Autoimmunity in systemic lupus erythematosus: Integrating genes and biology

Seminars in Immunology 18 (2006) 230–243

Review

Autoimmunity in systemic lupus erythematosus: Integrating genes and biology夽 Sandeep Krishnan ∗ , Bhabadeb Chowdhury, George C. Tsokos Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA

Abstract Susceptibility to the autoimmune phenotype of systemic lupus erythematosus (SLE) is heritable. Linkage analysis and recent advances in the field of single nucleotide polymorphisms (SNPs) have resulted in the identification of several genetic loci and functional allelic variants of signaling proteins which have become the mainstay of understanding disease susceptibility and exploring the basis of autoimmunity in SLE. However, genetic heterogeneity and possible epistatic interactions among genetic elements have precluded replication of these findings in multiple population groups and thus complicated their interpretation. In this regard, the discovery that a plethora of normal signaling proteins are expressed in abnormal amounts in immune cells from patients with SLE has gained significance. Thus, the key to precise elucidation of the pathologic basis of autoimmunity in SLE lies in tying genetics and disease biology. This review highlights recent discoveries of important functional genetic variants and altered expression of normal signaling proteins that network together to disrupt peripheral tolerance and initiate the autoimmune process in SLE. © 2006 Elsevier Ltd. All rights reserved. Keywords: SLE; Autoimmunity; Allelic variants; Biomarkers; Signal transduction

1. Introduction Systemic lupus erythematosus (SLE) is the prototype autoimmune disease with complex and unclear etiology. SLE can be best described as a complex genetic trait wherein combinatorial effects of alleles at multiple variant loci converge to break down the immune tolerance when a hypothetical disease liability threshold is reached, following influence from environmental, hormonal or stochastic factors [1,2]. Over the past few decades, the search for genes that predispose an individual to SLE has been made through association studies of candidate genes and genome wide linkage analysis, which have met with measurable success [1,3–5]. In addition, spontaneous SLE murine models such as (NZB × NZW)F1, MRL/lpr and BXSB among others have also provided important insight into the mechanisms of disease susceptibility loci and immunopathogenesis in SLE (reviewed in [6,7]). The recent genomic revolution has boosted these approaches with the completion of the sequencing of the human genome, cataloging of millions of single nucleotide polymorphisms (SNPs) [8], and initiation of genotyping of SNPs in

夽 The opinions expressed herein represent those of the authors and not those of the Department of Defense. ∗ Corresponding author. E-mail address: [email protected] (S. Krishnan).

1044-5323/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2006.03.011

individuals in the ongoing International HapMap project [9]. These advances are expected to eventually facilitate efficient selection of SNPs for genome-wide association studies that carry the potential to identify many variants that contribute to the disease. Already the results have begun to demonstrate the identification of several functional allelic variants of proteins that participate in abnormal signaling in cells participating in the immune response [1,3–5]. A major problem frequently faced by gene mapping studies for complex traits is the disappointing success with replication of initial findings possibly due to: (1) differences in the allele frequencies, (2) genetic heterogeneity and (3) nonobservance of Mendelian pattern of inheritance by the abnormal genetic elements (reviewed in [10]). Compounding these problems are complexities arising from possible epistatic interactions between genetic elements and possible involvement of the same genes in mediating multiple autoimmune phenotypes [2]. Interestingly, while the disease susceptibility genes earmark an individual for development of the disease at birth, the disease process in most cases starts much later in life with little clue about when and how the pathogenic process started. Ultimately, all the abnormal pathways are mediated by a complex interplay between several proteins that are turned on either as part of the disease process itself or as part of the body’s attempt to contain the inflammatory process. Thus, there is increasing support for characterization of biomarkers of the disease that shape

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various aspects of the autoimmune process [11–15]. A hand-inhand approach of identifying abnormal genetic determinants of SLE along with identification of the actual mediators of autoimmunity would thus be expected to complement each other and (1) expedite our understanding of the disease pathogenesis, (2) aid in determining an individual’s disease status and response to treatment, and (3) foster better treatment modalities against SLE. In this review, we attempt to piece together data from known genetic and biological markers of SLE with the objective of building a theme for the autoimmune basis of SLE. We begin by highlighting in brief the progress made in identifying genes conferring susceptibility to SLE, with focus on the abnormal gene products involved in signal transduction and their effects on disrupting immune tolerance. In addition, we discuss how altered expression of certain normal proteins contributes to abnormal immune cell responses in SLE. 2. Allelic variants in SLE Gene association studies in SLE have largely focused on genes coding for proteins that are involved in orchestrating and regulating the immune response. Studies in both humans and murine lupus models have revealed the association of several genes with SLE whose deficiency or allelic variations may be critical to initiation and/or perpetuation of the autoimmune process. The major findings from human studies are summarized in Table 1 and below we discuss a few important members among these groups in the context of their possible role in disease pathology. While we have broadly classified these candidates on the basis of their function into genes affecting particular arms

