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Systemic lupus erythematosus (SLE) is a common systemic autoimmune disease with complex etiology but strong clustering in families (lambda(S) = approximately 30). We performed a genome-wide association scan using 317,501 SNPs in 720 women of European ancestry with SLE and in 2,337 controls, and we genotyped consistently associated SNPs in two additional independent sample sets totaling 1,846 affected women and 1,825 controls. Aside from the expected strong association between SLE and the HLA region on chromosome 6p21 and the previously confirmed non-HLA locus IRF5 on chromosome 7q32, we found evidence of association with replication (1.1 x 10(-7) < P(overall) < 1.6 x 10(-23); odds ratio = 0.82-1.62) in four regions: 16p11.2 (ITGAM), 11p15.5 (KIAA1542), 3p14.3 (PXK) and 1q25.1 (rs10798269). We also found evidence for association (P < 1 x 10(-5)) at FCGR2A, PTPN22 and STAT4, regions previously associated with SLE and other autoimmune diseases, as well as at > or =9 other loci (P < 2 x 10(-7)). Our results show that numerous genes, some with known immune-related functions, predispose to SLE.

Fc receptors represent a distinct group of hematopoeitic cell surface glycoproteins that have a characterized role in affecting the efficiency of the mononuclear phagocyte system to clear IgG immune complexes. Functional genetic variations in this family of receptors have been identified as heritable susceptibility factors for SLE and lupus nephritis across diverse populations. In this review, we describe the roles of the classical Fc receptors for IgG (Fc gamma) and non-classical Fc-like receptors (FCR1-FCRL6L), Fc receptors for IgE (Fc epsilon RI) and IgA and IgM (Fc alpha/mu R) in SLE diathesis. The combined effects of these genes on SLE pathogenesis, either via linkage disequilibrium or epistasis with additional genetic or environmental factors, provide a challenge for future investigations. The pursuit of a polygenic SLE-profile that includes longitudinal evaluations of SLE and markers involved in the protean clinical manifestations associated with SLE will facilitate our understanding of the cascade of inflammatory events associated with the diathesis.

Objective: To determine whether there is an increase in the number of TLR7 gene copies in patients diagnosed as having systemic lupus erythematosus (SLE) and whether gene amplification influences the autoantibody profiles in SLE patients, as has recently been reported in the BXSB/Yaa mouse model of lupus., Methods: We used a modified real-time quantitative polymerase chain reaction protocol to calculate the relative TLR7 gene copy number according to the comparative 2(-DeltaDeltaC(t) ) method in 99 SLE patients and 91 healthy controls matched for sex and ethnicity. Autoantibody profiles were determined by standard methods., Results: The relative number of TLR7 gene copies in SLE patients and healthy controls varied; however, no significant concordance between the number of relative gene copies and the SLE phenotype was found. There was also no difference in variation by ethnic group. Comparison of the relative gene copy numbers according to the presence or absence of antinuclear antibodies (ANAs), the ANA staining patterns, and the presence or absence of anti-RNA-associated antigen antibody showed no statistically significant difference in the SLE patients., Conclusion: We determined that although the relative gene copy number of TLR7 varied in both SLE patients and healthy controls, it was not significantly increased among our SLE patients as compared with our controls. We found no detectable trend for an association between the relative gene copy number and the autoantibody profile in SLE patients.

An important characteristic of tumors is that they at some point in their development overcome the surveillance of the immune system. Tumors secrete exosomes, multivesicular bodies containing a distinct set of proteins that can fuse with cells of the circulating immune system. Purified exosomes from TS/A breast cancer cells, but not non-exosomal fractions, inhibit (at concentrations of nanograms per ml protein) IL-2-induced natural killer (NK) cell cytotoxicity. The dietary polyphenol, curcumin (diferuloylmethane), partially reverses tumor exosome-mediated inhibition of natural killer cell activation, which is mediated through the impairment of the ubiquitin-proteasome system. Exposure of mouse breast tumor cells to curcumin causes a dose-dependent increase in ubiquitinated exosomal proteins compared to those in untreated TS/A breast tumor cells. Furthermore, exosomes isolated from tumor cells pretreated with curcumin have a much attenuated inhibition of IL-2 stimulated NK cell activation. Jak3-mediated activation of Stat5 is required for tumor cytotoxicity of IL-2 stimulated NK cells. TS/A tumor exosomes strongly inhibit activation of Stat5, whereas the tumor exosomes isolated from curcumin-pretreated tumor cells have a lowered potency for inhibition of IL-2 stimulated NK cell cytotoxicity. These data suggest that partial reversal of tumor exosome-mediated inhibition of NK cell tumor cytotoxicity may account for the anti-cancer properties of curcumin.

