Genetic association of CD247 (CD3ζ) with SLE in a large-scale multiethnic study.

A classic T-cell phenotype in systemic lupus erythematosus (SLE) is the downregulation and replacement of the CD3ζ chain that alters T-cell receptor signaling. However, genetic associations with SLE in the human CD247 locus that encodes CD3ζ are not well established and require replication in independent cohorts. Our aim was therefore to examine, localize and validate CD247-SLE association in a large multiethnic population. We typed 44 contiguous CD247 single-nucleotide polymorphisms (SNPs) in 8922 SLE patients and 8077 controls from four ethnically distinct populations. The strongest associations were found in the Asian population (11 SNPs in intron 1, 4.99 × 10(-4) < P < 4.15 × 10(-2)), where we further identified a five-marker haplotype (rs12141731-rs2949655-rs16859085-rs12144621-rs858554; G-G-A-G-A; P(hap) = 2.12 × 10(-5)) that exceeded the most associated single SNP rs858554 (minor allele frequency in controls = 13%; P = 4.99 × 10(-4), odds ratio = 1.32) in significance. Imputation and subsequent association analysis showed evidence of association (P < 0.05) at 27 additional SNPs within intron 1. Cross-ethnic meta-analysis, assuming an additive genetic model adjusted for population proportions, showed five SNPs with significant P-values (1.40 × 10(-3) < P< 3.97 × 10(-2)), with one (rs704848) remaining significant after Bonferroni correction (P(meta) = 2.66 × 10(-2)). Our study independently confirms and extends the association of SLE with CD247, which is shared by various autoimmune disorders and supports a common T-cell-mediated mechanism.

A functional haplotype of UBE2L3 confers risk for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical manifestations characterized by the development of pathogenic autoantibodies manifesting in inflammation of target organs such as the kidneys, skin and joints. Genome-wide association studies have identified genetic variants in the UBE2L3 region that are associated with SLE in subjects of European and Asian ancestry. UBE2L3 encodes an ubiquitin-conjugating enzyme, UBCH7, involved in cell proliferation and immune function. In this study, we sought to further characterize the genetic association in the region of UBE2L3 and use molecular methods to determine the functional effect of the risk haplotype. We identified significant associations between variants in the region of UBE2L3 and SLE in individuals of European and Asian ancestry that exceeded a Bonferroni-corrected threshold (P<1 × 10(-4)). A single risk haplotype was observed in all associated populations. Individuals harboring the risk haplotype display a significant increase in both UBE2L3 mRNA expression (P=0.0004) and UBCH7 protein expression (P=0.0068). The results suggest that variants carried on the SLE-associated UBE2L3 risk haplotype influence autoimmunity by modulating UBCH7 expression.

Role of MYH9 and APOL1 in African and non-African populations with lupus nephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by autoantibody production and organ damage. Lupus nephritis (LN) is one of the most severe manifestations of SLE. Multiple studies reported associations between renal diseases and variants in the non-muscle myosin heavy chain 9 (MYH9) and the neighboring apolipoprotein L 1 (APOL1) genes. We evaluated 167 variants spanning MYH9 for association with LN in a multiethnic sample. The two previously identified risk variants in APOL1 were also tested for association with LN in European-Americans (EAs) (N = 579) and African-Americans (AAs) (N = 407). Multiple peaks of association exceeding a Bonferroni corrected P-value of P < 2.03 × 10(-3) were observed between LN and MYH9 in EAs (N = 4620), with the most pronounced association at rs2157257 (P = 4.7 × 10(-4), odds ratio (OR) = 1.205). A modest effect with MYH9 was also detected in Gullah (rs8136069, P = 0.0019, OR = 2.304). No association between LN and MYH9 was found in AAs, Asians, Amerindians or Hispanics. This study provides the first investigation of MYH9 in LN in non-Africans and of APOL1 in LN in any population, and presents novel insight into the potential role of MYH9 in LN in EAs.

Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disorder with a complex pathogenesis in which genetic, hormonal and environmental factors have a role. Rare mutations in the TREX1 gene, the major mammalian 3'-5' exonuclease, have been reported in sporadic SLE cases. Some of these mutations have also been identified in a rare pediatric neurological condition featuring an inflammatory encephalopathy known as Aicardi-Goutières syndrome (AGS). We sought to investigate the frequency of these mutations in a large multi-ancestral cohort of SLE cases and controls. A total of 40 single-nucleotide polymorphisms (SNPs), including both common and rare variants, across the TREX1 gene, were evaluated in ∼8370 patients with SLE and ∼7490 control subjects. Stringent quality control procedures were applied, and principal components and admixture proportions were calculated to identify outliers for removal from analysis. Population-based case-control association analyses were performed. P-values, false-discovery rate q values, and odds ratios (OR) with 95% confidence intervals (CI) were calculated. The estimated frequency of TREX1 mutations in our lupus cohort was 0.5%. Five heterozygous mutations were detected at the Y305C polymorphism in European lupus cases but none were observed in European controls. Five African cases incurred heterozygous mutations at the E266G polymorphism and, again, none were observed in the African controls. A rare homozygous R114H mutation was identified in one Asian SLE patient, whereas all genotypes at this mutation in previous reports for SLE were heterozygous. Analysis of common TREX1 SNPs (minor allele frequency (MAF)>10%) revealed a relatively common risk haplotype in European SLE patients with neurological manifestations, especially seizures, with a frequency of 58% in lupus cases compared with 45% in normal controls (P=0.0008, OR=1.73, 95% CI=1.25-2.39). Finally, the presence or absence of specific autoantibodies in certain populations produced significant genetic associations. For example, a strong association with anti-nRNP was observed in the European cohort at a coding synonymous variant rs56203834 (P=2.99E-13, OR=5.2, 95% CI=3.18-8.56). Our data confirm and expand previous reports and provide additional support for the involvement of TREX1 in lupus pathogenesis.

Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing.

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls, P=0.016, OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10, directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

Complement component c5a is integral to the febrile response of mice to lipopolysaccharide.

OBJECTIVES: The complement system is critical to the febrile response of mice to intraperitoneally administered lipopolysaccharide (LPS). We previously identified C3 and C5 as two components potentially involved in this response. This study was designed to examine whether the complement system is also pivotal in the response of mice to intravenously or intracerebroventricularly injected LPS, to distinguish between C3 and C5 and their cognate derivatives as the essential mediator(s), and to determine whether the failure of complement-deficient mice to develop a fever could be due to their possible inability to secrete pyrogenic cytokines. METHODS: Wild-type (WT; C57BL/6J) mice, hypocomplemented or not by intravenously injected cobra venom factor (10 U/mouse), and C3-, CR3- and C5-sufficient and -deficient mice were intravenously challenged with LPS (0.25 mug/mouse); WT and C3-/- mice pretreated with a C5a receptor antagonist (C5aRa) were similarly challenged. In addition, the serum levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and IL-6 were compared in LPS-treated C5+/+ and C5-/- mice. RESULTS: LPS induced a 1 degrees C rise in core temperature in all the mice, except C5-/- mice and those pretreated with C5aRa. C5+/+ and C5-/- mice challenged intracerebroventricularly with LPS exhibited identical febrile responses. LPS induced similar increases in the serum levels of IL-1beta, TNFalpha and IL-6 in C5+/+ and C5-/- mice. CONCLUSIONS: C5a is crucial for the development of febrile responses to LPS in mice; its site of action is peripheral, not central. The possibility that an inability to produce cytokines could account for the failure of C5-/- mice to develop a fever is not supported.

Modulation of mouse endotoxic fever by complement.

It was recently reported that the complement system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneally (i.p.) injected, lipopolysaccharides (LPS). The present study was designed to identify which component(s) of the complement cascade may be specifically critical. To this end, we used mice with C3, C5, and CR2 gene deletions. To assess preliminarily the suitability of mice for such a study, we replicated our earlier studies with guinea pigs. Thus, to verify initially whether complement is similarly involved in the febrile response of wild-type (C57BL/6J) mice to i.p. LPS (Escherichia coli, 1 microg/mouse), we depleted complement with cobra venom factor (CVF; 7 U/mouse, intravenously [i.v.]). These animals did not develop fever, whereas the core temperature (T(c)) of CVF vehicle-treated controls rose approximately 1 degrees C by 80 min postinjection and then gradually abated over the following 2.5 h, confirming the involvement of complement in fever production after i.p. LPS injection and the suitability of this species for these studies. C3- and C5-sufficient (C3(+/+) and C5(+/+)) mice also developed 1 degrees C fevers within 80 min after i.p. LPS (1 or 2 microg/mouse) injection. These fevers were totally prevented by CVF (10 U/mouse, i.v.) pretreatment. C3- and C5-deficient (C3(-/-) and C5(-/-)) mice were also unable to develop T(c) rises after i.p. LPS. Both CR2(+/+) and CR2(-/-) mice responded normally to i.p. LPS (1 microg/mouse). These data indicate that C5, but not C3d acting through CR2, may play a critical role in the febrile response of mice to i.p. LPS.

Cr2, a candidate gene in the murine Sle1c lupus susceptibility locus, encodes a dysfunctional protein.

