following CD40 engagement on B cells. Of note, JAK3 has been observed to bind CD40 directly (Hanissian and Geha, 1997), but the adapter molecules that mediate JAK3 phosphorylation following receptor engagement remain to be delineated. The role of TRAFs, if any, in the CD40-induced early biochemical events described above remain to be elucidated.

FIG. 27. CD40-mediated signaling events whose TRAF involvement is unclear. For abbreviations, see text.

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1. JAKs and STATs Controversy exists over the role of JAK–STAT pathways in CD40mediated signaling in B cells. STATs exist as latent cytoplasmic proteins that are translocated to the nucleus following phosphorylation by members of the JAK family of tyrosine kinases activated upon receptor engagement. JAKs bind receptors following engagement, are phosphorylated, and then undergo autophosphorylation, which leads to phosphorylation and activation of STATs. Whereas CD40 ligation has been reported to induce phosphorylation and nuclear translocation of STAT6 by a JAK1- to JAK3independent pathway in resting murine splenic B cells (Karras et al., 1997), CD40 engagement also induces activation of the JAK3–STAT3 pathway in EBV-negative B cell lines (BJAB, Daudi, and Ramos) and unseparated, activated tonsillar B cells (Hanissian and Geha, 1997). By contrast, activation of JAK1, JAK2, and Tyk2 was not observed in EBV-negative B cell lines (BJAB, Daudi, and Ramos) or in unseparated, activated tonsillar B cells (Hanissian and Geha, 1997). In contrast, activation of the JAK3– STAT3 pathway was not observed following CD40 ligation on small, resting B cells from murine spleen (Karras et al., 1997) or human tonsil (Revy et al., 1999). Of note, constitutive activation of STAT3 has been observed in EBV-positive B cell lines that express the functionally active version of CD40, LMP1 (Cherry, Namalva, Akata, and Daudi), but not in EBVnegative B cell lines (DG75 and BL41) that do not express LMP1 (WeberNordt et al., 1996). The presence of activated STAT6 was not examined, however. Binding of JAK3 to CD40 and activation of JAK3 as evidenced by phosphorylation may be two separate phenomena. For example, immunoprecipitation experiments demonstrated that JAK3 associates with CD40 in B cell lines, unseparated, activated tonsillar B cells (Hanissian and Geha, 1997), and purified small, resting tonsillar B cells (Revy et al., 1999), but that CD40 engagement only resulted in phosphorylation of JAK3 in activated B cells from the periphery, the tonsil, or EBV-negative B cell lines such as BJAB or Ramos (Hanissian and Geha, 1997; Matsumoto et al., 1999). In addition, JAK3 is often expressed at a lower level in resting B cells than in activated tonsillar B cells and can be induced following a variety of activation signals, including CD40 engagement (Tortolani et al., 1995). Mutational analysis of CD40 has demonstrated that substitutions for P227 or P229 abrogate, and substitutions for P202 decrease, JAK3 binding following receptor ligation (Hanissian and Geha, 1997). The relevance for this finding is supported by the observation that an amino acid sequence identical to the box1 JAK-binding motif is contained within amino acids 222–229 of


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the cytoplasmic tail of CD40 (222PTNKAPAP229). Suprisingly, mutating T254 that is essential for binding TRAFs 1, 2, 3, and 5 also abrogated association of JAK3 with CD40 following receptor engagement (Hanissian and Geha, 1997). This amino acid is proximal to a more membrane-distal stretch of amino acids (260–270; 260PVTQEDGKESR270) that contains homologous sequence to the box2 motif required for phosphorylation and activation of JAKs. Whether this box2-related sequence has the ability to mediate JAK phosphorylation when JAK3 is bound to the box1 motif of CD40 remains to be tested. An alternative explanation is provided by the finding that TRAF3 contains a consensus JAK phosphorylation site and that CD40induced phosphorylation of JAK3 is not observed in TRAF3-negative cells. These observations have led to the hypothesis that TRAF3 expression may be required for JAK3 to transduce downstream signals including phosphorylation and nuclear translocation of STATs (Revy et al., 1999). Further evidence for this possibility is supplied by the observation that mutation of T254 that is essential for binding of TRAF3 to the cytoplasmic tail of CD40 also affects binding of JAK3 to CD40 as well as JAK3 phosphorylation induced following CD40 engagement (Hanissian and Geha, 1997). The role of JAK3-induced signaling pathways in functional outcomes induced following ligation of CD40 is controversial. In this regard, CD40 engagement on BI-141 murine T lymphoma cells that overexpressed wildtype CD40 or a mutant CD40 molecule that cannot bind JAK3 because of a mutation at P229 (Hanissian and Geha, 1997) induced JNK activation and nuclear translocation of NF-␬B equivalently. In addition, stimulation of murine WEHI B cells overexpressing either wild-type human CD40 or a mutant CD40 molecule that cannot bind JAK3 with anti-human CD40 mAb (G28.5) induced equivalent expression of CD54. This result is in agreement with the finding that engagement of CD40 on the surface of B cells isolated from SCID patients genetically deficient in JAK3 expression resulted in induction of CD54 that was equivalent to that expressed by B cells isolated from control subjects ( Jabara et al., 1998). Moreover, serum Ig levels in these patients were comparable to those in normal control subjects. Furthermore, CD40-induced proliferation, isotype switching, and induction of surface expression of CD23, CD80/B7-1, and LT-움 on B cells from JAK3-deficient SCID patients were equivalent to those in B cells purified from normal control subjects. By contrast, these results are in apparent conflict with the initial finding in EBV-negative BJAB B cells that engagement of a transfected chimeric CD8–CD40 molecule consisting of the extracellular domain of CD8 fused to the intracellular domain of CD40 mutated at P229 was not able to induce CD54 expression observed when a chimeric CD8–CD40wild type molecule was engaged (Hanissian and Geha, 1997). This study also reported that abrogation of JAK3 binding to

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CD40 with the P229 mutant interfered with the ability of CD40 to induce surface expression of CD23 and LT-움. Apparently conflicting data from these studies have been interpreted in two ways. First, JAK3 may not have been expressed in the resting peripheral blood B cells isolated from JAK-deficient SCID patients or in WEHI murine B cells, since a previous study has shown that CD40 engagement induces JAK3 expression in resting cells (Tortolani et al., 1995). JAK3 expression was not examined in either of these B cell sources. Alternatively, examination of CD40-mediated signaling pathways in BJAB B cells transfected with a chimeric CD8–CD40 molecule but also expressing wild-type CD40 may be inherently flawed, since the chimeric molecule may not be able to effectively recruit signaling molecules such as JAK3 that may have a preference for binding the wild-type CD40. Definitive experiments designed to delineate the role of JAK3-mediated signaling pathways in B cell activation mediated following CD40 engagement remain to be carried out. 2. src, ZAP-70, and tec Family Kinases src, ZAP-70, and tek family kinases such as lyn, syk, and btk are activated following CD40 engagement on B cells (Faris et al., 1994; Ren et al., 1994; Uckun et al., 1991). Signaling cascades downstream of these kinases mediated by activated phospholipase C웂2 are also induced following CD40 engagement on B cells (Gulbins et al., 1996; van Kooten and Banchereau, 1996), but experiments to connect these two observations have not been carried out. Specifically, orchestrated activation of these kinases has been shown to induce activation of phospholipase C웂2, leading to production of IP3 and diacylglycerol, increased intracellular calcium, calcineurinmediated nuclear translocation of NF-ATc, and PKC-induced activation of the RAF1-mediated MKK1–ERK or IKK–NF-␬B pathway. In this regard, activated syk phosphorylates an adapter molecule, BLNK/SLP-65/ BASH (reviewed in Kurosaki and Tsukada, 2000), that has been shown to bring signaling molecules into close proximity for optimal activation of phospholipase C웂2. Moreover, two other initial events must occur besides BLNK/SLP-65/ BASH activation by syk. First, PI3K that is activated by a RAS-dependent mechanism following CD40 ligation (Aagaard-Tillery and Jelinek, 1996; Padmore et al., 1997; Ren et al., 1994) must convert PIP2 in the membrane to PIP3. In turn, PIP3 binds the pleckstrin homology domain of btk so that it is in close proximity to membrane-associated lyn. Second, CD40 must activate lyn by an undefined mechanism (Faris et al., 1994; Ren et al., 1994). Activated lyn associates with btk via its SH3 domain (Alexandropoulos et al., 1995; Cheng et al., 1994; Yang et al., 1995) and phosphorylates btk on its SH2 domain (Rawlings et al., 1996) so that it can associate with BLNK/


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SLP-65/BASH that has been phosphorylated by syk. This event brings phospholipase C웂2 bound to BLNK/SLP-65/BASH via its SH2 domains in close proximity with activated btk and activated syk that have both been shown to be essential for optimal activation of this phospholipase. Finally, it should be noted that it is not currently known whether CD40-induced activation of PI3K, tyrosine kinases, or phospholipase C웂2–mediated signaling cascades is mediated by known TRAFs or is TRAF independent. In addition, other signaling outcomes that are induced following CD40 ligation by an unknown mechanism and have been shown to be mediated by the BLNK/SLP-65/BASH adapter molecule, such as VAV–RAC–PAK– MEKK1 leading to MKK4–JNK or IKK–NF-␬B or SHC–GRB2–SOS– RAS–RAF1 leading to MKK1–ERK or IKK–NF-␬B, remain to be defined as TRAF dependent or TRAF independent. In summary, activation of src, ZAP-70, and tec family kinases following CD40 engagement on B cells may mediate signaling cascades, leading to NF-␬B, AP-1, or NF-AT, but the involvement of TRAFs in their activation remains to be defined. Activation of PI3K following CD40 ligation (Aagaard-Tillery and Jelinek, 1996; Padmore et al., 1997) by a RAS-dependent mechanism mediates the conversion of membrane-associated PIP2 to PIP3 that is essential to bring btk into close proximity with lyn. It should be noted that PI3K binds the 14-3-3 adapter protein that can associate with TRAF2 via A20. The relevance of this association to CD40-induced PI3K activation has not been determined. PI3K consists of a catalytic subunit of 앑110 kDa and a tightly associated regulatory subunit of 85 kDa that can be alternatively spliced to either 50 or 55 kDa. The functional relevance of PI3K to CD40-mediated B cell activation was first elucidated by the observation that CD154-induced proliferation and differentiation to Ig secreting cells were decreased in the presence of pharmacological inhibitors of PI3K, Ly290042 or Wortmannin (Aagaard-Tillery and Jelinek, 1996). In addition, serum levels of Ig were decreased in p85 PI3K knockout mice compared to their wild-type littermates, although isotype switching was intact. This finding was confirmed by the observation that CD40-induced proliferation was impaired in B cells isolated from p85 PI3K knockout mice, compared with wild-type littermates (Fruman et al., 1999). By contrast, CD40-mediated rescue of purified lymph node B cells from spontaneous apoptosis was unaffected by the absence of p85 PI3K. Following association with PIP3, btk is activated in a lyn-dependent manner after CD40 ligation on B cells. Of note, btk is expressed in freshly isolated tonsillar B cells as well as a variety of lymphoma (i.e., Ramos)– and EBV-infected lymphoblastoid B cell lines, representing all levels of maturity except for that of a differentiated plasmacytoma/myeloma cell line (Genevier et al., 1994). Optimal phospholipase C웂2 activation requires

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phosphorylation by btk as well as by syk. Of note, natural mutations of btk have been observed in the xid mouse and XLA humans (reviewed in Kawakami et al., 1999; Mohamed et al., 1999; Rawlings, 1999). Moreover, the point of disruption in CD40-mediated signaling in xid B cells may be at the stage of btk-mediated phospholipase C웂2 activation, since the pleckstrin homology domain of btk required for phospholipase C웂2 activation is mutated in these mice. XLA patients exhibit a variety of btk mutations that render the kinase nonfunctional. The role of btk in CD40-mediated signaling events has been studied using freshly isolated B cells or B cell lines generated from xid mice or XLA humans deficient in functional btk expression. Examination of the role of btk in induction of activation antigens following CD40 engagement has revealed that xid B cells are deficient in CD40-mediated up-regulation of either CD80 or CD86 (Hasbold and Klaus, 1994), whereas up-regulation of MHC class II following CD40 engagement is intact in xid B cells (Hasbold and Klaus, 1994) and CD40-induced expression of CD23 is intact in either xid or XLA B cells (Hasbold and Klaus, 1994; Nonoyama et al., 1998). Moreover, examination of the role of btk in CD40-mediated signals required for development of the humoral response has indicated that although xid mice or XLA humans produce lower amounts of antigenspecific antibody following immunization with the T cell–dependent antigen bacteriophage ␾X174 compared to normal controls, xid mice but not XLA humans underwent isotype switching following secondary immunization (Nonoyama et al., 1998). Finally, the role of btk in CD40-induced proliferation is controversial. Whereas four reports have demonstrated that xid B cells have a defective proliferative response to CD40 engagement (Fruman et al., 1999; Goldstein, et al., 1996; Hasbold and Klaus, 1994; Wu et al., 1995), two reports have shown that xid B cells proliferate following CD40 engagement as well as their wild-type counterparts (Anderson et al., 1996; Johnson-Leger et al., 1997). One study that examined CD40-mediated proliferation costimulated by IL-4 found that proliferation, of XLA B cells was similar to that of B cells isolated from control subjects (Nonoyama et al., 1998). A caveat to this result is that IL-4 may mask the CD40-signaling defect in proliferation, as has been seen in B cells isolated from xid mice (Fruman et al., 1999). In summary, btk plays a role in CD40-induced expression of CD80 and CD86, but is not involved in induction of MHC class II or CD23 following CD40 ligation on B cells. In addition, CD40-mediated signals required for development of the humoral response are impaired in the absence of btk. Specifically, Ig production is lower in animals or humans that have defective btk expression. Moreover, btk may mediate CD40-induced prolif-


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eration in some B cells; the role of btk may depend on the activation or differentiation state of the B cell. The src family kinase lyn has also been demonstrated to be activated following CD40 ligation (Ren et al., 1994) and to be critically involved in CD40-induced B cell proliferation (Nishizumi et al., 1995; J. Wang et al., 1996a). In addition to a defect in CD40-induced B cell proliferation, young mice genetically deficient in lyn have fewer B cells than wild-type mice and do not form GC structures in response to a T cell–dependent antigen such as NP–CGG (Nishizumi et al., 1995). Despite defects in CD40induced proliferation and in the formation of CD154–CD40–dependent GCs, lyn-deficient mice undergo a humoral response to T cell–dependent antigen that is largely intact. Specifically, the number and affinity of IgG1expressing splenic B cells specific for the immunizing antigen are comparable between lyn-deficient and wild-type mice. In addition, somatic hypermutation to the T cell–dependent antigen is intact in lyn knockout mice in that mutational frequency as well as R/S ratio in CDRs is equivalent to wild-type mice. Similar observations have been made for mice deficient in both lyn and fyn (Kato et al., 1998). These findings indicate that although lyn is involved in the CD40-mediated signaling pathway resulting in proliferation and GC formation, the signaling cascade utilized by CD40 for isotype switching, selection, somatic hypermutation, and secretion of IgG1 is independent of lyn activation. Together, these results suggest that CD40induced proliferation of B cells may be mediated by a lyn–btk–PI3K– dependent pathway. By contrast, the signaling cascade required for antigenspecific Ig production is independent of lyn activation. The roles of btk and PI3K in this functional outcome of CD40 engagement remain to be examined. It is notable that serum levels of IgM are elevated in lyn knockout mice compared to their wild-type littermates. In addition, aged lyn-deficient mice have increased numbers of splenic B cells that secrete IgM autoantibodies and contribute to a murine lupus-like syndrome (Nishizumi et al., 1995; J. Wang et al., 1996a). Of interest, induction of CD95 following CD40 ligation is partially impaired in mice genetically deficient in lyn ( J. Wang et al., 1996b). Although the relevance of CD95-induced apoptosis to B cell function in vivo has been questioned, the finding that mice deficient in lyn have elevated levels of IgM, including autoantibodies, suggests the possibility that CD40-induced growth arrest or apoptosis may be impaired in the absence of lyn signaling. In this regard, although lyn mediates sIg-induced growth arrest (Scheuermann et al., 1994), the role of this src family kinase in CD40-induced growth arrest or apoptosis has not been examined.

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3. cAMP Controversy exists over the role of cAMP-mediated signaling pathways in B cells following CD40 engagement. Whereas three studies demonstrated that CD40 stimulation of small, resting B cells from human tonsil or murine spleen increased levels of intracellular cAMP (Kato et al., 1994; Knox et al., 1993; Pollok et al., 1991), two studies did not observe changes in the levels of intracellular cAMP following stimulation of murine splenic B cells with CD154-expressing membranes from Th1 clones (Marshall et al., 1994) or a recombinant CD8–CD154 fusion protein (Baixeras et al., 1996). Further studies reported that a pharmacological inhibitor of protein kinase A (PKA), H-89, interfered with CD40-induced proliferation of murine splenic B cells after 3 days of culture but not with proliferation of human peripheral blood B cells after 10 days of culture. This result as well as the apparent contradictory data regarding whether CD40 ligation induced cAMP production may have to do with the timing of the experiment. For example, several reports have indicated that the optimal time to assay for CD40-induced proliferation of human peripheral blood B cells and the effects of various reagents on this phenomenon is at day 3 of culture. The effect of a pharmacological reagent on CD40-induced proliferation may not be apparent late after in vitro culture, such as at day 10. Further experiments examined the role of cAMP in functional outcomes induced following stimulation of the M12 murine lymphoma B cell line that had been transfected with a tetracycline-regulatable plasmid expressing a dominant negative form of the PKA regulatory subunit type I (PKA-RG324D ) with recombinant CD8–CD154 (Goldstein et al., 1997). Expression of this dominant negative PKA had no effect on CD40-mediated growth inhibition or induction of CD23, CD54, CD11a/CD18, CD80, or CD95 in M12 B cells. By contrast, addition of db-cAMP to CD40-stimulated M12 B cells costimulated CD80 expression and inhibited induction of CD23, CD54, and CD95. Finally, induction of cAMP with prostaglandin E2, forskolin, or db-cAMP in naive B cells isolated from cord blood inhibited CD154dependent Ig secretion, but had no effect on CD154-dependent proliferation. Similarly, agents that increase cAMP-inhibited Ig secretion, but not proliferation, of adult peripheral B cells induced by the human polyclonal activator SAC (Kimata et al., 1994; Simkin et al., 1987; Splawski and Lipsky, 1994), whose mechanism of action has been shown to be partially dependent upon homotypic CD154–CD40 interactions between B cells (Grammer et al., 1995). By contrast, agents that increase cAMP had no effect on Ig secretion or proliferation induced by high-level ligation of CD40 on adult peripheral B cells that contain both naive and memory cells (Splawski and Lipsky, 1994). Together, these results indicate that


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CD40 engagement may increase intracellular levels of cAMP in some circumstances that may depend on the activation and differentiation state of the cell. In addition, cAMP–PKA signaling does not play a role in CD40mediated induction of activation antigens (CD23, CD54, CD11a/CD18, CD80 and CD95) or growth inhibition but may play a role in CD40induced proliferation. Costimulatory signals that induce cAMP may play a role in regulating the functional outcomes of CD40 signaling by costimulating CD80 expression and inhibiting Ig secretion and induction of CD23, CD54, and CD95. By contrast, costimulatory signals that induce cAMP have no effect on CD40-induced proliferation. E. REVERSE SIGNALING THROUGH CD154 Experiments in a variety of systems with different cell types have demonstrated that CD154 functions not only as a ligand for CD40 but also as a direct signaling molecule that induces functional outcomes. The most recent evidence is provided by the finding that anti-CD154–conjugated Sepharose beads costimulate B cell responses induced by engaging Ig (Grammer et al., 1999a). By contrast, functional outcomes induced by CD40 ligation were not costimulated by anti-CD154–conjugated Sepharose beads even though CD154 was expressed on the surface of the B cell. Costimulation was not observed either, because the initial signaling was insufficient to induce this pathway of costimulation or because the CD40 and CD154 signaling pathways are redundant. The finding that CD154 is a direct signaling receptor on the surface of B cells is consistent with the previous finding that injection of a CD40.Ig construct into a mouse genetically deficient in CD40 induced small, but quantifiable, GCs after immunization and also enhanced in vivo production of IgM following immunization of normal mice (Gray et al., 1994; Van Essen et al., 1995). Moreover, some reports have documented the capacity of CD154 ligation to induce a variety of proximal signaling pathways in other cell types (Blotta et al., Brenner et al., 1997a,b; Cayabyab et al., 1994; Grammer et al., 1999a; 1996; Koppenhoefer et al., 1997; Macaulay et al., 1997). Finally, it should be noted that there is precedence for bidirectional signaling of B cells through other coreceptors of the TNF superfamily, such as OX40/ CD134–OX40L (Flynn et al., 1998; Stuber and Strober, 1995; Stuber et al., 1996) as well as CD70–CD27 (Lens et al., 1999). VI. Conclusion

The signaling and functional outcomes resulting from interactions between CD154 and CD40 are complex and are the result of the integration of a number of conditions, including the differentiation states of the cells

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involved, the expression level of each of the receptors, and the milieu in which the interactions occur. Delineation of these variables is essential to completely understand the role of CD154 and CD40 in normal and abnormal responses of the immune system. ACKNOWLEDGMENTS We thank Nancy Heard, Amrie Dowling-Otto, and Nicole Walters for skillful assistance in the preparation of the manuscript. A. C. Grammer was supported by an Arthritis Foundation postdoctoral fellowship and by National Institutes of Health IRP award RA-AR-0-1001.

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Yoshimoto, T., Nagase, H., Ishida, T., Inoue, J., and Nariuchi, H. (1997). Induction of interleukin-12 p40 transcript by CD40 ligation via activation of nuclear factor–kappaB. Eur. J. Immunol. 27, 3461–3470. Younes, A., Snell, V., Consoli, U., Clodi, K., Zhao, S., Palmer, J. L., Thomas, E. K., Armitage, R. J., and Andreeff, M. (1998). Elevated levels of biologically active soluble CD40 ligand in the serum of patients with chronic lymphocytic leukaemia. Br. J. Haematol. 100, 135–141. Young, L. S., Eliopoulos, A. G., Gallagher, N. J., and Dawson, C. W. (1998). CD40 and epithelial cells: across the great divide. Immunol. Today 19, 502–506. Yu, P. W., Huang, B. C., Shen, M., Quast, J., Chan, E., Xu, X., Nolan, G. P., Payan, D. G., and Luo, Y. (1999). Identification of RIP3, a RIP-like kinase that activates apoptosis and NFkappaB. Curr. Biol. 9, 539–542. Yuasa, T., Ohno, S., Kehrl, J. H., and Kyriakis, J. M. (1998). Tumor necrosis factor signaling to stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase ( JNK) and p38. Germinal center kinase couples TRAF2 to mitogen-activated protein kinase/ERK kinase kinase 1 and SAPK while receptor interacting protein associates with a mitogen-activated protein kinase kinase kinase upstream of MKK6 and p38. J. Biol. Chem. 273, 22681–22692. Zandi, E., and Karin, M. (1999). Bridging the gap: composition, regulation and physiological function of the IkappaB kinase complex. Mol. Cell. Biol. 19, 4547–4551. Zanke, B. W., Rubie, E. A., Winnett, E., Chan, J., Randall, S., Parsons, M., Boudreau, K., McInnis, M., Yan, M., Templeton, D. J., and Woodgett, J. R. (1996). Mammalian mitogenactivated protein kinase pathways are regulated through formation of specific-activator complexes. J. Biol. Chem. 271, 29876–29881. Zhang, Y., Cao, H. J., Graf, B., Meekins, H., Smith, T. J., and Phipps, R. P. (1998). CD40 engagement up-regulates cyclooxygenase-2 expression and prostaglandin E2 production in human lung fibroblasts. J. Immunol. 160, 1053–1057. Zhou, L., Ismaili, J., Stordeur, P., Thielemans, K., Goldman, M., and Pradier, O. (1999). Inhibition of the CD40 pathway of monocyte activation by triazolopyrimidine. Clin. Immunol. 93, 232–238. Zimmermann, S., and Moelling, K. (1999). Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286, 1741–1744. Zubler, R. H., Werner-Favre, C., Wen, L., Sekita, K.-I., and Straub, C. (1987). Theoretical and practical aspects of B-cell activation: murine and human systems. Immunol. Rev. 99, 281–299. Zupo, S., Dono, M., Azzoni, L., Chiorazzi, N., and Ferrerini, M. (1991). Evidence for differential responsiveness of human CD5⫹ and CD5⫺ subsets to T cell–independent mitogens. Eur. J. Immunol. 21, 351–359.


Cell Death Control in Lymphocytes KIM NEWTON AND ANDREAS STRASSER The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

I. Introduction

Lymphocyte apoptosis is crucial to proper immune function. It removes developing lymphocytes that fail to express an antigen receptor, thereby ensuring a functional repertoire of mature B and T cells, and it maintains tolerance toward self by eliminating lymphocytes with antigen receptors that recognize autoantigens. Apoptosis also regulates the size and duration of immune responses, activated lymphocytes being killed when an infection is cleared successfully. This chapter details the signaling pathways that are known to promote apoptosis as well as their impact on lymphocyte survival at different stages of development. Particular attention is paid to the roles of death receptors and members of the Bcl-2 protein family. II. Apoptosis and the Role of Caspases

Cells within multicellular organisms have a genetically determined suicide program that can be triggered by a range of physiological signals and experimentally applied stress conditions. A family of intracellular aspartatespecific cysteine proteases called caspases activates the effector phase of this suicide program (Thornberry and Lazebnik, 1998). Activation of caspase zymogens leads to proteolytic cleavage of vital cellular constituents, which culminates in cell death. This cell death mechanism is referred to as apoptosis (Kerr et al., 1972), and it is remarkably conserved, being found in species as distantly related as humans, Drosophila melanogaster, and Caenorhabditis elegans (Bergmann et al., 1998; Meier and Evan, 1998; Metzstein et al., 1998). Common morphological and biochemical features of apoptotic cells include chromatin condensation, internucleosomal DNA fragmentation, blebbing of the plasma membrane, and phosphatidylserine exposure on the outer leaflet of the plasma membrane (Fadok et al., 1992; Kerr et al., 1972; Wyllie et al., 1980). Current evidence indicates that caspase zymogens become activated in two ways. Caspases that are synthesized with long prodomains, such as mammalian caspases 2, 8, 9, and 10 and C. elegans Ced-3, have sufficient enzymatic activity to undergo autocatalytic activation when aggregated by cytoplasmic adapter molecules (Hu et al., 1998b; Martin et al., 1998; Muzio et al., 1998; Srinivasula et al., 1998; Yang et al., 1998). A death effector domain (DED) or caspase recruitment domain (CARD) in the caspase 179


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prodomain mediates homotypic interactions with analogous regions in adapter proteins. Thus, the CARD in procaspase 2 binds to the CARD in RAIDD (also called CRADD) (Ahmad et al., 1997; Duan and Dixit, 1997), the CARD in procaspase 9 binds to that in Apaf-1 (Hu et al., 1998b; Li et al., 1997; Pan et al., 1998c), and the CARD in Ced-3 binds to that in Ced-4 (Chinnaiyan et al., 1997; Irmler et al., 1997a; Wu et al., 1997a). Procaspases 8 and 10 each have two DEDs, and one of them interacts with the DED in FADD (also called MORT1) (Boldin et al., 1996; FernandesAlnemri et al., 1996; Muzio et al., 1996; Vincenz and Dixit, 1997). Autocatalytic cleavage removes the caspase prodomain and yields the small (앑10-kDa) and large (앑20-kDa) subunits that are assembled into the fully active form (p202p102 tetramer) of the caspase. Caspases activated in this manner can then cleave and activate caspases with short prodomains, such as caspases 3, 6, and 7 (Muzio et al., 1997; Slee et al., 1999; Srinivasula et al., 1996). Activation of these downstream caspases probably amplifies the process of caspase activation because caspase 3 can cleave procaspase 9, while caspase 6 can process procaspases 8 and 10 (Slee et al., 1999; Srinivasula et al., 1998). Given this complex hierarchy of caspase activation, the key issue is to identify not which caspases are activated in response to an apoptotic stimulus, but which caspase is the first to be activated and therefore essential for execution of the death stimulus (see below). Many caspase substrates have been identified (Stroh and SchulzeOsthoff, 1998), and these include structural proteins such as gelsolin (Kothakota et al., 1997), lamin (Orth et al., 1996), and fodrin ( Ja¨nicke et al., 1998) as well as proteins involved in DNA repair and cell cycle regulation, such as poly(ADP-ribose)polymerase (Lazebnik et al., 1994), replication factor C (Rheáume et al., 1997), and the retinoblastoma protein ( Ja¨nicke et al., 1996). Recent studies have shown that DNA fragmentation in apoptotic cells is a consequence of ICAD cleavage by caspase 3 (Enari et al., 1998; Liu et al., 1997; Sakahira et al., 1998). ICAD (also called DFF) binds to and inhibits the DNA endonuclease CAD (also called CPAN), but can no longer do so when cleaved (Enari et al., 1998; Halenbeck et al., 1998; Sakahira et al., 1998). Caspase 3 cleavage of the protein Acinus appears to be important for chromatin condensation (Sahara et al., 1999). Another substrate of caspase 3 is the serine–threonine kinase PAK2. Activation of PAK2 by caspase 3 cleavage is thought to be responsible for plasma membrane blebbing and the formation of apoptotic bodies because a dominant negative PAK2 mutant is able to block these morphological changes (Rudel and Bokoch, 1997). III. Apoptosis Signaling by Death Receptors

For most apoptotic stimuli, the precise sequence of events that leads to multiple caspase zymogens being brought together by their adapter

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molecules is still being elucidated. One exception, in which the route to caspase activation has been determined, is during apoptosis signaled by certain death receptors (Figure 1). Forming a subset of the tumor necrosis factor receptor (TNF-R) family, death receptors are type I transmembrane proteins characterized by cysteine-rich repeats in their extracellular ligand binding domains, but their defining characteristic is a cytoplasmic death domain (DD) (Ashkenazi and Dixit, 1999; Wallach et al., 1996). Death receptors include mammalian Fas (also called CD95 or APO-1) (Itoh and

FIG. 1. Apoptosis signaling by death receptors. Interactions between death ligands and their receptors initiate formation of the death-inducing signaling complex (DISC). The death domain (DD) in the cytoplasmic tail of the death receptor binds to DD-containing cytoplasmic adapter proteins such as TRADD and FADD, which provide the link between death receptors and caspases. FADD has a death effector domain (DED) that interacts with one of two DEDs in the prodomain of caspase 8. The induced proximity of caspase 8 zymogens brought into the DISC promotes their autocatalytic cleavage, and this yields the fully active form of caspase 8 that cleaves and activates downstream effector caspases. Vital cellular constituents are cleaved by the activated caspases, resulting in the demise of the cell. The caspase 8 inhibitor CrmA and the broad-specificity caspase inhibitor p35 are viral proteins that can prevent death receptor–induced apoptosis.

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Nagata, 1993; Itoh et al., 1991; Trauth et al., 1989; Yonehara et al., 1989), TNF-R1 (Tartaglia et al., 1993), DR3 (also called WSL-1, APO-3, TRAMP, or LARD) (Bodmer et al., 1997; Chinnaiyan et al., 1996a; Kitson et al., 1996; Marsters et al., 1996; Screaton et al., 1997b), DR4 (also called TRAIL-R1) (Pan et al., 1997b), DR5 (also called TRAIL-R2, KILLER, or TRICK2) (Chaudhary et al., 1997; Pan et al., 1997a; Schneider et al., 1997; Screaton et al., 1997a; Sheridan et al., 1997; Walczak et al., 1997; Wu et al., 1997c), and DR6 (Pan et al., 1998a), plus chicken CAR1 (Brojatsch et al., 1996). The DD is essential for ligated death receptors to signal apoptosis because it acts as the docking site for DD-containing cytoplasmic adapter proteins such as FADD (Boldin et al., 1995; Chinnaiyan et al., 1995) and TRADD (Hsu et al., 1995). FADD binds directly to Fas (Boldin et al., 1995; Chinnaiyan et al., 1995) and is recruited to TNF-R1 and DR3 via TRADD (Chinnaiyan et al., 1996a; Hsu et al., 1996). FADD, in turn, uses its DED to bring procaspase 8 into the death-inducing signaling complex (DISC), and the induced proximity of caspase 8 zymogens within the DISC facilitates their autocatalytic activation (Kischkel et al., 1995; Medema et al., 1997; Muzio et al., 1998). Clustering of multiple receptors by membraneanchored Fas ligand (FasL) is probably required to initiate DISC formation because soluble recombinant trimeric FasL is not cytotoxic to cells expressing Fas unless it is cross-linked (Schneider et al., 1998; Strasser and O’Connor, 1998; Tanaka et al., 1998). The mechanism by which caspases become activated upon ligation of DR4 and DR5 is controversial. The role of FADD in apoptosis induced by these receptors has been investigated by a number of groups using a dominant-negative mutant of FADD (FADD-DN) that lacks the DED. FADD-DN retains the ability to bind to Fas and TRADD, but being unable to bind to procaspase 8, it interferes with DISC formation and is a specific inhibitor of Fas-, DR3-, and TNF-R1–transduced apoptosis in tumor cell lines and nontransformed mouse T cells (Chinnaiyan et al., 1996a,c; Hsu et al., 1996; Newton et al., 1998). FADD-DN has been reported to block (Chaudhary et al., 1997; Schneider et al., 1997; Wajant et al., 1998; Walczak et al., 1997) or have no effect (Pan et al., 1997a,b; Sheridan et al., 1997) on apoptosis signaling by DR4 and DR5. Consistent with DR4 utilizing a distinct adapter molecule, FADD-deficient mouse embryo fibroblasts are resistant to apoptosis induced by overexpression of Fas, DR3, and TNF-R1, but are sensitive to killing by overexpression of DR4 (Yeh et al., 1998). More recently, however, FADD-deficient fibroblasts stably transfected with DR4 or DR5 were reported to be resistant to TRAIL-induced apoptosis (Kuang et al., 2000), as were FADD-deficient Jurkat cells (Bodmer et al., 2000; Sprick et al., 2000). FADD is recruited to the TRAIL DISC (Bodmer et al., 2000; Kischkel et al., 2000; Sprick et al.,


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2000) in support of a crucial role for FADD in TRAIL-induced apoptosis. Whether caspase 8 or caspase 10 is the critical initiator caspase in TRAILinduced apoptosis has not been resolved (Chaudhary et al., 1997; Pan et al., 1997a; Schneider et al., 1997; Sheridan et al., 1997; Walczak et al., 1997). Caspase 8–deficient mouse cells are resistant to Fas-, DR3-, and TNF-R1–transduced apoptosis, indicating that in mice, caspase 8 has a nonredundant role in these apoptosis signaling pathways (Varfolomeev et al., 1998). The effect of caspase 8 deficiency on TRAIL-induced apoptosis of mouse cells has not been examined. In humans, both caspases 8 and 10 have been implicated in apoptosis signaling by death receptors. For example, a patient with inherited autoimmune lymphoproliferative syndrome type II was found to express a dominant-interfering mutant of caspase 10, and T cells and dendritic cells from this person were abnormally resistant to apoptosis induced by TRAIL or agonist anti-Fas antibodies ( J. Wang et al., 1999). Nevertheless, in the human Jurkat cell line, which expresses TRAIL-R2, caspase 8 deficiency confers resistance to TRAILinduced apoptosis, indicating an essential role for caspase 8 downstream of TRAIL-R2 (Bodmer et al., 2000; Sprick et al., 2000). Apoptosis signaling by death ligands can be regulated in several ways. In humans, and possibly other species as well, secreted and membraneanchored decoy receptors may prevent apoptosis signaling by neutralizing FasL or TRAIL. DcR3 (also called TR6) and osteoprotegerin are secreted receptors for FasL (Pitti et al., 1998; Yu et al., 1999) and TRAIL (Emery et al., 1998), respectively. DcR1 (also called TRAIL-R3, TRID, or LIT) and DcR2 (also called TRAIL-R4 or TRUNDD) are membrane-bound receptors for TRAIL that lack a DD and do not signal apoptosis (DegliEsposti et al., 1997a,b; Marsters et al., 1997; Mongkolsapaya et al., 1998; Pan et al., 1997a, 1998b; Sheridan et al., 1997). Shedding of membraneanchored death ligands from the cell surface by matrix metalloproteinases may also regulate apoptosis signaling, since soluble trimeric FasL is not cytotoxic to cells and, at high concentrations, can even act as an antagonist of Fas signaling (Huang et al., 1999; Schneider et al., 1998; Suda et al., 1997; Tanaka et al., 1998). In natural killer cells and T cells, delivery of FasL to the cell surface is under strict control; FasL is stored in lytic granules, and degranulation in response to target cell recognition results in its rapid delivery to the cell surface (Bossi and Griffiths, 1999). Intracellular inhibitors of DISC formation can also prevent apoptosis signaling by death receptors. FLIP (also called Casper, I-FLICE, FLAME-1, CASH, MRIT, CLARP, or Usurpin) blocks apoptosis signaling by Fas, DR3, and TNF-R1 because it has two DEDs, like procaspase 8, and can be incorporated into the DISC via FADD (Goltsev et al., 1997; Han et al., 1997; Hu et al., 1997; Inohara et al., 1997b; Irmler et al., 1997b;

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Rasper et al., 1998; Shu et al., 1997; Srinivasula et al., 1997). However, unlike procaspase 8, FLIP lacks enzymatic activity. Cleavage of FLIP by caspase 8 within the DISC appears to prevent further recruitment and activation of procaspase 8 (Scaffidi et al., 1999). Consistent with a protective role for FLIP in death receptor-induced apoptosis, FLIP-deficient mouse embryo fibroblasts are highly sensitive to FasL and TNF-움 (Yeh et al., 2000). SODD is another cytoplasmic protein that may limit apoptosis signaling by death receptors. It has a DD and was identified by virtue of its ability to interact with DR3 and TNF-R1. In cell lines, endogenous SODD is found complexed to TNF-R1 and is displaced by TRADD after stimulation with TNF-움. However, it soon reassociates with TNF-R1, suggesting that it may act to switch off signaling ( Jiang et al., 1999). IV. Apoptosis Signaling through Apaf-1 and Caspase 9

While caspase 8 is the initial caspase to be activated in mammalian cells treated with FasL, TNF-움, and DR3L, other treatments may activate caspase 9 first (Figure 2). The role of caspase 9 as a critical initiator caspase is suggested by its long prodomain and by the phenotypes of mice lacking caspase 9 (Hakem et al., 1998; Kuida et al., 1998) or its adapter molecule Apaf-1 (Cecconi et al., 1998; Yoshida et al., 1998). Both mutant strains of mice exhibit neuronal hyperplasia attributed to a lack of apoptosis, and their fibroblasts and lymphocytes are abnormally resistant to apoptosis induced by ionizing radiation or cytotoxic drugs such as dexamethasone, etoposide, and doxorubicin. Unlike caspase 8–deficient cells, caspase 9– or Apaf-1–deficient cells are killed normally by Fas and TNF-R1 signaling (Cecconi et al., 1998; Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998). Interestingly, mice lacking Apaf-1 have more severe defects than those lacking caspase 9, indicating that Apaf-1 might regulate additional caspases. The intracellular events that lead to Apaf-1–mediated caspase 9 activation within the animal are still being determined. In the test tube, caspase 9 can be activated in the presence of Apaf-1, cytochrome c and either ATP or dATP (Li et al., 1997). Biochemical analysis has revealed that Apaf-1 can hydrolyze ATP/dATP and thereby initiate the formation of a 1.3- to 1.4-MDa complex composed of Apaf-1 and cytochrome c. This complex contains at least eight Apaf-1 molecules, which can then bind to and promote the autocatalytic activation of an equivalent number of procaspase 9 molecules (Saleh et al., 1999; Zou et al., 1999). Since cytochrome c is located in the mitochondrial intermembrane space in healthy cells and is released into the cytoplasm in cells undergoing apoptosis (Kluck et al., 1997; Liu et al., 1996; Yang et al., 1997), this complex may also be


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FIG. 2. Regulation of apoptosis by members of the Bcl-2 protein family. Prosurvival members of the Bcl-2 protein family can inhibit apoptosis induced by growth factor deprivation and various cytotoxic treatments, but do not block apoptosis signaled by death receptors such as Fas and TNF-R1. Bcl-2 and its homologues appear to prevent caspase activation in response to the former death stimuli by directly or indirectly inhibiting the function of Ced-4–like adapter molecules such as Apaf-1. The pro-apoptotic ‘‘BH3-only’’ members of the Bcl-2 protein family can promote apoptosis by neutralizing the prosurvival Bcl-2–like proteins. Caspase 8 can cleave and activate the BH3-only protein Bid following death receptor activation, but studies using Bid-deficient mice demonstrate that this is dispensable for Fas-mediated apoptosis in lymphocytes (Yin et al., 1999).

formed in cells. However, the events that lead to cytochrome c release and the mechanism of this release are unclear. It is possible that release of cytochrome c into the cytoplasm is a consequence of caspase activation rather than a triggering event, and merely serves to amplify caspase activation (Hengartner, 1998). For example, Bid, a pro-apoptotic member of the Bcl-2 protein family (see below), can promote cytochrome c release from isolated mitochondria but only after it has been cleaved by caspase 8 (Gross et al., 1999; Li et al., 1998; Luo et al., 1998). Mitochondrial depolarization is unlikely to be the mechanism of cytochrome c release because kinetic analyses indicate that cytochrome c release occurs prior to a reduction in mitochondrial membrane potential (Bossy-Wetzel et al., 1998; Vander Heiden et al., 1997; Yang et al., 1997). Another pro-apoptotic Bcl-2 family member, called Bax, has been found to have channel-forming activity at physiological pH, and this has prompted speculation that Bax might alter mitochondrial membrane permeability, thereby contributing to cytochrome c release (Antonsson et al., 1997). More recently, the pro-

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apoptotic proteins Bax and Bak and the anti-apoptotic protein Bcl-xL were shown to interact with the voltage-dependent anion channel (VDAC) that is a component of the mitochondrial permeability transition pore (Shimizu et al., 1999). Since Bax and Bak enabled cytochrome c to pass through VDAC reconstituted into liposomes, but Bcl-xL could block this activity of Bax and Bak, the authors proposed that interactions between Bcl-2 family members and VDAC may regulate cytochrome c release within cells (Shimizu et al., 1999). However, based on genetic and biochemical analyses, we prefer the idea that regulation of cell survival by Bcl-2 and its homologues is achieved mainly through their interaction with Ced-4/Apaf-1–like adapter proteins (Strasser et al., in press; see below). V. Apoptosis and Its Regulation by Members of the Bcl-2 Family

Proteins belonging to the Bcl-2 family are critical regulators of apoptosis (Adams and Cory, 1998). Members of this family have at least one of four Bcl-2 homology domains (BH1–4), and they can be divided into two groups based on their ability to either promote apoptosis (Bad, Bak, Bax, Bcl-xs, Bid, Bik, Bim/Bod, Blk, Bok, and Harakiri/DP5) or suppress it (A1/Blf-1, Bcl-2, Bcl-w, Bcl-xL, Boo/Diva, and Mcl-1) (Boise et al., 1993; Boyd et al., 1995; Chittenden et al., 1995; Farrow et al., 1995; Gibson et al., 1996; Han et al., 1996; Hegde et al., 1998; Hsu et al., 1997; Imaizumi et al., 1997; Inohara et al., 1997a, 1998; Kiefer et al., 1995; Kozopas et al., 1993; O’Connor et al., 1998; Oltvai et al., 1993; Song et al., 1999; Wang et al., 1996; Yang et al., 1995). Genetic analysis of C. elegans has provided many clues as to how these proteins influence cell survival (Metzstein et al., 1998). Normal C. elegans development involves the death of 131 somatic cells, and three genes, ced-3, ced-4, and egl-1, are required for these cell deaths to occur. These three genes, plus a fourth, ced-9, act in the order egl-1 —l ced-9 —l ced-4 씮 ced-3, whereby ced-9 suppresses the effects of ced-3 and ced-4, while ced-9 is itself suppressed by egl-1 (Conradt and Horvitz, 1998; Metzstein et al., 1998; Shaham and Horvitz, 1996). The mammalian counterparts of Egl-1, Ced-9, and Ced-4 that have been identified to date are the pro-apoptotic ‘‘BH3-only’’ Bcl-2 family members, the anti-apoptotic Bcl-2 family members, and Apaf-1, respectively (Conradt and Horvitz, 1998; Hengartner and Horvitz, 1994; Vaux et al., 1992; Zou et al., 1997). Additional mammalian Ced-4–like adapter proteins likely also exist. As mentioned, Ced-3 is a caspase (Xue et al., 1996; Yuan et al., 1993). In accordance with the model established for C. elegans, Bcl-2 and Bcl-xL can prevent caspase activation in mammalian cells in response to certain stimuli (Chinnaiyan et al., 1996b; Erhardt and Cooper, 1996; Estop-


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pey et al., 1997; Orth et al., 1996), and their activity is antagonized by proapoptotic members of the Bcl-2 protein family (Adams and Cory, 1998). Anti-apoptotic Bcl-2 family members may prevent caspase activation by interfering with adapter molecules such as Ced-4 and Apaf-1 so that they are unable to form the oligomeric complexes required for caspase activation. Such a mechanism is suggested by the ability of Ced-4 to interact directly with Ced-9, Bcl-xL, and the caspases, Ced-3, caspase 1, and caspase 8 (Chinnaiyan et al., 1997; James et al., 1997; Seshagiri and Miller, 1997; Spector et al., 1997; Wu et al., 1997b). The ability of Ced-4 to interact with mammalian homologues of Ced-3 and Ced-9 indicates that these interactions are conserved evolutionarily, and therefore are probably of functional significance. Indeed, the survival function of Ced-9 correlates with its ability to bind to Ced-4, as mutations in Ced-9 that block this interaction are loss-of-function mutations (Chinnaiyan et al., 1997; Spector et al., 1997; Wu et al., 1997b). Reports describing interactions between Boo and Apaf-1, or Bcl-xL and Apaf-1, infer that similar interactions may prevent caspase activation in mammalian cells (Hu et al., 1998a; Pan et al., 1998c; Song et al., 1999). Inhibition of the Bcl-xL –Ced-4, Bcl-xL – Apaf-1, and Boo–Apaf-1 interactions by the pro-apoptotic proteins Bik, Bak, and Bax suggests that pro-apoptotic Bcl-2 proteins might act simply as antagonists of anti-apoptotic Bcl-2 proteins (Chinnaiyan et al., 1997; Pan et al., 1998c; Song et al., 1999). However, others have been unable to detect interactions between anti-apoptotic Bcl-2 proteins and Apaf-1 (Moriishi et al., 1999). Formation of a multiprotein complex might depend on the presence of additional components. For example, the C. elegans ATPase MAC-1 can bind to Ced-4 and under certain circumstances may act in concert with Ced-9 to promote cell survival (Wu et al., 1999). It has also been reported that Bcl-2 and Bcl-xL can block cytochrome c release from the mitochondria (Kharbanda et al., 1997; Kluck et al., 1997; Vander Heiden et al., 1997; Yang et al., 1997). The mechanism by which these anti-apoptotic proteins prevent cytochrome c release is unclear. As discussed, it has been suggested that interactions between Bcl-2 family members and VDAC may regulate the exit of cytochrome c from the mitochondria (Shimizu et al., 1999). In preventing cytochrome c release, Bcl-2 and Bcl-xL might block Apaf-1–mediated activation of caspase 9. However, this cannot be their sole function because Bcl-2 protects cells from apoptosis induced by microinjection of cytochrome c, which bypasses the need for cytochrome c release from the mitochondria (Zhivotovsky and Brustugun, 1998). Similarly, Bcl-2 can delay Bax-induced apoptosis without blocking the accumulation of cytochrome c in the cytoplasm (Rosse´ et al., 1998).

