Search

OBJECTIVE Elevated interleukin 10 (IL10) in SLE patients has B-cell promoting effects, contributing to autoantibody production and tissue damage. We aimed to characterize upregulated IL10+ B-cell subsets and dysregulated IL10 expression in SLE B cells for new therapeutic options. METHODS Proportions of Th10 and IL10+ B-cell subsets in PBMCs were assessed using flow cytometry. The IL10-3'UTR dual-luciferase vector was constructed and co-transfected with siRNA, miRNA mimics or inhibitors into RAJI cell line. Transcript levels were quantified using Taqman assays. RESULTS Culture conditions that induced IL10+ Breg in healthy controls (HC) resulted in expansion of IL10+ DN2 (IgD- CD27- CD21- CD11c+ ) B-cells in SLE PBMCs. Proportions of IL10+ DN2, but not those of IL10- DN2, correlated with disease activity, levels of antibodies to dsDNA, and associated with high levels or seropositivity of anti-Smith and anti-cardiolipin IgG in SLE patients from two cohorts of mainly African Americans and Asians, respectively. Proportions of Th10 (CD45RA- CXCR5- CXCR3+ PD1hi CD4+ ) cells correlated with IL10+ DN2 frequencies, ANA titers and proteinuria levels in SLE patients. Screening predicted IL10 3'UTR-targeting miRNAs in SLE B cells identified miR-17-5p and miR-20a-5p with their levels inversely correlated with IL10 and transcription factor E2F2. In RAJI cells, knockdown of E2F2 expression resulted in increased levels of miR-17-5p and miR-20a-5p and decreased IL10 mRNA and protein levels, and overexpression and inhibition of miR-17-5p down-regulated and up-regulated IL10 mRNA levels respectively; suggesting regulation of IL10 expression by E2F2-miR-17-5p loop. CONCLUSION IL10 promotes extrafollicular autoimmune responses in active SLE patients, which might be dampened by targeting the E2F2-miR-17-5p circuitry.

Objective Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a strong genetic component. We undertook the present work to perform the first genome-wide association study on individuals from the Americas who are enriched for Native American heritage. Methods We analyzed 3,710 individuals from the US and 4 countries of Latin America who were diagnosed as having SLE, and healthy controls. Samples were genotyped with HumanOmni1 BeadChip. Data on out-of-study controls genotyped with HumanOmni2.5 were also included. Statistical analyses were performed using SNPtest and SNPGWA. Data were adjusted for genomic control and false discovery rate. Imputation was performed using Impute2 and, for classic HLA alleles, HiBag. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Results The IRF5–TNPO3 region showed the strongest association and largest OR for SLE (rs10488631: genomic control–adjusted P [Pgcadj] = 2.61 × 10−29, OR 2.12 [95% CI 1.88–2.39]), followed by HLA class II on the DQA2–DQB1 loci (rs9275572: Pgcadj = 1.11 × 10−16, OR 1.62 [95% CI 1.46–1.80] and rs9271366: Pgcadj = 6.46 × 10−12, OR 2.06 [95% CI 1.71–2.50]). Other known SLE loci found to be associated in this population were ITGAM, STAT4, TNIP1, NCF2, and IRAK1. We identified a novel locus on 10q24.33 (rs4917385: Pgcadj = 1.39 × 10−8) with an expression quantitative trait locus (eQTL) effect (Peqtl = 8.0 × 10−37 at USMG5/miR1307), and several new suggestive loci. SLE risk loci previously identified in Europeans and Asians were corroborated. Local ancestry estimation showed that the HLA allele risk contribution is of European ancestral origin. Imputation of HLA alleles suggested that autochthonous Native American haplotypes provide protection against development of SLE. Conclusion Our results demonstrate that studying admixed populations provides new insights in the delineation of the genetic architecture that underlies autoimmune and complex diseases.