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of the immune response in reality there may be considerable overlap in their actions. 2.1. Deficiencies and allelic variants shaping innate immunity 2.1.1. Complement genes Complement genes are components of the innate immunity and play an important role in mediating host resistance to microbial infection and also participate in the prevention of autoimmunity. The latter function is mediated by the role of complement proteins in clearing apoptotic fragments and cell debris that can otherwise form a source for autoantigens [16–18]. Individuals with complete deficiency of complement components C1q, C4 and C2 have a high risk of developing SLE, with the risks being 90% for C1q, 75% for C4 and 10% for C2 [19–21]. Results from studies in mice parallel these observations [6,22]. Additionally, deficiencies of C1r/s, C5 and C8 may also predispose to SLE or SLE like syndromes (reviewed in [4]). The complement receptor CR1 (CD35) expressed on the surface of erythrocytes and that binds C3b and C4b is significant in clearing circulating immune complexes containing C3 and C4. Several studies have identified structural and functional polymorphisms of CR1 gene with variable results among SLE patients. Significantly, a meta-analysis of these polymorphisms among 18 such studies suggests an association of CR1 S, a structural variant CR1, and SLE in Caucasians [23]. 2.1.2. Fcγ receptors The different types of Fc␥ receptors display different affinities for IgG subclasses and display cell type specific expression

Table 1 Major genes associated with human SLEa Immune response

Gene

Innate

Complement C4 Fc␥ receptor Fc␥RIIa Fc␥RIIb Fc␥RIIIa CRP MBL

Adaptive

Apoptosis

a

MHC class II DR-3 DR-2 CTLA-4 PDCD-1 MCP-1 PTPN22 TYK2 Cytokines IL-10 TNF ␣ TNF ␤ FAS FASL Bcl-2

Associated alleles

Proposed functional defect

AQ0

Defective clearance of immune complexes/apoptotic debris

R131 T232 F176 +1846A D54

Defective clearance of immune complexes Defective clearance of immune complexes Defective clearance of immune complexes Defective clearance of immune complexes/apoptotic debris Defective clearance of immune complexes

DRB1*0301 DRB1*1501 +49G PD-1.3A −2518G W620 F362,S384

Defective antigen presentation Defective antigen presentation Defective control of T cell activation Defective control of activation induced cell death Recruitment of inflammatory cells Unregulated activation of T cells Interference with actions of cytokines

Multiple TNF2 TNFB*2

Altered immune response Altered immune response Altered immune response

230A −822C Multiple

Defective apoptosis Defective apoptosis Increased survival of autoimmune cells

This table lists only important genetic variants. See text for references.

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patterns. Allelic variants bearing single amino acid substitutions within the extracellular domains of stimulatory Fc␥ receptors can alter their binding affinity to respective subclasses of IgG and affect important functions of the immune system such as phagocytosis, clearance of immune complexes and antibodydependent cellular cytotoxicity (ADCC) thus adding to the risk of developing autoimmunity. Amongst these receptors, genetic polymorphisms of FcγRIIa, FcγRIIIa, FcγRIIIb and FcγRIIb, a cluster of four genes at 1q23 encoding for low affinity IgG receptors have been found to be associated with SLE (reviewed in [3]). Of these genes, FcγRIIa and FcγRIIIa are involved in signal transduction in different cellular contexts by virtue of bearing immuno-receptor tyrosine-based activation motifs (ITAMs) on their cytoplasmic tails, and which have the strongest association with SLE [4,24]. Fc␥RIIa (CD32) is expressed on polymorphonuclear cells, mononuclear phagocytes and platelets. It has two codominantly expressed alleles as a result of a SNP in the genomic DNA resulting in differences in the amino acid at position 131 with distinct outcome on the ability to bind ligands. For example, the presence of histidine at position 131 (H131) confers greater ability to bind IgG2 than arginine at position 131 (A131) [25]. IgG2 is a poor activator of classical complement pathway. Therefore, expression of Fc␥RIIa bearing H131 in its extracellular domain is essential to clearing immune complexes containing IgG2. Studies across multiple ethnic groups support a positive role of the polymorphism that results in expression of Fc␥RIIa-A131 in conferring susceptibility to SLE (reviewed in [3,5]). However, these results have been inconsistent possibly stemming from several factors including differences in the statistical and genotyping methods used in the studies [3,5]. Meta-analyses of data derived from several association studies have strongly supported the role of this polymorphism in conferring SLE susceptibility but not lupus nephritis [26,27]. Fc␥RIIIa (CD16) is expressed on NK cells and mononuclear phagocytes. Similar to Fc␥RIIa, Fc␥RIIIa also has two codominantly expressed allelic variants differing in a single amino acid at position 176 (or 158, excluding the leader sequence) with the presence of either valine (V176) or phenylalanine (F176). The avidity for IgG1 and IgG3 containing immune complexes is greater for V176 homozygotes than F176 homozygotes [25]. Here too there is variability in reported findings with regard to the relationship between Fc␥RIIIa-F176 and SLE susceptibility (reviewed in [3]). Meta-analysis of data derived from 11 independent studies also failed to find an association for susceptibility to SLE by this polymorphism but strongly supported an influence on development of lupus nephritis in subjects of African, Asian and European origin [28]. Because studies in some population show that FcγRIIa and FcγRIIIa may be in linkage disequilibrium but not in others, it remains to be determined if their association with SLE is dependent or independent of each other [4,24,28]. Polymorphisms of FcγRIIb, another member of the FcR gene family, have been also associated with SLE. FcγRIIb maps to the same region on chromosome 1q23. Fc␥RIIb is unique in that among the classical IgG Fc-receptor binding family, it is