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Cytochrome c release may simply act to amplify caspase activation rather than be a triggering event (Hengartner, 1998). Consistent with this notion, Fas-induced cytochrome c release is a late event (Adachi et al., 1998) which, in hepatocytes, requires caspase 3 activity (Woo et al., 1999). That caspase activation occurs prior to cytochrome c release is also suggested by the fact that loss of caspase function not only prevents apoptosis in C. elegans or D. melanogaster, it also permits cells that would normally die to continue to function normally. For example, loss-of-function mutations in ced-3 rescue cells destined to die in C. elegans (Ellis and Horvitz, 1986). Similarly, expression of the baculovirus caspase inhibitor p35 in the eyes of D. melanogaster mutants that develop retinal degeneration can reduce excessive apoptosis in the eye so that the flies retain visual function (Davidson and Steller, 1998). Cytochrome c is essential for normal mitochondrial function, so it seems reasonable to assume that if it were released in cells lacking caspase activity, then cell function and viability would still be compromised because of impaired energy production. Additional evidence is provided by Apaf-1– or caspase 9–deficient mouse cells, which are protected from only some of the apoptotic deaths that can be blocked by Bcl-2 (Cecconi et al., 1998; Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998). For example, the death of myeloid and pre-B cells following cytokine withdrawal is delayed but not prevented by loss of Apaf1 or caspase 9 (V. Marsden, L. O’Connor, D. Metcalf, L. O’Reilly, D. Huang, and A. Strasser, unpublished observations). This suggests that cytochrome c–mediated activation of caspase 9 contributes to apoptosis signaling but is not essential for apoptosis induction. It also raises the possibility that a novel adapter protein and initiator caspase combination regulates this physiologically important death stimulus. VI. Transcriptional and Posttranslational Control of Bcl-2 Family Members

Members of the Bcl-2 family can be regulated at the level of transcription. For example, transcripts for the pro-apoptotic protein DP5 are induced in neurons after nerve growth factor withdrawal (Imaizumi et al., 1997), whereas lymphocytes treated with mitogens increase transcription of antiapoptotic genes. A1 induction in mitogen-stimulated B and T cells is dependent on the NF-␬B transcription factor Rel (Grumont et al., 1999), while Bcl-xL is induced in T cells stimulated with cross-linking antibodies to CD3 plus CD28 (Boise et al., 1995; Van Parijs et al., 1996). There is also evidence that posttranslational modifications of Bcl-2 family members can regulate cell survival. For example, when hematopoietic cell lines or neurons are cultured under favorable conditions, the pro-apoptotic activity of Bad appears to be neutralized because it is phosphorylated by


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the kinase Akt and protein kinase A, and then sequestered away from anti-apoptotic Bcl-2 proteins by the phoshoserine-binding protein 14-3-3 (Blume-Jensen et al., 1998; Datta et al., 1997; del Peso et al., 1997; Harada et al., 1999; Zha et al., 1996). This model suggests that the ability of Bad to enhance apoptosis in cells deprived of growth factors is dependent on its dephosphorylation. One phosphatase that may dephosphorylate Bad upon apoptosis induction is calcineurin (H.-G. Wang et al., 1999). Whether Bad is essential for apoptosis induced by growth factor withdrawal is not clear. For example, activated B and T lymphocytes from Bim-deficient mice are resistant to cytokine deprivation, indicating that Bad cannot compensate for loss of Bim function in this cell type (Bouillet et al., 1999). The pro-apoptotic protein Bim appears to be held in check by its interaction with LC8, a component of the dynein motor complex. Upon apoptosis induction, Bim and LC8 are released from the dynein motor complex, and Bim can interact with Bcl-2 and its homologues (Puthalakath et al., 1999). Presumably, Bim and other BH3-only proteins promote apoptosis by antagonizing the effects of anti-apoptotic Bcl-2 proteins. The posttranslational modifications that cause Bim and LC8 to dissociate from the dynein motor complex are not known. Phosphorylation of Bcl-2 has been described, but how this affects Bcl-2 function is not clear (Chang et al., 1997; Haldar et al., 1995; Ito et al., 1997). VII. Apoptosis and the Immune System

Apoptosis plays a prominent role in the development and homeostasis of the immune system. It contributes to (i) the generation of a functional repertoire of mature B and T cells by eliminating developing lymphocytes that fail to express an antigen receptor, (ii) tolerance toward self by removing lymphocytes with antigen receptors that recognize self antigens, and (iii) immune homeostasis by culling activated lymphocytes when an infection is cleared successfully (Strasser, 1995). The importance of apoptosis in the immune system is emphasized by mutations that prevent lymphocyte apoptosis and thereby contribute to the development of either cancer or autoimmune disease. For example, constitutive expression of Bcl-2 in B cells due to translocation of the bcl-2 gene next to the immunoglobulin heavy-chain gene is associated with follicular lymphoma in humans (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto and Croce, 1986; Tsujimoto et al., 1984). Similarly, constitutive expression of a bcl-2 transgene in B cells predisposes mice to immature B cell lymphomas and plasmacytomas (McDonnell and Korsmeyer, 1991; Strasser et al., 1993). When cell cycle control is also impaired, as in bcl-2/c-myc or bcl2/pim-1 doubly transgenic mice, the incidence of these tumors is increased

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significantly (Acton et al., 1992; Strasser et al., 1990). Depending on their genetic background, bcl-2 transgenic mice can also develop autoimmune disease (Strasser et al., 1991b). Bim-deficient mice exhibit a similar autoimmune disease (Bouillet et al., 1999) identifying this BH3-only pro-apoptotic protein as a critical regulator of normal immune function. Mutations that affect the activity of Fas and FasL also prevent lymphocyte apoptosis. These mutations produce lymphadenopathy, splenomegaly, and autoimmunity in both mice (Fas gene knockout, Fas mutant lpr or FasL mutant gld mice) and humans (Cohen and Eisenberg, 1993; Fisher et al., 1995; Nagata and Golstein, 1995; Rieux-Laucat et al., 1995). Mutations in Fas can also predispose mice to plasmacytoma development (Davidson et al., 1998; Peng et al., 1996) and have been implicated in the development of some cases of human multiple myeloma (Landowski et al., 1997). Signaling by other death receptors can also suppress tumorigenesis. We have used a FADD-DN transgene to inhibit death receptor signaling in the T lymphocytes of rag-1–deficient mice, which are unable to rearrange their antigen receptor genes, and all FADD-DN rag-1⫺/⫺ mice developed thymic lymphoma. In contrast, we have never observed thymic tumors in rag-1⫺/⫺ or lpr rag-1⫺/⫺ mice, indicating that Fas is not the only death receptor to suppress tumor development through FADD (Newton et al., 2000). The availability of mice that are deficient for components of the cell death machinery or that express transgenes encoding inhibitors of apoptosis (e.g., Bcl-2) has enabled researchers to determine which apoptosis signaling pathways operate at different stages in lymphocyte differentiation. Signaling pathways that prevent lymphocyte apoptosis have also been identified. These findings are reviewed in the following sections. VIII. T Cell Development—Apoptosis at the Pre-TCR Checkpoint

Mouse T lymphocytes of the T cell receptor (TCR)-움/웁 lineage develop in the thymus from bone marrow– or fetal liver–derived multipotential stem cells. Glycoproteins expressed at the cell surface and the rearrangement status of the TCR-움 and TCR-웁 gene loci identify distinct stages of thymocyte differentiation (Figure 3). Early T cell progenitors (here called pro-T cells) are CD4⫺8⫺ and can be subdivided into four populations according to expression of CD25 (interleukin 2 receptor [IL-2R] 움 chain) and CD44 (Pgp-1). The presently accepted developmental sequence is CD25⫺44⫹ (pro-T1) 씮 CD25⫹44⫹ (pro-T2) 씮 CD25⫹44⫺ (pro-T3) 씮 CD25⫺44⫺ (pro-T4) (Godfrey and Zlotnik, 1993). Signals through the IL-7 receptor (IL-7R/웂c), c-Kit (stem cell factor receptor), and Flk2/Flt3 are essential for cell proliferation and survival during the pro-T1 to proT3 stages of development (Akashi et al., 1997; Kondo et al., 1997; Mack-


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FIG. 3. Apoptosis during TCR-움/웁 T cell development. Early T cell progenitors require interleukin 7 (IL-7) for their survival since expression of a bcl-2 transgene can rescue T cell development in IL-7–, IL-7R– or 웂c-deficient mice. Later in T cell development, essential survival signals are provided first by expression of a pre–T cell receptor (pre-TCR) and later by the complete TCR. Apoptosis at the pre-TCR checkpoint involves p53 and death receptor signaling, since pre-T cells are produced in rag-deficient or scid mice lacking either p53 or FADD function. Survival and maturation beyond the pre-T stage require expression of a complete TCR. Cells that fail to express a complete TCR that can bind to the major histocompatibility complex (MHC) undergo apoptosis, as do those bearing an autoreactive TCR (negative selection). The former can be rescued by expression of a bcl-2 transgene, although this is not sufficient for further maturation and emigration from the thymus. How apoptosis is signaled during negative selection remains unclear, but Bcl-2 can delay this death to a certain extent.

arehtschian et al., 1995; Marashovsky et al., 1997; Rodewald et al., 1995; Rodewald et al., 1997). Notably, expression of a bcl-2 transgene can substitute for the anti-apoptotic activity of the IL-7R/웂c receptor (Akashi et al., 1997; Kondo et al., 1997; Maraskovsky et al., 1997). Rearrangement of TCR-웁 genes is initiated during transition from the pro-T2 to the pro-T3 stage (Capone et al., 1998). A productive gene rearrangement and expression of a pre-TCR composed of the TCR-웁 chain, the pT움 chain, and CD3 signal-transducing proteins (Groettrup et al., 1993) results in progression to the pro-T4 and CD4⫹8⫹ pre-T stages (Fehling et al., 1995). Thymocytes that survive the pre-TCR checkpoint proliferate and differentiate to yield the numerically dominant CD4⫹8⫹ population. Pro-T3 cells that lack a pre-TCR (or a suitable substitute, e.g., TCR-움/웁 or TCR-웂/␦) die after 3–4 days, presumably because pre-TCR expression generates an essential survival signal (Penit et al., 1995). The death of pro-T3 cells that fail to express a pre-TCR is evident in the small thymi of mutant scid mice and mice lacking either of the recombinase-

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activating genes, rag-1 or rag-2. These mice are defective in antigen receptor gene rearrangement and do not produce pro-T4 cells, pre-T cells, or mature TCR-움/웁 T cells because differentiation is arrested at the pro-T3 stage (Habu et al., 1987; Mombaerts et al., 1992; Shinkai et al., 1992; Shores et al., 1990). A requirement for CD3␧ in signal transduction at the pre-TCR checkpoint is demonstrated by the lack of pro-T4, pre-T, and mature T cells in CD3␧-deficient mice (Malissen et al., 1995). Furthermore, ligation of surface-bound CD3␧ with cross-linking antibodies is sufficient to trigger pre-T cell generation in rag-1⫺/⫺, rag-2⫺/⫺, pT움⫺/⫺, or TCR-웁⫺/⫺ mice (Fehling et al., 1997; Jacobs et al., 1994; Levelt et al., 1993; Shinkai and Alt, 1994). The mechanism by which pre-TCR/CD3 signaling controls survival and proliferation of thymocytes remains unclear. Overexpression of the antiapoptotic protein Bcl-2 rescues pro-T cells from a lack of IL-7R/웂c signaling (Akashi et al., 1997; Kondo et al., 1997; Maraskovsky et al., 1997), but it does not promote survival of pre-TCR–deficient pro-T3 cells in scid (Strasser et al., 1994a) or rag-1⫺/⫺ mice (Maraskovsky et al., 1997). Some CD4⫹8⫹ preT cells are observed in scid, rag-1⫺/⫺ and rag-2⫺/⫺ mice lacking the tumor suppressor p53 (Bogue et al., 1996; Guidos et al., 1996; Jiang et al., 1996; Nacht and Jacks, 1998; Nacht et al., 1996). Interestingly, there is large variation between individual animals in the number of CD4⫹8⫹ thymocytes produced, and most mice rapidly develop lymphoid malignancy (Nacht et al., 1996). p53 deficiency also restores pre-T cell content in CD3웂⫺/⫺ mice (Haks et al., 1999). A report describing CD4⫹8⫹ pre-T cells and even mature CD3⫹4⫹8⫺ and CD3⫹4⫺8⫹ thymocytes in scid mice homozygous for the Fas loss-of-function lpr mutation (Yasutomo et al., 1997) implied that Fas might deliver the death signal to pro-T3 cells that lack a preTCR. However, this result is at odds with flow cytometric data suggesting that surface expression of Fas is restricted to more mature pre-T cells (Nishimura et al., 1995; Ogasawara et al., 1995). We have never observed pre-T and mature T cells in lpr rag-1⫺/⫺ mice (Newton et al., 2000), so the lpr scid pre-T cells probably developed because the scid mutation imposes only a partial block on antigen receptor gene rearrangement (Bosma and Carroll, 1991). Chance in-frame TCR-웁 gene rearrangements in a few lpr scid thymocytes could have allowed their differentiation to the CD4⫹8⫹ pre-T stage. These pre-T cells may then have accumulated because their life span was extended in the absence of Fas. In contrast to lpr rag-1⫺/⫺ mice, we have seen pro-T4 and pre-T cells in FADD-DN transgenic rag-1⫺/⫺ mice (Newton et al., 2000), suggesting that other death receptors deliver the death signal via FADD to pro-T3 cells without a pre-TCR.


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IX. Positive and Negative Selection at the Pre-T Stage of Development

Maturation to the pre-T stage coincides with TCR-움 gene rearrangement (Petrie et al., 1993), and thymocytes expressing a complete TCR-움/웁–CD3 complex become subject to immunological selection based on their TCR움/웁 specificity (von Boehmer, 1994). Pre-T cells that lack a functional TCR fail to receive a survival signal and die by apoptosis, while cells bearing TCRs that can recognize self major histocompatibility complex (MHC) plus peptide antigens undergo positive selection. However, if the avidity of a TCR for MHC plus self antigen is too great, a pre-T cell undergoes apoptosis in a process referred to as negative selection. Bcl-2 protein is expressed at high levels in pro-T2 cells, presumably as a consequence of IL-7R/웂c signaling, and at lower levels in pro-T3 and pro-T4 cells (von Freeden-Jeffry et al., 1997). It is not detected in pre-T cells, but it is again expressed in mature CD3⫹4⫺8⫹ and CD3⫹4⫹8⫺ thymocytes (Gratiot-Deans et al., 1993; Veis et al., 1993a), suggesting that Bcl-2 may contribute to the survival of positively selected T cells. The enhanced survival of unselected thymocytes in TCR transgenic or MHCdeficient mice that constitutively express a bcl-2 transgene supports this notion (Linette et al., 1994; Strasser et al., 1994b). Failure of the unselected bcl-2 transgenic cells to differentiate further indicates that additional signals must be required for maturation and emigration from the thymus. Mice that lack Bcl-2 display reduced thymus cellularity but all thymocyte subsets are present (Nakayama et al., 1994; Veis et al., 1993b). This phenotype probably reflects the reduced survival of lymphoid progenitor cells (Matsuzaki et al., 1997) compounded by the poor viability of T cells at later stages during development. Thymus cellularity is higher in bcl-2⫺/⫺/bax⫺/⫺ doubly deficient mice than in bcl-2⫺/⫺ mice, so one function of Bcl-2 may be to neutralize the pro-apoptotic effects of Bax (Knudson and Korsmeyer, 1997). The prosurvival protein Bcl-xL is also expressed in the thymus but its expression is limited to pre-T cells (Ma et al., 1995), and its nonredundant role in T cell development has not yet been determined. Evidence that negative selection involves caspase activation and apoptosis is provided primarily by two studies. First, mice expressing a selfreactive transgenic TCR have increased numbers of thymocytes with fragmented DNA (Surh and Sprent, 1994). Second, in fetal thymic organ culture, peptide-specific deletion of thymocytes expressing a transgenic TCR can be blocked with the caspase inhibitor zVAD.fmk (Clayton et al., 1997). Transgenic mice that express the baculovirus caspase inhibitor p35 in T cells appear to be resistant to thymocyte deletion after injection of exogenous peptides or superantigens (Izquierdo et al., 1999), but this form of deletion may not be a physiologically relevant model of negative

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selection. Specifically, excessive mature T cell activation in peripheral lymphoid organs may result in cytokine and steroid levels that are toxic to thymocytes regardless of the specificity of their TCR (Page et al., 1998). Crosses between p35 transgenic mice and TCR transgenic mice that express TCRs specific for endogenous antigens would probably be more informative and would represent the first direct test of whether caspase activation is required for negative selection within the animal. The genes that effect thymocyte negative selection appear to be regulated by the transcription factors Nur77 and Nor-1 (Cheng et al., 1997). Nur77 was first identified as a potential regulator of T cell apoptosis because of its induction in T cell hybridomas and thymocytes undergoing apoptosis in response to TCR engagement (Liu et al., 1994; Woronicz et al., 1994). Thymocytes and mature T cells from mice lacking Nur77 are normally sensitive to TCR-mediated apoptosis (Lee et al., 1995), but this may be because Nor-1 is still present and able to perform similar functions to Nur77 (Cheng et al., 1997). Evidence that Nor-1 or Nur77 is required for negative selection comes from the study of transgenic mice that express in their T cells a dominant-negative mutant of Nur77 (Nur77-DN), which is able to block transactivation by both Nur77 and Nor-1. Nur77-DN transgenic mice display impaired deletion in the anti-HY TCR transgenic model of negative selection, which relies on deletion mediated by the male HY antigen when it is presented by the MHC class I molecule H-2Db (Zhou et al., 1996a). This indicates that Nur77 and Nor-1 function in negative selection either by inducing cell death genes or by repressing cell survival genes. Regulation of critical genes could occur by a direct or indirect transcriptional mechanism. Negative selection of autoreactive thymocytes is not dependent on death receptors that signal apoptosis via FADD and caspase 8. Mice that express in their T cells a crmA transgene to inhibit caspase 8, or a FADD-DN transgene, can still delete thymocytes with TCRs that recognize endogenous peptides or superantigens (Newton et al., 1998; Smith et al., 1996; Walsh et al., 1998). Two members of the TNF-R family that lack a DD and have been implicated as regulators of thymocyte negative selection are CD30 and CD40. CD30 deficiency impairs deletion of autoreactive thymocytes in anti-HY TCR transgenic mice, but does not prevent deletion of thymocytes that recognize endogenous Mls superantigens (Amakawa et al., 1996). CD40 has been implicated in thymocyte negative selection because neutralizing antibodies to CD40L perturb both deletion of selfreactive T cells by endogenous superantigens and deletion of TCR transgenic T cells by endogenous peptide antigens (Foy et al., 1995). However, it is not clear whether CD30 and CD40 deliver a death signal to doomed thymocytes or function within antigen-presenting cells. CD30 and CD40


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can signal apoptosis in certain cell lines, but this may be an indirect effect of TNF-R1 signaling due to induction of membrane-anchored TNF-움 expression (Grell et al., 1999) and/or the sequestration and degradation of TRAF2 (Duckett and Thompson, 1997). TRAF2 is a cytoplasmic adapter molecule recruited to ligated TNF-R1 via TRADD (Hsu et al., 1996; Shu et al., 1996). It is required for JNK activation plus recruitment of IKK and the inhibitor of apoptosis protein c-IAP1, and it is thought to promote cell survival rather than apoptosis (Devin et al., 2000; Lee et al., 1997; Shu et al., 1996; Yeh et al., 1997). Analysis of chimeric mice in which only thymocytes lack CD30 or CD40 would clarify whether these receptors function in thymocytes or in antigen-presenting cells. Constitutive expression of a bcl-2 or bcl-xL transgene in T cells can partially perturb the deletion of thymocytes with TCRs that recognize endogenous superantigens. However, their removal is only delayed rather than prevented because self-reactive T cells are not evident in peripheral lymphoid organs (Grillot et al., 1995; Sentman et al., 1991; Strasser et al., 1991a). Bcl-2 and Bcl-xL are also poor inhibitors of negative selection when thymocytes bear MHC class I or II–restricted transgenic TCRs that recognize endogenous peptide antigens (Chao and Korsmeyer, 1997; Grillot et al., 1995; Strasser et al., 1994b; Tao et al., 1994; Van Parijs et al., 1998a). Expression of a bcl-2 transgene together with a crmA or FADD-DN transgene does not perturb thymocyte negative selection any more than expression of a bcl-2 transgene alone (Newton et al., 1998; Smith et al., 1996). Since negative selection is so crucial for the maintenance of self-tolerance, it may be safeguarded by many mechanisms. For instance, autoreactive cells with enhanced survival might still be subject to a development arrest such that they are unable to leave the thymus or are rendered nonfunctional. X. T Cell Apoptosis in Peripheral Lymphoid Organs

Mature T cells in peripheral lymphoid organs require continued interaction with MHC molecules for their survival in vivo (Kirberg et al., 1997; Tanchot et al., 1997). Stimulation of the TCR may promote T cell survival by maintaining Bcl-2 protein levels within the cell because loss of Bcl-2, and not Bcl-xL, increases the spontaneous death of mature T cells in culture (Ma et al., 1995; Nakayama et al., 1993, 1994; Veis et al., 1993b). The different roles identified for the various anti-apoptotic Bcl-2–like proteins probably reflect their different patterns of expression rather than a unique function for each protein, because in stably transfected cell lines they promote cell survival in an identical manner (Gibson et al., 1996; Huang et al., 1997).

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The two major reasons for apoptosis of activated mature T cells in vivo are likely to be limited availability of cytokines and repeated engagement of the TCR (Strasser, 1995) (Figure 4). Apoptosis induced by the latter, referred to as activation-induced cell death (AICD), is mediated predominantly by Fas and appears to be a mechanism of maintaining tolerance toward self antigens expressed in the periphery. The first clues that Fas and FasL are involved in AICD came from experiments showing that activated T cells from FasL mutant gld or Fas-deficient lpr mice undergo less apoptosis after restimulation with mitogens in vitro compared to control T cells (Russell and Wang, 1993; Russell et al., 1993; Singer and Abbas, 1994). Similar resistance to apoptosis was observed when normal activated T cells were cultured with soluble Fas decoy receptors or neutralizing antibodies to Fas. These experiments established that interactions between Fas and FasL on the same cell or on neighboring cells are required for AICD (Alderson et al., 1995; Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995; Strasser, 1995). Susceptibility to apoptosis signaling by Fas is enhanced in activated T cells because levels of FasL and Fas on the T cell surface increase after TCR engagement (Alderson et al., 1995; Brunner et al., 1995; Dhein et al., 1995; Ju et al., 1995; Nishimura et al., 1995; Suda et al., 1993).

FIG. 4. Apoptosis of mature T cells. Interactions between the T cell receptor (TCR) and major histocompatibility complex (MHC) molecules are required for mature T cells to persist in peripheral lymphoid organs. Whether T cell apoptosis in the absence of these interactions occurs via a Bcl-2–inhibitable pathway or involves death receptor signaling is unknown. T cells that are stimulated by mitogens or antigens to become T cell blasts are dependent on cytokines for their survival and proliferation. When cytokines become limiting, apoptosis is signaled via the pro-apoptotic ‘‘BH3-only’’ protein Bim and can be inhibited by overexpression of Bcl-2 or a homologue. Repeated TCR engagement on activated T cells increases expression of Fas and FasL, resulting in autocrine or paracrine apoptosis signaling that can be blocked by the caspase 8 inhibitor CrmA.


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Many studies have shown that Fas signaling can contribute to removal of activated T cells in vivo. For example, injection of bacterial superantigens into lpr mice induces normal proliferation of specific T cell subsets, but subsequent deletion of these cells is impaired (Mogil et al., 1995; Scott et al., 1993; Strasser et al., 1995; Van Parijs et al., 1996). Similar observations were made when gld or lpr mice expressing a MHC class II–restricted transgenic TCR specific for hen egg lysozyme (HEL) were injected with HEL peptide (Van Parijs et al., 1998a) or when T cells from these mice were transferred into HEL-expressing transgenic mice (Van Parijs et al., 1998b). A lack of apoptosis following proliferation was also seen when T cells from lpr mice expressing a MHC class I–restricted transgenic TCR specific for ovalbumin (OVA) were transferred into transgenic mice expressing membrane-bound OVA (Kurts et al., 1998). Failure to delete chronically activated T cells would explain the accumulation of T cells in gld and lpr mice as they age, although the reason most of the T cells bear the unusual surface phenotype CD3⫹4⫺8⫺ B220⫹ is not known (Cohen and Eisenberg, 1993). Lymphadenopathy in lpr mice is reported to be prevented by expression of a transgenic TCR in some but not all instances (Sidman et al., 1992; Sytwu et al., 1996; Van Parijs et al., 1998a). The absence of lymphadenopathy in some TCR transgenic lpr mice likely reflects the reduced number of T cells that can recognize and be activated by self antigens. The best evidence that lymphoproliferation in lpr mice is induced by self antigens rather than by exogenous antigens, either infectious or dietary, is that lpr mice fed an antigen-free diet in a germ-free environment still develop significant lymphadenopathy (Maldonado et al., 1999). Some studies provide evidence that in certain settings other death receptors (e.g., TNF-R1) and TNF-R family members that lack a DD (e.g., TNF-R2) can contribute to the elimination of activated T cells. For example, neutralizing antibodies to TNF-움 were shown to enhance the resistance of lpr T cells to AICD in vitro, and using T cells from mice deficient for TNF-R1 or TNF-R2, this resistance was attributed to impaired TNF-R2 signaling in CD4⫺8⫹ cells (Zheng et al., 1995). This result contrasts with in vivo experiments in which proliferation and deletion of OVA-specific TCR transgenic CD4⫺8⫹ cells transferred into OVA-expressing mice were not affected by loss of TNF-R2 (Kurts et al., 1998). Neutralizing antibodies to TNF-움 did inhibit deletion of CD4⫹8⫺ T cells in lpr mice expressing a MHC class II–restricted transgenic TCR specific for influenza hemagglutinin (HA) when the mice were injected with HA peptide. In this system, the lpr mutation alone had no effect on T cell deletion (Sytwu et al., 1996). In a further TCR transgenic model involving a class I MHC–restricted TCR specific for a lymphocytic choriomeningitis virus (LCMV) peptide,

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T cell deletion after LCMV peptide injection was delayed in the absence of TNF-R1 (Speiser et al., 1996) but not Fas (Zimmermann et al., 1996). Consistent with a role for TNF-R1 in the removal of activated T cells, it has been reported that development of lymphadenopathy in lpr mice is accelerated by loss of TNF-R1 (Zhou et al., 1996b). However, the genetic background of the lpr TNF-R1⫺/⫺ mice complicates the interpretation of this result. The magnitude and onset of lpr-induced lymphadenopathy vary between mouse strains (Nagata and Golstein, 1995), so if the mice were insufficiently backcrossed, differences shown by the lpr TNF-R1⫺/⫺ mice might just reflect their mixed genetic background. Mice deficient in TNF-R1 alone do not develop lymphadenopathy, indicating that impaired TNF-R1 signaling itself is not sufficient for disease development (Pfeffer et al., 1993; Rothe et al., 1993). The role of TNF-R family members in AICD is difficult to dissect because they are also involved in T cell activation (Grell et al., 1998; see below). Lymphadenopathy in lpr or gld mice is enhanced by constitutive expression of a bcl-2 transgene in T cells (Reap et al., 1995; Strasser et al., 1995; Tamura et al., 1996). Unlike Fas deficiency, expression of a bcl-2 or bcl-xL transgene protects resting and cycling T cells against apoptosis due to growth factor withdrawal (Grillot et al., 1995; Sentman et al., 1991; Strasser et al., 1991a, 1994c, 1995). These observations indicate that when growth factors become limiting, T cell apoptosis occurs via a Bcl-2–sensitive, Fasindependent pathway. Therefore, the more severe phenotype of bcl-2 transgenic lpr mice is probably due to the fact that their activated T cells are resistant to a broader range of apoptotic stimuli (Strasser et al., 1995). Bcl-xL probably plays a role in the survival of activated T cells, because antibody-mediated cross-linking of CD28 in addition to CD3 results in T cell proliferation plus increased Bcl-xL expression (Boise et al., 1995; Van Parijs et al., 1996). Cytokines such as IL-4 and IL-7 enhance Bcl-2 expression in T cells (Akashi et al., 1997; Vella et al., 1998). Nevertheless, bcl-2⫺/⫺ T cells can proliferate in response to mitogens in culture (Nakayama et al., 1994), although they may die prematurely after blast formation. Bcl-2–sensitive pathways to apoptosis also remove activated T cells in vivo. For example, T cell numbers remained elevated for longer in bcl-2 transgenic mice that have been injected with bacterial superantigens compared with injected control animals (Strasser et al., 1991a). Apoptosis involving both Fas-dependent and Bcl-2–sensitive pathways appears to operate in this experimental system because activated T cells persist even longer in lpr mice that express a bcl-2 transgene (Strasser et al., 1995). In a second system, Bcl-2 (or Bcl-xL) expression can inhibit the deletion of LCMV-specific TCR transgenic CD4⫺8⫹ T cells when mice are injected with LCMV peptide (Petschner et al., 1998). Expression of a bcl-2 trans-


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gene also prevents deletion of T cells expressing a HEL-specific transgenic TCR when the cells are transferred into mice that are immunized with HEL (Van Parijs et al., 1998b). A similar block in deletion is observed when T cells from female mice expressing bcl-2 and anti-HY TCR transgenes are transferred into male mice (Teh et al., 1996). Whether Bcl-2–sensitive or –insensitive (e.g., Fas-activated) pathways to apoptosis dominate in the deletion of activated T cells in vivo may depend on the nature, amount, and mode of delivery of the antigen. These factors could influence the number of T cells that become activated, the amount of T cell help and therefore cytokine availability, the extent of TCR engagement, and whether T cells receive appropriate costimulatory signals from antigen-presenting cells. The Bcl-2–sensitive cell death pathways triggered in activated T cells by limited cytokine availability probably involve the pro-apoptotic protein Bim, because T lymphoblasts lacking Bim are markedly resistant to cytokine withdrawal in culture (Bouillet et al., 1999). Bim deficiency also enhances the survival of resting T cells in culture. Both features of bim⫺/⫺ T cells are believed to contribute to the fact that bim⫺/⫺ mice have 2- to 3-fold more small, noncycling T cells than their wild-type counterparts (Bouillet et al., 1999). It has been reported that mouse and human T cells stimulated with mitogens in vitro are initially resistant to FasL killing (Irmler et al., 1997b; Suda et al., 1996). However, this resistance is not observed after several days of stimulation when the amount of FLIPL protein in the T cells is reduced (Irmler et al., 1997b). FLIP levels may therefore determine whether or not T cells undergo Fas-dependent AICD. Reduced FLIP expression in T cells activated for several days is proposed to be a consequence of IL-2 signaling, because cross-linking of CD3 on activated IL-2⫺/⫺ T cells in vitro does not induce apoptosis as it does in normal activated T cells (Refaeli et al., 1998). However, IL-2⫺/⫺ T cells do not proliferate normally after mitogenic stimulation in vitro, so defective AICD could simply be a consequence of the proliferation defect. IL-2R웁⫺/⫺ T cells display a similar proliferation defect, but there is disagreement as to whether these cells are defective at AICD (Suzuki et al., 1997; Van Parijs et al., 1999). The identification of a mutant human IL-2R웁 chain that can signal IL-2–induced proliferation in retrovirus-infected IL-2R웁⫺/⫺ mouse T cells but cannot restore AICD to normal levels provides the strongest evidence that IL-2 signaling is directly required for AICD (Van Parijs et al., 1999). IL-2⫺/⫺, IL-2R움⫺/⫺, and IL-2R웁⫺/⫺ mice do develop lymphadenopathy and autoimmunity (Sadlack et al., 1993; Suzuki et al., 1995; Willerford et al., 1995), but they differ quite significantly from lpr and gld mice in that they do not accumulate unusual CD3⫺4⫺8⫺B220⫹ T cells. Since loss of essential IL-2 signaling components does not recapitulate the lpr

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and gld phenotype, mechanisms other than impaired Fas signaling may be responsible for lymphocyte accumulation in IL-2⫺/⫺, IL-2R움⫺/⫺, and IL-2R웁⫺/⫺ mice. The finding that CTLA-4–deficient mice develop a lymphoproliferative disorder that does not resemble lpr/gld-like lymphadenopathy indicates that there are several mechanisms to negatively regulate lymphocyte expansion (Tivol et al., 1995; Waterhouse et al., 1995). XI. The TNF-R Family and T Cell Proliferation

Members of the TNF-R family can promote cell proliferation (Alderson et al., 1993, 1994; Grell et al., 1998; Yamada et al., 1997), and recent findings implicate death receptors in the proliferation of T cells. Studies using FADD-DN transgenic or FADD⫺/⫺ T cells indicate that the cytoplasmic adapter FADD is required not only for apoptosis signaling by Fas but also for T cell proliferation (Newton et al., 1998; Zhang et al., 1998). FADD presumably engages molecules other than caspase 8 to provide the proliferative signal, because crmA transgenic T cells can proliferate normally (Newton et al., 1998; Smith et al., 1996). The death receptor or receptors that use FADD to signal T cell proliferation have not been determined. Studies using lpr and TNF-R1⫺/⫺ mice indicate that Fas and TNF-R1 are dispensable for normal T cell proliferation (Newton et al., 1998; Pfeffer et al., 1993; Rothe et al., 1993). Interestingly, transformed T cell lines that stably express FADD-DN do not have a proliferation defect (Huang et al., 1999). Since these cells are cycling continuously, whereas normal T cells are quiescent prior to activation, FADD may act as T cells exit G0 and enter the G1 phase of the cell cycle (Newton et al., 1998). XII. B Cell Development—Apoptosis at the Pre-BCR Checkpoint

B cell development in the bone marrow resembles T cell development in that glycoproteins expressed at the cell surface and the rearrangement status of the immunoglobulin (Ig) heavy- and light-chain gene loci identify distinct stages of B cell differentiation (Figure 5). Early B cell progenitors (here called pro-B cells) are B220⫹CD43⫹c-Kit⫹ and their Ig heavy-chain gene loci are in the germline configuration. Rearrangement of the VH, DH, and JH variable heavy-chain gene segments and expression of a pre-B cell receptor (pre-BCR) are required for progression to the B220⫹CD43⫺ preB stage (Benschop and Cambier, 1999). The pre-BCR is composed of the Ig 애 heavy chain, the ␭5 and Vpre-B surrogate light chains, plus the Ig움 and Ig웁 signal-transducing proteins (Karasuyama and Rolink, 1994). Just as pro-T3 cells lacking a pre-TCR in the thymus undergo apoptosis, so do


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FIG. 5. Apoptosis during B cell development. Pro-B cells require interleukin 7 (IL-7) for their survival and differentiation. While a bcl-2 transgene can substitute for a lack of IL-7R/웂c signaling in pro-T cells, it fails to rescue B cell production in IL-7⫺/⫺, IL-7R⫺/⫺, or 웂c⫺/⫺ mice. Bcl-2 can inhibit apoptosis of B cell progenitors that fail to make a pre–B cell receptor (pre-BCR) or that cannot express a complete BCR, but importantly this is not sufficient for the cells to differentiate further. Bcl-2 can also prevent the deletion of immature B cells with an autoreactive BCR to a significant extent if the antigen is soluble, and to a lesser extent when it is membrane bound. These effects of Bcl-2 likely explain the expanded populations of B cell progenitors in the bone marrow of bcl-2 transgenic mice ( Janani et al., 1998). Ig, Immunoglobulin.

pro-B cells that fail to express a pre-BCR (Osmond, 1993). Hence, scid, rag-1⫺/⫺, or rag-2⫺/⫺ mice that are defective in antigen receptor gene rearrangement do not possess pre-B or mature B cells, and B cell development is arrested at the pro-B stage (Bosma and Carroll, 1991; Mombaerts et al., 1992; Shinkai et al., 1992). Constitutive expression of a bcl-2 transgene in rag-deficient B cells enhances pro-B cell survival to the extent that substantial numbers accumulate in the periphery. However, enhanced proB cell survival does not lead to differentiation and the acquisition of features associated with later stages of B cell development (Tarlinton et al., 1997; Young et al., 1997), indicating that additional signals must be required for normal pro-B cell differentiation. Consistent with these observations, expression of a bcl-xL transgene in mouse B cells improves the survival of pro-B cells with nonproductive Ig heavy-chain gene rearrangements, but does not permit their differentiation to the pre-B stage (Fang et al., 1996). Mutant scid mice expressing a bcl-2 transgene contain an unusual B cell population bearing more mature surface markers (e.g., CD21, CD22, and CD23) than pro-B cells, but this appears to be because the scid mutation does not impose a complete block on Ig heavy-chain gene rearrangement (Strasser et al., 1994a; Tarlinton et al., 1997). Expression of truncated D애 proteins resulting from DHJH rearrangements in some scid pro-B cells

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presumably allows formation of a pre-BCR (Tarlinton et al., 1997). The same unusual B cell population is observed in bcl-2 transgenic rag-2⫺/⫺ mice that express an Ig heavy-chain transgene (Young et al., 1997) and in bcl-2 transgenic 애MT mutant mice, which lack the membrane-spanning region of the Ig 애 heavy chain (Tarlinton et al., 1997). 애MT B cells can rearrange their Ig heavy-chain genes, so isotype switching by alternative splicing or by switch recombination is suggested to allow expression of a surrogate pre-BCR (e.g., sIgG) that can promote B cell differentiation (Tarlinton et al., 1997). Bcl-xL protein is expressed at high levels in pre-B cells as compared to Bcl-2 (Fang et al., 1996; Grillot et al., 1996; Merino et al., 1994) and is important for pre-B cell survival in vivo (Motoyama et al., 1995). Therefore, one function of the pre-BCR may be to promote pre-B cell survival by increasing levels of Bcl-xL. Transition from the pro-B to the pre-B stage of B cell development also requires signaling by the IL-7R/웂c complex (Cao et al., 1995; Peschon et al., 1994; von Freeden-Jeffry et al., 1995). Whereas expression of a bcl-2 transgene can rescue pro-T cells from a lack of IL-7R/웂c signaling (Akashi et al., 1997; Kondo et al., 1997; Maraskovsky et al., 1997), it cannot restore normal B cell production (Kondo et al., 1997; Maraskovsky et al., 1998). Thus, signals triggered by IL-7 or the pre-BCR/pre-TCR appear to differ in developing B and T cells. Enforced Bcl-2 expression blocks pro-T but not pro-B cell death in the absence of IL-7R/웂c signaling, and it blocks pro-B but not pro-T cell death in the absence of a pre-BCR or pre-TCR. XIII. Selection of Immature B Cells in the Bone Marrow