Objective To evaluate whether olfactory 1/early B cell factor–associated zinc-finger protein (OAZ), a candidate lupus susceptibility gene involved in antinuclear antibody (ANA) production, plays a role in the regulation of B cells by mesenchymal stem cells (MSCs). Methods MSCs derived from the bone marrow of patients with systemic lupus erythematosus (SLE) and healthy control subjects were expanded and incubated with small interfering RNAs specific for OAZ or a nontargeting sequence. Knockdown of messenger RNA levels of OAZ and its downstream genes was measured using real-time polymerase chain reaction, and protein levels of chemokine/cytokine and immunoglobulins were determined by enzyme-linked immunosorbent assay or Western blotting. The effects of modulating the OAZ levels in MSCs, by either silencing or overexpression, on B cell proliferation and terminal differentiation were assessed by coculturing MSCs with mouse spleen cells. Results OAZ gene expression was highly enriched in MSCs compared with peripheral blood leukocytes and was increased in patients with SLE compared with control subjects. After the silencing of OAZ expression, SLE MSCs could regain the ability to inhibit B cell proliferation and terminal differentiation, as indicated by decreased percentages of bromodeoxyuridine-positive cells and CD138+ cells as well as decreased levels of IgG, IgM, and ANAs. The level of CCL2 was increased after OAZ knockdown, while anti-CCL2 antibodies completely counteracted the effect of OAZ silencing. Umbilical cord–derived normal MSCs that overexpressed OAZ had a diminished ability to inhibit B cell proliferation and terminal differentiation. Conclusion OAZ down-regulation could restore the impaired function of SLE MSCs in the immune regulation of B cells, contributing to a reduction in ANA levels. OAZ might represent a new target for therapy in patients with SLE.

Objective Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE) that exhibits familial aggregation and may progress to end-stage renal disease (ESRD). LN is more prevalent among African Americans than among European Americans. This study was undertaken to investigate the hypothesis that the apolipoprotein L1 gene (APOL1) nephropathy risk alleles G1/G2, common in African Americans and rare in European Americans, contribute to the ethnic disparity in risk.

Objective The increased risk of thrombosis in systemic lupus erythematosus (SLE) may be partially explained by interrelated genetic pathways for thrombosis and SLE. The present study was undertaken to investigate whether 33 established and novel single-nucleotide polymorphisms (SNPs) in 20 genes involved in hemostasis pathways that have been associated with deep venous thrombosis (DVT) in the general population are risk factors for SLE among Asian subjects. Methods Patients in the discovery cohort were enrolled in 1 of 2 North American SLE cohorts. Patients in the replication cohort were enrolled in 1 of 4 Asian or 2 North American cohorts. We first genotyped 263 Asian patients with SLE and 357 healthy Asian control subjects for 33 SNPs in the discovery phase, and then genotyped 5 SNPs in up to an additional 1,496 patients and 993 controls in the replication phase. Patients were compared to controls for bivariate association with minor alleles. Principal components analysis was used to control for intra-Asian ancestry in the replication cohort. Results Two genetic variants in the gene VKORC1 were highly significant in both the discovery and replication cohorts: rs9934438 (in the discovery cohort, odds ratio [OR] 2.45, P = 2 × 10−9; in the replication cohort, OR 1.54, P = 4 × 10−6) and rs9923231 (in the discovery cohort, OR 2.40, P = 6 × 10−9; in the replication cohort, OR 1.53, P = 5 × 10−6). These associations were significant in the replication cohort after adjustment for intra-Asian ancestry: for rs9934438, OR 1.34, P = 0.0029; for rs9923231, OR 1.34, P = 0.0032. Conclusion Genetic variants in VKORC1, which are involved in vitamin K reduction and associated with DVT, correlate with SLE development in Asian subjects. These results suggest that there may be intersecting genetic pathways for the development of SLE and thrombosis.