The human IgA FcR (FcalphaRI; CD89) mediates a variety of immune system functions including degranulation, endocytosis, phagocytosis, cytokine synthesis, and cytokine release. We have identified a common, nonsynonymous, single nucleotide polymorphism (SNP) in the coding region of CD89 (844A-->G) (rs16986050), which changes codon 248 from AGC (Ser(248)) to GGC (Gly(248)) in the cytoplasmic domain of the receptor. The two different alleles demonstrate significantly different FcalphaRI-mediated intracellular calcium mobilization and degranulation in rat basophilic leukemia cells and cytokine production (IL-6 and TNF-alpha) in murine macrophage P388D1 cells. In the absence of FcR gamma-chain association in P388D1 cells, the Ser(248)-FcalphaRI allele does not mediate cytokine production, but the Gly(248)-FcalphaRI allele retains the capacity to mediate a robust production of proinflammatory cytokine. This allele-dependent difference is also seen with FcalphaRI-mediated IL-6 cytokine release by human neutrophils ex vivo. These findings and the enrichment of the proinflammatory Gly(248)-FcalphaRI allele in systemic lupus erythematosus populations in two ethnic groups compared with their respective non-systemic lupus erythematosus controls suggest that FcalphaRI (CD89) alpha-chain alleles may affect receptor-mediated signaling and play an important role in the modulation of immune responses in inflammatory diseases.

FcgammaRIIb (CD32B, Online Mendelian Inheritance in Man 604590), an IgG FcR with a tyrosine-based inhibitory motif, plays a critical role in the balance of tolerance and autoimmunity in murine models. However, the high degree of homology between FcgammaRIIb and FcgammaRIIa in humans and the lack of specific Abs to differentiate them have hampered study of the normal expression profile of FcgammaRIIb and its potential dysregulation in autoimmune diseases such as systemic lupus erythematosus (SLE). Using our newly developed anti-FcgammaRIIb mAb 4F5 which does not react with FcgammaRIIa, we found that FcgammaRIIb is expressed on the cell surface of circulating B lymphocytes, monocytes, neutrophils, myeloid dendritic cells (DCs), and at very low levels on plasmacytoid DCs from some donors. Normal donors with the less frequent 2B.4 promoter haplotype have higher FcgammaRIIb expression on monocytes, neutrophils, and myeloid DCs similar to that reported for B lymphocytes, indicating that FcgammaRIIb expression on both myeloid and lymphoid cells is regulated by the naturally occurring regulatory single nucleotide polymorphisms in the FCGR2B promoter. FcgammaRIIb expression in normal controls is up-regulated on memory B lymphocytes compared with naive B lymphocytes. In contrast, in active SLE, FcgammaRIIb is significantly down-regulated on both memory and plasma B lymphocytes compared with naive and memory/plasma B lymphocytes from normals. Similar down-regulation of FcgammaRIIb on myeloid-lineage cells in SLE was not seen. Our studies demonstrate the constitutive regulation of FcgammaRIIb by natural gene polymorphisms and the acquired dysregulation in SLE autoimmunity, which may identify opportunities for using this receptor as a therapeutic target.

Antigen presenting cells recognize pathogens via pattern recognition receptors (PRR), which upon ligation transduce intracellular signals that can induce innate immune responses. Because some C-type lectin-like receptors (e.g. dectin-1 and DCSIGN) were shown to act as PRR for particular microbes, we considered a similar role for dectin-2. Binding assays using soluble dectin-2 receptors showed the extracellular domain to bind preferentially to hyphal (rather than yeast/conidial) components of Candida albicans, Microsporum audouinii, and Trichophyton rubrum. Selective binding for hyphae was also observed using RAW macrophages expressing dectin-2, the ligation of which by hyphae or cross-linking with dectin-2-specific antibody led to protein tyrosine phosphorylation. Because dectin-2 lacks an intracellular signaling motif, we searched for a signal adaptor that permits it to transduce intracellular signals. First, we found that the Fc receptor gamma (FcRgamma) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcRgamma, activation of NF-kappaB, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor alpha and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcRgamma chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcRgamma to induce innate immune responses.

Objective: To investigate the possible association of the Fcgamma receptor IIb (FcgammaRIIb) Ile/Thr187 transmembrane domain polymorphism, which significantly affects receptor signaling, with susceptibility to systemic lupus erythematosus (SLE) in Taiwanese patients., Methods: We used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to genotype 351 Taiwanese SLE patients and 372 age- and sex-matched healthy individuals from the same geographic area. Allele frequencies and genotype distributions were compared between the patients and controls, both as an aggregate and as stratified by sex, autoantibody profile, and clinical parameters. A combined analysis was conducted to assess the FCGR2B Thr187 allele as a common risk factor in different ethnic populations., Results: The minor Thr187 allele was significantly associated with SLE in Taiwanese subjects (P = 0.017, odds ratio [OR] 1.989 [95% confidence interval (95% CI) 1.119-3.553]). Interestingly, male SLE patients showed enrichment of the Thr/Thr187 genotype (24%; 7 of 29) as compared with female SLE patients (10%; 32 of 322) (P = 0.043, OR 2.884 [95% CI 1.028-7.839]). Additionally, SLE patients with Thr/Thr187 and Ile/Thr187 genotypes were more likely to have pleural effusions (P = 0.038, OR 1.874 [95% CI 1.033-3.411]) and anti-SSA/Ro antibody production (P = 0.046, OR 2.221 [95% CI 1.013-4.897]). Combined analysis of 4 groups of Asian patients strongly supported the association of the FCGR2B Thr187 allele with the lupus phenotype (P = 0.000159)., Conclusion: The FcgammaRIIb transmembrane polymorphism is a strong disease susceptibility candidate in epistasis with other genetic effects in Taiwanese and other Asian populations. It may also play a more prominent role in male patients with SLE.