Expression of a pre-BCR, like expression of a pre-TCR during thymocyte development, promotes proliferation as well as survival and differentiation of developing B cells. After an estimated four to six cell divisions (Osmond, 1993), pre-B cells become small and rearrange their Ig light-chain genes (Benschop and Cambier, 1999). Nonproductive rearrangement of the ␬ and ␭ Ig light-chain genes yields a B cell that is unable to express surface IgM. Such B cells probably die by a Bcl-2–inhibitable pathway to apoptosis, because bcl-2 transgenic rag-2⫺/⫺ mice that express an Ig heavy-chain transgene, or bcl-2 transgenic scid mice (by virtue of chance productive Ig gene rearrangements and D애 protein expression in some pre-B cells), possess an abnormal pre-B cell population that has some of the characteristics of more mature B cells (Strasser et al., 1994a; Tarlinton et al., 1997; Young et al., 1997). Expression of surface IgM defines the virgin B cell stage (Benschop and Cambier, 1999). At this point in their development, B cells become subject to immunological selection on the basis of their BCR specificity. While it


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is clear that negative selection is used to remove autoreactive B cells (Hartley et al., 1993; Nemazee and Burki, 1989), the role of positive selection in the generation of the B cell repertoire is uncertain. From studies that have used inducible gene targeting to remove the BCR on B cells, it is clear that B cell survival is dependent on BCR expression (Lam et al., 1997), but it is not known whether this involves stimulation of the BCR by a ligand, akin to the TCR–MHC interactions required for T cell survival (Kirberg et al., 1997; Tanchot et al., 1997). Mice expressing an Ig heavy-chain transgene from an anti–Thy-1 autoantibody were shown to contain CD5⫹ B cells specific for Thy-1 only in the presence of Thy-1 antigen (Hayakawa et al., 1999). However, it remains to be determined whether positive selection of B cells is a general phenomenon or is peculiar to this particular class of B cell. B cell death in the absence of a complete BCR may involve a Bcl-2–inhibitable pathway to apoptosis, because Bcell loss in mice following Cre-LoxP–mediated deletion of the BCR was delayed by a bcl-2 transgene (Lam et al., 1997). Investigation of the apoptosis signaling pathways responsible for negative selection at the immature B cell stage has relied on mutant lpr or bcl-2 transgenic mice expressing transgenic BCRs specific for endogenous self antigens. Enforced Bcl-2 expression can inhibit deletion of autoreactive immature B cells that recognize soluble self antigens, but is less effective at blocking deletion induced by membrane-bound self antigens (Hartley et al., 1993; Lang et al., 1997). Nevertheless, tolerance toward soluble and membrane-bound self antigens is maintained because autoreactive B cells that accumulate in the periphery retain their immature phenotype and are functionally inactive (a state referred to as anergy) (Hartley et al., 1993; Lang et al., 1997). Expression of a bcl-xL transgene was reported to allow mature autoreactive B cells to develop, but again, these cells were functionally inactive (Fang et al., 1998). By contrast, Fas-deficient lpr mice appear to delete autoreactive immature B cells normally (Rathmell and Goodnow, 1994; Rubio et al., 1996). These studies indicate that deletion of autoreactive immature B cells involves Bcl-2–sensitive pathways to apoptosis, but in the absence of apoptosis, developmental arrest and induction of clonal anergy can still achieve tolerance toward self antigens. Cells that survive long enough may also escape deletion by undergoing further Ig heavychain gene rearrangements, leading to replacement of the autoreactive BCR by another BCR. This process is referred to as receptor editing (Fang et al., 1998; Lang et al., 1997). XIV. B Cell Apoptosis in Peripheral Lymphoid Organs

Some autoreactive B cells may be deleted in peripheral lymphoid organs (Figure 6). In mice that express a soluble form of HEL, for example, B

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FIG. 6. Apoptosis of mature B and plasma cells. Expression of a B cell receptor (BCR) generates an essential survival signal in mature B cells. Following stimulation with mitogens or antigen, B cell blasts and also antibody-forming cells are dependent on cytokines for their survival. When cytokines are less abundant, the pro-apoptotic ‘‘BH3-only’’ protein Bim promotes apoptosis which can be blocked by overexpression of Bcl-2. Interactions between CD40L on activated T cells and CD40 on B cells increase Fas expression on the B cell surface and thereby render B cells susceptible to killing by activated T cells expressing FasL. However, B7.2 on activated B cells may signal T cells via CD28 to produce costimulatory molecules that can signal inhibition of Fas-transduced apoptosis in B cells.

cells specific for HEL are not deleted in the bone marrow and can be found in a functionally inactive state in peripheral lymphoid organs. These cells get deleted in vivo when they present antigen to HEL-specific CD4⫹8⫺ T cells; FasL on the T cell surface engages Fas on the B cell surface, causing apoptosis (Rathmell et al., 1995). When Fas signaling is prevented by the lpr mutation, anergic B cells that encounter HEL-specific T cells are able to proliferate (Rathmell et al., 1995). This result might explain the development of autoimmunity in lpr mice. The outcome of B cell–T cell interactions is determined by BCR signaling. CD40L expressed on the T cell surface engages CD40 on the B cell surface to promote B cell proliferation, but it also increases expression of Fas, thereby rendering the B cell susceptible to killing by FasL. Signals from the BCR of normal B cells can increase FLIP expression and neutralize the apoptotic signals from Fas (Lagresle et al., 1996; Rathmell et al., 1996; Rothstein et al., 1995; Wang et al., 2000), but anergic B cells, which have abnormal BCR signaling (Healy et al., 1997), are not protected. Failure to express high levels of B7.2 following BCR engagement may be a key factor in the sensitivity of anergic B cells to FasL-dependent T cell killing, because anergic B cells expressing a B7.2 transgene are not deleted in vivo. In B7.2 transgenic mice, B cell–T cell interactions lead instead to B cell proliferation and autoantibody secretion (Rathmell et al., 1998). B7.2 normally engages CD28 on the T cell surface, and it is suggested


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that this is needed for T cells to produce stimulatory molecules which can activate signals in B cells that override the Fas death signal (Rathmell et al., 1998). Whether naive B cells that encounter self antigen are rendered anergic and deleted in a Fas-dependent manner by CD4⫹8⫺ T cells is uncertain. lpr mice that express a transgenic BCR to a self antigen expressed only in the liver do not contain autoreactive B cells, indicating that these B cells are deleted normally in the absence of Fas (Kench et al., 1998). In contrast, deletion is impaired by expression of a bcl-2 transgene (Lang et al., 1997). Enforced Bcl-2 expression also reduced but did not completely block deletion of mature autoreactive B cells when Cre-LoxP–mediated gene inversion was used to alter BCR specificity in peripheral lymphoid organs (Lam and Rajewsky, 1998). However, in these systems, artificially high numbers of B cells are responsive to self antigen, so they may not reflect what happens when a normal B cell within a polyclonal repertoire encounters self antigen. Apoptosis prevented by Bcl-2 may just be a consequence of limited T cell help. In addition to its role in maintaining B cell tolerance toward self antigens, apoptosis is involved in terminating humoral immune responses. This aspect of immune homeostasis is disrupted in bcl-2 transgenic mice. Antibody responses to foreign antigens are prolonged and elevated (up to 200-fold) in bcl-2 transgenic mice (Strasser et al., 1991b), due to extrafollicular antibody-forming cells (AFCs) and germinal center cells having an abnormally long life span (Smith et al., 1994). The pro-apoptotic protein Bim appears to be important for the death of AFCs because bim⫺/⫺ mice also have 30- to 200-fold more AFCs than their wild-type counterparts (Bouillet et al., 1999). Failure of AFCs to die normally in bcl-2 transgenic or bim⫺/⫺ mice is probably a contributing factor in the development of systemic lupus erythematosus–like autoimmune disease in these mice (Strasser et al., 1991b; Bouillet et al., 1999). Fas deficiency, in contrast, has no effect on the removal of antibody-forming B cells (Smith et al., 1995). XV. Conclusion

Transgenic mice expressing inhibitors of apoptosis in their lymphocytes and mutant mice that lack apoptosis signaling molecules have provided significant insight into the mechanisms by which nonfunctional, dangerous, or effete lymphocytes are killed. They have provided evidence that death receptors and the Bcl-2 protein family regulate distinct pathways to apoptosis, and that these pathways are engaged in response to different physiological stimuli. However, the details of lymphocyte apoptosis that can be inhibited by anti-apoptotic Bcl-2 family members remain sketchy. For

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example, which of the pro-apoptotic proteins are being countered in bcl-2 or bcl-xL transgenic lymphocytes, and how are these pro-apoptotic proteins being activated by physiological death stimuli? Given that there may be considerable functional overlap between the different pro-apoptotic proteins, an answer to the former question may require the analysis of mice deficient for several of these proteins. Another unresolved question is how apoptosis is signaled in thymocytes during negative selection. Neither Bcl-2, FADD-DN, nor CrmA expression can abrogate this process so vital to proper immune function, and it remains an area of considerable interest. ACKNOWLEDGMENTS Work in our laboratory is supported by fellowships from the Leukemia Society of America, the University of Melbourne, and the Anti-Cancer Council of Victoria (Melbourne) and by grants from the National Health and Medical Research Council (Canberra), the Dr. Josef Steiner Cancer Research Foundation (Bern), the Cancer Research Institute (New York), and the Anti-Cancer Council of Victoria.

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Systemic Lupus Erythematosus, Complement Deficiency, and Apoptosis M. C. PICKERING,* M. BOTTO,* P. R. TAYLOR,* P. J. LACHMANN,† AND M. J. WALPORT* *Rheumatology Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London; †Microbial Immunology Group, Center for Veterinary Science, Cambridge, England

I. Introduction

There are many links between the complement system and the autoimmune disease systemic lupus erythematosus (SLE). Soon after the identification of antinuclear antibodies, the major serological hallmark of the disease, it was discovered that complement proteins are deposited in the tissues of patients. An association was found between the degree of complement activation in blood samples from patients and the level of disease activity. Complement proteins were discovered to be co-located with antibodies in inflamed tissues, such as the glomeruli of patients with glomerulonephritis. These data suggested that the formation or deposition of immune complexes in tissues leading to complement and leukocyte activation could cause the pathogenesis of the tissue injury of SLE. However, it was also discovered that the presence of antibodies and complement in tissues was not sufficient to cause inflammatory injury. Clinically normal tissues from patients with SLE (e.g., the skin) also contained deposited antibodies and complement proteins. Indeed, the presence of these proteins at the dermoepidermal junction in nonlesional skin was found to be a moderately specific diagnostic test for SLE, named the lupus band test. As the spectrum of autoantibodies characterized in SLE increased, it was discovered that about one third of patients with the disease have autoantibodies to complement proteins, especially to C1q. A reduction was also discovered in the expression of a complement receptor on erythrocytes, CR1, which plays a role in binding immune complexes in the circulation. Each of these links between complement and SLE, which we discuss in detail below, may be explained on the basis that the autoantibodies of SLE, on binding autoantigen, activate complement. Downstream events following complement activation could explain the development of autoantibodies to complement and to erythrocyte CR1 consumption. However, the links between complement and SLE are much more complicated and curious. It was found that inherited complement deficiency is strongly associated with the development of SLE. Indeed, the very rare inherited homozygous deficiency of the first protein of the classical 227


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pathway of complement, C1q, is almost invariably associated with the development of severe disease. Thus, on the one hand, complement deficiency causes SLE. On the other, SLE causes complement consumption and tissue injury. Even more surprisingly, C1q deficiency causes SLE, yet SLE commonly causes autoantibodies to C1q to develop. In this chapter, we attempt to resolve these paradoxes and, in doing so, discuss each of the associations between abnormalities of complement and SLE. First, we describe and tabulate the links between complement deficiency and SLE. Second, we describe some of the phenotypic abnormalities in mice with engineered mutations in complement genes. Third, we review the links between complement activation and inflammation in SLE. Fourth, we describe the autoantibody response to complement proteins and the significance of these autoantibodies. Finally, we develop the hypothesis that a major physiological activity of the complement system is to promote the clearance of autoantigens and mask them from the immune system. If this activity is deficient, in the presence of other disease susceptibility genes, autoantigens may drive a pathological autoantibody response leading to the development of SLE. II. Description of the Associations between Complement and SLE in Humans

A. GENETIC DEFICIENCIES OF COMPLEMENT AND SLE 1. Homozygous Classical Pathway Deficiency Homozygous hereditary deficiency of each of the classical pathway components (C1q, C1r, C1s, C4, and C2) is associated with greatly increased susceptibility to SLE. There is a hierarchy of severity and susceptibility to the development of disease according to the position of the deficient complement protein in the activation sequence of the classical pathway of complement. The primary publications reporting these associations are cited in Tables I–V, which tabulate the reported cases of deficiencies of the classical pathway proteins and C3. Thus, 39 of the 42 (93%) described individuals with homozygous C1q deficiency had SLE, which was frequently very severe. Next in the hierarchy comes C1r and C1s deficiency (usually combined) [SLE prevalence: 8 of 14 subjects (57%)], then C4 deficiency [SLE prevalence: 18 of 24 subjects (75%)]. There is then a significant step change in the strength of the association of SLE with deficiency of the next protein in the classical pathway, C2. Deficiency of C2 is the most common hereditary complement deficiency in western European white populations and is associated with the development of SLE in 앑10% of cases. Finally, there is C3 deficiency, which, although strongly associated with the development of rashes and

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glomerulonephritis, is typically not associated with the development of lupus autoantibodies. These clinical observations strongly suggest that there is a physiological function of the classical pathway of complement activation that protects against the development of SLE. Furthermore, the hierarchy of susceptibility and severity of lupus, according to the missing classical pathway protein (C1q ⬎ C4 Ⰷ C2), suggests that an activity of the early part of the classical pathway plays a key protective role against the disease. We now review the associations between inherited homozygous complement deficiency and SLE in detail. We consider each protein in turn and tabulate the published associations of hereditary complement deficiency and SLE, with the exception of C2 deficiency, for which the abundance of published cases would make a table summarizing all the case histories too cumbersome. 2. C1q Deficiency Thirty-nine of the 42 recorded patients with homozygous C1q deficiency have developed a clinical syndrome similar to SLE (reviewed in Walport et al., 1998). These cases are summarized in Table I. In the affected patients, rash occurred in 37, glomerulonephritis in 16, and cerebral disease in 8. Antinuclear antibodies were reported in 24 of the 35 patients tested, and antibodies to extractable nuclear antigens were present in 15 of 24 patients assessed. Notably, the incidence of anti–double-stranded DNA antibodies was low; only 5 of the 25 patients tested were positive. No clinical phenotype has been observed among any of the heterozygous C1qdeficient relatives of the homozygous deficient subjects. Among C1q-deficient individuals, further analysis of their complement profile typically showed raised levels of C3 and C4 and elevated C1 inhibitor activity compared with serum samples from normal subjects. This demonstrates that in normal subjects there is significant physiological turnover of the classical pathway resulting from C1 activation. In the absence of C1 function, C4 and C3 levels, together with C1 inhibitor, the inhibitor of activated C1r and C1s, are elevated in concentration due to reduced turnover. The molecular basis of C1q deficiency has been characterized in 12 families and is tabulated in Table I. Three genes, organized in tandem, encode C1q. In each family studied, a single mutation affecting one of these three genes has been found and mutations in all three genes have been characterized in different families. In every case, no functional C1q activity was detected. However, in some families, no C1q protein was detected, while in others, antigenic C1q which showed no functional activity could be detected.

TABLE I HOMOZYGOUS C1q DEFICIENCY

Family

Age at Onset (yr), Gender, Race/ Ethnic Group

230

1

Absent C1q 37, M, Japanese

2

5, F, Japanese

3

10, M, Turkish parents, consanguineous

4a

9, M, Spanish (Canary Islands)

Clinical Features Discoid rash, erythema multiforme on soles and palms

SLE, glomerulonephritis; died at age 6 Deformed fingernail and toenail with monilia, hyperkeratotic desquamative rash, fits, mouth monilia and aphthae, otitis media; died of septicemia at age 10

Rash, hair loss, MPGN, Rothmund–Thompson syndrome (poikiloderma congenitale), bilateral posterior cataract

Laboratory Tests ANA⫹ 1/20, DNA⫹, LE⫺, ENA⫹ (RNase resistant); IF lesional skin: negative for Ig and complement; 0.4 애g/mL C1q detected ANA⫺, DNA⫺, latex positive, RNP⫹ ANA⫺, DNA⫺, latex⫺. ENA⫺, anti–smooth muscle positive, antiHBsAg⫹; skin IF: positive for IgG and C3; necropsy: MCGN

ANA⫺, DNA⫺, latex positive 1/640; renal IF: positive for IgG, IgM, C3, and C3PA

Notes FFP infusions: rash improved at 10 days, worse by 1 mo Brother died of SLE at age 10

References Nishino et al. (1981)

Komatsu et al. (1982), Orihara et al. (1987) Response to Berkel et al. (1977, plasmapheresis and 1979), Loos et al. FFP in 10 days (1980), Petry et al. Genetic analysis of the (1997a) family: C 씮 T transition at position 186 of the A chain, Gln to stop codon, no DNA available from propositus Mampaso et al. (1981)

8, F, ?

4c

5, M, ?

5

29, M, Japanese

Discoid lupus

6

21, M, Japanese

7

1, F, Greek, white

8a

1, M, Yugoslav, white

Itchy, spreading rash on upper limbs and shoulder; leukopenia; discoid lupus Light-sensitive cutaneous vasculitis since 1 yr of age, mild alopecia, Raynaud’s phenomenon, hyperkeratotic and atrophic skin, aphthous ulceration Purulent otitis media, febrile then nonfebrile convulsions, rash on palms and soles, photosensitivity, ‘‘butterfly’’ rash, MPGN; died at age ?

231

4b

Rothmund–Thompson syndrome, bilateral posterior cataract, rash, hematuria, MPGN Rothmund–Thompson syndrome, bilateral posterior cataract, rash, hematuria, MPGN

ANA⫹ 1/80, DNA⫺, latex positive 1/640; renal IF: positive for IgG, IgM, C3, and C3PA ANA⫹ 1/30, DNA⫺, anti–smooth muscle positive 1/30, latex positive 1/80; renal IF: positive for IgA, IgG, IgM, C3, and C3PA ANA⫺, DNA⫺, ENA⫺

Nagaki et al. (1982), Orihara et al. (1987) Uenaka et al. (1982)

ANA⫺, LE⫺, latex negative ANA⫺, LE⫺; renal biopsy: mild mesangial proliferation; renal IF: capillary IgG, mesangial IgM and C3; skin IF: positive for IgG, IgM, C3, and C5 ANA⫹ 1/80 speckled, DNA 94% (⬍30%), anti-Sm⫹, anti-RNP⫹, anti-Ro⫹, latex positive 1/40; IgG2 deficiency

Minta et al. (1982)

Transient improvement following plasma exchange and FFP

Mikuska et al. (1983), Slingsby et al. (1996)

Deletion of C at codon 43 in the C chain (continues)

TABLE I (Continued )

Family


8b

3, M, Yugoslav, white

9

13, F, Saudi Arabian

232

10

7, F, Turkish

11a

4, F, Turkish

11b

6, F, Turkish

Clinical Features ‘‘Butterfly’’ rash, cutaneous vasculitis on palms and toes, fever Grand mal seizures at ages 13 and 15; meningitis, polyarthritis, fever, extensive rash, alopecia, oral thrush, leukopenia, thrombocytopenia Rash: crusted lesions on erythematous base; died of meningitis at age 9 Malar rash, facial swelling, stomatitis, extensive macular eruption with erythema and desquamation; died of sepsis at age 6 Facial and truncal edema, hematuria

Laboratory Tests

Notes

References

⫹

ANA 1/80 speckled; lupus band test: IgM⫹ ANA⫹ 1/16, DNA⫺, ENA⫺; lupus band test: positive for IgG, IgM, and C3; skin biopsy: hyperkeratosis, basal vacuolation

Steinsson et al. (1983)

ANA⫹, DNA⫺, ENA⫺, LE⫹, latex negative; skin IF: C3 at dermoepidermal junction ANA⫹, DNA⫺, ENA⫺, LE⫹, cryoglobulins negative; skin IF: IgG, IgM, and C3 in vessel walls

Berkel et al. (1981)

ANA⫺, antimitochondrial negative

Abnormal immune response to T cell–dependent antigens

Berkel (1993), Berkel et al. (1981)

C 씮 T transition at position 186 of the A chain, Gln to stop codon

Petry et al. (1997a)

233

12a

?, M, Slovakian

12b

?, M, Slovakian

12c

?, F, Slovakian

13

9, F, white

14a

5, M, Turkish parents, unrelated

14b

?, F, Turkish


Malar rash, arthritis, photosensitivity, necrotizing vasculitis, recurrent bacterial infections Malar rash, diffuse discoid lupus, arthritis, pericarditis, photosensitivity, vasculitis, alopecia, recurrent bacterial infections SLE-like syndrome, recurrent bacterial infections Widespread rash, alopecia, photosensitivity, onychomycosis, fits, cerebral atrophy, basal ganglion calcification, cytomegalovirus retinitis; died at age 28 Erythematous desquamated skin lesions, aphthae, otitis media, bronchopneumonia; died of renal failure at age 9 Asymptomatic at age 22

Petry et al. (1995), Toth et al. (1989)


ANA⫹ 1/2560, antiRNP⫹, anti-Sm⫹, antiRo⫹; latex positive 1/160, DNA⫺; skin IF: positive for IgG, IgM, and C3 at the epidermal basement membrane ANA⫺, DNA⫺, ENA⫺, latex negative, cryoglobulins negative, progressive renal failure and DNA⫹; renal biopsy: ‘‘consistent with SLE’’

C 씮 T transition at position 6 of the A chain, Gln to stop codon C 씮 T transition at position 41 of the C chain, Arg to stop codon

No response to FFP therapy No DNA available

Bowness et al. (1994), Slingsby et al. (1996)

Berkel et al. (1997)

C 씮 T transition of the A chain, Gln to stop codon (continues)


Family


Clinical Features

Laboratory Tests ⫺

⫺

Notes

References

No response to FFP or IVIG therapy Normal immune response to hepatitis B vaccine C 씮 T transition of the A chain, Gln to stop codon Normal immune response to hepatitis B vaccine C 씮 T transition of the A chain, Gln to stop codon

Topaloglu et al. (1996)

3, F, Turkish

Arthralgia, photosensitive rash, oral monilia and aphthae, deformed finger and toe with monilia

ANA , DNA , latex positive 1/80, antiRo⫹, ACA IgG⫹ high titer but ACA IgM⫺; renal biopsy: MPGN

15b

15, F, Turkish

ANA⫺, DNA⫺, ENA⫺, latex negative, ACA IgG weakly positive but ACA IgM⫺, renal IF: mesangial IgA, IgM, C3, and C5b-9

16

16, F, Pakistani parents, consanguineous

Photosensitive rash, 2 episodes of macroscopic hematuria, IgA nephropathy associated with MPGN Vasculitic rash, recurrent urinary tract infections, cerebral lupus

ANA⫺, DNA⫺, ENA⫺, latex negative

Clinical improvement with weekly FFP infusions administered for 8 wk

Unpublished case

Fever, MCGN, discoid facial lesions; died at age 8

ANA⫹, DNA⫹, latex positive; renal IF: positive for IgG, IgM, C3, and C5 but negative for C1q and C4

Point mutation resulting in premature stop codon in the B chain

McAdam et al. (1988), Reid and Thompson (1983), Thompson et al. (1980)

234

15a

Dysfunctional C1q detected 17a 4, M, Pakistani parents, consanguineous

1.5, F, Pakistani

17c

0.5, F, Pakistani

18a 18b

?, M, Moroccan parents, consanguineous 3, M, Moroccan

18c

16, F, Moroccan

18d

23, M, Moroccan

19a

7, F, Dutch, white

19b

4, F, Dutch, white

Vasculitic rash on digits, face, and trunk; oral ulcers; pneumonia; died at age 7 Vasculitis with oral and facial lesions; died at age 2.5 Asymptomatic at age 42 Rash, widespread discoid lupus at age 15, thrombocytopenia (45 ⫻ 109/L), growth retardation

235

17b

Subacute cutaneous lupus erythematosus, arthralgia Subacute cutaneous lupus erythematosus Glomerulonephritis at ages 7 and 20: fever, aphthae, ‘‘butterfly’’ rash, alopecia, myositis, grand mal seizure, somnolence; died of sepsis at age 20 Nephritis at ages 4 and 23: discoid rash, arthralgia, alopecia, fever, ‘‘butterfly’’ rash, aphthae

ANA⫹, DNA weakly positive, latex positive

Mucocutaneous lesions improved with thalidomide

ANA⫹, DNA⫺ initially, no subsequent tests performed

R. Thompson (personal communication) Chapuis et al. (1982), Meyer et al. (1985), Petry et al. (1997b)

ANA⫹, DNA weakly positive, anti-Ro⫹; skin biopsy: lymphocytic infiltration of the upper epidermis with exocytosis; skin IF: positive for IgG and IgM but C3⫺ ANA⫹ 1/160, DNA⫺, anti-Ro⫹

Gly to Asp at position 15 of the B chain

ANA⫹ 1/320, anti-Sm⫹


ANA⫹, DNA⫺, antiRNP⫹, latex positive, LE⫺; skin IF: positive for IgG and C3; renal IF: granular IgA, IgG, IgM, and C3


Hannema et al. (1984)

ANA⫹ 1/20,000, DNA⫺, anti-RNP⫹, latex positive, LE⫺, leukopenia (continues)


Family


Clinical Features

Laboratory Tests

Notes

References

⫹

42, M, Dutch, white

Membranous glomerulonephritis

20

6, F, German

Photosensitive rash, facial erythema, arthritis, Libman–Sacks endocarditis, fits and psychosis, peritonitis, MCGN, pneumonitis, generalized skin lesions; died at age 29

21a

Discoid rash, facial erythema, parotitis

ANA⫹, anti-Ro⫹, ACA⫹

21b

19 mo, M, Indian parents, consanguineous 4, F, Indian

22a

5, F, Saudi Arabian

Discoid rash, photosensitivity Severe mucocutaneous lesions, alopecia, photosensitivity, renal disease, cerebral atrophy

ANA⫹ 1/80, ENA weakly positive ANA⫹, anti-Ro⫹, antiLa⫹, anti-Sm⫹

236

19c

ANA 1/80; renal IF: positive for IgA, IgG, and IgM but negative for C1q and C3 ANA⫹ high titer, DNA⫹, anti-Sm⫹

150-kDa C1q molecule found comprising a single structural subunit containing two A chain–B chain dimers and a C–C chain dimer G to A at position 6 of the C chain, Gly to Arg G to A at position 6 of the C chain, Gly to Arg Sibling died at age 12, possibly of SLE G to A at position 6 of the C chain, Gly to Arg

Kirschfink et al. (1993), Petry et al. (1995)

Slingby et al. (1996)

Suwairi et al. (1997)

22b

14, M, Saudi Arabian

22c

?, M, Saudi Arabian

Low levels of C1q detected 23 1.5, F, ?

24 237

10, F, Japanese parents, consanguineous

Discoid lupus, photosensitivity Asymptomatic at age 5

ANA⫹, anti-Ro⫹

G to A at position 6 of the C chain G to A at position 6 of the C chain

Photosensitive rash, arthritis, ‘‘butterfly’’ rash, vasculitis on palms and soles, monilial stomatitis, staphylococcal meningitis, pneumococcal sepsis Rash, photosensitivity, aphthae, discoid lupus, syncope, peripheral numbness, basal ganglion and temporal lobe calcification

ANA⫹ speckled, DNA⫺, lupus band test positive, ENA⫹ 1/2 million (RNase resistant), latex positive 1/6400

C1q 44 애g/mL (normal range, 160 ⫾ 50)

Wara et al. (1975)

ANA⫹ 1/160, latex positive 1/320, antiRNP⫹, anti-Sm⫹, antiRo⫹; skin IF: negative at the epidermal junction

C1q 5% of normal levels FFP transiently improved Sister died at age 7 with mucocutaneous candidiasis

Orihara et al. (1987)

A, Adenine; ACA, anticardiolipin antibodies; ANA, antinuclear antibody; Arg, arginine; Asp, aspartic acid; C, cytosine; ENA, extractable nuclear antigens; FFP, fresh-frozen plasma; G, guanine; Gln, glutamine; Gly, glycine; HBsAg, hepatitis B surface antigen; IF, immunofluorescence; Ig, immunoglobulin; IVIG, intravenous immunoglobulin; LE, lupus erythematosus; MCGN, mesangiocapillary glomerulonephritis; MPGN, mesangioproliferative glomerulonephritis; RNP, ribonucleoprotein; SLE, systemic lupus erythematosus; T, thymine. Modified from Walport et al. (1998).

238

PICKERING et al.

3. C1s and C1r Deficiency Hereditary deficiency of C1s and C1r is rarer than that of C1q deficiency and, of the 14 reported cases, 8 have developed a lupus-like illness and only 2 are healthy (Table II). One young Japanese boy suffered a virusassociated hemophagocytic syndrome and later developed a fatal unexplained febrile illness (Endo et al., 1999). A further patient has suffered from severe recurrent pyogenic infections, another had chronic glomerulonephritis, and screening of a series of patients with disseminated gonococcal infection revealed a further case of C1r deficiency (Ellison et al., 1987). In the majority of cases, deficiencies of both components coincided (Loos and Heinz, 1986), probably explained by the close proximity of the C1r and C1s genes on the short arm of chromosome 12 (Kusumoto et al., 1988). In these cases, C1r levels were usually absent and C1s levels were 앑50% of normal. Among these individuals, further analysis of their complement profile typically showed raised levels of C3 and C4 and elevated C1 esterase inhibitor activity, as seen in association with C1q deficiency. Selective deficiency of C1s has been reported (Endo et al., 1999; Suzuki et al., 1992), and molecular analysis of one case has shown that homozygous C1s deficiency was the result of compound heterozygosity at the C1s loci. This individual possessed a 4–base pair deletion in exon 10 of the paternal C1s gene that resulted in a premature stop codon 90 base pairs downstream of the deletion. On the maternal gene, a single nucleotide substitution (guanine for thymine) in codon 608 of exon 12 was detected which results in the generation of a stop codon. Although both mutations would be predicted to result in truncated C1s proteins, no detectable C1s protein was found on Western blot analysis of the patient’s serum (Endo et al., 1999). Homozygosity for the 4–base pair deletion in exon 10 was demonstrated in the other reported case of selected C1s deficiency (Table II) (Inoue et al., 1998). 4. C4 Deficiency C4 is present in normal serum of humans as two isotypes, C4A and C4B, encoded by tandemly duplicated genes within the class III region of the major histocompatibility complex (MHC). Total C4 deficiency therefore requires the presence of mutations in both the C4A and C4B genes. Mutants (or null alleles) of the two isotypes of C4, associated with no expressed protein, are designated C4AQ*0 and C4BQ*0 (where Q*0 designates ‘‘quantity zero’’). The individual frequency of C4AQ*0 and C4BQ*0 alleles among healthy populations of many different ethnic origins is high (see Section II,B,1). However, HLA haplotypes carrying null alleles at both the C4A and C4B loci are extremely rare. Individuals homozygous for C4AQ*0, C4BQ*0 haplotypes are totally deficient in C4.

TABLE II HOMOZYGOUS C1r AND C1s DEFICIENCY Family

Age (yr) at Onset, Gender, Race

Clinical Features

60, F, ?

Hypertension, rash, temporal artery vasculitis, death from intracerebral hemorrhage 3 yr after illness onset

2

2.5, M, Puerto Rican parents, unrelated

3a

1.5, F, Puerto Rican

Recurrent otitis media, complicated pneumonia (empyema, pneumatocele), purulent staphylococcal lymphadenitis, staphylococcal liver abscess, pneumococcal bacteremia Alopecia, facial rash, 3 episodes of meningitis, deforming polyarthropathy, febrile episode with seizures, hypertension

3b

12, M, Puerto Rican

239

1

Photosensitivity, severe discoid lupus, polyarthralgia, Raynaud’s phenomenon, alopecia, cellulitis, pneumonia, proteinuria, hematuria

Laboratory Tests ⫹

⫹

⫹

ANA 1/1000, DNA , anti-Ro , skin biopsy: anti-애⫹ only C1s 37% (72–124, RID), C1r ⬍12% (72–123, RID), CH50 ⬍5% NHP, C1q 125% (72–123, RID) C4 646 mg/L (127–331), C3 1130 mg/L (451–861), C1 esterase inhibitor 601 mg/L (206–348) by nephelometry C1r undetectable (Ouchterlony), C1s 50% normal levels (Ouchterlony), CH50 0% NHP, C1q detected (Ouchterlony) C4 85 mg/dL (16–44), C3 235 mg/dL (80–235), C1 esterase inhibitor activity not tested ANA⫺, DNA⫺, LE⫺, latex positive 1/320, skin biopsy: ‘‘lupus erythematosus’’ C1r ⬍0.01 OD units/mL (0.11, RID), C1s ⬍10 mg/mL (24, RID), C1 hemolytic activity 1% NHP, C1q 130 mg/mL (158) C4 50.2 ⫻ 103 CH50 units/mL (20.5), C3 18.5 ⫻ 103 CH50 units/ mL (13.5), C1 esterase inhibitor present ANA⫹ 1/80 speckled, DNA⫺, LE⫺, latex positive 1/640, immunofluorescence renal biopsy: positive for IgA, IgG, IgM, C3, C4, and C1q (mesangial)

Notes

References

CH50 partially corrected by addition of purified C1r Failure of C1r synthesis by patient’s cultured monocytes to rise in response to 웂-interferon despite normal rise in C1s Heterozygous for HLA-A1, B37, DR1/HLA-A2, B15

Chevailler et al. (1994)

Garty et al. (1987)

Improved with hydroxychloroquine, prednisolone, and methyldopa Heterozygous for HLA-A9, B5/ HLA-A19, B12

Lee et al. (1978), Blum et al. (1976), Chase et al. (1976)

Improved with hydroxychloroquine and prednisolone Heterozygous for HLA-A9, B5/ HLA-A29, B12

(continues)

TABLE II (Continued ) Family


Clinical Features

31, M, Puerto Rican

Asymptomatic at time of report, history of tuberculous lymphadenitis

3d

16, F, Puerto Rican

Asymptomatic

4

6, F, ?

Systemic lupus erythematosus

240

3c

Laboratory Tests C1r ⬍0.01 OD units/mL (0.11, RID), C1s ⬍10 mg/mL (24, RID). C1 hemolytic activity 1% NHP, C1q 100 mg/mL (158) C4 50.2 ⫻ 103 CH50 units/mL (20.5), C3 16.9 ⫻ 103 CH50 units/ mL (13.5), C1 esterase inhibitor present ANA weakly positive. latex positive 1/640 C1r ⬍0.01 OD units/mL (0.11, RID), C1s 12 mg/mL (24, RID), C1 hemolytic activity 1% NHP, C1q 150 mg/mL (158) C4 31 ⫻ 103 CH50 units/mL (20.5), C3 22.5 ⫻ 103 CH50 units/mL (13.5), C1 esterase inhibitor present ANA⫹ 1/20, latex positive 1/160 C1r ⬍0.01 OD units/mL (0.11, RID), C1s ⬍10 mg/mL (24, RID), C1 hemolytic activity 1% NHP, C1q 130 mg/mL (158) C4 40.8 ⫻ 103 CH50 units/mL (20.5), C3 16.6 ⫻ 103 CH50 units/ mL (13.5), C1 esterase inhibitor present ANA⫹ (initially negative), LE⫺, CH50 0% NHP, absence of C1, elevated C1 esterase inhibitor level, CH50 restored after addition of purified C1s whereas unchanged following addition of purified C1r or C1q

Notes

References

HLA typing not performed

Heterozygous for HLA-A2, B5/ HLA-A29, B12

Symptoms improved with corticosteroid therapy, but no change in complement component levels

Pondman et al. (1968)

241

5a

18, M, ?

Scaling, erythematous atrophic skin lesions at age 15, febrile episodes with polyarthritis, vasculitis, one brother died of ‘‘lupus illness’’ at age 12

5b

24, F, ?

Recurrent otitis media and upper respiratory tract infections since childhood, polyarthralgia, recurrent rash, one brother died of ‘‘lupus illness’’ at age 12

6

13, F, ?

Chronic glomerulonephritis

7

14.5, F, Puerto Rican

Arthralgia, mesangioproliferative glomerulonephritis, history of rheumatic fever, varicella infection and postvaricella encephalitis

C1r undetectable (Ouchterlony), C1s 11.7 애g protein/mL (30, RID), CH50 ⬍12 units/mL (29–61), C1q 16 애g/mL (17–20) C4 hemolytic activity 401,800 units/ mL (43,000–449,000), C3 hemolytic activity 4500 units/mL (1536–3664), C1 esterase inhibitor activity 180% NHP C1r undetectable (Ouchterlony), C1s 12.8 애g protein/mL (30, RID), CH50 ⬍12 units/mL (29–61), C1q 20.8 애g/mL (17–20) C4 hemolytic activity 2,460,000 units/mL (43,000–449,000), C3 hemolytic activity 7800 units/mL (1536–3664), C1 esterase inhibitor activity 178% NHP CH50 ⬍1 unit/mL (45–58), C1 hemolytic activity 100 units/ 0.5 mL (21,000–30,000), C1s 16 애g/mL (20.4–45.2), C1q level normal C4 950 애g/mL (300–410), C3 1250 애g/mL (1100–1550), elevated C1 esterase inhibitor level ANA⫹ 1/320–640, DNA⫺, anti-Sm⫺, latex positive 1/640, immunofluorescence renal biopsy: positive for IgG and C3 CH50 ⬍1% NHP, C1 hemolytic activity ⬍0.5% NHP, C1r undetectable (RID), C1s 40% normal (RID), C1q 18.5% normal (RID)

Reduced C1 hemolytic activity restored following addition of purified C1r Serum bactericidal activity against Escherichia coli in vitro markedly impaired

Day et al. (1972), de Bracco et al. (1974)

Reduced C1 hemolytic activity restored following addition of purified C1r Serum bactericidal activity against E. coli in vitro markedly impaired

Day et al. (1972) de Bracco et al. (1974), Moncada et al. (1972)

C1r not measured, but addition of purified C1r alone reconstituted total hemolytic and C1 hemolytic activity

Pickering et al. (1970)

HLA-Aw24, A10, Bw35, B18 haplotype C1 hemolytic activity partially restored after addition of purified C1r (⬍0.5% NHP to 39% NHP)

Rich et al. (1979)

(continues)

TABLE II (Continued ) Family


35, F, African American

9

11, M, Japanese

10

6, M, Japanese

242

8

Clinical Features

Disseminated gonococcal infection, alcohol and intravenous drug abuse Malar rash, glomerulonephritis with proteinuria and hematuria since age 11, previous pulmonary tuberculosis, previous nephrectomy for hydronephrosis, cardiomyopathy, renal replacement therapy commenced at age 26

Virus-associated hemophagocytic syndrome at age 4, pyrexia of undetermined origin at age 6, unconcious following convulsion during this illness and died 6 mo later without regaining consciousness

Laboratory Tests

Notes

C4 881 애g/mL (200–800), C3 860 애g/mL (800–1800), normal C1 esterase inhibitor level Complete C1r deficiency

ANA⫹ 1/160, DNA⫹, anti-Sm⫺, latex positive, LE⫺, anti-RNP⫺, immunofluorescence renal biopsy: positive for lgG and C3 CH50 ⬍1 unit/mL, C1 hemolytic activity 3 units/mL (87,000), C1s undetectable but C1r present (Ouchterlony), C1r 45% normal (RIE), C1q 16.7 mg/dL (8.8–15.3) (RID) C4 hemolytic activity 155,000 units/ mL (66,000), C3 hemolytic activity 5100 units/mL (6000), C1 inhibitor hemolytic activity 23,710 units/mL (14,170) C1s undetectable (RIE), parents and one sibling possessed 50% normal C1s levels

References

Ellison et al. (1987)

Homozygous 4–base pair deletion on exon 10 of C1s gene (TTTG resulting in a premature stop codon), heterozygous deletion present in patient’s mother

Suzuki et al. (1992), Inoue et al.(1998)

C1s deficiency due to compound heterozygosity: 4–base pair deletion on paternal C1s gene (TTTG resulting in a premature stop codon), maternal nonsense mutation in codon 608 in exon 12; no truncated proteins detected on Western blot analysis of patient’s serum

Endo et al. (1999)

Values in parentheses represent the reported normal range or normal mean. ANA, Antinuclear antibody; Ig, immunoglobulin; LE, lupus erythematosus; NHP, normal human pool; OD, optical density; RID, radial immunodiffusion; RIE, rocket immunoelectrophoresis, RNP, ribonucleoprotein.


243

The first report of complete C4 deficiency was published in 1974 (Hauptmann et al., 1974). Twenty-four cases have now been reported, among whom 18 suffered from lupus-like illness, often developing at an early age and associated with increased frequency of pyogenic infections (Table III). Only 2 of the 24 recorded cases of C4 deficiency were entirely healthy at the time of reporting (Table III). Antinuclear antibodies (ANAs) were positive in 15 of the 20 cases tested, although often present at low titer, while anti–double-stranded DNA antibodies were found in only 2 of the 11 tested. Anti-Ro antibodies were typically positive (7 of 10 tested), while anti-La antibodies, which frequently accompany anti-Ro antibodies, were not detected in any of these patients. Molecular analysis has shown that 6 cases have a homozygous deletion containing the C4B and adjacent 21-hydroxylase A (CYP21A) genes, although the mechanism of C4A nonexpression was not elucidated (Fredrikson et al., 1998; Fremeaux-Bacchi et al., 1994; Lhotta et al., 1996; Nordin Fredrikson et al., 1991; Uring-Lambert et al., 1989). Other work has shown that mutations resulting in C4A nonexpression may be due to either a 2–base pair insertion in exon 29 or a single base pair deletion in exon 20 of the C4A gene (Table III) (Fredrikson et al., 1998; Lokki et al., 1999). An identical 2–base pair insertion on exon 29 of the C4B gene also results in nonexpression, providing the first molecular evidence of a C4B pseudogene (Lokki et al., 1999). A unique patient had C4 deficiency caused by uniparental isodisomy of an MHC haplotype containing null alleles of C4A and C4B (Welch et al., 1990). 5. C2 Deficiency Homozygous C2 deficiency is the most common inherited classical pathway complement deficiency, with an approximate prevalence in western European white populations of 1 : 20,000. In contrast to homozygous C1q deficiency, the majority of deficient individuals are probably healthy. SLE has been thought to occur in up to 33% of C2-deficient individuals (Agnello, 1978), although this figure is highly likely to be an overestimate due to ascertainment artifact. This is evident from the following argument. Using data from population studies, the frequency of the C2Q*0 allele in healthy western European white populations is 앑6 ⫻ 10⫺3 (Table IV) and therefore the predicted homozygote frequency is 4.8 ⫻ 10⫺5, or 1 in 20,000. The population of the United Kingdom (UK) is presently estimated at 59 million. Using this figure, the number of homozygous C2-deficient individuals in the UK may be estimated to be 앑2950. SLE in C2-deficient individuals is strongly biased toward female patients, in contrast to C1q and C4 deficiency (see Table VI). Therefore, approximately half of the homozygous C2-deficient subjects in the population form the major at risk

TABLE III HOMOZYGOUS C4 DEFICIENCY

Family

Age (yr) at Onset, Gender, Race/ Ethnic Group

Clinical Features

18, F, ?