Objective American Indian-Europeans, Asians, and African Americans have an excess morbidity from systemic lupus erythematosus (SLE) and a higher prevalence of lupus nephritis than do Caucasians. The aim of this study was to analyze the relationship between genetic ancestry and sociodemographic characteristics and clinical features in a large cohort of American Indian-European SLE patients. Methods A total of 2,116 SLE patients of American Indian-European origin and 4,001 SLE patients of European descent for whom we had clinical data were included in the study. Genotyping of 253 continental ancestry-informative markers was performed on the Illumina platform. Structure and Admixture software were used to determine genetic ancestry proportions of each individual. Logistic regression was used to test the association between genetic ancestry and sociodemographic and clinical characteristics. Odds ratios (ORs) were calculated with 95% confidence intervals (95% CIs). Results The average American Indian genetic ancestry of 2,116 SLE patients was 40.7%. American Indian genetic ancestry conferred increased risks of renal involvement (P less than 0.0001, OR 3.50 [95% CI 2.63- 4.63]) and early age at onset (P less than 0.0001). American Indian ancestry protected against photosensitivity (P less than 0.0001, OR 0.58 [95% CI 0.44-0.76]), oral ulcers (P less than 0.0001, OR 0.55 [95% CI 0.42-0.72]), and serositis (P less than 0.0001, OR 0.56 [95% CI 0.41-0.75]) after adjustment for age, sex, and age at onset. However, age and sex had stronger effects than genetic ancestry on malar rash, discoid rash, arthritis, and neurologic involvement. Conclusion In general, American Indian genetic ancestry correlates with lower sociodemographic status and increases the risk of developing renal involvement and SLE at an earlier age. Copyright © 2012 by the American College of Rheumatology.

Objective Candidate gene and genome-wide association studies have identified several disease susceptibility loci in lupus patients. These studies have largely been performed in lupus patients who are Asian or of European ancestry. This study was undertaken to examine whether some of these same susceptibility loci increase lupus risk in African American individuals. Methods Single-nucleotide polymorphisms tagging 15 independent lupus susceptibility loci were genotyped in a set of 1,724 lupus patients and 2,024 healthy controls of African American descent. The loci examined included PTPN22, FCGR2A, TNFSF4, STAT4, CTLA4, PDCD1, PXK, BANK1, MSH5 (HLA region), CFB (HLA region), C8orf13-BLK region, MBL2, KIAA1542, ITGAM, and MECP2/IRAK1. Results We found the first evidence of genetic association between lupus in African American patients and 5 susceptibility loci (C8orf13-BLK, BANK1, TNFSF4, KIAA1542, and CTLA4; P = 8.0 × 10−6, P = 1.9 × 10−5, P = 5.7 × 10−5, P = 0.00099, and P = 0.0045, respectively). Further, we confirmed the genetic association between lupus and 5 additional lupus susceptibility loci (ITGAM, MSH5, CFB, STAT4, and FCGR2A; P = 7.5 × 10−11, P = 5.2 × 10−8, P = 8.7 × 10−7, P = 0.0058, and P = 0.0070, respectively), and provided evidence, for the first time, of genome-wide significance for the association between lupus in African American patients and ITGAM and MSH5 (HLA region). Conclusion These findings provide evidence of novel genetic susceptibility loci for lupus in African Americans and demonstrate that the majority of lupus susceptibility loci examined confer lupus risk across multiple ethnicities.

Objective A previous genome-wide association study conducted in a population of European ancestry identified rs4963128, a KIAA1542 single-nucleotide polymorphism (SNP) 23 kb telomeric to IRF7 (the gene for interferon regulatory factor 7 [IRF-7]), to be strongly associated with systemic lupus erythematosus (SLE). This study was undertaken to investigate whether genetic polymorphism within IRF7 is a risk factor for the development of SLE. Methods We genotyped one KIAA1542 SNP (rs4963128) and one IRF7 SNP (rs1131665 [Q412R]) in an Asian population (1,302 cases, 1,479 controls), to assess their association with SLE. Subsequently, rs1131665 was further genotyped in independent panels of Chinese subjects (528 cases, 527 controls), European American subjects (446 cases, 461 controls), and African American subjects (159 cases, 115 controls) by TaqMan genotyping assay, to seek confirmation of association in various ethnic groups. A luciferase reporter assay was used to assess the effect of Q412R polymorphism on the activation of IRF-7. Results Consistent association of rs1131665 (Q412R) with SLE was identified in Asian, European American, and African American populations (total 2,435 cases and 2,582 controls) (Pmeta = 6.18 × 10−6, odds ratio 1.42 [95% confidence interval 1.22–1.65]). Expression of the IRF7 412Q risk allele resulted in a 2-fold increase in interferon-stimulated response element transcriptional activity compared with expression of IRF7 412R (P = 0.0003), suggesting that IRF7 412Q confers elevated IRF-7 activity and may therefore affect a downstream interferon pathway. Conclusion These findings show that the major allele of a nonsynonymous SNP, rs1131665 (412Q) in IRF7, confers elevated activation of IRF-7 and predisposes to the development of SLE in multiple ethnic groups. This result provides direct genetic evidence that IRF7 may be a risk gene for human SLE.