Background: Renal involvement is a serious manifestation of systemic lupus erythematosus (SLE); it may portend a poor prognosis as it may lead to end-stage renal disease (ESRD). The purpose of this study was to determine the factors predicting the development of renal involvement and its progression to ESRD in a multi-ethnic SLE cohort (PROFILE)., Methods and Findings: PROFILE includes SLE patients from five different United States institutions. We examined at baseline the socioeconomic-demographic, clinical, and genetic variables associated with the development of renal involvement and its progression to ESRD by univariable and multivariable Cox proportional hazards regression analyses. Analyses of onset of renal involvement included only patients with renal involvement after SLE diagnosis (n = 229). Analyses of ESRD included all patients, regardless of whether renal involvement occurred before, at, or after SLE diagnosis (34 of 438 patients). In addition, we performed a multivariable logistic regression analysis of the variables associated with the development of renal involvement at any time during the course of SLE. In the time-dependent multivariable analysis, patients developing renal involvement were more likely to have more American College of Rheumatology criteria for SLE, and to be younger, hypertensive, and of African-American or Hispanic (from Texas) ethnicity. Alternative regression models were consistent with these results. In addition to greater accrued disease damage (renal damage excluded), younger age, and Hispanic ethnicity (from Texas), homozygosity for the valine allele of FcgammaRIIIa (FCGR3A*GG) was a significant predictor of ESRD. Results from the multivariable logistic regression model that included all cases of renal involvement were consistent with those from the Cox model., Conclusions: Fcgamma receptor genotype is a risk factor for progression of renal disease to ESRD. Since the frequency distribution of FCGR3A alleles does not vary significantly among the ethnic groups studied, the additional factors underlying the ethnic disparities in renal disease progression remain to be elucidated.

Fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis (RA FLSs) exhibit prosurvival, rather than apoptotic, response to tumor necrosis factor (TNF)-alpha stimulation. Here, we show that JAB1 is a critical regulator of the TNF-alpha-mediated anti-apo-ptosis pathways in RA FLSs. We found that knockdown of JAB1 using small interfering (si)RNA led to restoration of the TNF-alpha-induced apoptosis response, reduction of nuclear factor-kappaB activity, delayed degradation of IkappaB-alpha, and inhibited phosphorylation of JNK. Analysis of the interactions of JAB1 by reciprocal co-immunoprecipitations and confocal microscopy revealed that JAB1 interacts with TNF receptor-associated-factor 2 (TRAF2). The generation of the anti-apoptotic signal on binding of TNF-alpha to the TNF receptor (TNFR)1 has been shown to be associated with the recruitment of TRAF2 to the TNFR1 in a process that requires ubiquitination of TRAF2 with lysine-63-linked polyubiquitin chains. We found that TNF-alpha stimulation of JAB1 siRNA-transfected RA FLSs failed to stimulate ubiquitination of TRAF2. Thus, we conclude that JAB1-regulated ubiquitination of TRAF2 is a novel mechanism whereby TNF-alpha can induce anti-apoptosis signaling and production of matrix metalloproteinases through activation of nuclear factor-kappaB and JNK in RA FLSs.

Individual genetic admixture estimates, determined both across the genome and at specific genomic regions, have been proposed for use in identifying specific genomic regions harboring loci influencing phenotypes in regional admixture mapping (RAM). Estimates of individual ancestry can be used in structured association tests (SAT) to reduce confounding induced by various forms of population substructure. Although presented as two distinct approaches, we provide a conceptual framework in which both RAM and SAT are special cases of a more general linear model. We clarify which variables are sufficient to condition upon in order to prevent spurious associations and also provide a simple closed form "semiparametric" method of evaluating the reliability of individual admixture estimates. An estimate of the reliability of individual admixture estimates is required to make an inherent errors-in-variables problem tractable. Casting RAM and SAT methods as a general linear model offers enormous flexibility enabling application to a rich set of phenotypes, populations, covariates, and situations, including interaction terms and multilocus models. This approach should allow far wider use of RAM and SAT, often using standard software, in addressing admixture as either a confounder of association studies or a tool for finding loci influencing complex phenotypes in species as diverse as plants, humans, and nonhuman animals., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

In common with many other cell types, synovial fibroblasts produce exosomes. In this study, we show that the exosomes produced by synovial fibroblasts obtained from individuals with rheumatoid arthritis (RASF), but not exosomes produced by synovial fibroblasts obtained from individuals with osteoarthritis, contain a membrane bound form of TNF-alpha as demonstrated by colloidal gold immunostaining of TNF-alpha and confirmed by both Western blot and mass spectrometry. The RASF-derived exosomes, but not exosomes derived from fibroblasts obtained from individuals with osteoarthritis, are cytotoxic for the L929 cell, a TNF-alpha-sensitive cell line, and stimulate activation of NF-kappaB and induction of collagenase-1 in RASF. These effects are blocked by addition of soluble TNFR1 (sTNFbp), suggesting that a TNF-alpha-signaling pathway mediates these biological activities. sTNFbp also reduced the production of exosomes by RASF, suggesting the interruption of a positive amplification loop. Exosomes can transmit signals between cells, and RASF exosomes, effectively taken up by anti-CD3-activated T cells, activated AKT and NF-kappaB and rendered these activated T cells resistant to apoptosis. Neutralization of exosomal membrane TNF-alpha by sTNFbp partially reversed this resistance, suggesting that not only TNF-alpha but also additional exosomal proteins may contribute to the development of apoptosis resistance.