the only receptor that bears an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain [29]. It plays an important role in immune homeostasis by downmodulating B cell receptor signaling and thus regulating B cell survival and proliferation, by augmenting antigen presentation by Langerhans cells and by decreasing antibody-mediated phagocytosis by macrophages. Studies on Fc␥RIIb-I/T 232 polymorphism that alters the inhibitory function of this receptor have revealed an association with SLE susceptibility in Asian populations but not in African American, US Caucasian and Swedish populations (reviewed in [3,5]). Recently, Kimberly and colleagues have demonstrated that the promoter for human FcγR2b is polymorphic and the less frequent 2B.4 functional promoter haplotype is associated with SLE [30]. This promoter haplotype has increased binding capacity for GATA4 and Yin-Yang1 (YY1) transcription factors in both B lymphocytes and monocytes that leads to increased expression of Fc␥RIIb receptor and may contribute to the autoimmune pathology in SLE [31]. The association between polymorphisms of FcγRIIIb gene and SLE remains unclear [32,33]. 2.1.3. C reactive protein (CRP) CRP, a component of innate immune response, is a pentraxin normally involved in phagocytosis of apoptotic debris and other immune complexes. During the active phase of SLE, despite the presence of marked tissue inflammation, there is defective CRP response [34]. The CRP gene is located on chromosome 1 within an interval linked with SLE in multiple populations. While there is evidence that gene polymorphisms can contribute to reductions in the basal level of CRP [35,36], family-based studies of association and linkage have identified one such single nucleotide polymorphism CRP-4 in the 3 region of the CRP gene that is associated with development of SLE [36]. The resulting defect in clearing of products of apoptosis, such as chromatin and nuclear proteins, may provide potential sources of autoantigens in the pathogenesis of SLE. It could be argued that the defect in clearing nucleosomes bound to CRP may also be enhanced by allelic polymorphisms of Fc␥RIIa with it being the main receptor for CRP [37]. 2.1.4. Mannose binding lectin (MBL) MBL is an acute phase lectin with structure and functions similar to that of complement C1q. The lectin pathway of complement activation is initiated by binding of MBL to mannose groups of microbial surface carbohydrates. Efficacy of this pathway is crucial to opsonization and clearance of microbes in an antibody-independent manner. Several studies have reported that deficiency or low levels of serum MBL as a result of polymorphisms in the promoter or coding region of MBL gene might be associated with development of SLE (reviewed in [4]). While several polymorphisms have been reported for MBL gene, the most significant ones are the codon 52, 54 and 57 polymorphisms studied in the Spanish and Chinese populations [38–41]. The presence of any of the less common alleles results in significant reduction in the serum MBL concentrations and may affect disease severity.

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2.2.1. Major histocompatibility complex (MHC) The MHC region on the short arm of chromosome 6 is highly polymorphic and has several functions the most important being the regulation of the ability of the immune system to differentiate self from non-self. Genes of MHC have been associated with SLE for more than three decades. In the Caucasian population, the DR2 (HLA-DRB1*1501) and DR3 (HLA-DRB1*0301) have been associated with a two-to three-fold relative risk conferred by each allele while in the other population groups these findings are not well established [3,4,42,43]. Alterations in the nature of antigen presentation to helper T cells leading to abnormal T cell responses may be one mechanism by which these alleles contribute to the disease pathology.

functions by two ways, by competitively blocking binding of activating molecule CD28 by B7-1 and B7-2 and by transducing inhibitory signals possibly via activation of serine/threonine phosphatases [45] (Fig. 1A). CTLA-4 knock-out mice develop a fatal lymphoproliferative phenotype [46,47]. Several studies have found a strong association of CTLA-4 gene polymorphisms with susceptibility to SLE [48–50]. Among several polymorphisms associated with CTLA-4 gene, allelic variation characterized by T/C substitution at the −1722 site has been shown to specifically influence susceptibility to SLE [48]. Several other autoimmune diseases such as type 1 diabetes and Graves’ disease have been associated with a CTLA-4 polymorphism that results in reduced production of a splice variant possessing the inhibitory activity, thus highlighting the importance of CTLA-4 in providing protection against autoimmunity [51].