Objective We previously observed the association of the co-occurrence of the HLA–DRB1 shared epitope (SE) and RANKL single-nucleotide polymorphisms (SNPs) with younger age at the onset of rheumatoid arthritis (RA) in 182 rheumatoid factor (RF)–positive European American patients with early-onset RA. The aim of this study was to fine-map the 48-kb RANKL region in the extended cohort of 210 European American RF-positive patients with early RA, to seek replication of RA-associated SNPs in additional RA cohorts of 501 European Americans and 298 African Americans, and to explore the functional consequences of RA-associated SNPs. Methods SNP genotyping was conducted using pyrosequencing or TaqMan polymerase chain reaction (PCR) assays. Associations of rs7984870 with RANKL expression in plasma, peripheral blood mononuclear cells, and isolated T cells were quantified using enzyme-linked immunosorbent assay and reverse transcription–PCR. Site-directed mutagenesis of rs7984870 within the 2-kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay. Results A single promoter SNP, rs7984870, was consistently significantly associated with earlier age at the onset of RA in 3 independent seropositive (RF or anti–cyclic citrullinated peptide antibody) RA cohorts but not in seronegative RA patients. The C risk allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive patients with RA, significantly elevated RANKL messenger RNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to the transcription factor SOX5. Conclusion The RANKL promoter allele that increased transcription levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to a younger age at the onset of RA in seropositive European American and African American patients.

Supported by grants from the NIH (5P01-AI-083194 to Drs. Lessard, Rasmussen, Gaffney, Alarcon-Riquelme, Criswell, Jacob, Kimberly, Vyse, Harley, Sivils, Langefeld, and Tsao; 5P01-AR-049084 to Drs. Kimberly and Langefeld; and R01-AR-043814 and R21-AR-065626 to Dr. Tsao), the Oklahoma Medical Research Foundation (to Drs. Lessard, Gaffney, and Sivils), the Alliance for Lupus Research (to Drs. Criswell and Tsao), and the Wake Forest School of Medicine Center for Public Health Genomics (to Dr. Langefeld). Dr. Criswell is recipient of a Kirkland Scholar Award. Sample collection and phenotyping of the subjects utilized in this study was funded by the Ministry for Health and Welfare, Republic of Korea (Korea Healthcare Technology R&D Project grant HI13C2124 to Dr. Bae, and HI13C1754 to Dr. Song). The out-of-study Korean control data were provided by the Korean Biobank Project, which is supported by the Korea Centers for Disease Control and Prevention at the Korea National Institute of Health.