The major murine systemic lupus erythematosus (SLE) susceptibility locus, Sle1, corresponds to three loci independently affecting loss of tolerance to chromatin in the NZM2410 mouse. The congenic interval corresponding to Sle1c contains Cr2, which encodes complement receptors 1 and 2 (CR1/CR2, CD35/CD21). NZM2410/NZW Cr2 exhibits a single nucleotide polymorphism that introduces a novel glycosylation site, resulting in higher molecular weight proteins. This polymorphism, located in the C3d binding domain, reduces ligand binding and receptor-mediated cell signaling. Molecular modeling based on the recently solved CR2 structure in complex with C3d reveals that this glycosylation interferes with receptor dimerization. These data demonstrate a functionally significant phenotype for the NZM2410 Cr2 allele and strongly support its role as a lupus susceptibility gene.

Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function.

CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement. We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21. A sequence coding for a biotinylation signal peptide was added to the 3' end of the human C3dg cDNA. The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme. Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly. Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor. Two assays were used to assess the functional capacity of the recombinant C3dg. First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells. Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody. These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.

Complement component C3 is not required for full expression of immune complex glomerulonephritis in MRL/lpr mice.

Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g., Factor B) and infusion of complement inhibitors (e.g., Crry, anti-C5 Ab) protect against inflammatory renal disease. Paradoxically, genetic deficiencies of early components of the classical complement pathway (e.g., C1q, C4, and C2) are associated with an increased incidence of lupus in humans and lupus-like disease in murine knockout strains. Complement protein C3 is the converging point for activation of all three complement pathways and thus plays a critical role in biologic processes mediated by complement activation. To define the role of C3 in lupus nephritis, mice rendered C3 deficient by targeted deletion were backcrossed for eight generations to MRL/lpr mice, a mouse strain that spontaneously develops lupus-like disease. We derived homozygous knockout (C3(-/-)), heterozygous (C3(+/-)), and C3 wild-type (C3(+/+)) MRL/lpr mice. Serum levels of autoantibodies and circulating immune complexes were similar among the three groups. However, there was earlier and significantly greater albuminuria in the C3(-/-) mice compared with the other two groups. Glomerular IgG deposition was also significantly greater in the C3(-/-) mice than in the other two groups, although overall pathologic renal scores were similar. These results indicate that C3 and/or activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice and may in fact play a beneficial role via clearance of immune complexes.

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B.

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.

Complement opsonization is required for presentation of immune complexes by resting peripheral blood B cells.

Complement receptor 2 (CD21, CR2) is a B cell receptor for complement degradation products bound to Ag or immune complexes. The role of CD21 in mediating Ag presentation of soluble immune complexes by resting B cells was studied. Complement-coated immune complexes were formed by the incubation of influenza virus with serum from immune donors. These complexes bound to peripheral blood B cells in a complement-dependent manner. The binding required CD21 or, to a lesser extent, complement receptor 1 (CR1, CD35). B cells pulsed with immune complexes containing complement elicited a response from a panel of influenza-specific T cell clones, while those pulsed with immune complexes formed in the absence of complement did not. The expression of the early activation marker CD69 and the costimulatory molecule CD86 were not induced by CD21 ligation alone, suggesting that CD21-mediated Ag presentation occurs independently of B cell activation. Up-regulation of these markers required exposure to T cell factors elicited by the recognition of Ag derived from complement-containing immune complexes. These findings suggest that binding of Ag to CD21 enables Ag-nonspecific B cells to participate in the activation of Ag-specific T cells in a process that occurs independently of well-characterized B cell activation events.

CD21 augments antigen presentation in immune individuals.

CD21 (complement receptor 2, CR2) binds the C3 degradation products, iC3b and C3d, which are covalently linked to antigen or immune complexes in the process of complement activation. The ability of antigen-nonspecific B cells to present immune complexes containing high titers of acquired antibodies was tested. Influenza virus was incubated with serum from immune donors to create complement-containing complexes. These bound specifically to CD21 on transfected fibroblasts and B cell lines, as measured by microcytofluorimetry. Binding of immune complexes was ablated by inactivation of serum complement. In addition, the immunoglobulin in immune human serum blocked influenza binding to cells in the absence of complement, implying a minimal role for immunoglobulin-Fc receptor interactions in this system. Significant immune complex binding required a threshold level of CD21 expression, suggesting that only those cells with the highest levels of CD21 are likely to participate in the processing of macromolecular antigens. B cells pulsed with complement-influenza complexes elicited an augmented response from a panel of influenza-specific, class II-restricted T cell clones, as compared with those which had bound immunoglobulin-influenza complexes lacking complement. This enhanced response did not require CD35. In addition, B cell lines expressing higher levels of CD21 were more efficient in processing antigen than those with lower levels. These findings suggest that presentation of antigen by B cells in immune individuals is dependent on the binding of complement-antigen immune complexes to CD21.