Rash, alopecia, photosensitivity, hematuria, proteinuria at 2 mo gestation

2

1.5, M, white

Rash, transient arthritis, fever, developed nephrotic syndrome; renal biopsy: mesangial sclerosis

3a

17, M, white

3b

12, M, white

Severe HSP at age 17 responsive to penicillin and corticosteroid, hypertension and nephrotic syndrome 6 yr later with renal failure, 2 yr after renal transplant recurrence of HSP in transplanted kidney Asymptomatic

244

1

Laboratory Tests

Notes

References

ANA⫺, DNA⫺, anti-Ro⫺, anti-La⫺, cryoglobulins detected, LE test negative, lupus band test negative ANA⫹ 1/40, DNA binding raised, anti-Ro⫹, anti-La⫺; persistent lymphopenia and reduced neutrophil chemotaxis; immunofluorescence skin biopsy: negative; immunofluorescence renal biopsy: positive for IgG, IgM, IgA, and C3

Homozygous for HLA-A2, B40, Cw3 Homozygous deletion of C4B and 21-hydroxylase A genes

Hauptmann et al. (1974), Meyer et al. (1985), Uring-Lambert et al. (1989) Awdeh et al. (1981a), Clark and Klebanoff (1978), Jackson et al. (1979), Meyer et al. (1985), Ochs et al. (1977), Schaller et al. (1977)

Immunofluorescence renal biopsy: positive for IgA, IgG, IgM, and C3; identical findings in transplanted kidney

Not reported

Required chlorambucil therapy and prednisolone to control nephritis Reduced antibody response following immunization with bacteriophage ␾X174 and no IgM–IgG class switch Reduced lymphocyte response to mitogens and allogeneic cells Phagocytosis and bactericidal activity in the presence of C4-deficient serum reduced particularly in suboptimal conditions; latter reversed by addition of purified C4 Heterozygous for HLA-A2, B12, Dw2/HLA-A2, B15, Dw8 Homozygous for HLA-A30, B18, DR7

Homozygous for HLA-A30, B18, DR7

Lhotta et al. (1990, 1993), Tappeiner et al. (1978)

Lhotta et al. (1990), Tappeiner et al. (1978)

24, F, ?

Discoid lupus, oral ulceration, leukopenia, malaise

ANA⫹

5

19, F, ?

Photosensitive rash, arthralgia, dystrophic nail changes, proteinuria, thrombocytopenia

ANA⫹, LE⫹, DNA⫹, lupus band test negative, anti-Chido⫺, antiRodgers⫺

6a

2, F, ?

ANA⫺, DNA⫺, anti-Ro⫹, anti-La⫺; immunofluorescence renal biopsy: positive for IgG, IgA, IgM, and C3

6b

5, M, ?

Scarring atrophic skin lesions, reduced creatinine clearance at age 24; renal biopsy: mild focal and segmental mesangial expansion Acute oliguric renal failure; renal biopsy: mild mesangial expansion, mild rash

6c

2.5, M, ?

7

2, F, ?

8a

6, F, ?

245

4

Severe rash, fevers; died of septicemia and cerebral vasculitis at age 3; necropsy: normal renal histology on microscopy Atypical rash, recurrent otitis media and purulent parotitis, polyarthritis, glomerulonephritis

Rash, proteinuria, hematuria, hypertension, mesangioproliferative glomerulonephritis

Homozygous for HLA-A26, Bw49, DR2 No evidence of C4B gene deletion 2 sisters died of SLE at ages 14 and 17 Homozygous for HLA-A2, B40, Cw3, DR1 Rash improved with corticosteroids and chloroquine Homozygous for HLA-A24, B38, DR13

Ballow et al. (1979), Goldstein et al. (1988) Minta et al. (1981), Urowitz et al. (1981)

Lhotta et al. (1993), Meyer et al. (1985), Tappeiner et al. (1982)

ANA⫹ 1/640; immunofluorescence renal biopsy: positive for IgA, IgM, and C3 ANA⫹ 1/40

Nephritis resolved with prednisolone Homozygous for HLA-A24, B38, DR13


Homozygous for HLA-A24, B38, DR13


ANA⫹ ⬍1/25, DNA⫺, RF⫹, antiChido⫺, anti-Rodgers⫺

Heterozygous for HLA-A2, B40, Cw3, DR6/HLA-A30, B18, DR3 Homozygous deletion of C4B genes, C4A nonexpression due to a 2–base pair insertion in exon 29 in paternal gene (HLA-A2, B40, Cw3, DR6) and single nucleotide deletion in exon 20 in maternal gene (HLA-A30, B18, DR3)—both mutations generate premature stop codons Nephritis improved with prednisolone and azathioprine Homozygous for HLA-A30, B18, DR7

Fredrikson et al. (1998), Kjellman et al. (1982), Nordin Fredrikson et al. (1991)

ANA⫹ 1/320, anti-Ro⫹, anti-La⫺, immunofluorescence renal biopsy: positive for IgG, IgM, IgA, and C3

Lhotta et al. (1993), Meyer et al. (1985), Tappeiner et al. (1982)

(continues)

TABLE III (Continued )

Family


Clinical Features

Laboratory Tests

Notes Required pulse cyclophosphamide therapy in addition to prednisolone and azathioprine to control nephritis Homozygous for HLA-A30, B18, DR7 Nephritis improved with prednisolone and azathioprine Homozygous for HLA-A30, B18, DR7 IgG antibody response to tetanus, rubella, and EBV vaccination normal In vitro lymphocyte responses to concanavalin A, phytohemagglutinin, and allogeneic cells normala C4-deficient neutrophil phagocytosis and bactericidal activity normala Phagocytosis and bactericidal activity in the presence of C4-deficient serum normal in optimal but not suboptimal conditions; latter reversed by addition of purified C4a Homozygous for HLA-A11, Bw35, Cw4, DR1 Homozygous deletion of C4B and 21-hydroxylase A genes IgG antibody response to tetanus vaccination normal Homozygous for HLA-A11, Bw35, Cw4, DR1 (see also case 9a)

5, M, ?

Minor rash, proteinuria, hematuria, hypertension, mesangioproliferative glomerulonephritis

ANA⫹ 1/320; immunofluorescence renal biopsy: positive for IgG, IgM, IgA, and C3

8c

5, F, ?

Nephrotic syndrome, mesangioproliferative glomerulonephritis

ANA⫹ 1/40; immunofluorescence renal biopsy: positive for IgG and C3

9a

2, F, Moroccan parents, consanguineous

Recurrent severe respiratory tract infections, photosensitive rash

ANA⫺, DNA⫺, RF⫹ 1/996, cryoglobulins negative, antiRo⫺, anti-La⫺, anti-Sm⫺, antiRNP⫺, anti-Chido⫺, antiRodgers⫺; raised IgM 310 (82–33) mg/dL; reduced Thelper subset and increased Tsuppressor/ cytotoxic subset; immunofluorescence skin biopsy: negative

9b

7, F, Moroccan parents, consanguineous

Recurrent respiratory tract infections, pneumonia with pleurisy at age 18 mo, skin lesions similar to those in case 9a

Raised IgM 240 (82–333) mg/dL; normal lymphocyte subset numbers, increased B lymphocytes 25.2% (8–17%)

246

8b

References Lhotta et al. (1993), Tappeiner et al. (1982)

Lhotta et al. (1993)

Mascart-Lemone et al. (1983), Meyer et al. (1985), UringLambert et al. (1989)

Mascart-Lemone et al. (1983)

247

10

6, F, German

11

44, F, ?

12

2, F, Algerian

13

9, F, ?, nonconsanguineous

14a

16, M, North African parents, consanguineous 12, M, North African parents, consanguineous

14b

Photosensitivity, rash, Raynaud’s phenomenon Positive test for syphilis at ages 17 and 23 treated with penicillin, developed photosensitive rash at age 41, polyarthritis, Coombs-positive hemolytic anemia, oral ulcers Bacterial meningitis, rash, Raynaud’s phenomenon, cicatricial atrophy, glomerulonephritis, later developed osteomyelitis of left femur, recurrent pulmonary infections, died of cardiopulmonary complications (Uring-Lambert et al., 1989) Malar rash, vasculitic rash, photosensitivity

Malar rash at age 6, hematuria, photosensitivity, polyarthralgia at age 14 Asymptomatic

ANA⫹, DNA⫺, anti-Sm⫹, antiRNP⫹ ANA⫹, biological false–positive test for syphilis, anti-Ro⫹, antiLa⫹; sister died of lupus nephritis, complotype unknown

Homozygous for HLA-A30, B17, DR7 Heterozygous for HLA-A11, B18, Cw5, DR2/HLA-A1, B7, Cw7, DR2 No evidence of C4B gene deletion

Klein et al. (1984)

ANA⫹ 1/1024 speckled, DNA⫺, anti-Sm⫹, anti-Ro⫹, anti-La⫺; immunofluorescence renal biopsy: positive for IgM and C1q mesangial deposits

Homozygous for HLA-A1, B17 No evidence of C4B gene deletion

Dumas et al. (1986), Meyer et al. (1985), Uring-Lambert et al. (1989)

ANA⫹, anti-DNA⫺, anti-Ro⫹, anti-Sm⫹

Unilateral paternal isodisomy (identical paternal chromosomes without a maternal chromosome, in this case chromosome 6) Single paternal haplotype: HLAA28, B40, Cw3, DR6, DRw52, DQw1 Homozygous deletion of C4B and 21-hydroxylase A genes Homozygous for HLA-A2, B17, DR7 Homozygous deletion of C4B and 21-hydroxylase A genes Homozygous for HLA-A2, B17, DR7 Homozygous deletion of C4B and 21-hydroxylase A genes

Welch et al. (1990)

ANA⫺, DNA⫺ anti-Ro⫹, anti-La⫺, anti-Sm⫺; immunofluorescence renal biopsy: positive for IgG and C1q mesangial deposits ANA⫺, no antibodies to extractable nuclear antigens detected

Goldstein et al. (1988), Reveille et al. (1985)

Fremeaux-Bacchi et al. (1994)

(continues)

TABLE III (Continued )

Family 15

b


Clinical Features


⫺

Notes ⫺

Recurrent fever, vomiting, hematuria, mesangioproliferative glomerulonephritis

ANA 1/20, DNA , anti-Chido , anti-Rodgers⫺; immunofluorescence renal biopsy: positive for IgG, IgM, (IgA), C3, and C1q

16a

30, F, Finnish

Photosensitivity, malar rash, polyarthritis, leukopenia, exacerbations during pregnancies

ANA⫹ 1/320, RF weakly positive, anti-Sm⫹ 1/1280; immunofluorescence skin biopsy: positive for IgM and C3

16b

?, M, Finnish

Photosensitivity only

248

10, M, ?

Attacks responded to alternate-day prednisolone therapy Homozygous for HLA-A24, B38, Cw7, DR13, DQ6 Homozygous deletion of both C4B and 21-hydroxylase A genes Heterozygous for HLA-A2, B39, Cw7, DR2 (DRB1*1501, DRB5*0101, DQB1*0602)/HLAA2, B40, Cw3, DR2 (DRB1*1501, DRB5*0101, DQB1*0601) C4B deletion on maternal gene (HLA-A2, B40, Cw3, DR2), C4A gene on maternal haplotype, and C4A and C4B gene on paternal haplotype all contained identical 2–base pair insertion in exon 29 resulting in a premature stop codon in exon 30 No truncated C4 polypetide detected in culture Heterozygous for HLA-A2, B39, Cw7, DRB1*1501, DRB5*0101, DQB1*0602/HLA-A2, B40, Cw3, DRB1*1501, DRB5*0101, DQB1*0601

References Lhotta et al. (1996)

Lokki et al. (1999)

ANA, Antinuclear antibody; EBV, Epstein–Barr virus; Ig, immunoglobulin; HSP, Henoch–Scho¨nlein purpura; LE, lupus erythematosus; RF, rheumatoid factor; RNP, ribonucleoprotein. a Also demonstrated for case 9b. b Related to family 6.

TABLE IV C2Q*0 ALLELES AND SYSTEMIC LUPUS ERYTHEMATOSUS C2Q* Allele Frequency C2Q*0 Deficiency % (No.) No. of Reference

249

Glass et al. (1976) Christiansen et al. (1983) Fielder et al. (1983) Hartung et al. (1989) Christiansen et al. (1991) Truedsson et al. (1993) Sullivan et al. (1994) Unpublished data

Race White White White White White White White African American White

Patients 137 43 29 248 62 86 122 127 219

Homozygous

Controls 509 176 42 2163 76 100 427 194 406

Patients 0.7 (1) 0 0 0.4 (1) 0 0 1.6 (2) — 0

Controls 0 0 0 —b 0 0 0 — 0

Heterozygous Patients 5.1 (7) 0 3.4 (1) 2.0 (5) 1.6 (1) 5.8 (5)c 1.6 (2) — 3.6 (8)

Controls 1.2 (6) 1.7 (3) 0 —b 1.3 (1) 1 (1)c 1.4 (6) — 1.7 (7)

Detection of the 28–Base Pair Genomic Deletion by PCR Analysis

HLA-A25, B18, DR2 Haplotype Analysis Patients a

0.0328 0 0.0208 0.0141 0.0080 — — — —

Controls a

0.0058 0.0088 0 —b 0.0065 — — — —

Patients

Controls

— — — — — 0.0291 0.0246d 0 0.0183

— — — — — 0.0050 0.0070d 0 0.0086

PCR, Polymerase chain reaction. a Of the C2-deficient individuals HLA typed, all possessed either HLA-A25 or HLA-B18 or both. b The haplotype frequency was not stated in the control group, but the individual antigen frequencies (%) in patients and controls were: HLA-A 25—6.5% versus 4.7%, HLA-B 18—11.3% versus 10.7%, HLA-DR2—40.6% versus 29.1% ( p ⫽ 0.006, relative risk ⫽ 1.7). c p ⫽ 0.0999 using Fisher’s exact test. d p ⬍ 0.05.

250

PICKERING et al.

group (i.e., 1475). If the incidence of SLE among C2-deficient individuals was as high as 33%, there would be 앑500 C2-deficient lupus patients in the UK. The prevalence of SLE in the UK is 앑1 in 3000, giving a national total of 19,600 cases. If 33% of C2-deficient individuals developed lupus, then such cases would represent 2.5% of the lupus population. However, there are data showing that the frequency of homozygous C2deficient individuals among patients with SLE is, at most, 1% (Table IV). Therefore, of the 19,600 UK lupus patients, a maximum of 196 might be C2 deficient. This gives a maximum prevalence of SLE among the UK C2-deficient population of 앑13%. It is clear from these figures that the incidence of SLE among C2-deficient women is not 33%, but much more likely to be 앑10%. Consistent with the fact that the majority of homozygous C2-deficient individuals are well was the finding that the first 8 reported individuals with homozygous C2 deficiency (from 4 families) were all healthy (Agnello, 1978; Cooper et al., 1968; Klemperer et al., 1966, 1967) and, indeed, 2 of them were immunologists! However, following these initial reports, many cases were subsequently recorded of patients with C2 deficiency and SLE (Agnello, 1978). At present, at least 100 such cases have been reported. The severity of SLE associated with homozygous C2 deficiency is comparable to ‘‘idiopathic’’ SLE, but certain phenotypic differences exist. Renal and cerebral involvement appears less common, while arthralgia is more frequent (Agnello, 1978; Ruddy, 1986). Cutaneous involvement, typically widespread erythematous annular lesions, is common. Serological differences include the rarity of antibodies to doublestranded DNA and ANAs, while the frequency of anti-Ro antibodies appears to be high compared with idiopathic lupus (Agnello, 1978; Provost et al., 1983). For example, in a study of 9 homozygous C2deficient female lupus patients, 7 were anti-Ro antibody positive, while only 5 possessed low-titer ANAs and only 3 were anti-DNA antibody positive (Provost et al., 1983). Skin immunofluorescence studies in individuals with homozygous C2 deficiency and SLE typically do not show the presence of either complement or immunoglobulin at the dermoepidermal junction (Agnello, 1978). There are also reports of associations of C2 deficiency with recurrent infections, although these are less frequent than the reports of the association of SLE and C2 deficiency (Borzy et al., 1984; Hyatt et al., 1981; Leggiadro et al., 1983; Newman et al., 1978; Sampson et al., 1982; Thong et al., 1980). The reason for this association, in some cases, may be partly explained by coexistent abnormalities of the alternative pathway function in C2-deficient individuals. Two C2-deficient children with recurrent septi-


251

cemia were shown to have 50% normal factor B levels and reduced alternative pathway hemolytic activity, the latter normalized upon addition of purified factor B (Newman et al., 1978). Deficiency of C2 may be either due to a failure to synthesize the protein (termed type I deficiency) or due to a selective defect in its secretion (type II deficiency). Type I C2 deficiency is by far the most common cause of C2 deficiency and, in at least 90% of cases, is associated with the extended haplotype: HLA-25, B18, DR2, C2Q*0, C4A4, C4B2, BfS (Agnello, 1978; Hauptmann et al., 1982). The molecular basis for type I deficiency, associated with this haplotype, is due to a 28–base pair genomic deletion which causes skipping of exon 6 during RNA splicing, resulting in generation of a premature termination codon ( Johnson et al., 1992). Molecular analysis of a 3-year-old white boy with complete C2 deficiency has elucidated a further cause of type I C2 deficiency. On one allele, the 28–base pair genomic deletion was detected, while on the other, a novel mutation consisting of a single base pair deletion in exon 2 that caused a frameshift mutation and premature stop codon was present (Wang et al., 1998). This deletion was present on the haplotype HLA-A3, B35, DR4, C2Q*0, C4A32, C4BQ*0, BfS. Type II C2 deficiency is due to missense mutations at highly conserved residues in the C2Q*0 allele (Wetsel et al., 1996). A cytosineto-thymine substitution in exon 5 (C566T, Ser189Phe) was associated with the haplotype HLA-A11, B35, DR1, C2Q*0, C4AQ*0, C4B1, BfS. The second is a guanine-to-adenine substitution (G1930A, Gly444Arg) associated with the haplotype HLA-A2, B5, DR41, C2Q*0, C4A3, C4B1, BfS. A third mutation associated with type II C2 deficiency has been described (G392A, Cys111Tyr) on another haplotype: HLA-A28, B58, DR12. The precise mechanisms by which these mutations result in the failure to secrete the C2 protein are not known. 6. C3 Deficiency Homozygous C3 deficiency is strongly associated with recurrent and severe bacterial infections, particularly those caused by encapsulated organisms such as Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. These infections illustrate the important role of C3 as a bacterial opsonin. Major infections in patients with C3 deficiency are most prominent in childhood and are less of a clinical problem in adults. This presumably reflects the lesser importance of complement in host defense to pyogenic bacteria as antibody responses mature in response to repeated infectious challenges. Among C3-deficient subjects, there is also an increased susceptibility to ‘‘immune complex’’–mediated disease, particularly glomerulonephritis.

252

PICKERING et al.

Twenty-three individuals from 16 families have been reported to date (Table V). Seventeen of these suffered from severe and recurrent pyogenic infections. Only 2 were apparently healthy, while 1 individual developed mesangiocapillary glomerulonephritis (MCGN) but did not have a history of recurrent infection. In contrast to homozygous classical pathway component deficiency, complete absence of C3 has been associated with an SLElike illness in only 3 individuals, all of whom were ANA negative (Imai et al., 1991; Sano et al., 1981). MCGN has been reported in 4 of the 23 cases, while 3 individuals had clinical evidence of nephritis (e.g., proteinuria and hematuria) and 1 individual developed immunoglobulin A (IgA) nephropathy. Many family studies have shown that heterozygotes possess 50% normal C3 levels. With the exception of 1 heterozygous sibling who developed MCGN (Pussell et al., 1980), heterozygote individuals are healthy (Botto and Walport, 1993). An additional family has been described containing 3 female siblings who expressed a dysfunctional C3 protein, among whom 1 suffered from SLE (Nilsson et al., 1992). The first molecular analysis of human homozygous C3 deficiency demonstrated the presence of a GT-to-AT mutation at the 5⬘donor splice site in intron 18 (Botto et al., 1990). This splice site mutation caused a 61–base pair deletion in exon 18 that resulted in a frameshift mutation and premature stop codon in exon 18. Further characterized mutations have included the presence of a 5⬘ donor splice site mutation in intron 10 (Huang and Lin, 1994), the presence of an 800–base pair deletion which included exons 22 and 23 (Botto et al., 1992), and a point mutation that affected a factor I cleavage site (Watanabe et al., 1993). An individual with C3 deficiency but normal C3 cDNA has also been reported (Katz et al., 1994; Peleg et al., 1992; Singer et al., 1994). In this case, a maternally inherited point mutation in exon 13 resulted in a single amino acid substitution in the 웁-chain and caused impaired C3 secretion (Singer et al., 1994). 7. Terminal Pathway Component Deficiency There is a small number of patients with SLE and homozygous deficiency of the membrane attack complex proteins. These include isolated case reports of SLE in individuals with deficiencies of C5 (Ross and Densen, 1984), C6 (Tedesco et al., 1981; Trapp et al., 1987), C7 (Segurado et al., 1992; Zeitz et al., 1981), C8 ( Jasin, 1977), and C9 (Kawai et al., 1989). These associations are more likely to be explained by ascertainment artifact than by a causal link between deficiency of the membrane attack complex protein and the development of SLE. The reasons for this are set out in Section II,A,9. 8. Mannose-Binding Lectin Deficiency The third pathway for activation of the complement system is the mannose-binding lectin (MBL) pathway, which is homologous to the classi-

TABLE V HOMOZYGOUS C3 DEFICIENCY Clinical Features Family

Age Onset of Infection, Gender, Race/Ethnic Group

Infections

Other Complications

253

1

15 yr, infancy, F, South African, white, parents consanguineous

Pneumonia ⫻4; meningitis: Neisseria meningitidis; otitis media

Erythema gyratum perstans, Sweet’s syndrome

2

4 yr, during first year, F, American but? race (adopted)

MCGN

3

4 yr, 1 yr, F, South African, white, parents unrelated

Otitis media: Haemophilus influenzae b; UTI; Escherichia coli; septicemia: Streptococcus pneumoniae Meningitis: S. pneumoniae ⫻3; lobar pneumonia: S. pneumoniae

4

3 yr, N/A, M, white, parents consanguineous 5 yr, 5 mo, F, American but? race

5

6a

13 yr, ?, F, Lebanese

6b 6c

7 yr, 6.5 yr, F, Lebanese 5 yr, 3 yr, M, Lebanese

7a

19 yr, early childhood, F, Japanese parents, consanguineous 14 yr, N/A, F, Japanese parents, consanguineous

7b

No infections Pneumonia, septic arthritis, otitis media, febrile convulsions Frequent earache, sore throat Peritonitis: S. Pneumoniae Peritonitis Bronchitis

No infections

Died at age 7.5; necropsy: purulent meningitis with polymorphs present in subarachnoid space Maculopapular rash, fever, arthralgia in wrist

Notes/Laboratory Tests 800–base pair deletion involving exons 22 and 23 No blood neutrophilia during infection Normal leukocytosis to infection Normal Rebuck window

Alper et al. (1972, 1976), Botto et al. (1992), Weiss and Schulz (1989) Ballow et al. (1975), Berger et al. (1983)

Peripheral lymphoid tissues: barely discernible germinal centers, low IgG levels (3.8–6 g/L) Illness resolved following whole blood transfusion Blunted leukocytosis to infection

Grace et al. (1976)

SLE-like illness at age 16: erythematous rash, fever, arthralgia, photosensitivity SLE-like illness at age 10: photosensitive facial rash, arthralgia

Osofsky et al. (1977) Davis et al. (1977)

Pussell et al. (1980)

Proteinuria, microhematuria Proteinuria, microhematuria Proteinuria

References

Left renal artery stenosis Heterozygous sibling with MCGN ANA⫺, LE⫺

Sano et al. (1981)

ANA⫺, LE⫺

(continues)

TABLE V (Continued ) Clinical Features Family

Age Onset of Infection, Gender, Race/Ethnic Group

Infections

Other Complications

Notes/Laboratory Tests

References

Rash during infection, arthralgia Elder sister died of pneumonia and meningitis at age 6 mo

Normal leukocytosis to infection GT–TT mutation at the 5⬘ donor splice site of intervening sequence 10 Elder sister died of pneumonia and meningitis at age 6 mo

Hsieh et al. (1981), Huang and Lin (1994)

254

8

10 yr, 8 mo, F, Aborigine of the Atayal tribe in Taiwan, parents unrelated

Pneumonia, septic arthritis; otitis media: H. influenzae

9a

26 yr, 7 mo, F, Dutch

9b


9c


Meningitis: N. meningitidis; meningitis: S. pneumoniae; sepsis: Staphylococcus aureus Meningitis: S. pneumoniae; otitis media Osteomyelitis, otitis media

10

7 yr, 5 mo, M, Laotian parents, unrelated

Lobar pneumonia; meningitis: S. pneumoniae ⫻2

11

12 yr, N/A, F, Kuwaiti:

No infections

Roord et al. (1983)

Transient maculopapular rash Transient maculopapular rash, MCGN type I MCGN C3 앑4 애g/ml (0.3% normal)

Microhematuria, nephrotic syndrome, renal failure, MCGN type I

Administration of FFP of no benefit Acute administration of FFP not associated with renal deterioration Normal size but greatly reduced C3 mRNA (1% normal)—defect unknown

Roord et al. (1983, 1989) Borzy and Houghton (1985), Borzy et al. (1988), Singer et al. (1996)

Cozma et al. (1987)

12

6 yr, 3 mo, M, Brazilian parents, consanguineous

13

10 yr, ?, M, English parents, consanguineous

14a

23 yr, 4 yr, M, Japanese parents, consanguineous 14 yr, N/A, F, Japanese parents, consanguineous 19 yr, childhood, M, New Zealand

14ba 15a

Meningitis: N. meningitidis ⫻3; bronchopneumonia ⫻4, otitis media, osteomyelitis ⫻2 Otitis media; URTI: Streptococcus pyogenes

Transient erythema multiforme at time of infection

Meningitis

IgA nephropathy

No infections

Lupus-like illness

Meningitis: N. meningitidis; periorbital cellulitis, pneumonia, 2 episodes of impetigo, recurrent cold sores in childhood

255 15b 16

7 yr, N/A, F, New Zealand 4 yr, ?, F, ?

Asthma, rhinitis Meningitis ⫻4, recurrent otitis

2 male siblings died with infections at age 6 mo

Normal leukocytosis to infection

Grumach et al. (1988)

GT–AT mutation at the 5⬘ donor splice site of intervening sequence 18

Botto et al. (1990)

Imai et al. (1991) ANA⫺ ANA⫹ C3 mRNA normal size and quantity Normal-sized proC3 molecule but aberrant trypsin cleavage profile Impaired C3 synthesis due to nucleotide substitution in exon 13 resulting in single amino acid change in 웁 chain of C3 Paternal C3 gene defect uncharacterized

Katz et al. (1994), Peleg et al. (1992), Singer et al. (1994)

C3 0.8% normal 2 male siblings died with infections at age 6 mo

Sanal et al. (1992)

ANA, Antinuclear antibody; FFP, fresh-frozen plasma; Ig, immunoglobulin; LE, lupus erythematosus; MCGN, mesangiocapillary glomerulonephritis; N/A, not applicable; SLE, systemic lupus erythematosus; URTI, upper respiratory tract infection; UTI, urinary tract infection. Modified from Botto and Walport (1993). a First-cousin of case 14a.

256

PICKERING et al.

cal pathway. MBL is homologous to C1q but binds to terminal mannose groups on the surface of many pathogens. Following ligand binding, MBL activates two serine esterases, MASP-1 and MASP-2, which are homologous to C1r and C1s, and these in turn cleave C4 and C3 (Lu et al., 1990; Matsushita and Fujita, 1992). MBL is structurally and functionally analogous to complement C1q and this led to the hypothesis that individuals with deficiency of MBL might be predisposed to the development of lupus. The MBL gene comprises four exons and is located on chromosome 1. Five polymorphisms that result in reduced serum MBL have been identified (Lipscombe et al., 1995). Three point mutations have been described in exon 1 that result in reduced MBL serum levels: Asp54, Glu57, and Cys52 mutations (Lipscombe et al., 1995). Two linked promoter polymorphisms, at nucleotide positions ⫺550 (H/L variants) and ⫺221 (X/Y variants), have also been described (Madsen et al., 1995). These polymorphisms occur on three haplotypes: HY, LY, and LX, which are associated with high, intermediate, and low levels of serum MBL, respectively. A point mutation (guanine to adenine) at nucleotide 230 of exon 1 that results in the substitution of aspartic acid for glycine in codon 54 (Asp54 mutation) is associated with severe, recurrent infections in children and adults (Summerfield et al., 1995, 1997), and the mutant MBL protein is unable to activate complement (Super et al., 1992). In a study of 102 white lupus patients, both the frequency of this allele (41% versus 30%) and the number of homozygous individuals (10% versus 7%) were increased in patients compared to 136 healthy controls, although this did not reach statistical significance (Davies et al., 1995a). However, in a small study of 50 Spanish lupus patients, the frequency of the Asp54 allele was significantly increased in the patient group (52% versus 31%, p ⫽ 0.03) (Davies et al., 1997). In both of these studies, the combination of a C4 null allele and a dysfunctional MBL allele was more strongly associated with SLE than either allele alone (Davies et al., 1995a, 1997). An association between low levels of serum MBL and SLE has been reported in Chinese lupus patients, among whom an increased frequency of the Asp54 mutation was found (0.33 versus 0.23) (Lau et al., 1996). In a study of 92 African American lupus patients, the frequencies of both the Asp54 mutation (0.163 versus 0.087, p ⫽ 0.0225) and the Glu57 mutation (0.125 versus 0.047, p ⫽ 0.0067) were significantly increased compared with 86 healthy controls (Sullivan et al., 1996). Furthermore, the frequency of the promoter polymorphisms associated with high levels of MBL were significantly decreased among the lupus patients (HY haplotype frequency: 0.078 versus 0.164, p ⫽ 0.0115). Although the increase in the frequency of the LX haplotype among lupus patients did not reach statistical significance, the number of LX/LX homozygotes was higher among the


257

patient group (11% versus 2.6%, p ⫽ 0.0324). Moreover, an association between the LX haplotype and SLE has been demonstrated in a study of 112 Chinese lupus patients (LX haplotype frequency: 0.259 versus 0.154, p ⫽ 0.019) (Ip et al., 1998). In summary, the data show that both structural and promoter polymorphisms associated with low serum MBL are increased among patients with SLE from different ethnic backgrounds. These results suggest that MBL may play a similar role to C1q in conferring protection against the development of SLE, though with a much weaker protective effect. 9. Complement Deficiency, SLE, and Ascertainment Artifacts Ascertainment artifact is a common trap in studies of associations in medicine. Thus, as described above, the first two individuals identified with C2 deficiency were both immunologists. Similarly, the first two humans with IgA deficiency were both immunologists working in Dr. Henry Kunkel’s laboratory in New York. It is implausible that C2 deficiency is a disease susceptibility gene for becoming an immunologist or that IgA deficiency was a cause for working in Henry Kunkel’s laboratory! There are analogous reasons for worrying that the association of complement deficiency with SLE is a similar though less extreme type of artifact induced by the selective assay of complement levels among patients with the disease. However, there are compelling data that the link between deficiency of classical pathway complement proteins and SLE is causal rather than artifactual. First, two large population surveys in Switzerland (of 4000 consecutive recruits into the army) and Japan (of 145,640 consecutive blood donors) identified no individuals with homozygous deficiency of any classical pathway protein or of C3 (Hassig et al., 1964; Inai et al., 1989). Second, surveys of the inbred populations local to some of the C1q-deficient patients in Turkey have failed to reveal any asymptomatic C1q-deficient individuals (Berkel et al., 2000). Third, among families in which a C1q- or C4-deficient proband was identified, the great majority of the sibships who were also found to be homozygous complement deficient also had SLE or later developed disease (Table VI). The concordance of SLE between siblings with C1q, C1r/C1s, and C4 deficiency is 90%, 67%, and 80%, respectively. This very high concordance provides additional evidence against the association of complement deficiency with SLE being due to ascertainment artifact. It is interesting to contrast these concordance data with the best published study of twin concordance of the expression of SLE, which showed concordance of disease of 2% among dizygotic twins and 24% among monozygotic twins (Deapen et al., 1992).

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PICKERING et al.

TABLE VI HOMOZYGOUS COMPLEMENT DEFICIENCY AND SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Homozygous Complement Deficiency Incidence of SLE Total no. of reported cases Individuals with SLE No. % Sex ratio All cases F : M (no.) F : M (ratio) Individuals with SLE F : M (no.) F : M (ratio) Sibling concordance data for SLE No. of families with SLE and ⬎1 deficient sibling Total no. of siblings from these families Sibling concordance for SLE (%)

C1q

C1r/C1s

C4

C2a

C3

42

14

24

77

23

39 93

8 57

18 75

24 32b

3 13

23 : 19 1.2 : 1

8:6 1.3 : 1

14 : 10 1.4 : 1

43 : 30c 1.4 : 1

16 : 7 2.3 : 1

22 : 17 1.3 : 1

5:3 1.7 : 1

12 : 6 2:1

21 : 3 7:1

3:0 —

11

2

4

8

1

29

6

10

17

2

90

67

80

58

N/A

N/A, Not applicable. a From Ross and Densen (1984). b See also text. c Gender not reported for four cases.

Fourth, several conditions in which there is chronic acquired deficiency of complement, for example, caused by deficiency of C1 inhibitor (hereditary angioedema) or autoantibodies to the C3 convertase enzyme (C3 nephritic factor), show a markedly raised prevalence of SLE. We review this association in the next section. Finally, mice in which a gene-targeted mutation of C1q has been engineered developed spontaneous lupus-like disease (reviewed in Section III). However, it remains likely that there is still some ascertainment artifact that may tend to overestimate the strength of the association between complement deficiency and the development of SLE. This is most obvious in the case of C2 deficiency (discussed above), in which simple mendelian calculations using the Hardy–Weinberg equation show that 1 : 20,000 of western European white populations have homozygous C2 deficiency. Only a minority of these individuals can have symptomatic SLE, or lupus clinics would be swamped with C2-deficient patients! It is likely that ascertainment artifact is the explanation for the very rare reported cases of patients with SLE and inherited deficiency of a membrane


259

attack complex protein (reviewed in Section II,A,7). A survey of Japanese blood donors identified 138 subjects of 145,640 with a homozygous deficiency of a membrane attack complex protein, mainly of C9; none of these subjects had SLE (Inai et al., 1989). In this same population, no subjects were identified with a classical pathway protein deficiency (Fukumori et al., 1989). By contrast, 5 Japanese patients with SLE and C1q deficiency have been reported, but only 1 with SLE and C9 deficiency (Kawai et al., 1989). 10. Acquired Complement Deficiency and SLE Patients with heterozygous deficiency of C1 inhibitor suffer from the disease hereditary angioedema. The angioedema in this disease is caused by a failure of the reduced levels of C1 inhibitor to regulate the activity of kallikrein, C1r, and C1s, leading to the production of kinins which increase vascular permeability. This failure to regulate C1r and C1s is also associated with increased turnover of C4 and C2. Patients with this disease have chronically severely reduced levels of these complement proteins, even in the absence of attacks of angioedema. It is of great interest that there are now a number of reports of patients with hereditary angioedema developing SLE (Table VII). This association reinforces the data that complement deficiency is a cause of SLE and illustrates that acquired, as well as inherited, deficiency of classical pathway proteins may be a cause of disease. There are several autoantibodies to complement proteins that interfere with the physiological regulation of complement activation in vivo, and each of these has been associated with the development of SLE. These antibodies are C3 nephritic factor, anti-C1 inhibitor autoantibodies, and anti-C1q antibodies. In each of these cases, there is a ‘‘chicken and egg’’ dispute, since it could be argued that development of the anticomplement autoantibody is itself part of the SLE process. However, in the case of C3 nephritic factor, which stabilizes the C3bBb C3 convertase enzyme of the alternative pathway, 8 cases of SLE have been described (Table VIII) (Cronin et al., 1995; Font et al., 1990; Jasin, 1979; Sheeran et al., 1995; Walport et al., 1994). In each of these, the onset of SLE occurred many years after the development of the partial lipodystrophy or dense-deposit MCGN (the main clinical phenotypes associated with the presence of C3 nephritic factor), supporting the idea that the C3 nephritic factor was the ‘‘egg’’ rather than the ‘‘chicken’’. B. COMPLEMENT NULL ALLELES Because of the strong association between hereditary homozygous classical pathway complement component deficiency and SLE, it was hypothe-

TABLE VII HEREDITARY C1 INHIBITOR DEFICIENCY AND SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) OR DISCOID LUPUS ERYTHEMATOSUS (DLE) Age (yr) at Diagnosis Clinical Features Case No.

Age (yr), Gender, Race/Ethnic Group

C1 Inhibitor Deficiency

SLE/DLE


SLE/DLE

260

a

1a

18, M, ?

17

8

Angioedema, recurrent abdominal pain and vomiting

Discoid rash, alopecia, photosensitivity, splenomegaly

1ba

18, M, ?

8

5

Facial and scrotal angioedema, abdominal pains

Severe discoid rash, alopecia, photosensitivity, splenomegaly

1cb

?, F, ?

16

10

Angioedema affecting the face and extremities, recurrent abdominal pain and vomiting

2

?, F, ?

23

21

Angioedema

Malar rash, polyarthralgia, fever, grand mal seizure at age 25, died 4 yr later from cardiac failure; necropsy: atherosclerosis and inflammatory arteritis Malar rash, photosensitivity

3

?, F, ?

?

?

Angioedema

Photosensitivity, discoid rash

4

?, F, ?

30

48

Angioedema

Photosensitivity, discoid rash

5

?, F, ?

6

14

Angioedema

Proliferative glomerulonephritis

Laboratory Tests/Notes ⫹

⫹

⫺

References

ANA , ssDNA , dsDNA , C1q 0.166 mg/mL (0.134–0.245), C4 0.054 mg/mL (0.258–0.780), C1, inhibitor level 0.037 mg/mL (0.094–0.318) ANA⫹, ssDNA⫹, dsDNA⫺, C1q 0.212 mg/mL (0.134–0.245), C4 0.108 mg/mL (0.258–0.780), C1 inhibitor level 0.043 mg/mL (0.094–0.318) Not available (normal levels of C1 inhibitor present in father and maternal grandparents)

Kohler et al. (1974)

ANA⫹, DNA⫹, C4 1–2.3 mg/dL (30–70) (mother and brother have hereditary angioedema but not SLE) ANA⫹, DNA⫹, C4 5.8 mg/dL (30–70), functional but not antigenic C1 inhibitor deficiency (son has hereditary angioedema but not SLE) ANA⫹, C4 5.5 mg/dL (30–70), (3 relatives have hereditary angioedema but not SLE) ANA⫹, DNA⫹, (identical twin has C1 inhibitor deficiency but no symptoms of either SLE or hereditary angioedema)

Donaldson et al. (1977)

Rosenfeld et al. (1974), Donaldson et al. (1977)

6

43, F, white

6

43

261

Angioedema, including laryngeal attacks Recurrent angioedema

Polyarthralgia, proliferative glomerulonephritis Fever, polyarthralgia, generalized rash, proteinuria, pulmonary fibrosis

7

38, F, Ojibwa Indian and German

?

38

8

48, F, Japanese

20

48

Angioedema affecting the face and extremities

Fever, facial rash, polyarthralgia, photosensitivity

9

24, F, white

22

24

Recurrent abdominal pain

Facial rash, photosensitivity

10

17, M, French

—

—

Angioedema

Proliferative lupus glomerulonephritis

11

33, F, ?

—

17

No symptoms attributable to hereditary angioedema

Polyarthralgia, rash, pericarditis, membranoproliferative glomerulonephritis

12

24, F, white

15

—

Recurrent abdominal pains

13

24, F, white

14

14

Angioedema of the extremities, abdominal pain

Facial rash, photosensitivity (sister, who had C1 inhibitor deficiency, developed photosensitive facial rash during danazol therapy) Nonscarring rash in sunexposed areas

14

33, F, Japanese

4

20

Angioedema affecting the face and extremities, abdominal pain

Photosensitivity, malar rash

ANA⫹, DNA⫹, C1 inhibitor ⬎5% normal ANA⫹, DNA⫺, anti-RNP⫹, anti-Sm⫹, C4 ⬍7 mg/dL (12–72), C1 inhibitor level 6.2 mg/dL (14.8–26.1) (several sisters, a nephew, and 1 child have hereditary angioedema but not SLE) ANA⫺, C4 3 mg/dL, C1 inhibitor levels 10% normal (4 family members have hereditary angioedema but not SLE) ANA⫺, DNA⫺, C1 inhibitor level undetectable (mother and 4 siblings have hereditary angioedema but not SLE) ANA⫺, DNA⫺

ANA⫹, DNA⫹, C4 2 mg/dL (12–60), C1 inhibitor level 1.2 mg/dL (21–41) (mother, brother, and aunt have hereditary angioedema but not SLE) ANA⫺, DNA⫺, anti-Ro⫹ (mother and 2 brothers have hereditary angioedema but not SLE)

ANA⫺, anti-Ro⫹, C4 6 mg/dL (20–50), C1 inhibitor level 4.8 mg/dL (15–35) (father and sister have hereditary angioedema but not SLE) ANA⫹, reduced C1q and C4 levels, reduced C1 inhibitor levels (mother and sister have hereditary angioedema but not SLE)

Young et al. (1980) Massa and Connolly (1982)

Shiraishi et al. (1982)

Youinou et al. (1983)

Hory et al. (1981), Hory et al. (1983) Suzuki et al. (1986)

Guillet et al. (1988)

Gudat and Bork (1989)

Horiuchi et al. (1989)

(continues)

TABLE VII (Continued ) Age (yr) at Diagnosis Clinical Features Case No.

Age (yr), Gender, Race/Ethnic Group


SLE/DLE


SLE/DLE

38, F, ?

16

22

Angioedema affecting the face, larynx, and extremities; abdominal pain

Scarring facial rash; skin biopsy: DLE

16

26, F, ?

3

19

Angioedema affecting the extremities, abdominal pain

DLE in sun-exposed areas, alopecia, polyarthralgia

17

9, F, ?

?

1

Intermittent facial swelling

Photosensitivity

18

44, F, white

20

?