Objective More than 80% of autoimmune disease predominantly affects females, but the mechanism for this female bias is poorly understood. We suspected that an X chromosome dose effect accounts for this, and we undertook this study to test our hypothesis that trisomy X (47,XXX; occurring in ∼1 in 1,000 live female births) would be increased in patients with female-predominant diseases (systemic lupus erythematosus [SLE], primary Sjogren's syndrome [SS], primary biliary cirrhosis, and rheumatoid arthritis [RA]) compared to patients with diseases without female predominance (sarcoidosis) and compared to controls. Methods All subjects in this study were female. We identified subjects with 47,XXX using aggregate data from single-nucleotide polymorphism arrays, and, when possible, we confirmed the presence of 47,XXX using fluorescence in situ hybridization or quantitative polymerase chain reaction. Results We found 47,XXX in 7 of 2,826 SLE patients and in 3 of 1,033 SS patients, but in only 2 of 7,074 controls (odds ratio in the SLE and primary SS groups 8.78 [95% confidence interval 1.67–86.79], P = 0.003 and odds ratio 10.29 [95% confidence interval 1.18–123.47], P = 0.02, respectively). One in 404 women with SLE and 1 in 344 women with SS had 47,XXX. There was an excess of 47,XXX among SLE and SS patients. Conclusion The estimated prevalence of SLE and SS in women with 47,XXX was ∼2.5 and ∼2.9 times higher, respectively, than that in women with 46,XX and ∼25 and ∼41 times higher, respectively, than that in men with 46,XY. No statistically significant increase of 47,XXX was observed in other female-biased diseases (primary biliary cirrhosis or RA), supporting the idea of multiple pathways to sex bias in autoimmunity.

Objective Maternal–fetal cell transfer during pregnancy can lead to long-lasting microchimerism, which raises the question of whether microchimerism sometimes contributes to autoimmune disease later in life. In an experimental model, transfusion of parental lymphocytes homozygous for major histocompatibility complex alleles results in systemic lupus erythematosus (SLE). We identified male patients with SLE and healthy male subjects and their mothers in order to investigate the mother–son HLA relationship in SLE risk. Male subjects were selected in order to avoid confounding due to fetal microchimerism, which may occur in women. Methods HLA genotyping for DRB1, DQA1, and DQB1 was conducted for sons and their mothers. Thirty men with SLE and their mothers were compared with 76 healthy men and their mothers. Results Sons with SLE were HLA-identical with their mothers (bidirectionally compatible) for the basic HLA–DRB1 groups encoded by DRB1*01 through DRB1*14 more often than were healthy sons (odds ratio [OR] 5.0, P = 0.006). Each DRB1 group contains multiple allelic variants; male patients with SLE and their mothers often were identical for both DRB1 allelic variants (OR 3.2, P = 0.08). For DQA1 and DQB1, the ORs were 2.3 (P = 0.08) and 2.0 (P = 0.21), respectively. When analysis was limited to male subjects with SLE-associated HLA genes (encoding HLA–DR2 or HLA–DR3), the differences further increased for DRB1 basic groups (OR 7.2, P = 0.01), DRB1 alleles (OR 15.0, P = 0.018), DQA1 6.4 (P = 0.006), and DQB1 (OR 5.7, P = 0.027). No increase in (unidirectional) compatibility of the mother from the son's perspective was observed at any locus. Conclusion We observed increased bidirectional HLA class II compatibility of male SLE patients and their mothers compared with healthy men and their mothers. This observation implies that maternal microchimerism could sometimes be involved in SLE and therefore merits further investigation.

Objective Recent case–control studies show associations of the minor T allele (of the C1858T single-nucleotide polymorphism corresponding to the R620W amino acid substitution) of PTPN22 with multiple autoimmune diseases, including systemic lupus erythematosus (SLE). We performed family-based association studies of this polymorphism in 4 independent cohorts containing SLE patients and their parents and/or other family members. Methods A total of 2,689 individuals from 902 independent Caucasian families with SLE were genotyped using polymerase chain reaction pyrosequencing (cohorts 1 and 2) and the Sequenom MassArray system (cohorts 3 and 4). The transmission disequilibrium test (TDT) and the pedigree disequilibrium test (PDT) were conducted to assess the evidence of association. Results The 1858 C > T allele frequencies of the parents showed no deviation from Hardy-Weinberg equilibrium within each cohort. No evidence of preferential transmission of the T allele from heterozygous parents to their affected offspring was observed in each of the 4 cohorts or in the combined sample. Consistent with the TDT result, the PDT analysis revealed no significant association between the T allele and SLE. In 54 of the 661 SLE patients (cohorts 1 and 3) with documented autoimmune thyroid disease, the T allele frequency was higher than in individuals with SLE alone (16.7% versus 8.5%; P = 0.008, odds ratio 2.16 [95% confidence interval 1.25–3.72]). Conclusion The R620W polymorphism of the PTPN22 gene is not a major risk allele for SLE susceptibility in our sample of Caucasian individuals from northern America, the UK, or Finland, but it appears to be a risk factor for the concurrent autoimmune diseases of autoimmune thyroid disease and SLE.