2.2.2. Cytotoxic T lymphocyte antigen-4 (CTLA-4) CTLA-4, a structural homologue of CD28, is a negative regulator of T cells and plays an important role in preventing autoimmune diseases by promoting long-lived anergy [44]. CTLA-4

2.2.3. Programmed cell death-1 (PDCD-1) PDCD-1 is an immunoreceptor of the CD28 family that bears a tyrosine-based inhibitory motif. It is normally expressed on the surface of activated T and B cells and regulates peripheral toler-

2.2. Allelic variants shaping adaptive immunity

Fig. 1. Signaling pathways involving (A) CTLA-4, (B) LYP and (C) PDCD-1 in normal T cells. PD-1, PDCD-1; PD-1L, PDCD-1-ligand; (–) inhibition; question mark indicates unknown mechanisms; dotted lines indicate suppressed pathways. Only selected associations are shown. See text for references.

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ance in both cell types [52,53] (Fig. 1C). Deficiency of PDCD-1 leads to SLE-like glomerulonephritis and arthritis in a C57BL/6 mouse background and a fatal autoimmune dilated cardiomyopathy in a BALB/c mouse background [54,55]. Recently, an intronic SNP in PDCD-1 gene was found to be associated with SLE development in the European and Mexican population [56–58]. This variant affects a binding site of the runt-related transcription factor 1 (RUNX1) in an intronic enhancer causing aberrant regulation of PDCD-1 expression that in turn may induce the lymphocytic hyperactivity observed in SLE [56]. These and additional studies showing the association between distinct SNPs that affect a RUNX1 binding site and rheumatoid arthritis and psoriasis have led to the suggestion that the loss of this binding may be a common theme in susceptibility to multiple autoimmune diseases [59–61]. 2.2.4. Monocyte chemoattractant protein 1 (MCP-1) In the quest for determining factors that trigger autoimmune tissue injury by recruiting mononuclear leukocytes, attention was focused on the chemokine MCP-1 that has chemoattraction for monocytes and memory T cells and that modulates the expression of adhesion molecules and regulates T cell functions. Tissue expression of MCP-1 has been reported in human SLE, and the lupus-prone NZB/W mice [62]. Neutralization of MCP-1 via antibody inhibits arthritis in the MRL-lpr mouse model, and MCP-1 deficient MRL-lpr mice showed prolonged survival and reduced tissue inflammation [63,64]. Recently, a strong association of the SNP −2518 A/G with susceptibility to SLE and lupus nephritis was reported in the context of increased production of MCP-1 [65]. However, further studies are necessary to compare the role of this polymorphism in susceptibility in different races in light of observations that it may play no role in disease susceptibility in Spanish patients [66]. 2.2.5. Protein tyrosine phosphatase N22 (PTPN22) PTPN22 gene encodes lymphoid tyrosine phosphatase (LYP), which is an important protein that regulates TCR signaling in memory/effector T cells [67] (Fig. 1B). Recently, it was discovered that a missense polymorphism that occurs in the proximal proline-rich SH3-binding domain of PTPN22 resulting in substitution of a highly conserved arginine with tryptophan (R620W) was associated with human SLE in North American white individuals and Spanish Caucasians [68]. As a result of this polymorphism, the interaction between LYP and a negative regulator of TCR signaling C-terminal Src tyrosine kinase (CSK) becomes affected thereby limiting the action of LYP in preventing spontaneous T cell activation [69,70]. Interestingly, this polymorphism is also associated with rheumatoid arthritis (RA) and type I diabetes, leading investigators to hypothesize PTPN22 as a good candidate gene for autoimmunity [68,71–73]. 2.2.6. Tyrosine kinase 2 (TYK2) and interferon regulatory factor 5 (IFN5) Considering the pivotal role proposed for type I interferons (IFN) in the development and maintenance of the disease process in SLE, recently a joint linkage and association study on individual SNPs within several genes encoding type I IFN signaling