Subcutaneous edema, vomiting, abdominal pain

Photosensitivity, later hypertension, proteinuria, hematuria, cerebral vasculitis

262

15

Laboratory Tests/Notes ⫺

⫺

ANA , DNA , C4 2 mg/dL (12–60), C1 inhibitor level 0.02 g/L (0.18–0.26) (son had asymptomatic hereditary angioedema) ANA⫹, anti-Ro⫹, C4 0.06 g/L (0.14–0.42), C1 inhibitor level 0.04 g/L (0.18–0.54) ANA weakly positive, anti-Ro⫹, antiLa⫹, DNA⫺, C4 undetectable, functional but not antigenic C1 inhibitor deficiency, reduced levels of free protein S (mother and sister have hereditary angioedema but not SLE) ANA⫹ (ANA⫺ at age 20, when clinical symptoms of hereditary angioedema developed), DNA⫺, anti-Ro⫹, C4 undetectable, C1 inhibitor antigenically and functionally undetectable (mother and brother have hereditary angioedema but not SLE)

?, —, Information not reported; ANA, antinuclear antibody; ds, double-stranded; RNP, ribonucleoprotein; ss, single-stranded. a Identical twins. b Mother of cases 1a and 1b.

References Duhra et al. (1990)

Cox et al. (1991)

Perkins et al. (1994)

Donaldson et al. (1996)

TABLE VIII C3 NEPHRITIC FACTOR AND SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) Age (yr) at Onset Clinical Features Case No.

Age (yr), Gender, Race

C3 Nephritic Factor

SLE

C3 Nephritic Factor

263

1

34, F, ?

5

17

Partial lipodystrophy

2

35, F, ?

7

31

3

35, F, ?

19

21

Partial lipodystrophy, MPGN type II Partial lipodystrophy

4 5

20, M, ? 38, F, ?

9 7

16 23

MPGN type II Partial lipodystrophy

6

37, F, ?

7

35


7

13, F, Afro-Caribbean

13

25

MPGN type III

8

44, F, white

Childhood

43


SLE Polyarthritis, serositis, photosensitivity Polyarthritis, serositis, thrombocytopenia Discoid rash, photosensitivity Malar rash, discoid rash Polyarthritis, photosensitivity, discoid rash Polyarthritis, photosensitivity

Meningococcal septicemia polyarthralgia, Raynaud’s phenomenon, recurrent angioedema Fatigue, photosensitivity, polyarthralgia, Raynaud’s phenomenon, hepatosplenomegaly


ANA , LE

⫹

Reference Jasin (1979)

ANA⫹, DNA⫹,

Font et al. (1990)

ANA⫹, anti-Ro⫹, IgG anticardiolipin positive ANA⫹, anti-Ro⫹ ANA⫹, lymphopenia

Walport et al. (1994) Walport et al. (1994) Walport et al. (1994)

ANA⫹, anti-Ro⫹, lymphopenia, neutropenia ANA⫹ 1/6400, DNA⫹, antiRo⫹

Walport et al. (1994)

ANA⫹ 1/2560, DNA⫺, antiRo⫺

Cronin et al. (1995)

Sheeran et al. (1995)

ANA, Antinuclear antibody; IgG, immunoglobulin G; LE, lupus erythematosus. MPGN, membranoproliferative glomerulonephritis. Modified from Walport et al. (1994).

264

PICKERING et al.

sized that partial deficiencies of C4 or C2 may increase disease susceptibility. This has been an extremely difficult hypothesis to test, for two reasons. The first of these is the difficulty of accurately ascertaining individual C4 null alleles and the second is due to the phenomenon of linkage disequilibrium within the MHC region. There are three reasons that it is difficult to ascertain C4 null alleles with precision. The first is because there are two isotypes of the protein, which require resolution either by electrophoretic separation (Awdeh and Alper, 1980) [which is easier after removal of C-terminal basic amino acids with carboxypeptidase B (Sim and Cross, 1986)] or by using monoclonal antibodies (Chrispeels et al., 1989; O’Neill, 1984). The second, more fundamental difficulty is that the levels of expressed C4A and C4B show overlap in the presence of one or two functional alleles (Hammond et al., 1992; Moulds et al., 1993; Wilson et al., 1989). The third is that patients with SLE often have severely reduced C4 levels in plasma because of complement activation in vivo. Several strategies have been devised to get around these problems, but none are perfect. The ratio of C4A to C4B expression has been used to deal with the problem of variable total C4 turnover in serum. Family studies provide increased confidence, because low C4A or C4B levels can be shown to be heritable. The molecular basis for the common C4A and C4B null alleles has been identified which allows accurate genotyping for some, though not all, null alleles (Schneider et al., 1986). However, the majority of published studies have been based on protein phenotyping. The phenomenon of linkage disequilibrium in the MHC raises a more fundamental difficulty. There is no doubt at all that there is one or more disease susceptibility genes for SLE located within the MHC—but after more than 20 years of research in this area, it still remains uncertain what is the relevant gene or genes. The best approach to trying to identify the relevant gene within the MHC that causes disease susceptibility is to study populations of different ethnic origins, in which the combinations of alleles at the many genes within the MHC are different. However, at present, a level of agnosticism is necessary about which are the relevant loci in the MHC that are associated with disease susceptibility to SLE in the majority of patients. It is universally agreed that total deficiency of either C2 or C4 causes a powerful predisposition to the development of SLE, but only a tiny minority of cases can be explained on this basis. In the majority of patients with SLE, there are three main groups of candidate genes within the MHC. The first are the genes that encode MHC class II proteins and components of the antigen-processing machinery, which control the repertoire of peptide presentation to T cell receptors (Beck and Trowsdale, 1999). The second are the mutated genes, which are responsible for C4A


265

and C4B null alleles (which we review in the next section). The third are the allelic variants at the loci for the cytokines tumor necrosis factor (TNF-움) TNF- and lymphotoxin. Very many other genes have been discovered within the MHC, and some of these may turn out to be important in determining susceptibility to SLE. 1. C4 Null Alleles Null alleles at either the C4A or C4B locus are very common, although, as discussed above, haplotypes carrying null alleles at both loci are very rare, accounting for the extreme rarity of total C4 deficiency. A single C4 null allele may be seen in up to 30% of healthy white subjects, with 앑4% having homozygous C4A deficiency and 1% exhibiting homozygous C4B deficiency. Low levels of C4 are present on normal erythrocytes. It was found that polymorphic variation of C4A is responsible for the blood group named Rodgers, and likewise, variation of C4B is responsible for the Chido blood group (O’Neill et al., 1979). This means that the erythrocytes from individuals with C4A deficiency are negative for the Rodgers blood group and those from subjects with C4B deficiency are negative for the Chido blood group. There is a strong association between C4AQ*0 null alleles and SLE among patients of western European white origins (Table IX). Moreover, in these patients the association shows a gene ‘‘dose-dependent’’ effect. For example, one study of white patients demonstrated a relative risk in heterozygotes of 3.23, rising in homozygotes to 16.86 (Howard et al., 1986). However, in the majority of these studies, the association with C4AQ*0 occurs in conjunction with an increased incidence of the 8.1 ancestral haplotype: HLA-A1, C7, B8, C4AQ*0, C4B1, DR3, DQ2. This particular MHC haplotype is of special interest to immunologists because it is associated with other diseases of immune dysfunction, including type 1 diabetes, autoimmune thyroid disease, myasthenia gravis, dermatitis herpetiformis, and celiac disease (Price et al., 1999). The 8.1 ancestral haplotype is also associated with IgA deficiency, reduced responsiveness to vaccination by hepatitis B surface antigen (Alper et al., 1989), and accelerated progression of human immunodeficiency virus (HIV) (Cameron et al., 1990). It is therefore a matter of some frustration that, after many years of studies of MHC associations with disease, it is still uncertain which gene or genes within this, the most common haplotype in western European white populations, are responsible for these important associations. In this context, it is important to note that an association between C4AQ*0 alleles and SLE was also demonstrated in the majority of studies of patients from different ethnic groups in which an association with HLADR3 was not found (Table IX). For example, in African American patients,

TABLE IX C4AQ*0 ALLELE FREQUENCIES IN DIFFERENT POPULATIONS OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS Homozygous C4AO*0 Deficiency

C4AQ*0 Allele Frequency

Study Number Reference

Race Ethnic Group

266

Fielder et al. (1983) Christiansen et al. (1983) Reveille et al. (1985) Howard et al. (1986) Dunckley et al. (1987) Batchelor et al. (1987) Gougerot et al. (1987) Kemp et al. (1987)

White Whitea White White White White, DR3 negative White White

Hartung et al. (1989) So et al. (1990) Schur et al. (1990)

White White White English/Irish Other Europeans Southern Sweden White Mexican Australian Aborigine White White

Sturfelt et al. (1990) Kumar et al. (1991) Christiansen et al. (1991) Reveille et al. (1991) Hartung et al. (1992)

Goldstein and Sengar (1993) Cornillet et al. (1993) De Juan et al. (1993)

French Canadians Non-French Canadians French Spanish

Patients

Controls

Patients % (No.)

Controls % (No.)

p Value, Relative Risk

Patients

Controls


DR3 Association with SLE?

29 41 15 63 63 30 20 88

42 176 64c 63 197 60 108 236

13.8 (4) 12 (5) 13.3 (2) 11.1 (7) 7.9 (5) 0 — 10.2 (9)

0 0 6 (4) 0 1.5 (3) 0 — 1.7 (4)

— — — p ⬍ 0.006, 16.86 p ⬍ 0.05 — — p ⬍ 0.002

0.08 0.20b 0.281 0.095 0.169 0.05 0.106 0.008 — 12e 0.121d 0.176 0.22 0.12 0.024 — 0.389c 0.15 0.29g — 12e 0.181d 0.052h 0.06d

p ⬍ 0.01 — NS p ⫽ 0.003, 3.23 p ⬍ 0.01 — NS — — p ⬍ 0.001, 1.0 p ⬍ 0.005 NS p ⫽ 0.03 NS p ⬍ 0.001 — — — — — p ⬍ 10⫺6 p ⬍ 10⫺6 NS NS

Yes Not tested Yes and DR2 No, but DR2 increased Not tested All DR3 negative Yes Yes

— — — — — p ⬍ 0.001 — — — — — — — — —

0.38 0.32b 0.366 0.254 0.317 0.083 0.054 0.159 0.153d 19.8d 0.277d 0.188 0.41 0.11 0.1625 0.391 0.318 0.298 0.11 0.302d 29e 0.30d 0.055h 0.12d

196 54 101 27e 62e 80 32 11 62 9 48 396 310e 310e 43

204 62 1731 144e 310e 330 — 9c 133 — — 204 155e 155e 44

— 3.7 (2) — 7 3 16 (13) 6 (2) 9.1 (1) 12.9 (8) 0 6 (3) — — — 0

— 0 — 5 1 2.4 (8) — 11.1 (1) 0 — — — — — 0

43 74 58 53

36 130 69 52

7 (3) 2.7 (2) — —

0 0 — —

— — — —

0.31d 0.149 27e 17d, e

0.10d 0.031 34e 15d, e

p ⫽ 0.001, 4.3 — NS NS

Yes and DR2 Yes and DR2 No Yes No Not tested Yes All DR3 negative No No Yes Yes Yes No No, but DQ6 increased Yes Not tested Yes Yes

Reveille et al. (1995a) Reveille et al. (1995b) Davies et al. (1995) Skarsvag (1995) Reveille et al. (1998)

267

Naves et al. (1998) Steinsson et al. (1998) Sullivan et al. (1999)

Mexican Greek White Scandinavian White Hispanic Spanish Icelandic White

Howard et al. (1986) Wilson et al. (1988) Olsen et al. (1989) Reveille et al. (1998) Sullivan et al. (1999)

African African African African African

Hong et al. (1994)

Korean

60

72

0

0

—

0.208

Dunckley et al. (1987) Hawkins et al. (1987) Zhao et al. (1989) Dunckley et al. (1987) Yukiyama et al. (1988) Yamada et al. (1990)

Chinese Southern Chinese Chinese Japanese Japanese Japanese Japanese

75 72 58 51 53 59 59

76 61 89 50 166 166 159

2.7 (2) — 8.6 (5) 11.8 (6) 0 0 0

0 — 0 0 0 0 0

NS — — p ⬍ 0.05 — — —

0.304 0.307 0.397 0.347 0.339 0.220 0d

American American American American American

55 36 82 51 69 68 84 64 104

112 33 59 121 186 119 96 194 140

— 0 — 13.7 (7) — — — — —

— 0 — 1.6 (2) — — — — —

— — — p ⫽ 0.0028, 9.7 — — — — —

0.14 0.11 63e 0.294d 30e 19e 0.143 0.266 0.034i

0.07 0.07 41e 0.141d 20e 13e 0.156 0.126 0.004i

p ⬍ 0.005 NS p ⫽ 0.008 p ⫽ 0.0172, 2.3 NS NS NS p ⫽ 0.002 p ⫽ 0.012

Yes No Yes Yes Yes Yes and DR8 No Yes (DRB1*03) Noi

35 59 79 88 84

35 59 68 73 82

2.9 (1) 1.7 (1) 5.1 (4) — —

0 0 0 — —

— — — — —

0.200 0.177 0.12d 20e 0.018i

0.071 0.079 0.04d 20e 0i

p ⫽ 0.046, 3.25 p ⫽ 0.02 p ⫽ 0.05, 4 NS NS

No Not tested No No, but DR2 increased Noi

p ⬍ 0.05, 2.1

No, but DR2 and DR9

p ⬍ 0.05 p ⬍ 0.05 p ⬍ 0.001 p ⬍ 0.01 p ⬍ 0.004 p ⬍ 0.005 —

Not tested No, but DR2 increased Not tested Not tested Not tested Not tested Not tested

0.125 Increased 0.188 0.155 0.169 0.121 0.207 0.066 0.003d

NS, Not significant. a Two female Burmese and three Australian Aborigines were included in this patient group. b Minimal estimated C4A null allele frequency. c Healthy relatives. d Frequency of the deleted C4A gene. e Population frequency (%). f Number of haplotypes. g C4A gene frequency in healthy Darwin Aborigines was taken from Ranford et al. (1987). h Frequency of the silent, nondeleted C4A gene. i Frequency of C4AQ*0 due to a 2–base pair insertion in exon 29 which is associated with HLA-B60 and -DR6.

268

PICKERING et al.

the presence of a C4AQ*0 allele conferred a relative risk of 4.5 despite no association with HLA-DR3 (Olsen et al., 1989). Furthermore, in Japanese and other Asian populations in which HLA-DR3 is extremely rare, the association with C4AQ*0 persisted (Dunckley et al., 1987; Yamada et al., 1990). However, a number of studies in white (De Juan et al., 1993; Goldstein and Sengar, 1993; Reveille et al., 1995b, 1998) and African American (Reveille et al., 1998) populations have not found significant associations between the presence of SLE and C4AQ*0 alleles. Reported associations between C4BQ*0 alleles and SLE are much weaker than for C4AQ*0 alleles (Table X). Many studies have found no increase in C4BQ*0 gene frequency in white populations and in other ethnic groups. It is therefore necessary to explain why partial deficiency of C4A and not C4B might predispose an individual to SLE. There are functional differences between C4A and C4B that could account for this. The C4A isotype shows preferential binding to amino groups, forming amide bonds, and binds particularly to proteins, for example, in immune complexes (Schifferli et al., 1986). C4B shows preferential binding to hydroxyl groups, forming ester bonds, and binds predominantly to carbohydrates. C4B binds more effectively to the surface of erythrocytes than C4A, and for this reason is more active in hemolysis than C4A. Complement activation by immune complexes interferes with lattice formation, thereby maintaining complexes in solution (Heidelberger, 1941). Hence, deficiency of C4A may cause less effective processing of immune complexes with deposition in tissues and resultant damage. The role of complement in the processing of immune complexes is discussed in detail in VI,C. There is also increasing evidence that the complement pathway plays a role in the clearance of apoptotic cells (discussed in Section VI). It is not known whether the C4A and C4B isotypes show any difference between their abilities to bind to apoptotic cells. The molecular basis of a number of the more common C4AQ*0 and C4BQ*0 alleles has been characterized. The C4AQ*0 allele on the extended haplotype HLA-A1, B8, DR3 is most commonly caused by a large deletion involving C4A and the adjacent 21-hydroxylase A pseudogene locus (CYP21A) (Carroll et al., 1985; Goldstein et al., 1988; Kumar et al., 1991; So et al., 1990). The deleted C4AQ*0 allele is easy to ascertain using molecular techniques and—as would be expected from the association between this deletion and the HLA-A1, B8, DR3 haplotype—a significant association between this deletion and SLE has been reported in white lupus patients (Hartung et al., 1992; So et al., 1990). However, the deleted C4AQ*0 allele was also significantly increased in a study of African American lupus patients (24% versus 7.4%, p ⫽ 0.05, relative risk ⫽ 4), although all patients with the deletion were either HLA-DR2⫹ or HLA-DR3⫹ (Olsen


269

et al., 1989). Among individuals who possess C4AQ*0 alleles originating from haplotypes other than HLA-A1, B8, DR3, this gene deletion is rare (Goldstein et al., 1988; Hong et al., 1994; Kumar et al., 1991; Yamada et al., 1990). For example in Japanese patients, despite a significant increase in C4AQ*0 allele frequency (44.1% versus 13.3%, p ⬍ 0.005), no patient had a deletion of the C4A gene (Yamada et al., 1990). Deficiency of C4A may also be a consequence of nonexpression of the C4A gene. A C4A pseudogene can result from a 2–base pair insertion in exon 29 of the C4A gene, which results in the generation of a premature stop codon, most commonly in association with HLA-B60, DR6 (Barba et al., 1993) but also described on the HLA-A2, B40, Cw3, DR6 haplotype (Nordin Fredrikson et al., 1991). This non–DR3-associated mutation has also been found in the C4B gene on the haplotype HLA-A2, B39, Cw7, DR2 (Lokki et al., 1999). A recent study of 188 lupus patients and 222 healthy controls demonstrated that the 2–base pair C4A gene mutation was significantly increased compared to controls (gene frequency: 0.027 versus 0.002, p ⫽ 0.004) (Sullivan et al., 1999). When the patients were analyzed by ethnicity, the gene frequency remained significantly elevated in the 104 white lupus patients (gene frequency: 0.034 versus 0.004, p ⫽ 0.012). In the 84 African American lupus patients studied, the gene frequency was lower (0.018) compared to the white patient group, while none of the 82 African American control population possessed this mutation. This supports the hypothesis that C4A null alleles may be the relevant disease susceptibility for lupus. However, the molecular basis for the other C4AQ*0 alleles is not yet known and this prevents the molecular epidemiology work that will be essential in establishing whether C4AQ*0 alleles are important independent disease susceptibility genes in all populations. 2. C2 Null Alleles C2 deficiency is the most common inherited deficiency of the classical pathway of complement. The case that homozygous C2 deficiency is a powerful predisposing factor for SLE has been considered above. Is there any evidence that heterozygous C2 deficiency may also be a disease susceptibility gene for the development of lupus? As is the case for C4, it is extremely difficult to detect C2 null alleles by measuring protein levels in blood samples from patients with active SLE because of the complement activation in vivo associated with disease activity. However, the C2 null allele (C2Q*0) occurs, in the large majority of cases, in association with the haplotype HLA-A25, B18, Cw-, DR2, C4A*4, C4B*2, C2Q*0, Bf*S (Agnello, 1978; Awdeh et al., 1981b; Hauptmann et al., 1982). Identification of this haplotype, especially of the unusual

TABLE X C4BQ*0 ALLELE FREQUENCIES IN DIFFERENT POPULATIONS OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS Homozygous C4BQ*0 Deficiency

Frequency of C4BQ*0 Allele

No. of Reference Fielder et al. (1983) Christiansen et al. (1983) Howard et al. (1986) Dunckley et al. (1987) Batchelor et al. (1987) Gougerot et al. (1987) Hartung et al. (1989) So et al. (1990) Schur et al. (1990)

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Christiansen et al. (1991) Hartung et al. (1992) De Juan et al. (1993) Reveille et al. (1995a) Reveille et al. (1995b)

Race/Ethnic Group

Patients

Controls

White Whitea White White White, DR3 negative White White White White English/Irish Other Europeans Australian Aborigine White White Mexican Greek

29 41 63 63 30 19 196 54 101 27e 62e 62 9 396 58 55 36

42 176 63 197 60 108 204 62 1731 144e 310e 133 204 69 112 33

Patients % (No.)

Controls % (No.)


Patients

Controls


DR3 Association with SLE?

0 5 (2) — 3.2 (2) 0 — — — — 0 0 3.2 (2) 22.2 (2) — — — —

0 4 (7) — 4 (8) 0 — — — — 1 2 — — — — — —

— — — NS — — — — — — — — — — — — —

0.086 0.15b 0.087 0.231 0.25 0.368 c 0.065d 0.069 — — 0.12 0.33 10g 41g 0.09 0.055

0.14 0.10b 0.111 0.195 0.266 0.134 c 0.024d 0.147 — — 0.17 0.22f 14g 29g 0.05 0.106

— — NS NS NS p ⬍ 0.001 NS NS p ⬍ 0.04 — — NS — NS NS NS NS

Yes Not tested No but DR2 increased Not tested All DR3 negative Yes Yes and DR2 Yes and DR2 No Yes No No No Yes Yes Yes No

Naves et al. (1998) Steinsson et al. (1998)

White Hispanic Spanish Icelandic

69 8 84 64

186 19 96 194

— — — —

— — — —

— — — —

23g 21g 0.286 c

18g 8g 0.063 c

NS p ⫽ 0.03 p ⫽ 0.6 ⫻ 10⫺4 NS

Howard et al. (1986) Wilson et al. (1988) Reveille et al. (1998) Hong et al. (1994)

African American African American African American Korean

35 59 88 60

35 59 73 72

— 5.1 (3) — —

— 5.1 (3) — —

— NS — —

0.171 0.156 19g 0

0.071 0.127 19g 0.0208

NS NS NS NS

Dunckley et al. (1987) Hawkins et al. (1987) Zhao et al. (1989) Dunckley et al. (1987) Yukiyama et al. (1988)

Chinese Southern Chinese Chinese Japanese Japanese

75 72 58 51 53

76 61 89 50 166

1.3 (1) — 1.7 (1) 0 0

1.3 (1) — 2.2 (2) 2 (1) 2.4 (4)

NS — — NS —

0.143 0.168 0.216 0.142 0.321

0.126 0.147 0.73 0.193 0.298

NS NS p ⬍ 0.001 NS NS

Reveille et al. (1998)

Yes Yes and DR8 No Yes (DRBI*03) No Not tested No but DR2 increased No but DR2 and DR9 increased Not tested No, but DR2 increased Not tested Not tested Not tested

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NS, Not significant. a Two female Burmese and three Australian Aborigines were included in this patient group. b Minimal estimated C4B null allele frequency. c Numerical data were not reported, but the authors stated that the C4BQ*0 gene frequency did not differ between patients and controls. d Frequency of the deleted C4B gene. e Number of haplotypes. f C4B gene frequency in healthy Darwin Aborigines was taken from Ranford et al. (1987). g Population frequency (%).

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C4 allotypes in combination with the Bf*S allotype of factor B, is a reasonable surrogate for the detection of the C2 null allele. Results of the first study to test the hypothesis that C2 deficiency might be a disease susceptibility gene for autoimmune disease showed that there was a significant association between SLE and heterozygous C2 deficiency (5.9% versus 1.2%, p ⫽ 0.0009) (Glass et al., 1976). This result has not been confirmed by subsequent studies. The common haplotype containing the C2 null allele was not raised in a study of 248 central European lupus patients (Hartung et al., 1989). Similarly, none of the HLA-B18⫹ lupus patients in another white population possessed the complotype C4A*4, C4B*2, Bf*S (Christiansen et al., 1983), suggesting that a single null C2 allele does not confer increased susceptibility to disease. As discussed earlier, the molecular basis of C2 deficiency is most commonly due to a 28–base pair genomic deletion, associated with the HLAA25, B18 haplotype (type I C2 deficiency) ( Johnson et al., 1992). This has enabled precise identification of null alleles, and several groups have now measured the prevalence of the deleted C2 null allele using molecular techniques. Among 86 Swedish lupus patients and 100 controls, no homozygous C2-deficient individuals were found and the frequency of C2 null alleles did not differ significantly between the groups (5.8% versus 1%, not significant using Fisher’s exact test) (Truedsson et al., 1993). A further study of 122 white lupus patients and 427 North American white controls did not demonstrate a significant increase in C2 heterozygotes in the patient group compared to ethnically matched controls (1.6% versus 1.4%, respectively) (Sullivan et al., 1994). Furthermore, the 28–base pair deletion was not detected at all in 127 African American lupus patients or in 194 African American healthy controls (Sullivan et al., 1994). The MHC haplotype carrying the C2Q*0 allele is sufficiently common that it must carry a selective advantage to compensate for the disadvantage of the raised incidence of lupus among the homozygotes. While it is quite likely that such an advantage lies in other genes in the haplotype, it is also plausible that the C2 deficiency itself has a compensating advantage. Schorey et al. (1997) have reported that Mycobacterium tuberculosis makes a C4-like molecule that can scavenge C2a to generate an active C3 convertase, which in turn causes C3 deposition on the mycobacteria and facilitates their entry into macrophages through complement receptors. Absence of C2 would subvert this mechanism and could therefore result in increased resistance to tuberculosis (Lachmann, 1998). In conclusion, the majority of studies (Table IV) have failed to demonstrate either a significant increase in heterozygous C2 deficiency or an increase in its associated haplotype. Hence, partial C2 deficiency does not appear to be a disease susceptibility factor for the development of SLE.


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C. COMPLEMENT RECEPTOR TYPE 1 (CR1, CD35, C3b/C4b RECEPTOR) CR1 (the ‘‘immune adherence’’ receptor, C3b/C4b receptor, or CD35) is predominantly a cell surface protein with a transmembrane domain. It is distributed on B cells, neutrophils, monocytes, macrophages, erythrocytes, follicular dendritic cells, and glomerular epithelial cells. Its principal ligands are C3b, iC3b, and C4b. There are also data that suggest that CR1 may play a role as a C1q receptor (Klickstein et al., 1997; Tas et al., 1999). CR1 has several important physiological functions. First, it has a crucial role in the protection of host tissues from damage by autologous complement activation. CR1 present on autologous cell membranes promotes the dissociation of C3b from Bb and the dissociation of C2a from C4b, resulting in the ‘‘decay acceleration’’ of the alternative and classical pathway C3 convertases, respectively. CR1 also acts as a cofactor to the serine esterase factor I, which cleaves C3b to iC3b. Factor H, a plasma protein, acts as an alternative cofactor to CR1 for this cleavage. However, only CR1 acts as a cofactor to factor I in the subsequent cleavage of iC3b to C3dg with the release of C3c. Second, CR1 acts as an opsonic receptor on neutrophils and macrophages, enhancing phagocytic uptake of C3b-, C4b-, or iC3b-coated material. Third, in humans, CR1 present on erythrocytes enhances the physiological transport of opsonized bacteria and complement-fixing immune complexes from the bloodstream to the fixed mononuclear phagocytic system of the liver and spleen. Fourth, ligation of CR1 and CR2 (CD21, Epstein–Barr virus receptor) on B cells lowers the threshold for B cell activation in conjunction with the binding of the membrane-bound antigen receptor (antibody) by antigen. These last two functions explain the important accessory role of complement in the optimal induction of antibody responses. 1. Inherited Numerical Polymorphism of CR1 In 1981, Miyakawa and colleagues demonstrated that erythrocytes from the majority of patients with SLE failed to adhere to aggregated human Ig in the presence of complement. This finding was independent of disease activity and was also found in some of the relatives of the patients. It was suggested that reduced expression of CR1 might be an inherited disease susceptibility factor for the development of SLE. These findings were confirmed and amplified using antibodies in radioligand binding assays to quantitate CR1 numbers on erythrocytes (Iida et al., 1982). Among healthy individuals, it was possible to detect three numerical phenotypes consisting of high, intermediate, and low erythrocyte CR1 expression (Wilson et al., 1982). Analysis of pedigrees suggested that the defect in expression on the erythrocytes of SLE patients was inherited.

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After the identification and sequencing of CR1 cDNA (Wong et al., 1985, 1986), a CR1 genomic polymorphism that correlated with erythrocyte CR1 numbers was identified (Wilson et al., 1986). Hybridization of HindIII-digested human genomic DNA with a CR1 cDNA probe showed that individuals with high numbers of CR1 sites per erythrocyte possessed a single 7.4-kb restriction fragment, whereas individuals with low numbers per erythrocyte had a single 6.9-kb restriction fragment (Wilson et al., 1986). Individuals possessing both restriction fragments had intermediate erythrocyte CR1 numbers. This polymorphism of CR1 has been found in all populations studied so far and appears to be ancient in human phylogeny. It has been speculated that variation in CR1 expression may play a role in host defense against infectious and parasitic disease (Xiang et al., 1999). This hypothesis is considered further in Section II,C,3. The identification of an inherited numerical polymorphism of CR1, together with the discovery that CR1 numbers were reduced on erythrocytes from patients with SLE, led to the hypothesis that the reduced erythrocyte CR1 expression in SLE was inherited. The HindIII 6.9-kb restriction fragment length polymorphism (RFLP) could be a marker of a disease susceptibility allele for the disease (Wilson et al., 1982). A reduction in the number of patients homozygous for the 7.4-kb ‘‘high’’ allele was reported (Wilson et al., 1987) compared with normal subjects. All subsequent studies have confirmed the reduced expression of CR1 on erythrocytes from patients with SLE (Holme et al., 1986; Inada et al., 1982; Minota et al., 1984; Walport et al., 1985). However, the balance of the evidence now supports the hypothesis that there is an acquired loss of CR1 from the erythrocytes of patients with SLE. The evidence for this is considered in the next section. 2. The Reduction in Erythrocyte CR1 Numbers Associated with SLE Is Acquired, Not Inherited Although among consanguineous relatives a reduction in erythrocyte CR1 levels compared to healthy controls was reported (Wilson et al., 1982), a subsequent study did not detect a significant difference (Walport et al., 1985). The identification of the RFLP correlating with numerical expression of CR1 meant that robust molecular epidemiological studies could be performed. Several groups found no difference in the allele frequency of this polymorphism among SLE patients compared with control populations (Cohen et al., 1989; Moldenhauer et al., 1987; Satoh et al., 1991; Tebib et al., 1989). In three of these studies, patients with SLE, despite being homozygous for the 7.4-kb allele associated with high expression of CR1, had low erythrocyte CR1 numbers (Mitchell et al., 1989; Moldenhauer et al., 1987; Satoh et al., 1991).


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In addition, several studies have provided evidence for acquired loss of CR1 from the erythrocytes of patients with SLE. The reduction in erythrocyte CR1 numbers was correlated with measures of disease activity (Corvetta et al., 1991; Holme et al., 1986; Ross et al., 1985). Additional correlations were reported between low erythrocyte CR1 numbers and increased circulating immune complexes (Iida et al., 1982; Inada et al., 1982), increased erythrocyte C3dg expression (Ross et al., 1985), and reduced C4 levels (Iida et al., 1982). Erythrocyte CR1 numbers increased significantly during SLE disease remission (Holme et al., 1986; Iida et al., 1982). The correlation between reduced erythrocyte CR1 and increased erythrocytebound C3dg demonstrated during active lupus also reversed during remission (Ross et al., 1985). Direct in vivo evidence of acquired reduction in erythrocyte numbers was obtained by studying patients with SLE receiving blood transfusions (Walport et al., 1987). In patients with active disease, as much as 60% of erythrocyte CR1 was lost during the first 5 days following transfusion. Normal expression of CR1 was found on reticulocytes from patients with SLE, showing that loss of CR1 occurs from these cells during their time in the circulation (Lach-Trifilieff et al., 1999). Finally, a reduction in erythrocyte CR1 numbers has been found to be associated not only with SLE but also with other diseases characterized by the presence of immune complexes and systemic complement activation. These diseases include rheumatoid arthritis (Corvetta et al., 1991; Iida et al., 1982), autoimmune hemolytic anemia (Ross et al., 1985), primary Sjo¨gren’s syndrome (Ross et al., 1985; Thomsen et al., 1986), juvenile rheumatoid arthritis (Thomsen et al., 1987), paroxysmal nocturnal hemoglobinuria (Pangburn et al., 1983; Ross et al., 1985), hepatitis C infection (Kanto et al., 1996), acquired immunodeficiency syndrome (AIDS) (Tausk et al., 1986), and lepromatous leprosy (Tausk et al., 1985). It is not known how CR1 is lost from erythrocytes in SLE. As noted above, reduced CR1 numbers are found in diseases associated with antibody and complement deposition on erythrocytes, that is, autoimmune hemolytic anemias and diseases associated with the presence of immune complexes. Indeed, in blood samples from patients with SLE, an inverse correlation was found between mean numbers of bound C3dg fragments and CR1 numbers per erythrocyte (Ross et al., 1985). Erythrocytes bearing C3 fragments and immune complexes interact with mononuclear phagocytic cells in the liver and spleen (see Section VI,C). During these interactions, any erythrocyte-bound immune complexes are stripped from the cell and erythrocyte-bound iC3b is catabolized to C3dg. CR1 is exceptionally sensitive to proteolytic degradation (Ripoche and Sim, 1986). It is possible that CR1 is cleaved from erythrocytes by the action of proteolytic enzymes

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from macrophages during their interactions with erythrocytes bearing complement or immune complexes. 3. Inherited Structural Polymorphisms of CR1 In addition to the inherited numerical polymorphism, CR1 has a complicated structural polymorphism. There are four codominantly inherited allotypes showing large size variation (Dykman et al., 1983, 1984, 1985). These have molecular weights of 220 (CR1-A, or F allotype), 250 (CR1-B or S allotype), 190 (CR1-C, or F1 allotype) (Dykman et al., 1984), and 280 (CR1-D) (Dykman et al., 1985). The size differences reflect changes in the number of long homologous repeats (LHRs) in the extracellular domain of CR1, which in turn are accompanied by changes in the number of C3b and C4b binding sites (Klickstein et al., 1988; Wong et al., 1989). The most common allele, CR1-A, consists of four LHRs, termed A, B, C, and D. It possesses a single C4b binding site in the N-terminal two SCRs (short consensus repeats) of LHR-A, while the N-terminal two SCRs of LHR-B and LHR-C each contain a C3b binding site (Klickstein et al., 1988). The largest allotype, CR1-B, has five LHRs and is predicted to have a third C3b binding site (Wong et al., 1989). The smallest allotype, CR1-C, has three LHRs with only a single C4b and C3b binding site (Wong and Farrell, 1991). These allotypic structural variants are thought to have arisen from variable duplication of the gene sequences encoding the LHR (Wong et al., 1986). In addition, it has been discovered that a number of blood group antisera—Knops, McCoy, Swain-Langley, and York—recognize specificities on CR1 (Moulds et al., 1991). Of particular interest, a malaria-encoded erythrocyte surface molecule, PfEMP1, has been shown to mediate rosetting of erythrocytes, by adhesion to CR1 molecules on other erythrocytes (Rowe et al., 1997). A common African structural variant of CR1, Sl(a⫺), shows reduced binding to PfEMP1 and may have a protective role against severe malarial infection. Several studies have analyzed the prevalence of these structural variants of CR1 among patients with SLE (Cornillet et al., 1992; Dykman et al., 1984; Moldenhauer et al., 1988; Moulds et al., 1996; Van Dyne et al., 1987). The frequency of the A, B, and C alleles has not differed between lupus patients and ethnically matched healthy controls in most studies (Dykman et al., 1984; Moldenhauer et al., 1987; Moulds et al., 1996). One study of 63 French lupus patients demonstrated an increase in the frequency of the CR1-B (S allotype) allele when compared with ethnically matched healthy controls (51% versus 26%, p ⫽ ⬍ 0.001) (Cornillet et al., 1992).


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III. Animal Models of Complement Deficiency

C5 deficiency was discovered as a spontaneous mutant in laboratory mice many years ago (Nilsson and Muller-Eberhard, 1967), and many laboratory strains now in common use lack expression of C5 protein (Rosenberg and Tachibana, 1986). C8-deficient mice have also been described (Tanaka et al., 1991). However, there have been no natural examples of any other complement deficiency occurring in mice. The advent of gene-targeting technology has allowed the development of mouse strains deficient in many different complement proteins. From the investigation of these mice, significant advances have been made in the study of the role of complement in the pathogenesis of SLE, which we now describe. We focus on the data from the mouse, which is an excellent model species for the development of SLE. We also briefly describe the findings from other species in which spontaneous complement-deficient mutants have arisen. A. C1q-DEFICIENT MICE C1q-deficient (C1qa⫺/⫺) mice were generated by insertional mutagenesis of the first exon of the C1qA chain gene, C1qa, resulting in mice with no C1qa transcripts detectable on Northern blots and no circulating C1q protein detectable by Western blot or enzyme-linked immunosorbent assay (ELISA) (Botto et al., 1998). As expected, these mice lacked classical pathway-mediated lytic activity and the ability to opsonize immune complexes with C3, but retained a functional alternative pathway. To date, C1q deficiency is the only complement deficiency in mice which has been associated with the spontaneous development of an SLE-like autoimmune disease. A large cohort consisting of 226 C1qa⫺/⫺ and 108 wild-type control mice, on a hybrid 129 ⫻ C57BL/6 genetic background, were monitored for the development of autoimmune disease. By 8 months of age, more than half of the C1q-deficient animals had detectable levels of ANAs and 25% had histological evidence of proliferative glomerulonephritis (Botto et al., 1998). The glomerulonephritis was associated with the presence of electron-dense subendothelial and subepithelial immune deposits and the glomeruli stained positively for IgG and C3, suggesting activation of complement via the alternative pathway. The presence of a large number of apoptotic bodies was noted in the glomeruli of the nephritic animals. C1q-deficient mice with no histological evidence of glomerulonephritis also exhibited a significantly increased number of glomerular apoptotic bodies compared with the control animals. The expression of SLE-like disease was critically

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dependent on the genetic background of the knockout animals. Only C1qa⫺/⫺ mice of the hybrid 129 ⫻ C57BL/6 genetic background developed an SLE-like disease. It is relevant that mice of this genetic background are already predisposed to the development of autoimmunity (Botto et al., 1998; Obata et al., 1979), and the C1q deficiency augmented the development of autoimmunity in these mice. B. FACTOR B/C2-DEFICIENT MICE Generation of mice deficient in complement components factor B and C2 (H2-Bf/C2⫺/⫺) by gene targeting resulted in a loss of both classical and alternative pathway-mediated complement activity and antibody-mediated C3 opsonic activity in the serum of deficient animals (Taylor et al., 1998). However, in contrast to C1q-deficient mice, these animals did not develop spontaneous autoimmunity. This suggested that the protective role of complement from autoimmunity did not require C3 activation and that the early components of the classical pathway were sufficient in mice as well as in humans. When crossed with the C1q-deficient mice to generate a strain of mice deficient in C1q, factor B, and C2 (C1qa/H2-Bf/C2⫺/⫺), the spontaneous development of autoimmunity, characterized by the production of ANAs and proliferative glomerulonephritis, was again evident (Mitchell et al., 1999). The only obvious difference between the pathologies of the diseases observed in the C1qa⫺/⫺ and C1qa/H2-Bf/C2⫺/⫺ mice was the complete absence of C3 deposition in the glomeruli of the nephritic C1qa/H2-Bf/C2⫺/⫺ animals, consistent with the inability of these mice to activate C3. These observations supported the existence of a role for C1q, and possibly C4, in protection from autoimmune glomerulonephritis and suggested that activation of complement and deposition of C3 was not required for the expression of the disease. The existence of a hierarchy among the early classical pathway proteins in mice, with respect to their protective role against autoimmunity, recapitulates the findings in humans and shows that they represent valid models for studying the pathogenesis of SLE associated with primary complement deficiency. C. C3-, C4-

AND

CR1/2-DEFICIENT MICE

So far, there has been no report of a predisposition to the spontaneous development of autoimmunity in mice rendered deficient in C3 (Wessels et al., 1995), C4 (Fischer et al., 1996), or CR1/2 (Ahearn et al., 1996; Molina et al., 1996) in spite of substantial abnormalities in the antibody responses of these mice, which is discussed later. The effect of these deficiencies on the expression of autoimmunity caused by the mutant Fas (CD95) gene, lpr, have been studied. Prodeus and co-workers (1998) crossed C3-, C4-, and CR1/2-deficient mice on the mixed genetic back-


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ground with Fas-deficient C57BL/6.lpr/lpr mice. C57BL/6.lpr/lpr mice develop a mild lupus-like disease associated with autoantibody production but not with glomerulonephritis (Izui et al., 1984). CR1/2- and C4-deficient lpr/lpr mice exhibited increased lymphadenopathy (and splenomegaly in the case of the CR1/2-deficient mice), increased levels of ANAs and anti–double-stranded DNA autoantibodies, an increased presence of IgG and C3 in the glomeruli, and increased glomerular hypercellularity compared to the complement-sufficient control animals. Strikingly, the C3-deficient mice did not show a similar phenotype to the C4-deficient animals. Apart from a mild increase in the presence of IgG in the glomeruli of the C3-deficient animals, they were not notably different from the control animals (Prodeus et al., 1998). These results were attributed to a break in the maintenance of B cell tolerance in the CR1/2- and C4-deficient mice, which is discussed in more detail in Section VI,D. D. COMPLEMENT DEFICIENCY IN OTHER NONHUMAN SPECIES Colonies of guinea pigs are widely available with C4, C2, and C3 deficiencies (Bitter-Suermann and Burger, 1986). These animals do not display any spontaneous glomerulonephritis (Foltz et al., 1994). However, SLE has not been described in the guinea pig, which seems to be a resistant species with respect to this disease. C4-deficient guinea pigs were found to have increased IgM rheumatoid factor levels and an increased polyclonal antibody response (Bottger et al., 1986a). The clinical phenotype of C3 deficiency in humans and that of dysregulated C3 consumption, due to deficiency of factor H, have been recapitulated in dogs and pigs with similar inherited deficiencies. A colony of dogs with C3 deficiency developed an extremely similar pattern of MCGN to that seen in some humans with C3 deficiency (Cork et al., 1991). Factor H deficiency was identified among pigs of the Norwegian Yorkshire breed, which suffered from a runting disease caused by early onset of MCGN type II (Hogasen et al., 1995; Jansen et al., 1995). An effective breeding program has eliminated factor H deficiency from the Norwegian pig population, and this model of glomerulonephritis is no longer available for study (Hogasen et al., 1997). IV. Complement and Inflammation in SLE

Complement activity in sera from patients with SLE is reduced in relation to disease activity (Lloyd and Schur, 1981; Schur and Sandson, 1968; Townes et al., 1963) and increases following treatment (Vaughan et al., 1951). Complement deposition was identified in inflamed tissues (Lachmann et al., 1962; Tan and Kunkel, 1966). Levels of the classical pathway

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proteins (C1q, C2, and C4) are particularly reduced (Hanauer and Christian, 1967; Morse et al., 1962; Schur and Sandson, 1968). C3 levels are less frequently abnormal, and low C3 levels are a pointer to the presence of severe disease (Lloyd and Schur, 1981; Weinstein et al., 1983). However, studies of cohorts of patients with SLE show only weak (albeit highly significant) correlations between disease activity and reductions in the levels of individual complement proteins (Cameron et al., 1976; Valentijn et al., 1985). There are at least two important factors that may confuse the relationship between complement levels in serum and disease activity. The first is the effect of variation in rates of biosynthesis of complement proteins. There have been several studies of the turnover in vivo of radiolabeled complement proteins, from which it is possible to estimate synthetic and catabolic rates of turnover of individual complement proteins (Alper and Rosen, 1967; Hunsicker et al., 1972; Sliwinski and Zvaifler, 1972). Protein concentrations are a function of synthetic and catabolic rates. Among patients with SLE, increased catabolic rates for complement proteins were observed, but these were accompanied by great variability in synthetic rates, from reduced to increased. Such variation in synthetic rates may completely mask the effects of increased catabolism secondary to disease activity. The second important factor confounding the value of simple measurements of complement protein concentrations as a measure of disease activity is the effect of the presence of autoantibodies such as anti-C1q antibodies to complement proteins, discussed in Section V. As seen below, high titers of anti-C1q antibodies are commonly associated with profound reductions in the levels of the classical pathway proteins and C3, and also with severe disease activity. The presence of complement deposits in tissues is poorly correlated with the presence of tissue injury (Biesecker et al., 1981; Gilliam et al., 1974; Pohle and Tuffanelli, 1968). This raises the key question, Is complement deposition an important cause of inflammatory tissue injury in SLE? Evidence from animal models of SLE and glomerulonephritis suggests that immune complex–mediated injury to tissues is mediated mainly by ligation of Fc receptors and that complement may play a much lesser role. For example, Fc–웂 chain–deficient NZB/NZW mice were protected from developing severe nephritis, despite the presence of immune complexes in the kidney (Clynes et al., 1998). A reciprocal experiment that supported this result was the finding in C1q-deficient mice that the development of spontaneous glomerulonephritis was independent of C3 activation (Mitchell et al., 1999). The Arthus reaction is widely used as an experimental model of inflammatory injury mediated by immune complexes. Using this reaction, some


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experiments have suggested primacy of Fc receptor ligation in mediating tissue injury, while others have shown a clear role for the complement system. In experiments studying a murine model of the reverse passive cutaneous Arthus reaction, C3⫺/⫺ and C4⫺/⫺ mice developed inflammatory responses comparable to those seen in wild-type control mice, whereas the response of Fc–웂 chain–deficient mice was significantly reduced (Sylvestre et al., 1996). By contrast, in similar experiments on the reverse passive cutaneous Arthus reaction, although complement depletion did not affect the inflammatory response in wild-type animals, complete abolition of the response in Fc웂RIII-deficient animals occurred only following depletion of complement (Hazenbos et al., 1996). Furthermore, using similar experimental models, there were very clear data showing a role for complement in the induction of inflammatory injury. For example, mice deficient in the C5a anaphylatoxin receptor showed marked reduction in inflammation in both peritoneal and cutaneous reverse passive Arthus reactions (Hopken et al., 1997) and were protected from immune complex–mediated lung inflammation (Bozic et al., 1996). In addition, both complement activation and activation of Fc웂RI on peritoneal macrophages were required to initiate inflammation in a murine model of immune complex peritonitis (Heller et al., 1999). These findings show that the mechanism of induction of inflammation by immune complexes is, in some situations, complement independent, and in others, complement dependent. They serve to illustrate the complexity of mechanisms of inflammation in disease mediated by immune complexes. The size, composition, and location of immune complexes may each modify whether and how inflammation ensues. V. Lupus Causes Autoantibody Production to C1q

High titers of autoantibodies to C1q are found in two closely related conditions. The first is the uncommon disease hypocomplementemic urticarial vasculitis syndrome, often abbreviated HUVS. Patients with this condition, as its name implies, suffer from chronic cutaneous vasculitis and urticaria and have very low C1q, C4, and C2 levels. Additional clinical features include glomerulonephritis, chronic obstructive airways disease of the lungs, angioedema, and ocular inflammation (Wisnieski et al., 1995). The serological hallmark of HUVS is the presence of high titers of antiC1q autoantibodies, first identified as C1q precipitins (Agnello et al., 1971). The second disease in which anti-C1q antibodies are common is SLE, and there is a significant overlap between the clinical features of HUVS and SLE (Trendelenburg et al., 1999).