Many tumor cells shed specialized membrane vesicles known as exosomes. In this study, we show that pretreatment of mice with exosomes produced by TS/A or 4T.1 murine mammary tumor cells resulted in accelerated growth of implanted tumor cells in both syngeneic BALB/c mice and nude mice. As implanted TS/A tumor cells grew more rapidly in mice that had been depleted of NK cells, we analyzed the effects of the tumor-derived exosomes on NK cells. The tumor-derived exosomes inhibit NK cell cytotoxic activity ex vivo and in vitro as demonstrated by chromium release assays. The treatment of mice with TS/A tumor exosomes also led to a reduction in the percentages of NK cells, as determined by FACS analysis, in the lungs and spleens. Key features of NK cell activity were inhibited, including release of perforin but not granzyme B, as well as the expression of cyclin D3 and activation of the Jak3-mediated pathways. Human tumor cell lines also were found to produce exosomes that were capable of inhibiting IL-2-stimulated NK cell proliferation. Exosomes produced by dendritic cells or B cells did not. The presentation of tumor Ags by exosomes is under consideration as a cancer vaccine strategy; however, we found that pretreatment of mice with tumor exosomes blunted the protective effect of syngeneic dendritic cells pulsed ex vivo with tumor exosomes. We propose that tumor exosomes contribute to the growth of tumors by blocking IL-2-mediated activation of NK cells and their cytotoxic response to tumor cells.

Polyclonal B cell activation might be related to pathogenic over-expression of B-cell-activating factor (BAFF) in primary Sjögren's syndrome (pSS) and other autoimmune diseases. We therefore investigated whether BAFF over-expression in pSS could be a primary, genetically determined event that leads to the disease. The complete BAFF gene was sequenced in Caucasian pSS patients and control individuals. The only single nucleotide polymorphism frequently observed, namely -871 T/C in the promoter region, was then genotyped in 162 French patients with pSS and 90 French control individuals. No significant differences in allele (T allele frequency: 49.7% in patients with pSS versus 50% in controls; P = 0.94) and genotype frequencies of BAFF polymorphism were detected between pSS patients and control individuals. BAFF gene polymorphism was not associated with a specific pattern of antibody secretion either. T allele carriers had significantly increased BAFF protein serum levels (mean values of 8.6 and 5.7 ng/ml in patients with TT and TC genotypes, respectively, versus 3.3 ng/ml in patients with CC genotype; P = 0.01), although no correlation was observed between BAFF polymorphism and mRNA level. In conclusion, BAFF gene polymorphism is neither involved in genetic predisposition to pSS nor associated with a specific pattern of antibody production.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by immunological hyperactivity and multi-system organ damage. A complex genetic trait involving multiple genes, with both genetic heterogeneity and a threshold effect for disease expression, SLE involves abnormalities of both the innate and adaptive immune systems. Recognition of an 'interferon signature' in SLE leukocytes, of the role of B cells in promoting disease activity, and of FCGR3A alleles as a biomarker of end organ damage, provide important insights into disease pathogenesis. Nonetheless, coordinated studies in humans and model systems hold promise for an even more rapid advance in understanding pathways of disease development and strategies for intervention. More effective markers of disease risk, disease activity, severity of organ damage and outcomes would facilitate earlier diagnosis and guide appropriately targeted treatment.

Rheumatoid arthritis synovial fibroblasts (RASFs) contribute to arthritic cartilage degradation. Although RASFs are normally resistant to apoptosis, Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based gene therapy has been successfully used in a mouse model of arthritis. We investigated this further by treating human RASFs with nontoxic doses of the proteasome inhibitor lactacystin. Treatment induced cytosolic accumulation of p53 and enhanced the susceptibility of RASFs to apoptosis mediated by TRAIL-R2 (DR5) but not Fas. A specific role for p53 in TRAIL-R2-mediated apoptosis was indicated by the ability of p53 siRNA to significantly reduce RASF apoptosis and by the reduced apoptosis of RASFs bearing p53 mutations on treatment with anti-DR5 antibody or anti-DR5 antibody plus lactacystin. p53 immunoprecipitation followed by mass spectrometry identified a vimentin-p53 complex, an interaction that was confirmed by reciprocal vimentin-p53 immunoprecipitation and by co-immunofluorescence. Interestingly, human caspase-4 cleaved human vimentin, and blockade of caspase-4 with a chemical inhibitor or with specific siRNA significantly inhibited TRAIL-R2-mediated apoptosis of RASFs. Furthermore, blockade of caspase-4 was paralleled by persistence of a cytosolic pattern of p53 and absence of p53 translocation to the nucleus. Taken together, our findings suggest a unique role for caspase-4 in cleaving vimentin and releasing cytosolic p53 for nuclear translocation, events that may regulate the sensitivity of RASFs to receptor-mediated apoptosis.