molecules was conducted in Swedish, Finnish and Icelandic SLE patients with the aim of identifying SLE susceptibility genes [74]. This study identified strong associations of SNPs within the coding region of TYK2 with SLE susceptibility in Swedish patients. It is believed that apart from interfering with IFN-␣ signaling, the variants might also affect other cytokines including IL-10 that has been reported to be increased in SLE patients [75]. Another SNP was identified in the Finnish and Swedish population in the intron 1 of IRF5 which possibly alters splicing of exon 1 of IRF5 and thus may affect several signaling processes including apoptosis, cell signaling and cell-cycle regulation [74,76,77]. 2.3. Other abnormal variants and genetic defects 2.3.1. DNaseI Defective clearance of autoimmune cells and autoantigens derived from rapid cellular apoptosis remains a challenging problem in the treatment of SLE. Thus components of the complement cascade and enzymes such as DNaseI involved in clearing proteins or protein–DNA complexes respectively, could form excellent targets of gene therapy. Recently, SLE patients lacking DNaseI were reported [78]. Mice lacking DNaseI also develop an SLE-like syndrome [79] and administration of recombinant DNaseI was found to ameliorate disease symptoms in murine lupus models [80,81]. 2.3.2. Toll like receptor 9 (TLR9) Recently, attention has been focused on TLR9 because of its possible role in triggering autoimmunity against chromatin in SLE in the context of massive apoptosis triggered by environmental factors [82,83]. CpG-DNA has been demonstrated to be a strong activator of the innate immune system. Alongside, because of its binding to TLR9, it is also a modulator of adaptive immune responses through the activation of plasmocytoid dendritic cells (PDC). A cooperative interaction has been reported between DNA-autoantibody-bound Fc␥RIIa (CD32) and TLR9 [84]. Activated PDC secrete numerous immunomodulatory cytokines and chemokines to trigger activation of antigen presenting cells and T cells. In mice too, the activation of dendritic cells by immune complexes containing DNA has been shown to occur at least in part via dual engagement of Fc␥RIII and TLR9 [85]. Genetic variations within TLR9 genes have been associated with increased risk for inflammatory diseases in humans. Although initial studies have failed to show significant association of TLR9 gene polymorphisms with SLE susceptibility in a Korean population [86], it would be interesting to see if they have a role in other populations. 2.4. Clues from shared alleles In a polygenic disease such as SLE, some genetic loci may exert disease specific effects while others may be simply exerting a broad effect on the pathologic autoimmune process. Indeed there is mounting evidence that specific susceptibility alleles are associated with multiple autoimmune diseases. Comparative studies of linkage results from 23 autoimmune or immune-

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mediated diseases by Becker et al. revealed that mapped candidate loci are clustered within 18 regions of the genome suggesting that many complex traits share a common set of susceptibility genes [87]. Several such genes have been identified (listed in [5]). For example, the gene coding for CTLA-4 has been associated with SLE, type 1 diabetes, Graves’ disease and RA. Thus, it is possible that abnormal CTLA-4 gene regulation contributes to T cell activation in these diseases. Similarly PTPN22 gene has been associated with several diseases along with SLE such as Graves’ disease, type 1 diabetes and RA among others (reviewed in [88]). As noted above, the common observation of association between SNPs that affect RUNX1 transcription factor binding site and multiple autoimmune diseases such as SLE, psoriasis and rheumatoid arthritis is another noteworthy example of pathway sharing amongst distinct autoimmune diseases [59–61]. These observations provide a clue to understanding molecular mechanisms and key pathways of autoimmunity in multiple disease phenotypes and may help the identification of targets for gene therapy. 3. Altered expression of signaling proteins in SLE One striking observation in SLE is that in cells across the immune system, there is alteration in the expression levels (either increase or decrease) of several signaling molecules. The abnormal regulation of such proteins could occur either as part of the disease pathology or as part of the compensatory mechanisms of the immune system. Given the complexities associated with identifying genetic markers of SLE, identification of biomarkers and characterization of the alterations in the signaling pathways mediated by these molecules would be an alternative approach to gaining insight into mechanisms of autoimmunity in SLE. Here we only highlight abnormal signaling molecules in T and B lymphocytes that might contribute to the abnormal functioning of these cells. 3.1. Altered signal transduction in SLE T cells Abnormally activated T cells provide help to B cells to produce autoantibodies in SLE. In addition, T cells display a greater degree of apoptosis and resistance to activation-induced cell death (AICD) compared to normal T cells. At the biochemical level, SLE T cells display abnormal T cell receptor (TCR)mediated signaling responses that include a lowered excitation threshold and heightened intracellular calcium responses [89–92]. ‘Rewiring’ of the TCR induced by reduced expression of TCR ␨ chain and appearance and association of a more potent signaling molecule FcR␥ chain with the TCR is believed to be responsible at least in part for mediating increased excitability of SLE T cells [93] (Fig. 2). Alteration of expression of several other proteins has been reported in SLE (Table 2). These include cell surface molecules, signaling kinases and transcription factors whose altered expression affects TCR signaling from cell surface to the nucleus. While mechanisms leading to altered expression of many of these proteins are not clear, we will discuss here the regulation of the expression of TCR ␨ chain. It is noteworthy that sev-