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Approximately one third of patients with SLE have elevated levels of anti-C1q autoantibodies in their serum (Antes et al., 1988; Siegert et al., 1991). The presence of anti-C1q antibodies in lupus is typically accompanied by a number of clinical and serological features. The complement profile is similar to that seen in HUVS, with very low levels of C1q, C4, and C2, and, to a lesser extent, C3. Anti-C1q antibodies tend to remain positive in SLE for prolonged periods, and there is associated prolonged hypocomplementemia. This differs from anti–double-stranded DNA antibody levels, which tend to fluctuate in concentration, together with inverse changes in complement levels. Many studies have shown that anti-C1q antibodies are correlated with the presence of glomerulonephritis and that increases in the titer of anti-C1q antibodies have been associated with flares of disease activity (Siegert et al., 1993). These autoantibodies are typically of the IgG2 subclass (Prada and Strife, 1992; Wisnieski and Jones, 1992). They react with a neo-epitope in the collagen region of C1q, which is exposed when C1q is bound to a surface, such as an immune complex, and dissociated from C1r and C1s (Antes et al., 1988). One of the most popular assays for the measurement of circulating ‘‘immune complexes,’’ the solid-phase C1q binding assay, was discovered to be really a measure of anti-C1q antibodies in the majority of cases. There are two common approaches now for the specific measurement and quantitation of antiC1q antibodies. The first is a variant of the solid-phase C1q binding assay, in which the addition of 1 M NaCl prevents immune complexes from binding to immobilized C1q on a microtiter plate but allows the cognate binding of specific autoantibodies (Siegert et al., 1990). The second method uses pepsin-digested C1q as the antigenic substrate on a microtiter plate, which contains the collagenous region of C1q but lacks the globular heads that might bind immune complexes or Ig aggregates in serum samples to be assayed (Menzel et al., 1991). The mechanism of the hypocomplementemia associated with the presence of anti-C1q antibodies is not certain. Anti-C1q antibodies do not cause complement activation in the fluid phase when added to fresh serum samples. The most likely mechanism is that they amplify complement activation by immune complexes in tissues, by binding to C1q fixed to immune complexes, enlarging the complexes and promoting further complement activation. This mechanism could also amplify inflammation caused by immune complexes within tissues, accounting for the association of anti-C1q antibodies with the presence of glomerulonephritis (Mannik and Wener, 1997). The mechanism for the development of anti-C1q autoantibodies in SLE is not understood. A unifying hypothesis that could explain, on the one


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hand, how C1q deficiency causes SLE and, on the other, how SLE causes anti-C1q antibodies is presented in Section VI. Autoantibodies to C1q are also found in a smaller proportion of patients with rheumatoid arthritis and other autoimmune diseases. Among these patients, the presence of these antibodies is not associated with hypocomplementemia and the significance of this autoantibody in these diseases is uncertain. VI. Hypotheses for the Association between Complement Deficiency and SLE

A. INTRODUCTION Currently, there are three hypotheses to explain the link between complement deficiency and SLE. The first two, which are not mutually exclusive, relate to the role of complement in the physiological ‘‘waste disposal’’ mechanisms for dying cells and immune complexes. The third relates to the role of complement in the humoral immune response. We discuss each of these in turn here. We first consider the links between apoptosis and SLE, which set the stage for exploration of the hypothesis that failures in the pathways for clearance of apoptotic cells may predispose an individual to SLE. We then describe the role of complement in the clearance of immune complexes and how defects in this pathway might promote disease. Finally, we describe briefly the role of complement in the induction of humoral immune responses, reviewed elsewhere in this series (Carroll, 2000), and in the modulation of immune response to autoantigens. B. APOPTOSIS AND SLE Research into SLE has been facilitated by discovery that a number of strains of mice develop spontaneous lupus closely resembling its human counterpart. Two strains of mice, MRL/lpr (Andrews et al., 1978; Morse et al., 1982) and C3H/gld (Roths et al., 1984), revealed a close link between SLE and the apoptotic pathways for physiological cell death. The lpr gene was discovered to encode a mutant variant of fas (CD95) protein (Watanabe-Fukunaga et al., 1992) and gld encoded a mutant fas–ligand (CD95–ligand) (Lynch et al., 1994). These discoveries provided a satisfying explanation for the similar phenotype of these two strains of mice. As part of their phenotype, both mouse strains develop striking polyclonal lymphadenopathy with massive expansion of a T cell receptor 움/웁⫹, CD3⫹CD4⫺CD8⫺ population of lymphocytes. Since ligation of fas triggers death by apoptosis of lymphocytes, it seems likely that dysfunction of this pathway is responsible for the accumulation of lymphocytes, leading to massive lymphadenopathy and splenomegaly. Both MRL/lpr and gld mice develop many of the phenotypic features of SLE, including the full spec-

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trum of specificities of ANAs, anti-C1q antibodies, glomerulonephritis, vasculitis, and arthritis (reviewed in Theofilopoulos and Dixon, 1985). It is important to note that the lpr and gld genes are not, on their own, sufficient to induce the expression of disease. C57BL/6 mice expressing the lpr mutation have a much milder phenotype than do MRL mice expressing the same mutant (Izui et al., 1984; Kelley and Roths, 1985). They develop lymphadenopathy, splenomegaly, and moderate levels of autoantibodies (though very high levels of rheumatoid factor), without glomerulonephritis or other tissue injury. This shows that other gene products are necessary to modify the phenotype associated with null mutations in what are apparently critical cell surface proteins in an important signaling pathway for cell death. Some loci affecting the expression of autoimmunity in MRL/lpr mice have been mapped (Vidal et al., 1998; Watson et al., 1992). Following the discovery of the phenotype and molecular basis of the MRL/lpr and gld strains of mice, a small cohort of humans has been described with a similar phenotype to that of mice (Sneller et al., 1992). This condition is named Canale–Smith syndrome after the authors who first described the clinical syndrome (Canale and Smith, 1967) or, more descriptively, autoimmune lymphoproliferative syndrome (ALPS) (Fisher et al., 1995). Patients typically have early onset of massive lymphadenopathy and splenomegaly, with an excess of CD3⫹CD4⫺CD8⫺ lymphocytes. Three types of mutations have been identified. The first is a homozygous mutation in the fas gene, seen in a child with neonatal onset of very severe lymphadenopathy and splenomegaly (Rieux-Laucat et al., 1995). The second and most common mutation is expressed as a dominant disease with very incomplete penetrance within families. A variety of heterozygous mutations in the fas gene has been found to cause this syndrome (Fisher et al., 1995). The third is a single example of a mutation in the fas–ligand gene reported in a patient with lymphadenopathy and SLE (Wu et al., 1996). The typical pattern of autoimmunity is more restricted than in mice, typically with the expression of autoimmune hemolytic anemia, neutropenia, and thrombocytopenia (Vaishnaw et al., 1999). The main differences between humans and mice are, first, that disease is common in patients with heterozygous mutations, which have a dominant negative effect on signaling following ligation of fas protein and, second, the extreme variability of expression of disease among outbred human families, showing the important influence of modifying genes. In some families, certain individuals with a mutated fas allele develop lymphoma rather than autoimmunity (Gronbaek et al., 1998), showing the importance of fas-mediated mechanisms in controlling lymphocyte growth. The mechanism for autoimmunity has not been firmly established in either mice or humans with mutations in fas and lpr genes. The most


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favored hypothesis has been that they have defects in the mechanisms for negative selection of autoreactive lymphocytes, especially in the periphery (Fatenejad et al., 1998; Herron et al., 1993), due to failure of the apoptotic mechanisms in lymphocytes that are part of the process of negative selection. There is evidence from studies using adoptive transfer of lymphocytes that the fas pathway defect is necessary in both the T cell and B cell compartment for full-blown disease to ensue (Sobel et al., 1994). A feature of ALPS in humans is the presence of hemophagocytosis with macrophages containing large numbers of phagocytosed blood cells in the liver and spleen (Le Deist et al., 1996). It is of particular note that the autoimmune disease in humans with this syndrome is dominated by hemolytic anemia, neutropenia, and thrombocytopenia, each associated with autoantibodies to the respective cell type. This suggests the hypothesis that the phagocytosed blood cells may be presented as autoantigens. This brings us to the second hypothesis linking apoptosis and SLE: that apoptotic cells are the source of the autoantigens that drive autoantibody production in genetically susceptible individuals. 1. Apoptotic Cells as a Source of Autoantigens in SLE The most striking feature of SLE is the spectrum of autoantibodies to ubiquitous autoantigens. These may be categorized into three types. The first are the intracellular autoantigens, typically organized into intracellular clusters of antigens such as chromatin, the spliceosome complex, and the Ro/La small cytoplasmic ribonucleoprotein complex. The second are a series of cell surface antigens, including phospholipids of the cell membrane such as phosphatidylserine. The third category is plasma proteins such as 웁2 glycoprotein I and C1q. It is generally accepted that it is the autoantigens themselves that drive the mature autoantibody response. However, it is not known what antigen triggers the autoimmune process in the first place. There are three possibilities. It could be the autoantigen itself, a mimic of the autoantigen (e.g., a viral polynucleotide sequence or cross-reacting viral or bacterial protein), or a complex of the autoantigen with a foreign antigen such as a self protein attached to a viral polynucleotide sequence. There are two striking puzzles about the lupus autoantigens. The first is that they are ubiquitous and abundant in every cell and every compartment of the body in every individual. It is therefore extremely surprising that tolerance can break down to these autoantigens, which are the very essence of ‘‘self.’’ The question that follows is, What are the protective mechanisms that normally prevent autoreactivity to these autoantigens, and how do they fail in lupus? The second puzzle is that, of all the many

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thousands of potential intracellular, cell surface, and plasma autoantigens, it is only a particular spectrum of autoantigens that are targeted in SLE. The question that follows is, By what mechanism are these, but not others, selected? The answers to these questions are not known, but there are some good hypotheses. The answer to the question about the nature of the autoantigens targeted in the disease may provide clues for answering the first question, about the mechanisms that protect the vast majority of individuals from the development of SLE. There is a mounting body of evidence that apoptotic cells are the source of the autoantigens of lupus. This evidence may be divided into three categories. The first is that the structure of apoptotic cells is reorganized such that the lupus autoantigens become superficially accessible to recognition by antibodies. In a series of morphological studies, Rosen and collaborators have found that apoptotic cells express in surface blebs and apoptotic bodies many of the nuclear autoantigens of SLE (Casciola-Rosen et al., 1994; Rosen et al., 1995). The major antigen bound by the antiphospholipid autoantibodies found in approximately one third of patients with lupus is phosphatidylserine. This negatively charged phospholipid is found in the inner lamella of the cell membrane in healthy cells, but is actively translocated to the outer layer as part of the process of apoptosis (Casciola-Rosen et al., 1996; Fadok et al., 1992b). On apoptotic cells, phosphatidylserine acts as one of the recognition molecules for the physiological uptake and disposal of apoptotic cells, which are reviewed below. The second category of evidence is that many of the lupus autoantigens undergo posttranslational modification during the process of apoptosis and may be cleaved or phosphorylated. This process could generate neoepitopes of autoantigens, which might appear as ‘‘non-self’’ to the immune system (Casciola-Rosen et al., 1999; Rosen and Casciola-Rosen, 1999). However, the putative relevance of the cleavage and posttranslational modification of autoantigens would be more compelling if there were a demonstration that any lupus autoantibodies bound selectively to a neo-epitope in an antigen modified as part of apoptosis. Third, there have been many experiments trying to induce or accelerate the production of lupus autoantibodies using various immunization protocols. The published results have frequently been equivocally positive. Recent experiments have shown that injection of apoptotic cells into mouse strains not normally susceptible to the development of SLE induces an autoantibody response (Mevorach et al., 1998b). The discovery that the source of the autoantigens in SLE may be apoptotic cells then leads to a possible answer to the second question above, about the mechanisms that protect against the development of autoimmu-


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nity. Despite the massive turnover of apoptotic cells within the body, it is very rare to visualize an apoptotic cell within a section of tissue. This is because there are a large number of complementary mechanisms that result in the rapid removal and destruction of apoptotic cells by phagocytic pathways that do not normally lead to inflammation. One possible pathway that could promote autoimmunity could be a breakdown of the normal mechanisms of removal of apoptotic cells, which might then drive an autoantibody response. It is hard to think that autoimmunity could be caused by a simple increase in the load of autoantigens. However, if the increased load of apoptotic cells was accompanied also by inflammatory signals, antigen-presenting cells might present autoantigens to T cells, in the context of costimulatory signals, and thereby overcome tolerance to the ubiquitous autoantigens of SLE. In the next section, we briefly consider the normal pathways for the clearance of apoptotic cells before turning to a possible role of the complement pathway in this and related clearance functions. 2. Apoptotic Cell Clearance Apoptosis, or programmed cell death, is a fundamental process for the removal of damaged and effete cells during development and the maintenance of homeostasis (Savill, 1997). The apoptotic death process is rapid and characterized by cell shrinkage, condensation and fragmentation of the nucleus, cytoplasmic blebbing with maintenance of membrane integrity, and cell fragmentation into discrete apoptotic bodies. Apoptotic cells are rarely detected in healthy tissues, as they are swiftly subject to receptormediated ingestion by both professional and nonprofessional phagocytes, followed by intracellular degradation. The normal processes of clearance of apoptotic cells cause the elimination of cells in a ‘‘silent’’ manner that causes no tissue injury. This is extremely important, because many cells (e.g., neutrophils, which have enormous turnover rates), contain enzymes and other products that could be very harmful in the absence of protective mechanisms for waste disposal. Indeed, the process of phagocytosis of apoptotic cells by macrophages causes the production of anti-inflammatory mediators such as tumor growth factor 웁1, prostaglandin E2, and interleukin 10 (IL-10) while reducing proinflammatory mediators such as TNF-움 and IL-1웁 (Fadok et al., 1998; Voll et al., 1997), ensuring the ‘‘safe and quiet’’ removal of apoptotic cell debris. As might be expected, in view of the importance of efficient removal of apoptotic debris, many receptor–ligand systems play complementary roles in mediating apoptotic cell clearance in vitro. During the process of apoptosis, the cell surface is modified to allow recognition of apoptotic cells by

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phagocyte surface receptors. Translocation of phosphatidylserine to the outer lipid layer of the plasma membrane, exposure of altered glycosylation patterns, and the relocation of intracellular molecules to the outer surface of the dying cell may all serve as mechanisms by which a phagocyte can recognize an apoptotic cell from its healthy neighbor. Receptors that have been implicated in the phagocytic removal of apoptotic cells are the 움v웁3 vitronectin receptor (Savill et al., 1990), the phosphatidylserine receptor (Fadok et al., 1992a), CD36 (Ren et al., 1995), CD14 (Devitt et al., 1998), scavenger receptor A (Platt et al., 1996), receptors for low-density lipoprotein (Bird et al., 1999; Chang et al., 1999; Sambrano and Steinberg, 1995), and complement receptors 3 and 4 (Mevorach et al., 1998a). These different receptor–ligand systems may function in conjunction with one another, but individual types of phagocyte may show specific receptor–ligand preferences (Fadok et al., 1992a). 3. Immune Response to Apoptotic Cells There is much interest in the uptake and processing of apoptotic and necrotic cells by dendritic cells and the subsequent presentation to T cells of antigens derived from these dead cells. There are three key messages from the studies of the interactions of apoptotic and necrotic cells with dendritic cells. The first is that immature dendritic cells avidly take up both apoptotic and necrotic cells. The second is that the dendritic cells that have taken up these dead cells then require specific signals in order to mature to active antigen-presenting cells, characterized by up-regulated expression of MHC and costimulatory molecules. The third message is that a number of the specific signals that lead to maturation of dendritic cells have been identified as proinflammatory cytokines and products of infectious agents, such as lipopolysaccharide. From these results, it is possible to speculate how apoptotic cells may, under pathological circumstances, drive autoimmune responses. In this section, we describe experiments on the uptake of apoptotic and necrotic cells by dendritic cells. We also discuss the effects of defects in clearance mechanisms of apoptotic cells on their uptake by macrophages and dendritic cells, and how the presence of autoantibodies to apoptotic cells may perturb their fate. Finally, we speculate how impairment of the mechanisms for the clearance of dying cells may promote the development of autoimmunity. Immature dendritic cells have been shown to be able to phagocytose apoptotic cells via the 움v웁3 vitronectin receptor (Albert et al., 1998a) and subsequently present apoptotic cell–derived antigens to MHC class I– and class II–restricted T cells (Albert et al., 1998a,b; Inaba et al., 1998) in a dose-dependent manner (Rovere et al., 1998). There is a large body of evidence that suggests that the ingestion of apoptotic cells by immature


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dendritic cells is not sufficient to cause dendritic cell maturation and effective antigen presentation. Proinflammatory mediators are necessary to induce the maturation of dendritic cells, especially molecules associated with infection, such as lipopolysaccharide and pathogen-derived nucleic acids (Cella et al., 1999). Cytokines produced by the host in response to infection may also play an important role in driving dendritic cell maturation, such as TNF-움 and interferon 움 (Luft et al., 1998). This requirement for inflammatory mediators for the maturation of dendritic cells promotes antigen presentation in the context of inflammation. The corollary of this is that uptake of antigens by immature dendritic cells in the absence of inflammation is less likely to promote effective antigen presentation (Gallucci et al., 1999; Sauter et al., 2000). These mechanisms favor immune responses to infectious agents rather than autoimmune responses. A number of groups have studied the maturation of dendritic cells that have taken up apoptotic and necrotic cells. The results of one study showed that only necrotic tumor cells induced maturation of human dendritic cells in culture. Neither apoptotic tumor cell lines nor apoptotic or necrotic cells from primary cultures caused dendritic cell maturation. Soluble supernatants from the necrotic cells could also induce dendritic cell maturation, as could monocyte-conditioned medium (Sauter et al., 2000). Similar results were reported using a comparable murine system (Gallucci et al., 1999). Another study casts doubt on even the ability of necrotic cells to induce dendritic cell maturation, as it was found that only necrotic cells from mycoplasma-infected cultures would stimulate dendritic cell maturation. Antibiotic treatment of these lines abolished their ability to induce dendritic cell maturation, even when the cells were rendered necrotic (Salio et al., 2000). In this chapter, we develop the hypothesis that abnormalities in the physiological clearance mechanisms of autoantigens may promote the development of autoimmune disease. Are the data on the handling of apoptotic cells by dendritic cells compatible with this hypothesis? A delay in the clearance of apoptotic cells by macrophages may increase the likelihood that apoptotic cell debris could be efficiently captured and presented by dendritic cells. The existence of such a balance between macrophage- and dendritic cell–mediated capture of apoptotic tumor cells, which can influence the outcome of a subsequent immune response, has been demonstrated in vivo (Ronchetti et al., 1999). Ronchetti and colleagues immunized mice intraperitoneally with apoptotic sygeneic tumor cells to protect them from subsequent challenge with live tumor cells. Immunizing mice with bone marrow–derived macrophages and dendritic cells, which had been pulsed with apoptotic tumor cells, showed that only dendritic cells could induce a protective antitumor

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cytotoxic T lymphocyte. While the apoptotic cells were not as efficient as nonreplicating live cells at priming an antitumor response, they were rendered more effective at protecting the mice from live tumor cells by pretreatment of the animals with carrageenan to reduce phagocytosis by the peritoneal macrophages. Conversely, enhancement of macrophage phagocytosis with granulocyte–macrophage colony-stimulating factor (GM-CSF) rendered the animals more susceptible to rechallenge with live tumor cells (Ronchetti et al., 1999). However, it is clear from the data presented in this section that simply increasing the uptake of apoptotic cells by dendritic cells cannot be sufficient to drive dendritic cell maturation and autoantigen presentation. Additional inflammatory signals appear to be required. It has been postulated that ingestion of high numbers of apoptotic cells by dendritic cells may be adequate to instigate dendritic cell maturation and the presentation of antigen derived from apoptotic cells in the absence of additional stimuli (Rovere et al., 1998). The same scientists also found that autoantibodies (anti–웁2-glycoprotein I) bound to apoptotic cells caused dendritic cells to secrete inflammatory cytokines, including IL-1웁 and TNF-움, that may have autocrine and paracrine effects, enhancing dendritic cell maturation (Rovere et al., 1999). In the next section, we discuss the evidence that complement plays a role in the clearance of apoptotic cells and consider the hypothesis that complement deficiency may promote autoimmunity via a pathway involving impairment of the clearance of apoptotic cells. 4. The Role of Complement in the Clearance of Apoptotic Cells Complement was implicated first in the clearance of apoptotic cells by the observation by Korb and Ahearn (1997) that C1q could bind specifically and directly to the surface blebs of apoptotic keratinocytes. This interaction is thought to be mediated via the globular heads of the C1q molecule (Navratil et al., 1998). This led to the hypothesis that C1q may promote the clearance of apoptotic cells, and hence exposed autoantigen, preventing stimulation of the immune system. In vitro studies by Mevorach and colleagues (1998a) using complementdepleted sera and human monocyte-derived macrophages supported a role for both the classical and alternative pathways of complement in the phagocytosis of apoptotic cells. Blockade of complement receptors CR3 and CR4 impaired the phagocytosis of apoptotic cells. The presence of iC3b on the surface of apoptotic cells that had been incubated with serum suggested that the clearance was mediated by interactions between iC3b and CR3 and/or CR4. Binding of opsonized apoptotic cells to CR3transfected CHO cells confirmed that CR3 had the potential to interact


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directly with iC3b-coated apoptotic cells and could hence be an apoptotic cell clearance receptor (Mevorach et al., 1998a). To address the relevance of complement in vivo in the phagocytic removal of apoptotic cells, we studied apoptotic cell clearance in complement-deficient mice. We established a model of apoptotic cell phagocytosis during sterile peritonitis. Mice were injected intraperitoneally with thioglycolate to induce sterile peritonitis and the recruitment of inflammatory macrophages. Four days later, the mice were injected intraperitoneally with syngeneic apoptotic thymocytes. The phagocytic uptake of the apoptotic thymocytes by the elicited macrophages was significantly impaired in both C1q- and C4-deficient mice compared to wild-type control animals. However, perhaps more significantly, the defect in phagocytosis was significantly greater in the C1q-deficient animals than in the C4deficient mice. Furthermore, when apoptotic cells were injected into the peritoneum of untreated mice, only the C1q-deficient mice exhibited a defect in phagocytosis, while C4- and C3-deficient mice showed phagocytic uptake similar to that of control animals (Taylor et al., 2000). These observations indicate the existence of a hierarchy within the classical pathway with regard to the role of the components in the phagocytosis of apoptotic cells, which recapitulates the hierarchy of disease susceptibility in humans with complement deficiency. Hence, one possible explanation of the link between complement deficiency and the predisposition to the development of SLE may be the degree of impairment of the physiological clearance of apoptotic cells. It is interesting to note that monocyte-derived macrophages from humans with SLE also exhibited impaired phagocytosis of apoptotic cells in vitro (Herrmann et al., 1998). We have observed a similar phagocytic defect in macrophages derived from the monocytes of C1q-deficient humans cultured in autologous serum (Taylor et al., 2000). This defect was correctable with purified human C1q. All of these data are compatible with the hypothesis that C1q deficiency causes SLE by impairment of the clearance of apoptotic cells. These in turn may provide the source of the autoantigens that drive the autoimmune response of SLE. A reduced ability of macrophages to remove apoptotic cells at sites of inflammation may tip the balance of clearance of these cells toward clearance by dendritic cells. As we discussed in Section VI,B,3, it seems likely that increased uptake of apoptotic cells by dendritic cells is not sufficient to induce dendritic cell maturation and antigen presentation. However, if the apoptotic cells are cleared abnormally at sites of inflammation, the necessary cytokine milieu may drive dendritic cell maturation and initiate an autoimmune response.

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5. A Hypothesis for the Development of Anti-C1q Autoantibodies in SLE The binding of C1q to a major particle driving the autoimmune response in SLE could provide an explanation for the high frequency of autoantibodies to C1q, found in 앑33% of patients with the disease. The autoantibodies of SLE typically react with several components of a normal physiological complex (e.g., anti–double-stranded DNA and antihistones in nucleosomes, or anti-Ro and anti-La in a small cytoplasmic RNP complex) (Hardin, 1986). Antiphospholipid antibodies are frequently found to be associated with antibodies to phospholipid-binding proteins, including annexin V (Hirata et al., 1981), which binds to apoptotic but not normal cell surfaces and 웁2-glycoprotein I (Galli et al., 1990; McNeil et al., 1990). By analogy, it seems likely that anti-C1q antibodies develop as part of an autoantibody response to C1q complexed with other lupus autoantigens, which could be apoptotic cells or immune complexes. It is then an interesting question whether autoantibodies to C1q or to proteins such as 웁2glycoprotein I might then enhance the expression of SLE by further interfering with autoantigen processing. C. THE ROLE OF COMPLEMENT IN CLEARANCE OF IMMUNE COMPLEXES Fc웂 receptors and complement mediate the normal processing of immune complexes. The interaction of complement with immune complexes was first characterized in the 1940s by Heidelberger (1941), who observed increased ‘‘particulation’’ of immune complexes, formed in solution in the absence of complement. Subsequent work has shown that the role of complement in the processing of immune complexes can be divided into two main activities. The first of these is modification of the lattice structure of immune complexes, maintaining immune complex solubility; the second is promotion of the recognition and capture of immune complexes by Fc웂 and complement receptors on cells of the mononuclear phagocytic system. There are several potential final destinations for immune complexes. The first of these destinations reflects the role of antibody in promoting the clearance and killing of pathogens, that is, uptake by a macrophage leading to clearance and catabolism of the immune complex. The second fate reflects another key role of immune complexes in the adaptive immune response, that is, the promotion of immune responses by targeting antigens to B lymphocytes, other antigen-presenting cells, and cells that both present and store antigen, such as follicular dendritic cells within germinal centers. In the absence of complement, the clearance of immune complexes is abnormal and the enhancing activities of immune complexes in adaptive immune responses are muted.


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Complement prevents immune complex precipitation at successive steps during complement activation. First, the C1 complex has been shown to delay immune complex precipitation in a dose-dependent manner (Schifferli et al., 1985), an effect that is probably transient, as C1 inhibitor rapidly degrades the complex, leaving just C1q, which is known to promote immune complex precipitation (Muller-Eberhard and Kunkel, 1961). After the initial stages of immune complex formation, classical pathway activation causes the deposition of C4b and C3b. Deposition of C3b on the heavy chain of IgG interferes with Fc–Fc interactions (Hong et al., 1984), which have been shown to promote the aggregation of immune complexes (Moller, 1979). Deposition of C3 on the antigen also blocks sites of antigen– antibody interaction, reducing the effective valency of antigen and antibody (Lachmann and Walport, 1987). The demonstration that sera deficient in C3, C4, or C2 could not prevent immune complex precipitation showed that classical pathway C3b deposition was essential for this process (Schifferli and Peters, 1982; Schifferli et al., 1985). Amplification of C3b deposition by the alternative pathway amplification loop may assist this process; however, the contribution of the alternative pathway to the prevention of immune complex precipitation is much less significant than that of the classical pathway (Schifferli et al., 1982). The alternative pathway can solubilize immune complex precipitates, but this is a very inefficient process compared to the prevention of precipitation in the first place (Miller and Nussenzweig, 1975). Solubilization of immune complexes is accompanied by significant complement activation and the generation of substantial quantities of the anaphylatoxins and membrane attack complex formation. Complement activity thus maintains immune complexes in a soluble form, which can be carried in the circulation away from the site of formation. In the circulation, there are transport mechanisms for immune complexes that promote their safe delivery to the mononuclear phagocytic system of liver, spleen, and bone marrow. CR1 on erythrocytes is predominantly responsible for the binding of C3b-bearing immune complexes in the circulation of primates. CR1 on the surface of erythrocytes is clustered and this enhances the avidity of binding of immune complexes to these cells (Paccaud et al., 1988). Immune complex clearance from the circulation of patients with SLE is abnormal, and there is evidence for defects of both complement- and Fc-dependent clearance mechanisms for immune complexes. Two types of in vivo studies have been performed in humans and nonhuman primates. The first type of study was of the clearance of IgG- and C3-coated erythrocytes; these were used as models for the study of the mechanisms of hemolytic anemia. IgG-coated erythrocytes were cleared mainly by the spleen (Frank et al., 1983). Of particular interest, patients with SLE showed

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delayed clearance of IgG-coated erythrocytes, which was interpreted to show defects in Fc receptor–mediated activity associated with SLE. By contrast, erythrocytes coated with C3 in vitro, using IgM cold agglutinin antibodies as a sensitizing reagent for complement activation, were not cleared permanently from the circulation (Atkinson and Frank, 1974). These cells were removed transiently from the circulation in the liver, thought to be by interaction of C3b and iC3b with complement receptors on Kupffer cells. After a short delay in the liver, during which it is thought that the iC3b on the erythrocytes was catabolized to C3dg, erythrocytes were released back into the circulation with a normal life span, bearing C3dg fragments for which no clearance receptor exists. There was no abnormality of clearance of these cells in patients with SLE. By contrast with these immune complexes containing erythrocytes as antigen, soluble immune complexes showed different patterns of clearance in vivo in both humans (Davies et al., 1992, 1993; Lobatto et al., 1988; Schifferli et al., 1989) and nonhuman primates (Cornacoff et al., 1983; Waxman et al., 1984). Large, soluble complement-fixing immune complexes bound to erythrocytes during their transit through the circulation and were cleared in the liver and spleen. In the presence of complement deficiency, both inherited and acquired, these immune complexes showed absent or diminished binding to erythrocytes. They showed reduced splenic clearance and disappeared from the circulation rapidly in the liver (Schifferli et al., 1989). However, in patients with SLE or complement deficiency, the immune complexes were retained less effectively in the liver and a proportion were released back into the circulation, with the potential to be deposited in many organs throughout the body (Davies et al., 1992, 1993; Schifferli et al., 1989). These results suggest that the efficient removal of soluble immune complexes by the liver requires ligation of both complement and Fc receptors on Kupffer cells. In the absence of efficient complement fixation, immune complexes may escape efficient clearance, deposit in tissues, and cause tissue injury via ligation of Fc receptors on neutrophils and other leukocytes. The resulting tissue injury may cause the release of autoantigens in an inflammatory milieu and augment the lupus autoimmune response. D. THE ROLE OF COMPLEMENT IN THE HUMORAL IMMUNE RESPONSE Pepys (1974) first showed a link between the complement system and antibody responses. He depleted mice of C3 using cobra venom factor and found that the antibody response to T cell–dependent antigens was suppressed, as was the generation of B cell memory in germinal centers (Papamichail et al., 1975). However, the use of cobra venom factor only allows transient depletion of C3. The identification of animals and patients


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genetically deficient in complement offered further opportunities to investigate the role of the complement system in the induction of humoral immune response. Guinea pigs genetically deficient of C2, C4, and C3 were investigated for their ability to mount an antibody response to bacteriophage ␾X174, an experimental thymus-dependent immunogen. When only limited amounts of antigen were used for immunization, all three types of complement-deficient animals showed markedly impaired antibody formation. This reduced antibody response was strictly dose dependent; when the antigen dose was increased, the immune response returned to normal (Bottger et al., 1986b). A similar role for the classical pathway complement in enhancing antibody responses has also been shown in mice genetically deficient in C3, C4 (Fischer et al., 1996), and C1q (Cutler et al., 1998). Comparison of the primary and secondary immune responses of these mice with wildtype controls following challenge with T cell–dependent antigens (bacteriophage ␾X174, sheep erythrocytes, or keyhole limpet hemocycianindinitrophenyl [KLH-DNP]) demonstrated that while their T cell response was normal, their B cell response was impaired. Splenic B cells of the complement (C1q-, C3-, and C4-)–deficient mice responded normally in proliferation assays in vitro following cross-linking of the B cell antigen receptor or CD40 and in response to stimulation with lipopolysaccharide. These results show that the B cells showed normal responses to pathways of stimulation independent of the complement system. The obverse of these experiments showing defective antibody responses in the absence of complement is the study of Dempsey and colleagues (1996), who demonstrated that oligomers of C3dg coupled to antigen may act as an adjuvant, markedly enhancing antibody response and lowering the threshold of B cells for activation by ligation of the antigen receptor. A similar phenotype to that of mice deficient in C3 and C4 was seen in mice deficient in CD21 and CD35 (Cr2⫺/⫺) (Ahearn et al., 1996; Molina et al., 1996), confirming the critical role of the complement system in enhancing antibody responses. Can the results obtained in experimental animal models be extrapolated to humans with complement deficiency? A C4-deficient patient showed an impaired humoral immune response to bacteriophage ␾X174 and failed to class switch the antibody response ( Jackson et al., 1979). However, antibody responses to rubella, Epstein–Barr virus, and tetanus toxoid were normal in another C4-deficient individual (Mascart-Lemone et al., 1983). This discrepancy could be attributed to the antigen dose dependency that was observed in guinea pigs, described above. A patient with C3 deficiency was shown to have a reduced primary immune response to hemocyanin (Roord et al., 1983). However, the same subject showed normal titers of

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antibody following secondary immunization with tetanus, diphtheria, and pertussis vaccine (Roord et al., 1983). Two C3-deficient patients who developed normal titers of primary and secondary antibody after immunization with ␾X174 failed to switch the antibody response from IgM to IgG, a result similar to that seen in complement-deficient animals (Ochs et al., 1986). The majority of C3 patients have had total IgG levels within the normal range. However, it has been reported that patients with deficiency in the classical pathway proteins have reduced levels of IgG4 and, to a lesser extent, IgG2 (Bird and Lachmann, 1988). From these observations, it is clear that the complement system plays an important role in the generation of humoral responses to low doses of antigens. Two mechanisms to explain the increase in immunogenicity have been proposed. First, opsonization of the antigen by complement enhances the targeting of antigen to follicular dendritic cells, which express CD35 (CR1) and CD21 (CR2), leading to more efficient antigen presentation to B cells and germinal center formation (Klaus et al., 1980; Papamichail et al., 1975). Second, the binding of cognate antigen carrying complement (in the form of an immune complex) to B cells lowers the threshold of activation of the cell by co-ligation of CD21 and the B cell IgM antigen receptor (Fearon and Carter, 1995). However, these findings do not lead to a good hypothesis to explain how complement deficiency is associated with SLE. In summary, they show that a normal physiological activity of complement is to lower the threshold of activation of B cells and promote B cell memory. In the absence of complement, antibody formation is impaired and this should ameliorate, rather than promote, autoimmunity. Another approach to the study of the role of complement in self-tolerance is the use of transgenic models of self-tolerance. Using such models, the expression of a transgenic autoantibody, either to a natural autoantigen or to a transgenically expressed ‘‘pseudo-autoantigen’’ can be examined in the context of other mutations. Prodeus and colleagues (1998) used such a system, comprising soluble hen egg lysosyme (sHEL) as autoantigen and anti-HEL as autoantibody, studied in the presence and absence of C4, C3, or CD21/CD35. They found evidence that ‘‘self-tolerance’’ to the sHEL was depressed in the presence of C4 or CD21/CD35 deficiency but not in the presence of C3 deficiency. They argued that this showed a role for complement in self-tolerance and proposed that deficiency of this activity may promote SLE. However, we interbred C1q-deficient mice with the same transgenic model and found no defect in self-tolerance to the pseudo-autoantigen, sHEL in the absence of C1q (Cutler et al., 1999). sHEL does not represent a typical autoantigen of SLE, as a soluble secreted protein. It will be of interest to study the expression of a more representative


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transgenic autoantibody, such as an anti-Sm or anti-DNA autoantibody in the context of complement deficiency. VII. What Lessons Can Be Learned from Other Murine Models of Autoimmunity?

The existence of other models of murine SLE generated by both genetargeting and natural mutations has given substantial insight into different mechanisms by which self-tolerance is broken, resulting in the spontaneous development of an SLE-like phenotype. These mechanisms can be divided into at least three different categories, each of which may require a mixture of genetic and environmental factors for full expression. The first of these is defects in the clearance of autoantigen, on which this review has focused. The second is abnormalities in the regulation and thresholds of B and T cell activation. The third category are the determinants, as yet unidentified, which cause susceptibility to particular patterns of organ injury (e.g., glomerulonephritis). SLE is a syndrome in mice and humans in which a series of different predisposing genes and environmental stimuli end in a final common pathway of autoantibody production and tissue injury. Additional support for the hypothesis that defects in autoantigen clearance may stimulate the development of SLE comes from two further transgenic models of disease, mice with targeted deletions in serum amyloid P component (SAP) and those with isolated IgM deficiency. An in vivo demonstration linking spontaneous autoimmunity and the failure of the physiological clearance of chromatin was shown in mice deficient in SAP (Bickerstaff et al., 1999). SAP is known to be able to bind to chromatin and displace H1-type histones, aiding in chromatin solubilization (Butler et al., 1990). SAP has also been shown to bind in vivo to the surface of apoptotic cells and to nuclear debris released during cell necrosis (Hintner et al., 1988). Mice deficient in SAP spontaneously developed high levels of autoantibodies against chromatin, histones, and DNA and also developed glomerulonephritis. Mice have been developed with a targeted deletion of part of the secretory 애 tailpiece, which prevents the secretion of IgM, though allowing the production of normal IgG levels (Boes et al., 1998; Ehrenstein et al., 1998). These mice are an important model to explore the physiological role of secreted IgM in vivo. When crossed with the spontaneous autoimmune mouse strain, MRL/lpr, the progeny developed a higher frequency of ANAs and glomerulonephritis than in the parental strain (Boes et al., 2000). Mice lacking secreted IgM expression alone developed higher titers of anti–doublestranded DNA antibodies (Ehrenstein et al., 2000). The investigators speculated that the development of autoimmunity in these mice was compatible with a role for natural autoreactive IgM in the clearance of autoantigens.