Purpose of Review: The genetic basis of systemic lupus erythematosus, a complex genetic trait, may provide important insights into autoimmune disease. Innovation in both practical and theoretical approaches will assist in accelerating the pace of discovery and our understanding of pathogenesis., Recent Findings: Significant progress has been made in the last year with respect to the refinement of genetic intervals to promising candidate genes involved in systemic lupus erythematosus pathogenesis and specific phenotype susceptibility. This review highlights these discoveries and suggests platforms that may affect the future of analysis of this complex disease., Summary: Understanding the genetic basis for systemic lupus erythematosus disease and sub-phenotype susceptibility will have a substantial effect on the therapeutic interventions used to care for patients.

The cytoplasmic domain (CY) of the ligand-binding alpha-chain of the gamma-chain-associated FcRs can modulate receptor function such as phagocytosis, endocytosis, and intracellular trafficking of receptor-Ag complexes. To assess the potential role of the CY domain of human FcgammaRIa (CD64) alpha-chain in the transcriptional regulation of receptor-induced gene expression, we developed stably transfected murine macrophage cell lines expressing a full-length or a CY deletion mutant (tail-less) of human FcgammaRIa to analyze gene expression in response to receptor-specific cross-linking. Using the Affymetrix murine genome U74Av2 GeneChip array, we observed >100 candidate genes having > or =2-fold difference expression at 1.5 and 3 h after stimulation. Focusing on several immunologically related genes, we confirmed differential expression of M-CSF, macrophage inhibitory cytokine-1, leukocyte-specific protein 1, MIP-2, and IL-1R antagonist by RT-PCR and RNase protection assays. Analysis of mRNA stability indicated that the differential regulation of gene expression by the CY of the CD64 alpha-chain is at the level of gene transcription. Our results indicate that the CY of the CD64 alpha-chain modulates transcriptional activity induced by receptor-specific engagement in macrophages and provides a framework for understanding distinct expression profiles elicited by different Fc gamma-chain-associated receptors.

Objective: To examine the roles of specific genetic polymorphisms as predictors of response to treatment of early rheumatoid arthritis (RA)., Methods: Subjects included 457 patients with early RA (duration of < or =3 years) who participated in a randomized controlled trial comparing weekly methotrexate and 2 dosages of etanercept (10 mg twice weekly and 25 mg twice weekly). Our primary outcome measure was achievement of 50% improvement in disease activity according to the criteria of the American College of Rheumatology (ACR50 response) after 12 months of treatment. Subjects were genotyped for HLA-DRB1 alleles and polymorphisms in the following genes: TNF, LTA, TNFRSF1A, TNFRSF1B, FCGR2A, FCGR3A, and FCGR3B. Univariate and multivariate analyses were performed to define the impact of specific polymorphisms and haplotypes on response to treatment. Covariates for the multivariate analyses included sex, ethnicity, age, disease duration, and baseline values for rheumatoid factor and the tender and swollen joint counts., Results: The presence of 2 HLA-DRB1 alleles encoding the shared epitope (SE) (compared with 1 or 0 copies) was associated with response to treatment with standard-dose etanercept (odds ratio [OR] 4.3, 95% confidence interval [95% CI] 1.8-10.3). Among Caucasian patients, 2 extended haplotypes that included the HLA-DRB1 alleles *0404 and *0101 (both of which encode the SE) and 6 single-nucleotide polymorphisms in the LTA-TNF region were associated with response to treatment. In a multivariate model that included treatment received and the aforementioned covariates, the ORs for the association of these haplotypes with achievement of an ACR50 response at 12 months were 2.5 (95% CI 0.8-7.3) and 4.9 (95% CI 1.5-16.1) for the *0404- and *0101-containing haplotypes, respectively., Conclusion: Genetic variation in the HLA-DRB1 and the LTA-TNF regions is significantly associated with response to treatment of early RA. These findings may have clinical application through the identification of patients who are most likely to benefit from treatment with methotrexate or etanercept.

The immunoreceptor tyrosine-based inhibitory motif-containing FcgammaRIIb modulates immune function on multiple cell types including B cells, monocytes/macrophages, and dendritic cells. The promoter for the human FCGR2B is polymorphic, and the less frequent 2B.4 promoter haplotype is associated with the autoimmune phenotype of systemic lupus erythematosus. In the present study, we demonstrate that the 2B.4 promoter haplotype of FCGR2B has increased binding capacity for GATA4 and Yin-Yang1 (YY1) transcription factors in both B lymphocytes and monocytes, and that overexpression of GATA4 or YY1 enhances the FCGR2B promoter activity. The 2B.4 haplotype leads to elevated expression of the endogenous receptor in heterozygous donors by approximately 1.5-fold as assessed on EBV-transformed cells, primary B lymphocytes, and CD14(+) monocytes. This increased expression accentuates the inhibitory effect of FcgammaRIIb on B cell Ag receptor signaling, measured by Ca(2+) influx and cell viability in B cells. Our results indicate that transcription factors GATA4 and YY1 are involved in the regulation of FcgammaRIIb expression, and that the expression variants of FcgammaRIIb lead to altered cell signaling, which may contribute to autoimmune pathogenesis in humans.