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Table 2 Altered expression of major proteins in SLE T and B cellsa Cell type

Molecule

Expression status

References

T cell

CD40 L CD70 TCR ␨ chain FcR␥ chain LCK Syk Vav Protein kinase C MAP kinase NF-␬B, p65 subunit Elf-1-p98 CREM

Increased Increased Decreased Increased Decreased Increased Increased Decreased Decreased Decreased Decreased Increased

[116,117] [140] [89] [141] [142] [141] [102] [143] [144] [145] [97] [105,146]

B cell

CD21 (CR2) CD35 (CR1) CD40L CD80 CD86

Decreased Decreased Increased Increased Increased

[147] [147] [117] [148] [148]

a

Only selected proteins are included.

eral polymorphisms have been reported in the promoter and 3 untranslated regions (3 UTR) of TCR ␨ gene [94]. Recently, it was shown that SLE T cells express an alternatively spliced mRNA lacking nucleotides 672–1233 within exon VIII at the 3 UTR [95]. As a result, the stability of this transcript as well as the rate of its translation is reduced. Studies in T cell lines have shown that expression of the abnormal splice variant of TCR ␨ also affected the surface expression of TCR/CD3 complex and functionally affected the production of IL-2 by these cells [96]. These findings highlight the importance of alternative splicing of TCR ␨ in mediating its reduced expression as well as abnormal T cell cytokine production. In addition, reduced expression of TCR ␨ is also mediated at the transcriptional and post-translational levels [90,97]. Recently it was reported that SLE T cells display high levels of caspase-3 which is involved in excessive proteolytic cleavage of TCR ␨ [98]. This multistep regulation of reduced TCR ␨ expression in SLE highlights the mechanisms employed by the disease process to target this important signaling molecule. Because expression of FcR␥ chain has been reported in conditions where the expression of TCR ␨ is reduced [99,100], it leads to the question if the expression of these two molecules is interrelated in SLE. Indeed, introduction of FcR␥ into normal T cells results in reduction in the expression of TCR ␨ chain and induces a hyperexcitable phenotype in the transfected cells akin to SLE T cells [101]. Interestingly, addition of caspase-3 inhibitors that limit proteolytic cleavage of TCR ␨ induced a simultaneous decrease in the expression of FcR␥ suggesting the presence of an ‘on–off’ switch that reciprocally regulates the expression of these molecules in SLE T cells. Heightened TCR-induced intracellular calcium flux is also mediated by abnormal signaling through the TCR machinery. In this regard, contribution from abnormal signaling through specialized glycolipid enriched membrane compartments called lipid rafts has been demonstrated. Lipid rafts are pre-clustered in SLE T cells as against a homogenous membrane distribu-

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Fig. 2. Signaling pathways in SLE T and B cells. T cells and B cells display abnormal expression of proteins involved in cell signaling including surface proteins, intracellular signaling molecules such as tyrosine kinases/phosphatases, and transcription factors. As a result of these events, there is defective downstream signaling from TCR leading to defective activation of T cells and low levels of IL-2 production. SLE T cells are more resistant to AICD and provide help to B cells to promote autoantibody production. Only selected pathways are shown. See text for references. (−) Inhibition, (+) stimulation, question mark indicates unknown mechanisms [Ca2+ ]i , receptor-induced calcium flux into cytoplasm.

tion in normal T cells. Lipid raft expression is increased in SLE T cells and they are enriched in FcR␥, Syk, Vav, PLC-␥1 and the residual TCR ␨, all of which may be involved in augmenting calcium responses [102,103]. Supporting this hypothesis is the observed reduction in calcium flux following treatment of cells with the lipid raft disrupting agent methyl ␤-cyclodextrin [102]. However, heightened calcium responses fails to rescue IL2 production by human SLE T cells. Significant in this regard are recent experiments by Juang et al. [105] who compared the actions of serum derived from normal subjects and SLE patients on normal T cells. They observed that anti-TCR/CD3 auto-antibodies present within lupus serum induced binding of transcriptional repressor CREM at the −180 site of the IL-

2 through the action of Ca2+ /calmodulin-dependent kinase IV (CaMKIV), thus decreasing the production of IL-2 [105]. It must be noted that in the murine lupus models, the hyper-excitability of T cells is associated with an increased production of IL-2 [104]. Nevertheless, these observations show that TCR signaling is affected at various levels in SLE to produce a hyper-responsive T cell phenotype with possible help to B cells to produce autoantibodies. 3.2. Altered signal transduction in B cells Absence of B cells protects against the development of SLE [106]. While self-reactive T cells may aid B cells, certain inher-