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A series of mouse strains illustrate that particular mutations that affect thresholds for activation of B and T cells may cause the development of SLE. An autoimmune phenotype was reported in mice with genetic deficiency of CD22 (O’Keefe et al., 1996, 1999), Lyn (Hibbs et al., 1995), and SHP-1 (Shultz et al., 1993; Tsui et al., 1993). Of particular interest was a study showing that a combination of heterozygous deficiencies of CD22, Lyn, and SHP-1 behaved as a complex quantitative trait, causing in combination a reduction in B cell threshold for activation by a transgenic autoantigen (Cornall et al., 1998). In addition, a break in self-tolerance has been observed in mice with defects in cell cycle regulation (Balomenos et al., 2000) and in genes involved in Fas-mediated cell death (Di Cristofano et al., 1999). VIII. Conclusions

The evidence is overwhelming that deficiency of classical pathway complement proteins causes the development of SLE in humans and transgenic mice. The most important complement proteins for protection against SLE are C1q, C1r and C1s, and C4. It is an important challenge to understand the mechanism of this protective effect. Complement plays a key role in the clearance of immune complexes. Recent evidence supports a role for complement in the clearance of apoptotic cells. These data support the hypothesis that complement deficiency causes lupus by the impairment of physiological disposal of autoantigens, and apoptotic cells may be an important source of the autoantigens that drive the characteristic spectrum of autoantibodies found in SLE. The events needed to break tolerance to ubiquitous autoantigens in SLE must be more than a simple increase in autoantigen levels. It seems likely that there must be concomitant proinflammatory signals that induce dendritic cells to provide the necessary costimulatory signals to drive T cell responses, which in turn provide the necessary help to B cells to produce an autoantibody response. There is good evidence, at least in murine models of disease, that genetic abnormalities modifying lymphocyte activation thresholds may increase disease susceptibility. One of the major paradoxes in the relationships between the complement system and SLE is that C1q deficiency causes SLE, yet SLE causes autoantibodies to C1q in approximately one third of patients. This paradox might be explained if C1q binds to lupus autoantigens and promotes their physiological clearance. An absence of C1q could then modify the clearance pathways of autoantigens and trigger an autoimmune response. On the other hand, complex formation between C1q and autoantigens might ex-


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Vidal, S., Kono, D. H., and Theofilopoulos, A. N. (1998). Loci predisposing to autoimmunity in MRL-Faslpr and C57BL/6-Faslpr mice. J. Clin. Invest. 101, 696–702. Voll, R. E., Herrmann, M., Roth, E. A., Stach, C., Kalden, J. R., and Girkontaite, I. (1997). Immunosuppressive effects of apoptotic cells. Nature 390, 350–351. Walport, M. J., Ross, G. D., Mackworth-Young, C., Watson, J. V., Hogg, N., and Lachmann, P. J. (1985). Family studies of erythrocyte complement receptor type 1 levels: Reduced levels in patients with SLE are acquired, not inherited. Clin. Exp. Immunol. 59, 547–554. Walport, M., Ng, Y. C., and Lachmann, P. J. (1987). Erythrocytes transfused into patients with SLE and haemolytic anaemia lose complement receptor type 1 from their cell surface. Clin. Exp. Immunol. 69, 501–507. Walport, M. J., Davies, K. A., Botto, M., Naughton, M. A., Isenberg, D. A., Biasi, D., Powell, R. J., Cheung, N. T., and Struthers, G. R. (1994). C3 nephritic factor and SLE: Report of four cases and review of the literature. Q. J. Med. 87, 609–615. Walport, M. J., Davies, K. A., and Botto, M. (1998). C1q and systemic lupus erythematosus. Immunobiology 199, 265–285. Wang, X., Circolo, A., Lokki, M. L., Shackelford, P. G., Wetsel, R. A., and Colten, H. R. (1998). Molecular heterogeneity in deficiency of complement protein C2 type I. Immunology 93, 184–191. Wara, D. W., Reiter, E. O., Doyle, N. E., Gewurz, H., and Ammann, A. J. (1975). Persistent Clq deficiency in a patient with a systemic lupus erythematosus–like syndrome. J. Pediatr. 86, 743–745. Watanabe, Y., Matsui, N., Yan, K., Nishimukai, H., Tokunage, K., Juji, T., Kobayashi, N., and Kohsaka, T. (1993). A novel C3 allotype C3‘FO2’ has an amino acid substitution that may inhibit iC3b synthesis and cause C3-hypocomplementaemia. Mol. Immunol. 30, 62 (abstr.). Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A., and Nagata, S. (1992). Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356, 314–317. Watson, M. L., Rao, J. K., Gilkeson, G. S., Ruiz, P., Eicher, E. M., Pisetsky, D. S., Matsuzawa, A., Rochelle, J. M., and Seldin, M. F. (1992). Genetic analysis of MRL–lpr mice: Relationship of the Fas apoptosis gene to disease manifestations and renal disease–modifying loci. J. Exp. Med. 176, 1645–1656. Waxman, F. J., Hebert, L. A., Cornacoff, J. B., VanAman, M. E., Smead, W. L., Kraut, E. H., Birmingham, D. J., and Taguiam, J. M. (1984). Complement depletion accelerates the clearance of immune complexes from the circulation of primates. J. Clin. Invest. 74, 1329–1340. Weinstein, A., Bordwell, B., Stone, B., Tibbetts, C., and Rothfield, N. F. (1983). Antibodies to native DNA and serum complement (C3) levels. Application to diagnosis and classification of systemic lupus erythematosus. Am. J. Med. 74, 206–216. Weiss, R. M., and Schulz, E. J. (1989). Complement deficiency in Sweet’s syndrome. Br. J. Dermatol. 121, 413–415. Welch, T. R., Beischel, L. S., Choi, E., Balakrishnan, K., and Bishof, N. A. (1990). Uniparental isodisomy 6 associated with deficiency of the fourth component of complement. J. Clin. Invest. 86, 675–678. Wessels, M. R., Butko, P., Ma, M., Warren, H. B., Lage, A. L., and Carroll, M. C. (1995). Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity. Proc. Natl. Acad. Sci. USA 92, 11490–11494. Wetsel, R. A., Kulics, J., Lokki, M. L., Kiepiela, P., Akama, H., Johnson, C. A., Densen, P., and Colten, H. R. (1996). Type II human complement C2 deficiency. Allele-specific


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amino acid substitutions (Ser189 씮 Phe; Gly444 씮 Arg) cause impaired C2 secretion. J. Biol. Chem. 271, 5824–5831. Wilson, J. G., Wong, W. W., Schur, P. H., and Fearon, D. T. (1982). Mode of inheritance of decreased C3b receptors on erythrocytes of patients with systemic lupus erythematosus. N. Engl. J. Med. 307, 981–986. Wilson, J. G., Murphy, E. E., Wong, W. W., Klickstein, L. B., Weis, J. H., and Fearon, D. T. (1986). Identification of a restriction fragment length polymorphism by a CR1 cDNA that correlates with the number of CR1 on erythrocytes. J. Exp. Med. 164, 50–59. Wilson, J. G., Wong, W. W., Murphy, E. E. 3d., Schur, P. H., and Fearon, D. T. (1987). Deficiency of the C3b/C4b receptor (CR1) of erythrocytes in systemic lupus erythematosus: Analysis of the stability of the defect and of a restriction fragment length polymorphism of the CR1 gene. J. Immunol. 138, 2708–2710. Wilson, W. A., Perez, M. C., and Armatis, P. E. (1988). Partial C4A deficiency is associated with susceptibility to systemic lupus erythematosus in black Americans. Arthritis Rheum. 31, 1171–1175. Wilson, W. A., Armatis, P. E., and Perez, M. C. (1989). C4 concentrations and C4 deficiency alleles in systemic lupus erythematosus. Ann. Rheum. Dis. 48, 600–604. Wisnieski, J. J., and Jones, S. M. (1992). Comparison of autoantibodies to the collagen-like region of C1q in hypocomplementemic urticarial vasculitis syndrome and systemic lupus erythematosus. J. Immunol. 148, 1396–1403. Wisnieski, J. J., Baer, A. N., Christensen, J., Cupps, T. R., Flagg, D. N., Jones, J. V., Katzenstein, P. L., McFadden, E. R., McMillen, J. J., Pick, M. A., et al. (1995). Hypocomplementemic urticarial vasculitis syndrome. Clinical and serologic findings in 18 patients. Medicine 74, 24–41. Wong, W. W., and Farrell, S. A. (1991). Proposed structure of the F⬘ allotype of human CR1. Loss of a C3b binding site may be associated with altered function. J. Immunol. 146, 656–662. Wong, W. W., Klickstein, L. B., Smith, J. A., Weis, J. H., and Fearon, D. T. (1985). Identification of a partial cDNA clone for the human receptor for complement fragments C3b/C4b. Proc. Natl. Acad. Sci. USA 82, 7711–7715. Wong, W. W., Kennedy, C. A., Bonaccio, E. T., Wilson, J. G., Klickstein, L. B., Weis, J. H., and Fearon, D. T. (1986). Analysis of multiple restriction fragment length polymorphisms of the gene for the human complement receptor type I. Duplication of genomic sequences occurs in association with a high molecular mass receptor allotype. J. Exp. Med. 164, 1531–1546. Wong, W. W., Cahill, J. M., Rosen, M. D., Kennedy, C. A., Bonaccio, E. T., Morris, M. J., Wilson, J. G., Klickstein, L. B., and Fearon, D. T. (1989). Structure of the human CR1 gene. Molecular basis of the structural and quantitative polymorphisms and identification of a new CR1-like allele. J. Exp. Med. 169, 847–863. Wu, J., Wilson, J., He, J., Xiang, L., Schur, P. H., and Mountz, J. D. (1996). Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. J. Clin. Invest. 98, 1107–1113. Xiang, L., Rundles, J. R., Hamilton, D. R., and Wilson, J. G. (1999). Quantitative alleles of CR1: Coding sequence analysis and comparison of haplotypes in two ethnic groups. J. Immunol. 163, 4939–4945. Yamada, H., Watanabe, A., Mimori, A., Nakano, K., Takeuchi, F., Matsuta, K., Tanimoto, K., Miyamoto, T., Yukiyama, Y., Tokunaga, K., and Yokohari, R. (1990). Lack of gene deletion for complement C4A deficiency in Japanese patients with systemic lupus erythematosus. J. Rheumatol. 17, 1054–1057.

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Signal Transduction by the High-Affinity Immunoglobulin E Receptor Fc␧RI: Coupling Form to Function MONICA J. S. NADLER, SHARON A. MATTHEWS, HELEN TURNER, AND JEAN-PIERRE KINET Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts

I. Introduction

Mast cells are activated by cross-linking of the high-affinity Fc␧RI receptor, initiated by the binding of multivalent antigen to prebound IgE molecules in the receptor complex. Once activated, mast cells elicit three types of biological responses: (i) secretion of preformed granules, (ii) synthesis and secretion of cytokines, and (iii) synthesis of lipid mediators. These responses form the basis of one of the most potent effector systems in immunity—immediate hypersensitivity—which includes, in the most extreme cases, anaphylaxis. The speed and efficiency of this immune response are facilitated by immunoglobulin E (IgE) antibody, which is prebound by its Fc portion to the Fc␧RI receptor in a 1 : 1 ratio with high affinity (Kd ⫽ 10⫺9 to 10⫺10) (Keown et al., 1998; Metzger, 1992, 1999). The high-affinity Fc␧RI receptor is expressed on the surface of mast cells and basophils as a multimeric 움웁웂2 complex (Blank et al., 1989; Kuster et al., 1992; Ra et al., 1989b). The 움-chain, responsible for binding to the Fc portion of IgE, is extensively N-glycosylated and contains two extracellular Ig-related domains, a transmembrane region and a short cytoplasmic tail which has no signaling capacity (Alber et al., 1991; Kinet et al., 1987; Kochan et al., 1988; Letourneur et al., 1995; Ra et al., 1989b; Shimizu et al., 1988). The 웁-chain, which consists of four transmembrane domains with cytoplasmic N- and C-termini (Kinet et al., 1988; Kuster et al., 1992; Ra et al., 1989b), and a disulfide-linked 웂-chain homodimer constitute the signal-transducing portion of the receptor (Fig. 1) (Blank et al., 1989; Kuster et al., 1990; Ra et al., 1989b). Upon antigen challenge, the receptors are cross-linked and the 웁- and 웂-chains play a central role in the initiation of signaling cascades by serving as phosphoacceptor sites for intracellular tyrosine kinases. The high-affinity Fc␧RI receptor belongs to the Ig superfamily (Kinet, 1999; Metzger, 1991; Ravetch and Kinet, 1991; Turner and Kinet, 1999). The modular nature of the related Fc receptor and antigen receptor structures is evidenced by the presence of similar phosphoacceptor sequences found in the cytoplasmic tails of the receptor chains known as immunoreceptor tyrosine-based activation motifs (ITAMs) Fig. 1 (Cambier, 1995a,b; 325


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FIG. 1. ITAM-containing immunoreceptors. The tetrameric Fc␧RI is shown in the context to related immunoreceptor family members. The sequences of the Fc␧RI 웁- and 웂-chain ITAMs are also shown with canonical residues in bold type. TCR, T cell antigen receptor; BCR, B cell antigen receptor; IgM, immunoglobulin M; ITAM, immunoreceptor tyrosine-based activation motif. White boxes represent ITAMs; black ovals represent immunoglobulin domains.

Daeron, 1997; Isakov, 1997; Reth, 1989). Signals generated from these related antigen receptors involve many of the same secondary molecules found in Fc␧RI cascades and result in changes in calcium flux, gene activation, and cellular activation. Furthermore, the 웁- and 웂-chains of Fc␧RI are also shared by other receptor complexes. The 웁-chain is present as a subunit of the low-affinity IgG receptor (Fc웂RIII) (Dombrowicz et al., 1998; Kurosaki et al., 1992), while the 웂-chain is found in Fc웂RIII, Fc웂RI, Fc움R, and the T cell antigen receptor (TCR) (Anderson et al., 1990; Ernst et al., 1993; Hibbs et al., 1989; Kinet, 1992; Kurosaki and Ravetch, 1989; Lanier et al., 1989; Morton et al., 1995; Orloff et al., 1990; Pfefferkorn and Yeaman, 1994; Ra et al., 1989a; Scholl and Geha, 1993).

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Overall, these similarities in structure and the presence of 웁- or 웂-chains in other complexes suggest redundancies in the function of ITAMs in other cell types. II. Signal Initiation: The Central Importance of ITAM

The Fc␧RI receptor does not contain intrinsic catalytic activity. Instead, it associates with nontransmembrane protein tyrosine kinases (PTKs) that phosphorylate tyrosine residues found in the ITAMs of 웁- and 웂-chains (Bolen, 1995; Daeron et al., 1995; Paolini et al., 1991). The ITAM is present in both the 웁- and 웂-chains and is centrally important to signal initiation (Fig. 1). Phosphorylated tyrosine residues within the ITAMs serve as docking sites for secondary signaling proteins, which in turn propagate the signal, leading to increases in cellular tyrosine phosphorylation, calcium flux, secretion, and gene activation. Mutation of the tyrosine residues in the 웁- and 웂-chain ITAMs abolishes signaling ( Jouvin et al., 1994; Letourneur and Klausner, 1991; Lin et al., 1996). Furthermore, experiments have shown that the ITAM sequence of the 웂-chain can be attached to unrelated extracellular domains and engaged to activate intracellular signaling cascades (Letourneur and Klausner, 1991; Romeo and Seed, 1991). Taken together, these studies show that the ITAM portions of 웁- and 웂-chains are required for Fc␧RI signaling. Although there are many similarities among ITAMs, there are structural differences and nuances in the Fc␧RI complex that may contribute to the unique signaling events mediated by this receptor. Importantly, the 웁- and 웂-chain ITAMs differ: the 웁-chain has a shorter spacing region between the first and second tyrosines of the consensus motif and contains an additional third tyrosine that the 웂-chain ITAM lacks (Fig. 1). There are also functional differences between the 웁- and 웂-chains. Experiments have shown that the 웂-chain alone is sufficient to initiate Fc␧RI signaling cascades (Letourneur and Klausner, 1991; Romeo and Seed, 1991). Despite the fact that the 웁-chain also contains an ITAM, chimeras of the 웁-chain do not signal efficiently ( Jouvin et al., 1994). Instead, the 웁-chain serves as an amplifier of 웂-chain signaling (Dombrowicz et al., 1998; Lin et al., 1996). The unique structural features of the 웁-chain ITAM may contribute to this novel function. Indeed, LYN preferentially associates with the 웁chain compared to the 웂-chain, which suggests a molecular explanation for its role as an amplifying molecule: the addition of more LYN within the receptor complex by 웁-chain could increase tyrosine phosphorylation events of several substrates, which in turn would lead to a greater activation signal. Further structural features of the 웁-chain may also contribute to its unique function, since truncation of the non–ITAM-containing N-

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terminal tail of the 웁-chain diminishes its amplifying potential (Lin et al., 1996). To date, this amplifier property of the 웁-chain is unique among antigen receptors. III. Phosphate Transfer and PTKs

In the context of a receptor that lacks intrinsic catalytic activity, such as Fc␧RI, the initiation of an antigen receptor signal results in the recruitment of secondary signaling molecules through inducible protein–protein interactions. The phosphorylated tyrosine residues in the ITAMs act as docking sites for Src homology 2 (SH2) domain–containing proteins. Specifically, tyrosine phosphorylation of the Fc␧RI 웂-chain ITAM results in the recruitment of SYK. The subsequent recruitment of further secondary signaling molecules also involves SH2 domains. Some SH2 domain–containing proteins are enzymes, some are transcription factors, and other are adapters that lack intrinsic catalytic activity and consist mainly of SH2 and SH3 domains, which bind to proline-rich regions. Various adapter proteins that contain both SH2 and SH3 domains are critical in linking signaling molecules together. These interactions result in increased tyrosine phosphorylation events and in increased enzymatic activity of secondary signaling proteins at or near the receptor complex. In addition, protein–lipid interactions are also essential for signal propagation and are mediated in part by pleckstrin homology (PH) domains, which bind directly to lipids, localizing signaling proteins to the plasma membrane. Overviews of Fc␧RI receptor proximal signaling events and the signaling molecules involved are shown in Figs. 2 and 3. A. LYN

AND

SYK

Receptor aggregation establishes the earliest events in Fc␧RI signaling, which involve activation of two tyrosine kinases that act in a two-step process to initiate the signal. Several studies have come together to establish the following sequence of events. The Src family PTK LYN is the earliest kinase activated by Fc␧RI receptor engagement (Eiseman and Bolen, 1992). LYN associates with the 웁-chain and phosphorylates tyrosine residues within the ITAMs of both 웁- and 웂-chains upon activation ( Jouvin et al., 1994; Paolini et al., 1991; Yamashita et al., 1994). SYK then binds to the receptor complex at the phosphorylated ITAMs of the 웂-chain via its SH2 domain, where it is further activated by LYN (Scharenberg et al., 1995). The phosphorylation of SYK by LYN fully activates SYK, and it has been demonstrated that these tyrosine phosphorylation events, which occur in the activation loop of SYK, are essential for intracellular signaling (ElHillal et al., 1997; J. Zhang et al., 1998). The position of LYN upstream


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FIG. 2. Fc␧RI activation induces a complex network of proximal signaling events. Fc␧RI aggregation is induced by cross-linking of bound immunoglobulin E by polyvalent antigen. Information is then passed to the 웁 and 웂 signaling chains of the receptor, activating intracellular signaling pathways that control mast cell effector functions, ultimately leading to allergic responses. LYN phosphorylates the 웁- and 웂-chain ITAMs, recruiting SYK, which is in turn phosphorylated and activated by LYN. LYN and SYK phosphorylate and activate a number of adapter proteins and protein tyrosine kinases, including LAT, the p85 regulatory subunit of P13K, and the Tec family kinase BTK (Bruton’s tyrosine kinase). Fc␧RI activation of phosphatidylinositol-3-kinase (PI3K) elevates plasma membrane phosphatidylinositol3,4,5-trisphosphate (PIP3) levels, recruiting pleckstrin homology (PH) domain–containing proteins such as BTK to the plasma membrane. BTK contributes to the activation of phospholipase C웂 (PLC웂), which in turn is localized to the membrane through the interaction of its Src homology 2 domain with the phosphorylated LAT adapter. PLC웂 metabolizes the membrane inositol phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate inositol-1,4,5-trisposphate (IP3) and diacylglycerol (DAG), which regulate calcium mobilization and activate members of the protein kinase C (PKC) family of serine–threonine kinases, respectively. IP3 binds to the IP3 receptors (IP3R) on the surface of endoplasmic reticulum calcium stores, leading to store depletion and elevation of cytosolic calcium levels. Activation of the Fc␧RI also regulates signaling pathways controlled by GTPase proteins. The phosphorylated LAT adapter molecule anchors the Grb2 and SLP-76 adapters, which in turn couple to guanine nucleotide exchange factors for Ras and Rac GTPases (Sos and Vav, respectively). GTP loading of these GTPases induces a wide range of effector responses, including activation of various transcription factors and changes in cell morphology. Csk, c-Src kinase. Black diamonds represent PH domain–phospholipid interactions; stars indicate phosphorylation events.

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FIG. 3. Molecules involved in Fc␧RI signaling pathways. Schematic representation of the signaling molecules discussed in the text, depicted to highlight their modular structures. Domains the enzymatic or nucleotide exchange factor activity are shown in open rectangles. C1, cysteine-rich domain (diacylglycerol binding); C2, calcium binding domain; PH, pleckstrin homology domain (lipid interactions); PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PKD, protein kinase D; PLC웂, phospholipase C웂; Pro, proline-rich domain (SH3 binding); SH2, Src homology 2 domain (phosphotyrosine binding); SH3, Src homology 3 domain (proline-rich region binding); Tec, Tec kinase homology domain; TM, transmembrane domain.

of SYK has been confirmed by genetic studies: LYN⫺/⫺ mast cells exhibit neither 웁 nor 웂 phosphorylation and therefore do not recruit SYK to the 웂-chain following receptor activation (Nishizumi and Yamamoto, 1997). Importantly, the overall requirement for LYN in Fc␧RI signaling is illustrated by the finding that IgE-mediated systemic anaphylaxis, a measure of Fc␧RI function (Dombrowicz et al., 1993), is diminished in LYNdeficient mice (Hibbs et al., 1995). Furthermore, LYN-deficient mast cells have decreased calcium mobilization and tyrosine phosphorylation events (Nishizumi and Yamamoto, 1997). Taken together, these results demonstrate that LYN is indispensable in the Fc␧RI pathway. SYK is also essential for mast cell activation: in SYK⫺/⫺ mast cells 웁- and 웂-chain phosphorylation events are intact, but downstream events are abrogated (Costello et al., 1996; Zhang et al., 1996).


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The structures of the Fc␧RI 웁- and 웂-chain ITAMS are distinct. However, the similar roles of ITAMs within the signaling chains of other receptors would suggest a parallel redundant function. Indeed, engagement of these related receptors initiates similar signaling cascades that utilize the same or related signaling molecules. Interestingly, there is genetic evidence for a differential role for LYN in Fc␧RI versus B cell antigen receptor (BCR) signaling even though both antigen structures share a great deal of similarity. In B cells, the BCR complex is required for B cell activation events that lead to proliferation and secretion of antibodies. LYN plays an initiating role for the BCR similar to the role it plays for Fc␧RI: it is one of the earliest PTKs activated, and it phosphorylates and activates several positive signaling molecules, including SYK (Kurosaki, 1997; Yamamoto et al., 1993). In addition, LYN participates in the activation of Bruton’s tyrosine kinase (Park et al., 1996; Rawlings et al., 1996). However, in B cells, LYN has also been implicated in the phosphorylation of the BCR co-receptor CD22, which recruits the protein tyrosine phosphatase (PTP) SHP-1 (Smith et al., 1998). Interestingly, B cells from LYN-deficient mice show a hyperresponsive phenotype: these mice are IgM hyperglobulinemic (i.e., have increased serum IgM levels) and develop severe glomerulonephritis (Hibbs et al., 1995; Nishizumi et al., 1995). These abnormalities result from the loss of LYN’s function as a negative regulator of BCR activation signals. The negative signaling function of LYN is dependent on CD22 and is further compounded by loss of SHP-1 (Cornall et al., 1998). Therefore, in B cells, the predominant function of LYN is the phosphorylation of the co-receptor CD22, which results in subsequent recruitment of SHP-1 into the vicinity of the BCR. In contrast, LYN does not dominantly function as a negative regulator of Fc␧RI signaling in mast cells. The opposing effects of LYN in mast cells versus B cells are an example of how the molecular and cellular context determines whether the action of a PTK is positive or negative. Importantly, the differences between the related antigen receptor complexes should be understood to appreciate the impact that specific mutations can have. This is especially true for diseases with complex genetic origins in which there may be several mutations or differential effects caused by the same mutation in different cell types. B. BRUTON’S TYROSINE KINASE Another PTK activated by antigen receptor engagement is Bruton’s tyrosine kinase (BTK). BTK is a member of the Tec family of tyrosine kinases, which are distinguished by the presence of a PH domain at the N-terminus, an SH2 domain, and an SH3 domain (Fig. 3). PH domains

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are important in binding to lipids and thus link the critical upstream protein tyrosine phosphorylation events with lipid metabolism, and in the case of BTK, interaction with lipid localizes it to the antigen receptor complex. Several studies have unraveled the role of BTK in antigen receptor signaling. First, BTK is targeted to the membrane via binding of its PH domain to the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Fluckiger et al., 1998; Scharenberg and Kinet, 1998); which is produced by the action of phosphatidylinositol-3-kinase (PI3K) upon receptor engagement. Second, this brings BTK into the vicinity of LYN, which further phosphorylates BTK, providing full activation (Park et al., 1996; Rawlings et al., 1996). Finally, in turn, BTK phosphorylates and contributes to the activation of phospholipase C웂1 (PLC웂1), resulting in calcium mobilization (Scharenberg and Kinet, 1998). The importance of this pathway has been illustrated in chicken B cell lines that lack BTK. These cells fail to phosphorylate PLC웂 or release calcium upon BCR stimulation, indicating that BTK plays an essential role in antigen receptor signaling, linking upstream phosphorylation events with calcium release (Takata and Kurosaki, 1996). Genetic analysis has also demonstrated the function of BTK in Fc␧RI signaling. X-linked immunodeficient (Xid) mice are characterized by a mutation in BTK that lowers its affinity for PIP3, decreasing BTK activation (Rawlings et al., 1993). In these mice, B cell development is arrested at the pre-B cell stage, with resulting hypo- or agammaglobulinemia (Conley et al., 1994). In contrast, mast cell development from both Xid mutant and BTK-deficient mice appears to be normal (Hata et al., 1998). However, BTK-deficient mast cells show decreased IgE-mediated anaphylactic responses, impaired degranulation, and reduced cytokine production (Hata et al., 1998). These data suggest that the role BTK plays in Fc␧RI signaling is similar to its role in BCR signaling. However, there may be some differences in the dependence on BTK in mast cells. Mice deficient for both BTK and the related Tec family member ITK have a more severe Fc␧RI signaling deficiency than that found in single BTK-deficient mice (F. W. Alt, personal communication), suggesting functional redundancy between BTK and ITK in mast cells. The lesser dependence on BTK in Fc␧RI signaling in mast cells than in antigen signaling in B cells illustrates that overlapping specificities can lead to differential functional effects upon mutation or ablation of given signaling molecule in different cell types. IV. Phosphate Transfer and PTPs

Phosphate transfer is a bidirectional process, with the removal of phosphate as important to the regulation of protein function as the addition of phosphate. Importantly, PTPs are not solely antagonists of PTKs. Although


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some PTPs oppose PTK action by dephosphorylation of PTKs, PTPs can act as positive or negative regulators in any given pathway on the basis of which phosphotyrosine residues they target. For example, the PTP CD45 is viewed as a positive regulator since its activity is required to dephosphorylate Src family PTKs for the initiation of antigen receptor signaling. In contrast, the SH2 domain–containing PTP-1 (SHP-1) is viewed as a negative regulator, as illustrated by the phenotype of motheaten (SHP-1 mutant) mice, in which there are multiple defects in myeloid and lymphocyte populations, leading to hyperproliferation and eventual death (reviewed in Bignon and Siminovitch, 1994; Neel, 1993; Tsui and Tsui, 1994). The diverse impact of the PTPs on cellular signaling should thus be viewed as a regulatory mechanism that is separate but related to that of the PTKs. Like tyrosine phosphorylation, tyrosine dephosphorylation can be activating or deactivating, depending on the molecular context. The roles of PTPs in mast cells, specifically those involved in Fc␧RI signaling, are not well characterized. To date, only a few studies have focused on this important but relatively unexplored area of mast cell research. Although well over 70 mammalian PTPs have been identified, only a few have been implicated in immune receptor signaling. There are likely to be more PTPs involved in Fc␧RI signaling cascades, and as sequencing of the human genome and deduction of coding sequences proceed at an unprecedented pace, the exact size of the PTP family will become evident. A. CD45 The transmembrane PTP CD45 was one of the first PTPs to be identified and characterized. It contains a large extracellular domain that is variable because of alternate splicing and two tandem intracellular PTP domains, of which only the N-terminal domain shows significant activity. The function of the C-terminal PTP domain is unclear, although it may participate in binding to various signaling molecules (Mustelin et al., 1998; Plas and Thomas, 1998). In addition to a large body of CD45 literature from in vitro and in vivo studies, the overall importance of CD45 to immune receptor signaling is underscored by the recent report of a human severe combined immunodeficiency syndrome associated with mutation of CD45 (Kung et al., 2000). Classically, the role of CD45 in lymphocytes has been viewed as that of a positive regulator: CD45 opposes the action of c-Src kinase (CSK) by dephosphorylating Src family PTKs on their negative regulatory site. This allows the activation of Src family PTKs, initiating antigen receptor signaling. This mechanism appears to be the same for the Fc␧RI receptor: studies have shown that inhibiting the function of CD45 with blocking antibodies prevents the release of histamine from activated basophils (Hook et al., 1991). In addition, CD45-deficient mast

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cells show defective Fc␧RI-induced histamine release and are resistant to IgE-mediated systemic anaphylaxis (Berger et al., 1994). Furthermore, adequate PTK activity in Jurkat T cells that have been reconstituted with Fc␧RI requires the presence of CD45 (Adamczewski et al., 1995). Another recent study has also indicated a role for CD45 in the positive regulation of Fc␧RI-induced LYN activity (Murakami et al., 2000). Taken together, these results indicate that CD45 plays an important positive role in Fc␧RI signaling. B. SHP-1 In contrast, the SH2-containing PTP SHP-1 has been implicated as a negative regulator of several hematopoietic signaling pathways and in setting the antigen receptor threshold for B and T cell antigen signals (reviewed in Long, 1999; Plas and Thomas, 1998; Siminovitch and Neel, 1998). SHP-1 has a single phosphatase (PTP) domain with two N-terminal SH2 domains and a C-terminus that contains several tyrosine phosphorylation sites. The SH2 domains of SHP-1 bind to phosphotyrosine-containing acceptor motifs located in the cytoplasmic tails of several transmembrane inhibitory receptors. These motifs, known as immunoreceptor tyrosinebased inhibition motifs (ITIMs) serve to target SHP-1 into the vicinity of cellular substrates (Bolland and Ravetch, 1999; Daeron, 1997). Once activated, SHP-1 dephosphorylates target molecules involved in antigen receptor signaling. There is a natural SHP-1 mutant mouse, motheaten, which has mutations in SHP-1 that either abrogate its expression or impair its catalytic function (Shultz et al., 1993; Tsui et al., 1993). The overall importance of SHP-1 in lymphocyte signaling is well illustrated by the phenotype of this mouse, which shows prominent abnormalities in many lymphocyte- and myeloid-derived populations (reviewed in Bignon and Siminovitch, 1994; Neel, 1993; Tsui and Tsui, 1994). However, the specific roles of SHP-1 in Fc␧RI-mediated mast cell function are only beginning to be explored. One useful way to probe PTP function is to overexpress catalytically inactive forms of the enzyme that retain the ability to bind to and thus ‘‘trap’’ substrates. Such ‘‘trapping mutants’’ have been described comprehensively, but one caveat of their use is that they can have the unanticipated consequences, including acting as dominant positives by sequestering phosphorylated substrates (Siminovitch and Neel, 1998). SHP-1 has been implicated in Fc␧RI signaling with the use of such trapping mutants. Although expression of SHP-1 trapping mutants in mast cells results in increased tyrosine phosphorylation of 웁, 웂, and SYK, suggesting a negative signaling role for SHP-1, these changes do not correlate with enhanced downstream signaling events (Xie et al., 2000). Moreover, the identities of SHP-1 substrates in


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mast cells are unknown, although studies in T and B cells have implicated LYN, SYK, and ZAP-70 as substrates of SHP-1 (Dustin et al., 1999; Lorenz et al., 1996; Plas et al., 1996; Somani et al., 1997; Yang et al., 1998). Further work is required to untangle the complexities of SHP-1 action in mast cells. Many questions remain regarding SHP-1 function, and the complex nature of SHP-1 signaling may be even more involved than originally envisioned. It is interesting to note that a splice variant of SHP-1 was recently isolated from Jurkat T cells ( Jin et al., 1999). This variant, SHP-1L, differs from SHP-1 at the C-terminus. The last C-terminal 66 amino acids are different, and the enzyme is 29 amino acids longer. Interestingly, SHP-1L shows increased catalytic activity at physiological pH compared to SHP-1, suggesting that it has a unique role in basal signaling. Although the function of this variant is unknown, the unique features of its altered C-terminus may confer a difference in substrate specificity or localization. It will be interesting to determine whether mast cells utilize SHP-1L or a similar isoform in the Fc␧RI pathway. C. OTHER PHOSPHATASES The related PTP, SHP-2, is also expressed in mast cells and was initially reported to associate with the Fc␧RI, but subsequent plasmon resonance studies have shown that this is a very low-affinity interaction (Ottinger et al., 1998). It is therefore unclear what role SHP-2 plays in Fc␧RI signaling. There have also been reports of other PTP activities that are activated upon Fc␧RI engagement (Mao and Metzger, 1997; Swieter et al., 1995). Cloning and characterization of these PTPs will be of great interest. V. Other Signaling Molecules

In antigen receptor signaling, coupling of Fc␧RI proximal phosphorylation events to downstream events is accomplished by various adapter molecules that link these initial events with calcium flux and mitogenic signaling. Recent studies have demonstrated that PLC웂 phosphorylation represents an important regulatory point for the control of calcium flux, and several adapter proteins are now clearly implicated in linking upstream PTK activity to PLC웂 phosphorylation and activation in various cell types. Genetic evidence for adapter proteins involved in Fc␧RI signaling has emerged. In addition, the requirement for VAV in Fc␧RI signaling has been reported. A. THE ADAPTER PROTEIN LAT The protein LAT (linker for activation of T cell), which was originally defined in T cells, is essential for Fc␧RI signaling. LAT is a 36- to 38-kDa

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protein that serves as a critical link between activated PTKs and downstream substrates (W. Zhang et al., 1998a). In T cells, LAT becomes heavily tyrosine phosphorylated upon TCR activation, and it has been reported to bind to the SH2 domains of multiple signaling proteins, including Grb2, Gads, Grap, PLC웂, and the p85 subunit of PI3K (Schraven et al., 1999). Mice deficient for LAT are resistant to IgE-mediated passive systemic anaphylaxis, which illustrates the importance of LAT to Fc␧RI signaling (Saitoh et al., 2000). In addition, LAT-deficient bone marrow–derived mast cells display decreased Fc␧RI-induced tyrosine phosphorylation of SLP-76 and PLC웂, show reduced calcium mobilization, and have defective MAP kinase activation, whereas tyrosine phosphorylation of the 웁- and 웂chains, SYK, and VAV remains unaltered. Fc␧RI-mediated effector functions of degranulation and cytokine production are also diminished in LAT⫺/⫺ mast cells. Therefore, LAT links the upstream kinase phosphorylation events to calcium signaling via PLC웂 in an essential manner required for effector function. An analogous role for LAT also occurs in T cells, where it has been shown to be essential for T cell development and TCRmediated activation (Finco et al., 1998; Zhang et al., 1999a,b). B. THE SLP-76 ADAPTER FAMILY The SH2 domain–containing protein of 76 kDa (SLP-76) is a member of a family of adapter proteins expressed in leukocytes ( Jackman et al., 1995). Characterized members of this family are SLP-76 itself, which is expressed in T cells, mast cells, natural killer cells, and platelets, and BLNK (also known as SLP-65/BASH), which is expressed exclusively in B cells (Fu et al., 1998; Goitsuka et al., 1998; Jackman et al., 1995; Wienands et al., 1998). A newer family member, known as cytokine-dependent hematopoietic cell linker, CLNK, is also expressed in cytokine-stimulated hematopoietic cells and is thought to be involved in cross-talk signals between interleukin 2 (IL-2) and IL-3 cytokines and antigen receptors (Cao et al., 1999). This family of adapter proteins is characterized by a common primary structure that includes a C-terminal SH2 domain, multiple N-terminal tyrosine phosphorylation sites, and a central proline-rich region. SLP-76, which was initially characterized as a PTK substrate in T cells, is essential for T cell development (Clements et al., 1998; Pivniouk et al., 1998). It is a substrate for ZAP-70 and SYK and, once phosphorylated, binds to VAV, Gads, Nck, and other proteins, including SLAP-130 (SLP-76–associated phosphoprotein of 130 kDa) (da Silva et al., 1997; Law et al., 1999; Liu et al., 1999; Musci et al., 1997a; Tuosto et al., 1996; Wu et al., 1996; Wunderlich et al., 1999). SLP-76 has been implicated in PLC웂 phosphorylation, calcium mobilization, MAP kinase activation, and IL-2 production, as demonstrated by the analysis of SLP-76⫺/⫺ Jurkat cells (Yablonski et al.,


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1998). In addition, transient overexpression of SLP-76 augments IL-2 production in wild-type Jurkat cells (Musci et al., 1997b). Importantly, loss of SLP-76 arrests T cell development, resulting in an absence of CD4⫹8⫹ lymphocytes (Clements et al., 1998; Pivniouk et al., 1998). Together, these results demonstrate that SLP-76 is critical in linking upstream PTK events to PLC웂 phosphorylation and subsequent downstream signaling events in T cells. In addition, the B cell–specific member of the SLP-76 family, BLNK, has also been implicated in linking upstream PTK activation events to PLC웂 phosphorylation and calcium release. Notably, BLNK-deficient B cell lines show reduced PLC웂 phosphorylation, lowered inositol-1,4,5trisphosphate (IP3) production, decreased calcium flux, and reduced cJun NH2-terminal kinase ( JNK) activation (Ishiai et al., 1999). Moreover, BLNK-deficient mice exhibit defects in B cell development and BCRmediated calcium mobilization and proliferation (Hayashi et al., 2000; Jumaa et al., 1999; Pappu et al., 1999). Overall, these observations indicate a similar function for this family of adapters: SLP-76 and BLNK link PTK activation events to PLC웂 phosphorylation and subsequent calcium release. SLP-76 has also been implicated in playing a vital role in Fc␧RI mast cell signaling. SLP-76–deficient mast cells appear to develop normally and are present in normal numbers in skin and bronchi. This suggests that SLP-76 is not required for mast cell development as it is for T cell development. Similar to LAT-deficient mice, SLP-76⫺/⫺ mice are resistant to IgEmediated passive anaphylaxis (Pivniouk et al., 1999). Furthermore, bone marrow–derived mast cells from SLP-76 mice show reduced PLC웂 phosphorylation, demonstrate reduced calcium mobilization, and have defective degranulation responses (Pivniouk et al., 1999). These results indicate that SLP-76 plays an important role in Fc␧RI signaling in mast cells. C. VAV VAV, a substrate of PTKs in antigen receptor–activated T cells, B cells, and mast cells, is essential for coordinating upstream signals to downstream cellular events. VAV functions as a guanine nucleotide exchange factor and regulates the activity of the small GTPase Rac1, promoting the transition from the inactive GDP-bound state to the active GTP-bound form. There are two VAV proteins, VAV1 and VAV2, and they have an overall structure that includes a PH domain, a Dbl domain, a catalytic region specific for Rho family GTPases, one SH2 domain, and two SH3 domains (Cantrell, 1998). VAV contributes to efficient calcium signaling in lymphocytes, as illustrated by the phenotype of VAV-deficient mice which show perturbed antigen receptor–induced calcium mobilization (Fischer et al., 1998; Holsinger et al., 1998; O’Rourke et al., 1998; Turner et al., 1997). In B cells, recruitment of VAV1 to the plasma membrane via the coreceptor

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CD19 appears to regulate this process (O’Rourke et al., 1998). VAV has additional functions that are mediated by its various protein binding domains, and its overall importance is underscored by VAV1-deficient mice that have a number of abnormalities in T and B cell development (Turner et al., 1997). In bone marrow–derived mast cell cultures from VAV-deficient mice, early signaling events are not perturbed, since SYK, LAT, and PLC웂1 tyrosine phosphorylation is equivalent to that of wild type–derived cells (Manetz et al., 2000). However, VAV is required for mast cell degranulation and cytokine production (Manetz et al., 2000). Although the signaling mechanism responsible for these alterations is currently unknown, VAV appears to be required for efficient Fc␧RI signaling. VI. Integration of Lipid–Protein Interactions and Calcium Flux

Engagement of antigen receptors leads to an increase in cellular calcium levels that is required for many cellular processes leading to secretion and gene transcription. Upon receptor activation, the calcium that is released from intracellular stores is in a finite amount and is quickly depleted. However, many cellular responses require a sustained calcium flux that is maintained by the influx of extracellular calcium through plasma membrane channels. Emptying of intracellular stores drives the influx of extracellular calcium in a process known as store-operated calcium influx. The plasma membrane channels that underlie calcium influx are currently unknown. Importantly, there are two aspects of antigen receptor–mediated calcium flux that involve lipid metabolism: IP3 and PIP3. First, phosphorylation of PLC웂 by SYK is required for its activation, resulting in the breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into IP3 and diacylglycerol (DAG). IP3 generated by PLC웂 binds to the IP3 receptor calcium channels on the surface of intracellular stores. Second, sustained calcium flux is regulated by PIP3, a lipid product of PI3K. As noted earlier, PIP3 is required to localize BTK to the plasma membrane. BTK then phosphorylates PLC웂, which is important for maintaining a sustained calcium response: PIP3 – BTK kinase interactions increase PLC웂 phosphorylation, enhancing its activity and thus generation of IP3 (Scharenberg and Kinet, 1998). New genetic evidence is unraveling these signaling interactions in mast cells. PI3K, which generates PIP3 by phosphorylating the 3⬘ position of PIP2, is activated upon Fc␧RI engagement. PI3K is composed of a 110-kDa catalytic and an 85-kDa regulatory subunit. There are three isoforms of p85, and generation of mice deficient for p85움 has suggested a unique function of p85움 in mast cells. In B cells, p85움 deficiency results in a phenotype similar to that of BTK-deficient mice, implicating p85움 as a


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critical regulator of B cell antigen receptor signaling (Fruman et al., 1999). However, a recent study showed that p85움 deficiency does not alter Fc␧RImediated signaling events, whereas other signaling pathways in mast cells are affected (Lu-Kuo et al., 2000). This suggests a differential use of p85 isoforms in mast cells, and future work is required to explore this further. The inositol phosphatase SHIP breaks down PIP3 by dephosphorylating the 5⬘ phosphate of the lipid PIP3 (Huber et al., 1999; Krystal et al., 1999). This in turn decreases the amount of IP3 produced by PLC웂: decreased levels of PIP3 result in decreased recruitment of BTK and thus reduced phosphorylation and activation of PLC웂. SHIP is tyrosine phosphorylated and associates with the Fc␧RI following its activation (Kimura et al., 1997). In SHIP⫺/⫺ mast cells, the degranulation response is greater than that in wild-type cells (Huber et al., 1998). The lower threshold of Fc␧RI activation observed in SHIP⫺/⫺ mast cells suggests that SHIP functions to negatively regulate Fc␧RI in vivo. Similar studies have also implicated SHIP as having a negative signaling role in B cells. Notably, SHIP-deficient B cell lines exhibit increased BCR-induced calcium flux (Hashimoto et al., 1999). Interestingly, SHIP also plays a role in modulating Fc␧RI responses via the inhibitory Fc웂RIIb receptor. Coligation of Fc웂RIIb and Fc␧RI inhibits IgE-mediated release of inflammatory mediators and cytokines (Fong et al., 1996). This is also analogous to the role played by the Fc웂RIIb in the regulation of B cell antigen receptor signals: coligation of Fc웂RIIb with the BCR abrogates calcium influx by recruiting and activating SHIP to break down PIP3 (Ono et al., 1997; Scharenberg and Kinet, 1998; Scharenberg et al., 1998). VII. From Lipid Signaling to Serine–Threonine Protein Kinases

A. PROTEIN KINASE C The protein kinase C (PKC) family of serine–threonine kinases has long been implicated in the regulation of cell growth, differentiation, and apoptosis, and their activity is regulated by DAG, the second lipid product of PLC웂. To date, 10 mammalian PKC enzymes have been identified and are divided into three subgroups on the basis of their structure and regulation: classical PKCs (움, 웁I, 웁II, and 웂) are activated by DAG and phosphatidylserine in the presence of calcium, while the novel PKCs (␦, ␧, ␩, and ␪) are regulated by DAG and phosphatidylserine but not calcium. In contrast, the atypical PKC enzymes (␨ and ␭/␫) are unresponsive to DAG and are instead regulated by ceramide and PIP2. Mast cells are known to express at least five different PKC isoforms—움, 웁, ␦, ␧, and ␨ (Ozawa et al., 1993)—all of which could potentially function in Fc␧RIregulated signaling pathways.