Objective: Serum B lymphocyte stimulator (BLyS) is increased in autoimmune diseases, both in animal models and in humans. This study examined the effect of BLyS blockade in 3 animal models of lupus., Methods: Antibodies and lupus-like disease manifestations were examined in mice after administration of a single injection of an adenoviral construct for the transmembrane activator and CAML interactor receptor (AdTACI) that produces high serum levels of TACI-Fc fusion protein., Results: In C57BL/6 (B6) lpr/lpr mice (B6.lpr/lpr), which were used to model autoimmunity in the absence of severe disease, treatment of younger mice with AdTACI prevented the development of hypergammaglobulinemia. In contrast, use of AdTACI for BLyS blockade had only transient effects on the levels of IgG in normal B6 mice. AdTACI blocked the development of autoantibodies in younger B6.lpr/lpr mice and reversed the production of autoantibodies in older B6.lpr/lpr mice, and also reduced the numbers of splenic plasma cells. In MRL.lpr/lpr mice, which were used to examine disease manifestations, AdTACI reduced the extent of glomerulonephritis and proteinuria and improved survival, but had little effect on T cell infiltration and interstitial nephritis. However, in (NZB x NZW)F(1) mice, AdTACI induced neutralizing anti-TACI antibodies and failed to reduce the numbers of B cells., Conclusion: BLyS blockade has little effect on IgG levels in normal mice, but reverses the production of spontaneously produced IgM and IgG autoantibodies in the setting of established autoimmunity. Blockade of BLyS ameliorates B cell-dependent disease manifestations even in the MRL.lpr/lpr model, but its effectiveness on autonomous T cell aspects of the disease is limited. Moreover, its effectiveness is neutralized by anti-TACI antibodies when present. These results provide a basis for understanding the potential effects of BLyS blockade in human disease.

FcgammaRIIb, the immunoreceptor tyrosine-based inhibitory motif-containing receptor for IgG (Mendelian Inheritance in Man no. 604590), plays an important role in maintaining the homeostasis of immune responses. We have identified 10 novel single-nucleotide polymorphisms in the promoter region of human FCGR2B gene and characterized two functionally distinct haplotypes in its proximal promoter. In luciferase reporter assays, the less frequent promoter haplotype leads to increased expression of the reporter gene in both B lymphoid and myeloid cell lines under constitutive and stimulated conditions. Four independent genome-wide scans support linkage of the human FcgammaR region to the systemic lupus erythematosus (SLE; Online Mendelian Inheritance in Man no. 152700) phenotype. Our case-control study in 600 Caucasians indicates a significant association of the less frequent FCGR2B promoter haplotype with the SLE phenotype (odds ratio = 1.65; p = 0.0054). The FCGR2B haplotype has no linkage disequilibrium with previously identified FCGR2A and FCGR3A polymorphisms, and after adjustment for FCGR2A and FCGR3A, FCGR2B showed a persistent association with SLE (odds ratio = 1.72; p = 0.0083). These results suggest that an expression variant of FCGR2B is a risk factor for human lupus and implicate FCGR2B in disease pathogenesis.

Antineutrophil cytoplasmic Abs (ANCA) can activate neutrophils in an FcgammaR-dependent manner, but the link between this ANCA-induced effect and mononuclear cell activation with the characteristic granuloma formation of Wegener's granulomatosis is unclear. Human alpha-defensins, small cationic antimicrobial peptides, are found in neutrophils and have chemotactic activity for T cells, dendritic cells, and monocytes. In this study, we quantitated the release of alpha-defensins (human neutrophil peptides 1-3) from human neutrophils after targeted FcgammaR cross-linking (XL). Homotypic XL of FcgammaRIIa, FcgammaRIIIb, or heterotypic XL of both receptors resulted in significant release of alpha-defensins, an effect also induced by both human polyclonal and murine monoclonal cytoplasmic staining ANCA (anti-proteinase 3). This release of alpha-defensins, as well as of other granule constituents (ANCA targets anti-proteinase 3 and myeloperoxidase and elastase), was significantly greater in donors homozygous for the NA1 allele of FcgammaRIIIb than in donors homozygous for NA2. Interestingly, the ANCA-induced release was completely inhibited by the IgG Fc-binding peptide TG19320, which blocks the IgG-Fc region from binding to FcgammaR. Based on their chemotactic properties, alpha-defensins and their release by ANCA may contribute to modulation of the acquired immune response and to granuloma formation. The greater activity of the FcgammaRIIIB-NA1 genotype may also explain the greater severity of disease and its flare-ups in patients with this allele.