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ent defects of B cells are central to the pathologic production of autoantibodies by B cells (Table 2 and Fig. 2). Similar to T cells, B cells isolated from SLE patients also demonstrate greatly enhanced tyrosine phosphorylation and intracellular calcium flux following activation [107]. Gene knock-out studies with several tyrosine kinases and other positive and negative regulators of B cell receptor (BCR) signaling (such as CD45, CD19, CD22, CD72 and Fc␥RIIb) have revealed that loss of any one of these components might induce a lupus like state in mice [108]. Fc␥RIIb is a negative regulator of B cell receptor (BCR) signaling. As discussed above, polymorphisms in Fc␥RIIb region can affect this function and are thus associated with SLE in several populations. Reduced Fc␥RIIb expression on B cell surface has been reported in several spontaneous lupus mouse models, which may result in defective control of B cell activation [108]. Abnormal expression of other molecules on B cell surface that provide the co-stimulatory signals to T cells can also alter T cell function. Reduced expression of B7.1 (CD80) on the surface of B cells for example might be responsible for reduced recall responses demonstrated by SLE T cells [109]. Alternatively, reduced expression of CD80 may result in reduced signaling through CTLA-4 and thus failure of control of T cell activation. 4. Genetic and biochemical basis of perpetuation of autoimmunity in SLE While an individual inherits the genetic make up for developing SLE, the actual autoimmune process is triggered much later in life. It is unclear at present when exactly the point of breakage of peripheral tolerance is reached; however it results from defects in all departments of the immune system. Here, we consider the major mechanisms perpetuating the autoimmune process with attention to molecular events that alter immune cellular signaling and function (Fig. 3). 4.1. Defective innate immune response The role of innate immunity in protecting against autoimmunity is via clearing auto antigens and apoptotic cell debris. In SLE, deficiency of early complement proteins such as C2, C4 and C1q can result in abnormal clearance of apoptotic fragments [19–21]. C1q receptors expressed on the surface of phagocytes are involved the important function of clearing apoptotic cells. Both humans and mice lacking C1q demonstrate SLE-like syndrome [110,111]. CR1 expression on the surface of erythrocytes is also an important buffer for immune complexes containing C3 and C4, and functional polymorphisms in CR1 gene that affect its expression can also lead to defective clearance of these complexes [23]. Recently, it has been proposed that apart from functioning in clearing apoptotic debris, complement also cooperates with other components of innate immunity in presenting SLE-inducing self-antigens to maturing B cells, resulting in selection against B cells reacting against those antigens [112]. Functional polymorphisms of FcγR genes especially those coding for Fc␥RIIa, Fc␥RIIIa and Fc␥RIIb have also been associated with SLE and may impair binding of immune complexes

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containing IgG2 and IgG3 to these receptors resulting in their defective clearance (discussed above). Inherent abnormalities of macrophages that result in abnormal clearance of apoptotic cells have also been described in SLE [113]. In addition to these abnormalities, defective expression of enzymes such as DNaseI involved in clearing proteins or protein–DNA complexes respectively may also contribute to the build up of autoantigen pool in human SLE [78,79]. 4.2. Defective activation of T and B lymphocytes The possible role of gene products of functional polymorphisms of CTLA-4 and PTPN22 genes on T cell activation has been discussed above. From a biochemical point of view, freshly isolated SLE T cells share several similarities with effector T cells in that similar to effector cells, they also downregulate TCR ␨ and upregulate FcR␥ and display increased association of Syk with FcR␥ [100,102]. Higher amounts of PLC-␥1 localizes to cell membrane in both activated Jurkat T cells and SLE T cells [102,114]. Additionally, lipid rafts are pre-clustered in both effector cells and SLE T cells [100,102]. These observations suggest that the hyper-excitable phenotype of an SLE T cell is in fact due to perpetuation of effector cells by the continuous autoimmune process. The effector status of T cells would suggest that considerable help would be made available to B cells to induce their differentiation into plasma cells. T cells derived from active lupus patients stimulated IgG synthesis without addition of antigen or mitogens [115]. This function may be enhanced by increased expression of CD40L (CD154) by SLE T cells [116,117] that could bind to CD40 on the surface of B cells to support their activation. CD4 T cells treated with DNA methyltransferase inhibitors overexpress CD70, a costimulatory ligand for CD27 expressed on B cells, upon which B cells were overstimulated to produce IgG, thus suggesting a role for the DNA hypomethylation-driven activation of B cells by T cells [118]. As a result of these interactions, self-reactive T cells not only activate B cells but also promote class switch recombination and somatic hypermutation aiding autoantibody production. Apart from producing autoantibodies in response to T cells, B cells are also involved in mediating other functions such as presenting autoantigens and costimulation to activate T cells as well as regulating the responses of other immune cells such as dendritic cells and mediating tissue damage (reviewed in [108]). Moreover, as discussed above, through cooperation between DNA containing immune complex-bound Fc␥RIIa and TLR9, B cells are also implicated in initiating autoimmunity against chromatin. Role of regulatory T cells (CD25+ CD4+ T cells, designated as Tregs) in SLE deserve a special mention here because of their important role in preventing autoimmunity by suppressing selfreactive T cells. They play an important role in preventing several autoimmune disease conditions such as thyroiditis, inflammatory bowel disease and insulin-dependent diabetes mellitus (IDDM) [119]. Defective production of IL-2 by T cells derived from many SLE patients suggests a possible effect on production and functioning of Tregs in SLE. Recent studies have revealed