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In addition to lipid-induced activation, PKC enzymes are also regulated by specific phosphorylation events. Phosphorylation of three serine– threonine residues located in the kinase domains and C-terminal regions of classical, novel, and atypical PKC isoforms is critical to ensure maximal catalytic competency upon subsequent lipid cofactor binding. The PI3Kregulated protein kinase PDK1 (phosphoinositide-dependent kinase 1) and a complex containing atypical PKC␨ and a rapamycin-sensitive component have been demonstrated to regulate these phosphorylation events in vivo (reviewed in Parekh et al., 2000). Furthermore, tyrosine phosphorylation of PKC␦ by Src family kinases also occurs in response to a variety of stimuli, including engagement of the Fc␧RI (Haleem-Smith et al., 1995; Song et al., 1998). However, the function of these tyrosine phosphorylation events remains controversial, with conflicting reports of effects on PKC activity in vitro (Denning et al., 1996; Li et al., 1994, 1996), although tyrosine phosphorylation of PKC␪ by the Src kinase LCK has been implicated in TCR signaling (Liu et al., 2000). The overall importance of tyrosine phosphorylation of PKC␦ for mast cell function remains to be determined. Despite recent advances in understanding the upstream regulation of PKCs, the exact function of specific PKC isoforms in intracellular signaling cascades remains unclear. In mast cells, studies using tumor-promoting phorbol esters (which mimic the biological activity of DAG), pharmacological inhibitors, or constitutively active or dominant-negative mutants have implicated roles for classical or novel PKC enzymes in a variety of Fc␧RI effector responses. These include activation of MAP kinase cascades (Kawakami et al., 1998; Zhang et al., 1997); the induction of cytokine genes through activation of Elk, NFAT, and AP-1 transcription factors (Baranes and Razin, 1991; Lewin et al., 1996; Razin et al., 1994; Turner and Cantrell, 1997; Turner et al., 1998); and as regulators of mast cell degranulation (Buccione et al., 1994; Chang et al., 1997; Kimata et al., 1999; Ozawa et al., 1993). In addition, Fc␧RI activation also involves threonine phosphorylation of the 웂-chain ITAM (Paolini et al., 1991; Pribluda et al., 1997), which may be mediated by receptor-associated PKC␦ (Germano et al., 1994). Importantly, the 웂-chain threonine phosphorylation site, T60, is required for the full activation of SYK upon Fc␧RI engagement (Swann et al., 1999). Furthermore, PKC␦ phosphorylates the 웂-chain on T60 in vitro (Swann et al., 1999). Taken together, these data suggest that threonine and tyrosine phosphorylation events are interdependent and linked via PKC. It will be interesting to investigate whether PKC␦ phosphorylates the 웂-chain T60 site in vivo and to determine how this event links into the signaling pathway that regulates SYK and subsequent downstream events that culminate in DAG production and PKC activation.


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Recent studies have attempted to define the functional roles of particular PKC enzymes in vivo. In mast cells, the in vivo function of PKC웁 has been explored. Analysis of bone marrow–derived mast cells from PKC웁deficient mice has demonstrated significant inhibition of Fc␧RI- or calcium ionophore–induced degranulation responses, as well as reduced IL-6, but not IL-10, production compared to wild-type cells (Nechushtan et al., 2000). These data support earlier observations from reconstitution and overexpression studies that defined a role for PKC웁 in Fc␧RI-regulated exocytosis and cytokine production (Chang et al., 1997; Ozawa et al., 1993). Similarly, the B cell compartment in the PKC웁-deficient mice is affected, with reduced numbers of B1 cells, reduced serum IgM and IgG levels, and the inability to mount humoral responses to T cell–independent antigens in vivo (Leitges et al., 1996). In contrast, the PKC␪ isoform (which is restricted to the T cell lineage) is important for TCR signaling. T cell development is normal in PKC␪ knockout mice, but the mature peripheral T cells exhibit defects in AP-1 and NF-␬B activation, IL-2 production, and proliferation in response to TCR activation (Sun et al., 2000). Thus, the PKC웁 isoform has a critical, nonredundant role in both mast cells and B cells, while PKC␪ has a similar role in T cells. At present the in vivo significance or functional redundancy of other PKC isoforms in mast cell or lymphocyte effector responses is unknown. B. PROTEIN KINASE D The signaling events occurring downstream of individual PKC enzymes remain poorly defined, largely because most cell types express several PKC isoforms and because few downstream targets have been identified. Interestingly, one recently identified target for classical or novel PKC enzymes in mast cells is the serine kinase protein kinase D (PKD) (Valverde et al., 1994), also known as PKC애 ( Johannes et al., 1994). PKD was originally classified as a new member of the PKC family based on the presence of a DAG binding cysteine-rich (C1) domain within its regulatory domain (Dieterich et al., 1996; Valverde et al., 1994; Van Lint et al., 1995). However, unlike PKC enzymes, PKD does not possess an autoinhibitory pseudosubstrate motif upstream of its DAG binding domain nor a calcium binding domain. Furthermore, the kinase domain of PKD shows very low homology to the conserved kinase domains of the PKC family (Valverde et al., 1994), and PKD displays a unique peptide substrate specificity in vitro (Valverde et al., 1994; Van Lint et al., 1995). PKD, unlike all known PKC enzymes, also contains a PH domain that serves to negatively regulate its catalytic activity (Iglesias and Rozengurt, 1998). Thus, PKD, together with the closely related protein kinase PKC␯ (Hayashi et al., 1999), constitute a distinct protein kinase family.

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Phorbol esters and growth factors stimulate PKD activity in a variety of cells types (Van Lint et al., 1998; Zugaza et al., 1996, 1997). PKD has been implicated in the regulation of NF-␬B transcriptional activity and in the control of Golgi organization in epithelial or fibroblast cell lines ( Jamora et al., 1999; Johannes et al., 1998; Prestle et al., 1996), underscoring its overall importance for cellular processes. PKD is highly expressed in mast cells and lymphocytes and is involved in antigen receptor signaling cascades (Sidorenko et al., 1996). Specifically, Fc␧RI aggregation rapidly and potently stimulates PKD activity (Matthews et al., 2000), and phosphorylation of two serine residues within the PKD kinase domain is both required and sufficient for activation of PKD (Iglesias et al., 1998). Production of DAG (but not IP3) is essential for antigen receptor coupling to PKD, as demonstrated by analysis of PKD activity in SYK- and PLC웂-deficient DT40 B cells (Sidorenko et al., 1996). However, mutation of the PKD C1 domain reveals that direct binding of DAG is dispensable for the activation of PKD. Instead, biochemical studies have revealed that classical or novel PKC enzymes control the activation of PKD in response to Fc␧RI, BCR, or TCR engagement (Matthews et al., 2000). Therefore, the identification of PKD as a target for DAG–PKC signals reveals a novel aspect of this signaling cascade in mast cells and lymphocytes. Future work is required to address the functional importance of this PKC–PKD connection in mast cells. Almost invariably, the biological effects of DAG and phorbol esters in vivo are attributed to the action of classical or novel PKC enzymes. However, it is important to note that the recent identification of DAG binding domains present in proteins other than PKCs has increased the complexity of this signaling pathway (reviewed in Ron and Kazanietz, 1999). The novel Ras guanine nucleotide exchange factor (GEF) RasGRP is highly expressed in T and B cells, and its activity (as well as that of a related Rap1-specific GEF) is stimulated by DAG or phorbol esters (Kawasaki et al., 1998; O’Ebinu et al., 1998; Tognon et al., 1998). These exchange factors therefore constitute a novel link between DAG- and GTPase-regulated signaling pathways in lymphocytes. Given that many signaling molecules in mast cells parallel those present in lymphocytes, mast cells may also contain novel DAG-regulated proteins that could function in Fc␧RI-regulated signaling pathways. Thus, the identification of additional ‘‘non-PKC’’ DAGresponsive proteins highlights the need for caution in interpreting previous and future studies on this signaling pathway. Moreover, future research should be focused on exactly how specific PKC enzymes are selectively activated by DAG in distinct cellular or receptor contexts and also on the identity and function of downstream targets of DAG-regulated proteins.


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VIII. Lipid Rafts

Specialized plasma membrane microdomains known as lipid rafts have been proposed to regulate receptor signaling by serving as platforms that control the spatial organization of key signaling molecules ( Janes et al., 2000; Kurzchalia and Parton, 1999; Sheets et al., 1999b; Xavier and Seed, 1999). These detergent-insoluble structures contain a high density of sphingolipids and cholesterol, and in addition a higher prevalence of phosphotidylinositol-linked proteins, receptor and nonreceptor tyrosine kinases, G proteins, and various other signaling molecules compared to bulk plasma membrane (Simons and Ikonen, 1997; Xavier and Seed, 1999). Lipid rafts are thought to be crucial for optimal antigen receptor signaling, as illustrated by studies in T cells, where disruption of lipid rafts (using pharmacological agents) is associated with attenuated TCR signaling (Stulnig et al., 1998; Xavier et al., 1998). Indeed, chemical cross-linking of lipid rafts induces signaling events analogous to TCR stimulation ( Janes et al., 1999). In mast cells the Fc␧RI receptor is associated with both loosely and tightly bound membrane lipids and ester-linked fatty acids (Kinet et al., 1985) and, until activation, is uniformly distributed within the plasma membrane. Upon engagement, the Fc␧RI transiently redistributes into lipid rafts, along with coordinate spatial reorganization of signaling molecules, as illustrated by the movement of chimeric proteins containing the SH2 domains of SYK and PLC웂 fused to green fluorescent protein (Stauffer and Meyer, 1997). Activation-induced compartmentalization of signaling proteins in mast cells has also been observed biochemically: detergentinsoluble membrane fractions from Fc␧RI-stimulated cells are enriched for activated LYN and tyrosine-phosphorylated 웁- and 웂-receptor chains (Field et al., 1995, 1997). Moreover, cholesterol depletion reversibly disrupts Fc␧RI–LYN interactions and reduces early tyrosine phosphorylation events in activated mast cells (Sheets et al., 1999a). Together, these data support the hypothesis that Fc␧RI activation and proximal signaling events occur in specialized membrane microdomains. Similar observations have also been made for antigen receptor complexes in T and B cells (Cheng et al., 1999; Montixi et al., 1998; Xavier et al., 1998). The description of lipid rafts in mast cells and other cell types has clearly necessitated that the model of Fc␧RI signaling events include three-dimensional spatial and geometrical considerations. Spatial inclusion or exclusion of specific molecules from a raft or specific lipid modifications to signaling molecules may provide independent means of regulation. For example, observations that CD45 may be excluded from lipid rafts illustrate this type of topological regulation (reviewed

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in Thomas, 1999). CD45 initiates antigen receptor signaling by dephosphorylating the inhibitory site of Src kinases. However, why CD45 does not subsequently dephosphorylate activated kinases or adaptor proteins, thus turning off signaling cascades, is unclear. Exclusion of CD45 from activated antigen receptor complexes may provide the explanation. In addition, N-myristoylation and protein palmitoylation are well known to regulate membrane association of key signaling proteins and may facilitate efficient partitioning into rafts. Interestingly, mutation of the palmitoylated cysteine residues of the SRC kinases LYN and FYN and the adapter molecule LAT prevents the association of these proteins with lipid rafts (Shenoy-Scaria et al., 1994; W. Zhang et al., 1998b), although it is difficult to attribute such effects to the lack of palmitate per se. However, it is attractive to speculate that reversible acylation events, such as palmitoylation, may provide an additional level of control for antigen receptor signaling by regulating the spatial organization of signaling proteins. Taken together, mechanisms controlling the spatial organization of lipid rafts will likely represent a novel means for regulation of Fc␧RI signal transduction. IX. Conclusion

Our knowledge of the signaling events that underlie Fc␧RI-mediated activation of mast cells is becoming clearer. Most recently, these advances have led to a greater understanding of how upstream PTK activity is integrated into downstream events. Importantly, genetic evidence has illustrated the essential requirement of several signaling molecules and has contributed to a better understanding of the protein–lipid interactions involved in calcium mobilization. However, many questions still exist. Paramount among these is the complete lack of understanding of the plasma membrane calcium channels responsible for calcium influx. To date, the existence of these channels is known only from electrophysiological measurements. Cloning and characterization of calcium influx channels and the signaling pathways that regulate them will clearly be of great importance. Such work will provide a basis for understanding how Fc␧RI-mediated proximal signals are integrated into downstream responses leading to gene activation. Other important areas of investigation include characterization of the molecules that negatively regulate Fc␧RI signals and elucidating how effector functions are differentially induced and regulated. Future analysis of mast cells deficient for different signaling molecules will no doubt continue to provide critical insights into many of these issues.


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ACKNOWLEDGMENT We thank Marie-He´le`ne Jouvin for assistance in preparing the manuscript.

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INDEX

A

positive and negative selection, 192–195 pre-TCR checkpoint, 190–192 tumor necrosis factor-R family role, 199–200 systemic lupus erythematosus, 283–292 anti-C1q autoantibody development, 292 apoptotic cell clearance, 287–288, 290–291 autoantigen source, 285–287 complement role, 290–291 immune response, 288–290 TRAF-mediated regulation, 134–136

Adapter proteins, see specific proteins 움/웁 T cell receptor interactions, major histocompatibility complex class I, 1–5 Animal models, see Disease models Antigen presentation, CD40-mediated regulation, 76–78, 130–131 Antigens, see also Immune response; Major histocompatibility complex human leukocyte antigen complex disease susceptibility, 34–38 diversity, 25–26, 32–34, 41 systemic lupus erythematosus autoantigen source, 285–287 AP-1, B cell immune response regulation by CD40-CD154-signaling, 79, 87–92 Apaf-1 adapter molecule, lymphocyte apoptosis-signaling, 184–186 Apoptosis lymphocyte death, 179–205 B cell development immature cell selection, 202–203 peripheral lymphoid organs, 203–205 pre-BCR checkpoint, 200–202 caspase role, 179–180, 184–186 immune system function, 189–190, 288–290 overview, 179, 205 regulation Apaf-1 signals, 184–186 Bcl-2 family, 184–189 caspase 9 signals, 184–186 death receptor signals, 180–184 transcriptional control, 188–189 T cell development peripheral lymphoid organs, 195–199

B B cell receptors immunoreceptor tyrosine-based activation motifs, 326–328 pre-BCR apoptosis checkpoint, 200–202 B cells apoptosis, 179–205 caspase role, 179–180, 184–186 cell development immature cell selection, 202–203 peripheral lymphoid organs, 203–205 pre-BCR checkpoint, 200–202 immune system function, 189–190, 288–290 overview, 179, 205 regulation Apaf-1 signals, 184–186 Bcl-2 family, 184–189 caspase 9 signals, 184–186 death receptor signals, 180–184 transcriptional control, 188–189 357

358

INDEX

development, apoptosis role immature cell selection, 202–203 peripheral lymphoid organs, 203–205 pre-BCR checkpoint, 200–202 immune response regulation by CD40CD154-signaling, 86–145 activators, 80 AP-1 activation, 79, 87–92 CRE activation, 87, 92 functional outcomes, 126–136 adhesion, 129–130 antigen-presenting cell function, 130–131 apoptosis regulation, 134–136 cell survival and proliferation, 127–128 cytokine secretion, 131 IgH class switching, 133–134 immunoglobulin production regulation, 131–134 inhibitors, 80 NF-AT activation, 79, 88–92 NF-␬웁 activation, 87, 92–93, 100–103, 116–121 signaling cascades, 116–126 cAMP-mediated-signaling pathways, 144–145 ERK activation, 89–90, 120, 125–126 JAK–STAT pathways, 138–140 JNK activation, 90–91, 98–100, 120–124 NF-␬웁 activation, 87, 92–93, 100–103, 116–121 p38 activation, 92, 98, 100, 124–125 reverse-signaling through CD154, 145 src activation, 140–143 tec family kinase activation, 140–143 ZAP-70 activation, 140–143 T cell collaboration, 69 TRAF adapter molecule discovery, 93–115 transcription factor activation, 87–93 immune response regulation by CD40/ CD154-signaling, overview, 61, 69 Bcl-2 family proteins, lymphocyte apoptosis regulation, 184–189 Bone marrow, B cell selection, 202–203

Bruton’s tyrosine kinase, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 331–332

C C2, deficiency, systemic lupus erythematosus relationship deficient mouse model, 278 genetic deficiency, 243–251 null alleles, 269–272 C3, deficiency, systemic lupus erythematosus relationship deficient mouse model, 278–279 genetic deficiencies, 251–252 C4, deficiency, systemic lupus erythematosus relationship deficient mouse model, 278–279 genetic deficiency, 238–243 null alleles, 265–269 Calcium flux, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 338–339 cAMP, B cell immune response regulation, 144–145 Caspases, lymphocyte apoptosis, 179–180, 184–186 CD4⫹, major histocompatibility complex interactions, 1 CD8⫹, major histocompatibility complex interactions, 1 CD40 discovery, 61–69 immune response regulation, 61–146 antigen presentation, 76–78 B cell responses, 86–145 AP-1 activation, 79, 87–92 CRE activation, 87, 92 NF-AT activation, 79, 88–92 NF-␬웁 activation, 87, 92–93, 100–103, 116–121 T cell collaboration, 69 TRAF adapter molecule discovery, 93–115 transcription factor activation, 87–93 event delineation, 115–136 functional outcomes, 126–136 signaling cascades, 116–126

INDEX

expression regulation, 78–86 functional outcomes, 126–136 adhesion, 129–130 antigen-presenting cell function, 130–131 apoptosis regulation, 134–136 cell survival and proliferation, 127–128 cytokine secretion, 131 humoral immune responses, 70–76 T cell-dependent responses, 70–75 T cell-independent responses, 75–76 inflammatory responses, 76–78 mechanisms, 78–86 overview, 61, 145–146 signaling cascades, 116–126 cAMP-mediated-signaling pathways, 144–145 ERK activation, 89–90, 120, 125–126 JAK–STAT pathways, 138–140 JNK activation, 90–91, 98–100, 120–124 NF-␬웁 activation, 116–121 p38 activation, 92, 98, 100, 124–125 reverse-signaling through CD154, 145 src activation, 140–143 tec family kinase activation, 140–143 ZAP-70 activation, 140–143 thrombosis, 76–78 TRAF adapter molecule apoptosis regulation, 134–136 CD40 event mediation, 115–145 discovery, 93–115 expression regulation, 114–115 I-TRAF/TANK, 109–114 MAPK activation, 97–100 NF-␬웁 activation, 100–103 structure–function relationship, 95–104 TRAF–CD40 association, 65, 104–109 transcriptional regulation, 82–83 properties, 64, 69, 94 CD45, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 333–334 CD154 discovery, 61–69 disease role, 63 immune response regulation antigen presentation, 76–78 B cell responses, 86–87

359

B cell–T cell collaboration, 69 endothelial cell function modulation, 77–79 expression regulation, 78–86 humoral immune responses, 70–76 T cell-dependent responses, 70–75 T cell-independent responses, 75–76 inflammatory responses, 76–78 mechanisms, 78–86 thrombosis, 76–78 transcriptional regulation, 82–83 properties, 64, 69, 94 Cell communication, see Signal transduction Cell death, see Apoptosis Complement, deficiency, systemic lupus erythematosus relationship, 227–299 animal models autoimmunity, 297–298 B-deficient mouse, 278 C2-deficient mouse, 278 C3-deficient mouse, 278–279 C4-deficient mouse, 278–279 C1q-deficient mouse, 277–278 CR1-deficient mouse, 227, 278–279 CR2-deficient mouse, 278–279 other nonhuman species, 279 apoptosis, 283–292 anti-C1q autoantibody development, 292 apoptotic cell clearance, 287–288, 290–291 autoantigen source, 285–287 complement role, 290–291 immune response, 288–290 association hypotheses, 283–297 apoptosis, 283–292 humoral immune response, 294–297 immune complex clearance, 292–294 autoantibody production to C1q, 281–283 complement null alleles, 259–272 C2 null alleles, 269–272 C4 null alleles, 265–269 complement receptor type 1, 273–276 acquired numbers reduction, 274–276 inherited numerical polymorphisms, 273–274 inherited structural polymorphisms, 276 genetic deficiencies, 228–259 acquired complement deficiency, 259 ascertainment artifacts, 257–259 C2 deficiency, 243–251

360

INDEX

C3 deficiency, 251–252 C4 deficiency, 238–243 C1q deficiency, 227–237 C1r deficiency, 238 C1s deficiency, 238 homozygous classical pathway deficiency, 228–229 mannose-binding lectin deficiency, 252–257 terminal pathway component deficiency, 252 inflammation, 279–281 overview, 227–228, 298–299 C1q complement protein, deficiency, systemic lupus erythematosus anti-C1q autoantibody development, 292 autoantibody production to C1q, 281–283 C1q-deficient mouse, 277–278 genetic deficiencies, 227–237 CR1, complement deficiency, systemic lupus erythematosus relationship acquired numbers reduction, 274–276 animal models, 227, 278–279 inherited numerical polymorphisms, 273–274 inherited structural polymorphisms, 276 CR2, complement deficiency, systemic lupus erythematosus relationship, animal models, 278–279 C1r complement protein, deficiency, systemic lupus erythematosus relationship, 238 C1s complement protein, deficiency, systemic lupus erythematosus relationship, 238 CRE, B cell immune response regulation by CD40-CD154-signaling, 87, 92 Cytokines, CD40-mediated immune response regulation, 131

Disease models, complement deficiency–systemic lupus erythematosus relationship autoimmunity, 297–298 B-deficient mouse, 278 C2-deficient mouse, 278 C3-deficient mouse, 278–279 C4-deficient mouse, 278–279 C1q-deficient mouse, 277–278 CR1-deficient mouse, 227, 278–279 CR2-deficient mouse, 278–279 other nonhuman species, 279 Diseases, see specific diseases

E Endothelial cells, activation, CD40/CD154mediated regulation, 77–79 ERK, CD40-induced activation, 89–90, 120, 125–126

F Fc␧RI, see Immunoglobulin E Fibroblast-like stromal cells, CD40 engagement effects, 76

G 웂/␦ T cell receptor, major histocompatibility complex class I interactions, 19–22, 42 Gene expression, see Major histocompatibility complex; Transcription Germinal center reaction CD154-expressing B cells role, 74 T cell-dependent activation, 70, 132 Granulocyte/macrophage colony-stimulating factor, CD40 expression regulation, 80

D H Death receptors, lymphocyte apoptosissignaling, 180–184 Dendritic cells, CD40-mediated maturation, 72

Human leukocyte antigen complex disease susceptibility, 34–38 diversity, 25–26, 32–34, 41

INDEX

I Immune response apoptosis role, 189–190, 288–290 CD40-mediated regulation, 61–146 antigen presentation, 76–78 B cell responses, 86–145 AP-1 activation, 79, 87–92 CRE activation, 87, 92 NF-AT activation, 79, 88–92 NF-␬웁 activation, 87, 92–93, 100–103, 116–121 T cell collaboration, 69 TRAF adapter molecule discovery, 93–115 transcription factor activation, 87–93 event delineation, 115–136 functional outcomes, 126–136 signaling cascades, 116–126 functional outcomes, 126–136 adhesion, 129–130 antigen-presenting cell function, 130–131 apoptosis regulation, 134–136 cell survival and proliferation, 127–128 cytokine secretion, 131 IgH class switching, 133–134 immunoglobulin production regulation, 131–134 humoral immune responses, 70–76 T cell-dependent responses, 70–75 T cell-independent responses, 75–76 inflammatory responses, 76–78 mechanisms, 78–86 overview, 61, 145–146 signaling cascades, 116–126 cAMP-mediated-signaling pathways, 144–145 ERK activation, 89–90, 120, 125–126 JAK–STAT pathways, 138–140 JNK activation, 90–91, 98–100, 120–124 NF-␬웁 activation, 116–121 p38 activation, 92, 98, 100, 124–125 reverse-signaling through CD154, 145 src activation, 140–143 tec family kinase activation, 140–143 ZAP-70 activation, 140–143

361

thrombosis, 76–78 TRAF adapter molecule apoptosis regulation, 134–136 CD40 event mediation, 115–145 discovery, 93–115 expression regulation, 114–115 I-TRAF/TANK, 109–114 MAPK activation, 97–100 NF-␬웁 activation, 100–103 structure–function relationship, 95–104 TRAF–CD40 association, 65, 104–109 transcriptional regulation, 82–83 systemic lupus erythematosus, complement deficiency role, 288–290, 294–297 Immunoglobulin E, high-affinity receptor Fc␧RI signal transduction, 325–344 calcium flux integration, 338–339 ITAM role, 327–328 lipid–protein interactions, 338–339 lipid rafts, 343–344 overview, 325–327, 344 phosphate transfer Bruton’s tyrosine kinase, 331–332 CD45, 333–334 LYN transfer kinase, 328–331 phosphate transfer kinases, 328–332 protein tyrosine phosphatases, 332–335 SHP-1, 334–335 SHP-2, 335 SYK role, 328–331 serine–threonine protein kinases, 339–342 protein kinase C, 339–341 protein kinase D, 341–342 signaling molecules LAT adapter protein, 335–336 SLP-76 adapter family, 336–337 VAV, 337–338 Immunoglobulin G, CD40-mediated secretion regulation, 131–134 Immunoglobulin H, CD40-mediated regulation, class switching, 133–134 Immunoglobulin M, CD40-mediated secretion regulation, 131–134 Immunoreceptor tyrosine-based activation motifs, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 326–328 Inflammatory responses CD40/CD154-mediated regulation, 76–78

362

INDEX

systemic lupus erythematosus complement deficiency, 279–281 Interferons CD40 expression regulation, 80 immune response effects, 72 platelet activation, 77 Interleukin CD40 expression regulation, 80 CD154 expression regulation, 80 immune response effects, 72 platelet activation, 77

J JAK kinases CD40 association, 64–65 JAK–STAT-signaling pathways, 138–140 JNK, CD40-induced activation, 90–91, 98–100, 120–124

L LAT adapter protein, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 335–336 Lectin, mannose-binding lectin deficiency, systemic lupus erythematosus relationship, 252–257 Ligands, MIC ligands, 19–23 Lipids, high-affinity immunoglobulin E receptor Fc␧RI signal transduction lipid–protein interactions, 338–339 lipid rafts, 343–344 Lipopolysaccharides, CD154 expression regulation, 80 Lymphocytes, see B cells; Natural killer cells; T cells LYN transfer kinase, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 328–331

웂/␦ T cell receptor interactions, 19–22, 42 histocompatibility mechanisms, 38–39 MIC genes, see MIC genes natural killer cell receptor interactions, 21–23, 42 phylogeny, 9–14 seminal features, 2, 4, 41 class II 움/웁 T cell receptor interactions, 1–5 immune response effects, 72 Mannose-binding lectin deficiency, systemic lupus erythematosus relationship, 252–257 MIC genes, 1–46 biochemistry, 18–19 characteristics, 5–6 diversity, 25–39 disease susceptibility, 34–38 histocompatibility, 38–39 repertoire, 25–34 genomics, 6–9 ligands, 19–23 MICAB-/- individuals, 39–41 overview, 1–5, 41–42 phylogeny, 9–14 structure, 23–25 transcription, 15–18 mechanisms, 16–18 transcripts, 15–16 Mitogen-activated protein kinases, CD40induced activation ERK, 89–90, 120, 125–126 JNK, 90–91, 98–100, 120–124 p38 activation, 92, 98, 100, 124–125 CRE transcription, 92 Models, see Disease models Monocytes, CD40-mediated maturation, 72

N M Major histocompatibility complex class I 움/웁 T cell receptor interactions, 1–5 disease susceptibility, 37–38

Natural killer cells major histocompatibility complex class I interactions, 21–23, 42 properties, 64 NF-AT, B cell immune response regulation by CD40-CD154-signaling, 79, 88–92

INDEX

NF-␬웁, CD40-mediated regulation B cell responses, 87, 92–93, 100–103, 116–121 positive and negative regulation, 80 signaling cascades, 116–121 TRAF adapter molecule, 100–103

P p38 activation, 92, 98, 100, 124–125 CRE transcription, 92 Peripheral lymphoid organs, lymphocyte apoptosis B cell development, 203–205 T cell development, 195–199 Phagocytes, CD40-mediated maturation, 72 Phosphates, transfer, high-affinity immunoglobulin E receptor Fc␧RI signal transduction Bruton’s tyrosine kinase, 331–332 CD45 role, 333–334 LYN transfer kinase, 328–331 phosphate transfer kinases, 328–332 protein tyrosine phosphatases, 332–335 SHP-1 role, 334–335 SHP-2 role, 335 SYK role, 328–331 Platelets, activation, CD40/CD154-mediated regulation, 77 Protein kinase C, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 339–341 Protein kinase D, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 341–342 Protein tyrosine kinases, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 328–332 Protein tyrosine phosphatases, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 332–335

S Serine/threonine kinases, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 339–342 protein kinase C, 339–341 protein kinase D, 341–342

363

SHP-1, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 334–335 SHP-2, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 335 Signal transduction B cell immune response regulation by CD40-CD154-signaling, 86–145 activators and inhibitors, 80 AP-1 activation, 79, 87–92 CRE activation, 87, 92 functional outcomes, 126–136 adhesion, 129–130 antigen-presenting cell function, 130–131 apoptosis regulation, 134–136 cell survival and proliferation, 127–128 cytokine secretion, 131 IgH class switching, 133–134 immunoglobulin production regulation, 131–134 NF-AT activation, 79, 88–92 NF-␬웁 activation, 87, 92–93, 100–103, 116–121 overview, 61, 69 signaling cascades, 116–126 cAMP-mediated-signaling pathways, 144–145 ERK activation, 89–90, 120, 125–126 JAK–STAT pathways, 138–140 JNK activation, 90–91, 98–100, 120–124 NF-␬웁 activation, 116–121 p38 activation, 92, 98, 100, 124–125 reverse-signaling through CD154, 145 src activation, 140–143 tec family kinase activation, 140–143 ZAP-70 activation, 140–143 T cell collaboration, 69 TRAF adapter molecule discovery, 93–115 transcription factor activation, 87–93

364

INDEX

high-affinity immunoglobulin E receptor Fc␧RI, 325–344 calcium flux integration, 338–339 ITAM role, 327–328 lipid–protein interactions, 338–339 lipid rafts, 343–344 overview, 325–327, 344 phosphate transfer Bruton’s tyrosine kinase, 331–332 CD45 role, 333–334 LYN transfer kinase, 328–331 phosphate transfer kinases, 328–332 protein tyrosine phosphatases, 332–335 SHP-1 role, 334–335 SHP-2 role, 335 SYK role, 328–331 serine–threonine protein kinases, 339–342 protein kinase C, 339–341 protein kinase D, 341–342 signaling molecules LAT adapter protein, 335–336 SLP-76 adapter family, 336–337 VAV, 337–338 SLP-76 adapter family, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 336–337 src kinases, CD40-induced activation, 140–143 STAT proteins, JAK–STAT-signaling pathways, 138–140 Stromal cells, CD40 engagement effects, 76 SYK, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 328–331 Systemic lupus erythematosus, complement deficiency, 227–299 animal models autoimmunity, 297–298 B-deficient mouse, 278 C2-deficient mouse, 278 C3-deficient mouse, 278–279 C4-deficient mouse, 278–279 C1q-deficient mouse, 277–278 CR1-deficient mouse, 227, 278–279 CR2-deficient mouse, 278–279 other nonhuman species, 279

apoptosis, 283–292 anti-C1q autoantibody development, 292 apoptotic cell clearance, 287–288, 290–291 autoantigen source, 285–287 complement role, 290–291 immune response, 288–290 association hypotheses, 283–297 apoptosis, 283–292 complement role in humoral immune response, 294–297 complement role in immune complex clearance, 292–294 autoantibody production to C1q, 281–283 complement null alleles, 259–272 C2 null alleles, 269–272 C4 null alleles, 265–269 complement receptor type 1, 273–276 acquired numbers reduction, 274–276 inherited numerical polymorphisms, 273–274 inherited structural polymorphisms, 276 genetic deficiencies, 228–259 acquired complement deficiency, 259 ascertainment artifacts, 257–259 C2 deficiency, 243–251 C3 deficiency, 251–252 C4 deficiency, 238–243 C1q deficiency, 227–237 C1r deficiency, 238 C1s deficiency, 238 homozygous classical pathway deficiency, 228–229 mannose-binding lectin deficiency, 252–257 terminal pathway component deficiency, 252 inflammation, 279–281 overview, 227–228, 298–299

T TANK, immune response regulation, I-TRAF/ TANK, 109–114

INDEX

T cell receptors immunoreceptor tyrosine-based activation motifs, 326–328 major histocompatibility complex class I and II interactions, 1–5, 19–20, 42 pre-TCR apoptosis checkpoint, 190–192 T cells apoptosis, 179–205 caspase role, 179–180, 184–186 cell development peripheral lymphoid organs, 195–199 positive and negative selection, 192–195 pre-TCR checkpoint, 190–192 tumor necrosis factor-R family role, 199–200 immune system function, 189–190, 288–290 overview, 179, 205 regulation Apaf-1 signals, 184–186 Bcl-2 family, 184–189 caspase 9 signals, 184–186 death receptor signals, 180–184 transcriptional control, 188–189 development, apoptosis role peripheral lymphoid organs, 195–199 positive and negative selection, 192–195 pre-TCR checkpoint, 190–192 tumor necrosis factor-R family role, 199–200 germinal center reaction initiation, 70, 74, 132 immune response regulation activators and inhibitors, 80 B cell collaboration, 69 humoral immune responses T cell-dependent responses, 70–75 T cell-independent responses, 75–76 Ig secretion regulation, 131–133 tec kinases, CD40-induced activation, 140–143 Threonine protein kinases, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 339–342 Thrombosis, CD40/CD154-mediated regulation, 76–78

365

TRAF adapter molecule immune response regulation apoptosis regulation, 134–136 CD40 event mediation, 115–145 functional outcomes, 126–136 signaling cascades, 116–126 discovery, 93–115 expression regulation, 114–115 functional outcomes, 126–136 adhesion, 129–130 antigen-presenting cell function, 130–131 apoptosis regulation, 134–136 cell survival and proliferation, 127–128 cytokine secretion, 131 IgH class switching, 133–134 immunoglobulin production regulation, 131–134 I-TRAF/TANK, 109–114 MAPK activation, 97–100 NF-␬웁 activation, 100–103 signaling cascades, 116–126 cAMP-mediated-signaling pathways, 144–145 ERK activation, 89–90, 120, 125–126 JAK–STAT pathways, 138–140 JNK activation, 90–91, 98–100, 120–124 NF-␬웁 activation, 116–121 p38 activation, 92, 98, 100, 124–125 reverse-signaling through CD154, 145 src activation, 140–143 tec family kinase activation, 140–143 ZAP-70 activation, 140–143 structure–function relationship, 95–104 TRAF–CD40 association, 65, 104–109 properties, 93–94 Transcription CD40 expression regulation, 78–79, 82–83 CD154 expression regulation, 78–79, 83–86 CRE transcription, 92 lymphocyte apoptosis regulation by Bcl-2 family proteins, 188–189 MIC genes, 15–18 mechanisms, 16–18 transcripts, 15–16 Transcription factors, B cell immune response regulation by CD40-signaling AP-1 activation, 79, 87–92

366 CRE activation, 87, 92 NF-AT activation, 79, 88–92 NF-␬웁 activation, 87, 92–93, 100–103, 116–121 Tumor necrosis factor CD40, see CD40 CD154, see CD154 immune response effects, 72 platelet activation, 77 properties, 64 T cell proliferation, 199–200

INDEX

V VAV, high-affinity immunoglobulin E receptor Fc␧RI signal transduction, 337–338

Z ZAP-70 kinases, CD40-induced activation, 140–143

CONTENTS OF RECENT VOLUMES

Volume 72 The Function of Small GTPases in Signaling by Immune Recognition and Other Leukocyte Receptors AMNON ALTMAN AND MARCEL DECKERT

Integrins in the Immune System YOJI SHIMIZU, DAVID M. ROSE, AND MARK H. GINSBERG INDEX

Volume 73

Function of the CD3 Subunits of the Pre-TCR and TCR Complexes during T Cell Development BERNARD MALISSEN, LAURENCE ARDOUIN, SHIH-YAO LIN, ANNE GILLET, AND MARIE MALISSEN

Mechanisms of Exogenous Antigen Presentation by MHC Class I Molecules in Vitro and in Vivo: Implications for Generating CD8⫹ T Cell Responses to Infectious Agents, Tumors, Transplants, and Vaccines JONATHAN W. YEWDELL, CHRISTOPHER C. NORBURY, AND JACK R. BENNINK

Inhibitory Pathways Triggered by ITIMContaining Receptors SILVIA BOLLAND AND JEFFREY V. RAVETCH

Signal Transduction Pathways That Regulate the Fate of B Lymphocytes ANDREW CRAXTON, KEVIN OTIPOBY, AIMIN JIANG, AND EDWARD A. CLARK

ATM in Lymphoid Development and Tumorigenesis YANG XU

Oral Tolerance: Mechanisms and Therapeutic Applications ANA FARIA AND HOWARD L. WEINER

Comparison of Intact Antibody Structures and the Implications for Effector Function LISA J. HARRIS, STEVEN B. LARSON, AND ALEXANDER MCPHERSON

Caspases and Cytokines: Roles in Inflammation and Autoimmunity JOHN C. REED

Lymphocyte Trafficking and Regional Immunity EUGENE C. BUTCHER, MARNA WILLIAMS, KENNETH YOUNGMAN, LUSIJAH ROTT, AND MICHAEL BRISKIN

T Cell Dynamics in HIV-1 Infection DAWN R. CLARK, BOB J. DE BOER, KATJA C. WOLTHERS, AND FRANK MIEDEMA

Dendritic Cells DIANA BELL, JAMES W. YOUNG, AND JACQUES BANCHEREAU

Bacterial CpG DNA Activates Immune Cells to Signal Infectious Danger HERMANN WAGNER 367

368

INDEX

Neutrophil-Derived Proteins: Selling Cytokines by the Pound MARCO ANTONIO CASSATELLA Murine Models of Thymic Lymphomas: Premalignant Scenarios Amenable to Prophylactic Therapy EITAN YEFENOF INDEX

Volume 74 Biochemical Basis of Antigen-Specific Suppressor T Cell Factors: Controversies and Possible Answers KIMISHIGE ISHIZAKA, YASUYUKI ISHII, TATSUMI NAKANO, AND KATSUJI SUGIE The Role of Complement in B Cell Activation and Tolerance MICHAEL C. CARROLL Receptor Editing in B Cells DAVID NEMAZEE Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection PIUS LOETSCHER, BERNHARD MOSER, AND MARCO BAGGIOLINI Escape of Human Solid Tumors from T-Cell Recognition: Molecular Mechanisms and Functional Signifcance FRANCESCO M. MARINCOLA, ELIZABETH M. JAFFEE, DANIEL J. HICKLIN, AND SOLDANO FERRONE

Volume 75 Exploiting the Immune System: Toward New Vaccines against Intracellular Bacteria JU¨ RGEN HESS, ULRICH SCHAIBLE, BA¨ RBEL RAUPACH, AND STEFAN H. E. KAUFMANN The Cytoskeleton in Lymphocyte Signaling A. BAUCH, F. W. ALT, G. R. CRABTREE, AND S. B. SNAPPER TGF-웁 Signaling by Smad Proteins KOHEI MIYAZONO, PETER TEN DIJKE, AND CARL-HENRIK HELDIN MHC Class II-Restricted Antigen Processing and Presentation JEAN PIETERS T-Cell Receptor Crossreactivity and Autoimmune Disease HARVEY CANTOR Strategies for Immunotherapy of Cancer CORNELIS J. M. MELIEF, RENE´ E. M. TOES, JAN PAUL MEDEMA, SJOERD H. VAN DER BURG, FERRY OSSENDORP, AND RIENK OFFRINGA Tyrosine Kinase Activation in the Decision between Growth, Differentiation, and Death Responses Initiated from the B Cell Antigen Receptor ROBERT C. HSUEH AND RICHARD H. SCHEUERMANN

The Host Response to Leishmania Infection WERNER SOLBACH AND TAMA´ S LASKAY

The 3⬘ IgH Regulatory Region: A Complex Structure in a Search for a Function AHMED AMINE KHAMLICHI, ERIC PINAUD, CATHERINE DECOURT, CHRISTINE CHAUVEAU, AND MICHEL COGNE´

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INDEX

Advances in Immunology Volume 76

Advances in Immunology Volume 45

Advances in Immunology Volume 34

Advances in Immunology Volume 1

Advances in Immunology Volume 10

Advances in Immunology Volume 30

Advances in Immunology Volume 82

Advances in Immunology Volume 31

Advances in Immunology Volume 52

Advances in Immunology Volume 48

Advances in Immunology Volume 56

Advances in Immunology Volume 12

Advances in Immunology Volume 21

Advances in Immunology Volume 41

Advances in Immunology Volume 71

Advances in Immunology Volume 84

Advances in Immunology Volume 43

Advances in Immunology Volume 73

Advances in Immunology, Volume 61

Advances in Immunology Volume 14

Advances in Immunology Volume 46

Advances in Immunology, Volume 106

Advances in Immunology Volume 19

Advances in Immunology Volume 99

Advances in Immunology Volume 81

Advances in Immunology Volume 4

Advances in Immunology Volume 80

Advances in Immunology Volume 64

Advances in Immunology Volume 62

Advances in Immunology Volume 28

Advances in Immunology Volume 76

Advances in Immunology Volume 45

Advances in Immunology Volume 34

Advances in Immunology Volume 1

Advances in Immunology Volume 10

Advances in Immunology Volume 30

Advances in Immunology Volume 82

Advances in Immunology Volume 31

Advances in Immunology Volume 52

Advances in Immunology Volume 48

Advances in Immunology Volume 56

Advances in Immunology Volume 12

Advances in Immunology Volume 21

Advances in Immunology Volume 41

Advances in Immunology Volume 71

Advances in Immunology Volume 84

Advances in Immunology Volume 43

Advances in Immunology Volume 73

Advances in Immunology, Volume 61

Advances in Immunology Volume 14

Advances in Immunology Volume 46

Advances in Immunology, Volume 106

Advances in Immunology Volume 19

Advances in Immunology Volume 99

Advances in Immunology Volume 81

Advances in Immunology Volume 4

Advances in Immunology Volume 80

Advances in Immunology Volume 64

Advances in Immunology Volume 62

Advances in Immunology Volume 28

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