Objective: The low-affinity receptor Fcgamma receptor IIb (FcgammaRIIb), with an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain, down-regulates humoral immune responses and modulates the risk of autoimmunity in animal models. The transmembrane domain of FcgammaRIIb may also contribute to receptor signaling. Therefore, we investigated the biologic significance of single-nucleotide polymorphisms (SNPs) throughout the coding region., Methods: Discovery of SNPs in FCGR2B was performed by direct cycle sequencing of complementary DNA samples derived by reverse transcriptase-polymerase chain reaction. To assess the biologic significance of the nonsynonymous transmembrane SNP, we studied 3 functions influenced by the FcgammaRIIb transmembrane domain: tyrosine dephosphorylation of CD19, inhibition of B cell receptor (BCR)-induced calcium response, and modulation of BCR- or anti-Fas-induced apoptosis., Results: The nonsynonymous C-to-T transition in the first cytoplasmic exon, originally reported in the Raji cell line, was not found in either the African-American or the Caucasian population, but a nonsynonymous T-to-C transition at nucleotide 775 in exon 4 of FCGR2B, which changes isoleucine to threonine at residue 187 in the transmembrane domain, was significantly more common in African Americans. Using the FcgammaRIIb-negative mouse B cell line IIA1.6, we expressed both allelic forms as both full-length and truncated cytoplasmic domain constructs. The FCGR2B-187T allele mediated a higher level of CD19 dephosphorylation (P = 0.029) and a greater degree of inhibition of the calcium response (P = 0.003) when co-engaged with BCR than did FCGR2B-187I, independent of the presence of the ITIM. In contrast, FcgammaRIIb modulation of BCR-induced and anti-Fas antibody-induced cell death rates were similar in IIA1.6 cells expressing either the 187I or the 187T allelic form., Conclusion: The differential activity of FCGR2B alleles suggests a novel mechanism of FcgammaRIIb regulation that may influence the risk of autoimmune disease.

Development of anti-Fas Abs to treat diseases with insufficient Fas-mediated apoptosis has been limited by concern about hepatotoxicity. We report here that hepatotoxicity elicited by anti-Fas Ab Jo2 is dependent on FcgammaRIIB. Thus, following Jo2 treatment, all FcgammaRIIB(-/-) mice survived while 80% of wild-type and all FcR-gamma(-/-) mice died from acute liver failure. Microscopic examination suggests that FcgammaRIIB deficiency protects the hepatic sinusoidal endothelium, a cell type that normally coexpresses Fas and FcgammaRIIB. In vitro studies showed that FcgammaRIIB, but not FcgammaRI and FcgammaRIII, on neighboring macrophages substantially enhanced Jo2 mediated apoptosis of Fas expressing target cells. However, FcgammaRI and FcgammaRIII appeared essential for apoptosis-inducing activity of a non-hepatotoxic anti-Fas mAb HFE7A. These findings imply that by interacting with the Fc region of agonistic Abs, FcgammaRs can modulate both the desired and undesired consequences of Ab-based therapy. Recognizing this fact should facilitate development of safer and more efficacious agonistic Abs.

A single-nucleotide polymorphism (SNP), identified at nucleotide position -844 in the 5' promoter of the FasL gene, lies within a putative binding motif for CAAT/enhancer-binding protein beta (C/EBPbeta). Electrophoretic mobility shift and supershift assays confirmed that this element binds specifically to C/EBPbeta and demonstrated that the two alleles of this element have different affinities for C/EBPbeta. In luciferase reporter assays, the -844C genotype had twice the basal activity of the -844T construct, and basal expression of Fas ligand (FasL) on peripheral blood fibrocytes was also significantly higher in -844C than in -844T homozygous donors. FasL is located on human chromosome 1q23, a region that shows linkage to the systemic lupus autoimmune phenotype. Analysis of 211 African American systemic lupus erythematosus patients revealed enrichment of the -844C homozygous genotype in these systemic lupus erythematosus patients compared with 150 ethnically matched normal controls (p = 0.024). The -844C homozygous genotype may lead to the increased expression of FasL, to altered FasL-mediated signaling in lymphocytes, and to enhanced risk for autoimmunity. This functionally significant SNP demonstrates the potential importance of SNPs in regulatory regions and suggests that differences in the regulation of FasL expression may contribute to the development of the autoimmune phenotype.

Newfound relatives of the classical Fc receptors (FcR) have been provisionally named the Fc receptor homologs (FcRH). The recent identification of eight human and six mouse FcRH genes substantially increases the size and functional potential of the FcR family. The extended family of FcR and FcRH genes spans approximately 15 Mb of the human chromosome 1q21-23 region, whereas in mice this family is split between chromosomes 1 and 3. The FcRH genes encode molecules with variable combinations of five subtypes of immunoglobulin (Ig) domains. The presence of a conserved sequence motif in one Ig domain subtype implies Ig Fc binding capability for many FcRH family members that are preferentially expressed by B lineage cells. In addition, most FcRH family members have consensus tyrosine-based activating and inhibitory motifs in their cytoplasmic domains, while the others lack features typical of transmembrane receptors. The FcRH family members, like the classical FcRs, come in multiple isoforms and allelic variations. The unique individual and polymorphic properties of the FcR/FcRH members indicate a remarkably diverse Fc receptor gene family with immunoregulatory function.