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Fig. 3. Schematic of possible mechanisms of development of autoimmunity in SLE. Several genetic, environmental and stochastic factors combine to initiate massive cellular apoptosis that form the potential pool of auto-antigens. Defects at multiple levels including innate and adaptive arms of the immune response along with defects in clearance of self-reactive lymphocytes form the basis of autoimmunity in SLE. As a result of these processes, tissue damage is mediated by deposited IC, inflammatory cells and uncontrolled actions of abnormal cytokines.

a deficiency of Tregs in the murine models (NZB × NZW)F1 and (SN)F1 [120]. While studies have shown that SLE patients display reduced numbers of Tregs, precise contribution of this observation to the autoimmune pathology of SLE remains to be determined [121]. 4.3. Altered cytokine milieu in SLE Cytokines contribute to the pathology of SLE in several ways. For example, absence of regulatory cytokines such as IL-2 may

prevent effective activation and functioning of T cells as well as induction of activation-induced cell death in T cells [122]. Decreased production of IL-12 in SLE might affect differentiation of CD4 T cells into Th1 cells [123,124]. High levels of cytokines such as IL-6 and IL-10 may promote antibody production by B cells, where as low levels of anti-inflammatory lymphokine transforming growth factor-␤ (TGF-␤) might result in unregulated inflammation [125]. Polymorphisms of IL-10 gene promoter have been described that alter the levels of synthesis of IL-10. However, their association with SLE differs between

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different population groups and their significance remains to be determined [126,127]. An interesting observation that a majority of SLE patients with high disease activity demonstrate increased expression of genes regulated by IFN in peripheral blood cells led to the concept of ‘IFN signature’ [128]. Among this group, IFN-␣ was found to be the major cytokine in SLE that drives activation of dendritic cells [129]. As observed earlier, chromatin containing IC can activate PDC via TLR9 and Fc␥RIIa to produce IFN-␣. These observations have paved way for several targeted therapy involving cytokines. 4.4. Defective elimination of self-reactive lymphocytes Another powerful check point that regulates peripheral responses to self-antigens is the ability to delete self-reactive lymphocytes by apoptotic death and rapid clearance of apoptotic cells and fragments by phagocytes. In SLE, there is a mixture of processes that disrupt apoptosis resulting in long lived activated cells as well as those that trigger massive apoptosis of cells that simply overwhelms the scavenging system (Fig. 3). In mice, gene knock-out and transgenic models have implicated several molecules in disrupting either the apoptotic pathway or mechanisms involved in clearing the dead cells, cellular debris and immune complexes (Table 2). These include among others Fas/FasL, Bcl-2, members of complement cascade and DNase (reviewed in [6]). In humans, peripheral blood monocytes display increased rates of apoptosis [130]. Freshly isolated lymphocytes from SLE patients when cultured in vitro demonstrate higher degrees of apoptosis compared to those derived from normal individuals and a correlation was reported between disease activity and rates of apoptosis [131]. Accentuated Fas-mediated apoptosis is believed to contribute to death of lymphocytes and monocytes in human SLE [132–135]. Defects in either the expression of Fas/FasL or in some cases, genetic polymorphisms may contribute to these abnormalities [136,137]. Additionally, abnormally elevated mitochondrial transmembrane potential (Ψ m ) and increased baseline reactive oxygen intermediate (ROI) have also been shown to enhance apoptosis in peripheral blood lymphocytes in SLE [108]. At the other end of the spectrum, the SLE T cells also display resistance to TCR-mediated AICD relative to normal T cells. Prolonged survival of activated SLE T may contribute to increased autoantibody production by B cells. It has been suggested that this resistance to AICD is partly linked to diminished levels of TNF-␣ in T cells resulting from a gene polymorphism in SLE [138,139]. 5. Concluding remarks In the aftermath of completion of the human genome project, the gold rush for isolating genes conferring disease susceptibility to autoimmune diseases has ostensibly benefited only singlegene diseases. However, significant advances have also been made in the area of complex traits such as SLE. Identification of DNA sequence variants across the human genome, development of more accurate and cheaper genetic techniques for investigat-

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