Although the cytoplasmic domain of the human FcgammaRIa alpha-chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcgammaRIa is constitutively phosphorylated, and the inhibition of dephosphorylation with okadaic acid, an inhibitor of type 1 and type 2A protein serine/threonine phosphatase, inhibits both receptor-induced activation of the early tyrosine phosphorylation cascade and receptor-specific phagocytosis. To explore the basis for these effects of the cytoplasmic domain of FcgammaRIa, we developed a series of human FcgammaRIa molecular variants, expressed in the murine macrophage cell line P388D1, and demonstrate that serine phosphorylation of the cytoplasmic domain is an important regulatory mechanism. Truncation of the cytoplasmic domain and mutation of the cytoplasmic domain serine residues to alanine abolish the okadaic acid inhibition of phagocytic function. In contrast, the serine mutants did not recapitulate the selective effects of cytoplasmic domain truncation on cytokine production. These results demonstrate for the first time a direct functional role for serine phosphorylation in the alpha-chain of FcgammaRIa and suggest that the cytoplasmic domain of FcgammaRI regulates the different functional capacities of the FcgammaRIa-receptor complex.

Objective: Although low-affinity alleles of human Fcgamma receptor types IIA and IIIA (FcgammaRIIA and FcgammaRIIIA, respectively) polymorphisms have been associated with systemic lupus erythematosus (SLE) in case-control studies, the relative contribution of these genes to SLE susceptibility has not been resolved., Methods: We analyzed the distribution of alleles of FcgammaRIIA, FcgammaRIIIA, and FcgammaRIIIB in 126 multiplex-SLE pedigrees and FcgammaRIIA and FcgammaRIIIA in a case-control replication study, using allele-specific polymerase chain reaction and direct sequencing of genomic DNA. Statistical tests of association were performed to detect evidence of linkage between the single nucleotide polymorphisms and SLE., Results: We found evidence for linkage at both the FcgammaRIIIA (single-point nonparametric linkage [NPL] 1.8, P = 0.038; multipoint NPL 2.7, P = 0.004) and the FcgammaRIIA (single-point NPL 2.0, P = 0.021; multipoint NPL 2.6, P = 0.006) loci, but not the FcgammaRIIIB locus. Family-based tests of association demonstrated increased transmission of the low-affinity F176 allele at the FcgammaRIIIA locus (odds ratio [OR] 2.18, P = 0.0005 by transmission disequilibrium test and P = 0.002, by pedigree disequilibrium test [PDT]), but little evidence of preferential transmission of alleles at FcgammaRIIA (P = 0.089 by PDT). Stratification by ethnicity showed preferential transmission of the associated FcgammaRIIIA allele both in families of African American ancestry and in those of European American ancestry. Despite significant linkage disequilibrium between these genes, 2- and 3-locus haplotype analysis of the extended Fcgamma receptor cluster did not reveal any significant association beyond that observed with FcgammaRIIIA alone. In a large case-control replication study of 438 patients with SLE and 219 controls, FcgammaRIIIA provided the strongest evidence of an FcgammaR-SLE association (additive model: V/V 176 versus V/F 176 OR 1.51, V/V 176 versus F/F 176 OR 1.98, P = 0.007)., Conclusion: To our knowledge, these data are the first to demonstrate linkage and both family-based and case-control-based association of FcgammaRIIIA with SLE. These data provide genetic evidence supporting a role for the physiologically relevant single nucleotide polymorphism of the FcgammaRIIIA gene in the pathophysiology of this complex genetic disease.

The proteins of the tumor necrosis factor (TNF) receptor superfamily are a group of cell-surface receptors critically involved in the maintenance of homeostasis of the immune system. By interacting with their corresponding ligands, these receptors either induce cell death or promote cell survival of immune cells. The number of recognized members of the TNF receptor and ligand superfamily has expanded substantially in the last several years. More important, the biologic function of this group of proteins has been closely associated with the regulation of the immune response and the pathogenesis of autoimmune disease. Thus, the direct targeting of these receptors by either inducing apoptosis or blocking survival of autoimmune T and B cells may be an important therapeutic strategy in the treatment of autoimmune disease. This review summarizes the recent progress in immunobiology of the TNF receptor superfamily and focuses on our studies of three critical family members-FasL/Fas, TNF-related apoptosis-inducing ligand (TRAIL)/TRAIL-Rs, and B lymphocyte stimulator(BLyS)/BLyS-Rs--to demonstrate the therapeutic potential of targeting these receptors for the treatment of autoimmune disease.

Immune complex initiation of inflammatory diseases is mediated, to a large extent, by Fcgamma receptors. Recent data have underscored three essential points: (1) there is substantial structural diversity within three distinct families of Fcgamma receptors; (2) there are functionally significant alleles of different receptor isoforms; and (3) each cell type has a unique distribution of Fcgamma receptor isoforms. Structurally different Fcgamma receptors utilize distinct signal transduction pathways. Thus, in clinical immune complex disease, consideration of the predominant cell type and receptor isoform involved, as well as the genotype of the individual patient, will be important in developing and applying targeted downregulatory agents such as misoprostol.