Objective The C-terminal peptide of amino acids 83–119 of the SmD1 protein is a target of the autoimmune response in human and murine lupus. This study was undertaken to test the hypothesis that SmD183–119-reactive T cells play a crucial role in the generation of pathogenic anti–double-stranded DNA (anti-dsDNA) antibodies. Methods Splenic or lymph node T cells derived from unmanipulated as well as SmD183–119-immunized NZB/NZW mice were analyzed in vitro by enzyme-linked immunospot (ELISpot) assay to determine T cell help for anti-dsDNA generation induced by the SmD183–119 peptide. Cytokines expressed by these T cells were measured by ELISpot assay, enzyme-linked immunosorbent assay, and flow cytometry. SmD183–119- and ovalbumin-specific T cell lines were generated and characterized. Results The SmD183–119 peptide, but not the control peptides, significantly increased the in vitro generation of anti-dsDNA antibodies in cultures from unmanipulated NZB/NZW mice. Interferon-γ (IFNγ), interleukin-2 (IL-2), IL-4, transforming growth factor β, and IL-10 production increased in response to the peptide in young mice; only IFNγ and IL-2 were increased in older, diseased mice. Activation of SmD183–119-reactive T cells by immunization of NZB/NZW mice resulted in elevated anti-dsDNA synthesis and, later, increased antibodies to SmD183–119. Most cells in SmD183–119-specific CD4+ T cell lines helping both antibodies had increased intracellular expression of IFNγ, and most expressed both IFNγ and IL-4. Conclusion The SmD183–119 peptide plays an important role in generating T cell help for autoantibodies, including anti-dsDNA, and activates different subsets of T cells as defined by distinct cytokine expression. This peptide is an interesting target structure for the modulation of autoreactive T cells, and its characterization may contribute to our understanding of the role of autoantigen-reactive T cells in the pathogenesis of SLE.

Objective To evaluate familiality of 15 clinical and laboratory features in systemic lupus erythematosus (SLE)–affected sibpairs, and to estimate correlations with the age at SLE diagnosis in affected sibpairs and parent–offspring pairs. Methods Concordance rates and sibling risk ratios were used as indicators of familiality for 15 manifestations of SLE. Pearson's correlations and paired t-tests were used to compare the age at SLE diagnosis in affected sibpairs and in parent–offspring pairs. Results Increased sibling risk ratios (1.9–3.9) for thrombocytopenia, discoid rash, neurologic disorder (defined as seizure or psychosis), and hemolytic anemia were observed in 159 SLE-affected sibpairs. Among these clinical features, paired expression of hemolytic anemia plus thrombocytopenia and hemolytic anemia plus neurologic disorder appeared to be more frequent in 709 SLE patients than would be expected by chance (P < 0.00001 and P < 0.007, respectively). The ratio of the presence of both hemolytic anemia and neurologic disorder was ∼13 times higher in the younger affected sib than in the older affected sib (P < 0.02). Familiality of patient age at SLE diagnosis, as observed by relative correlations, was greater in 125 affected sibpairs (r = 0.67, P < 0.0001) than in 37 affected parent–offspring pairs (r = 0.47, P = 0.003). The median ± SD age at SLE diagnosis was significantly lower in offspring (21.5 ± 10.1 years) than in their parents (41.6 ± 15.8 years) (P < 0.0001) but was not different in sibpairs. The combined non-Caucasian sibpairs had a younger mean age at SLE diagnosis compared with Caucasian sibpairs (P = 0.014). Conclusion Evidence for familiality of thrombocytopenia, discoid rash, neurologic disorder, hemolytic anemia, and co-occurring neurologic disorder plus hemolytic anemia in SLE was observed in 159 affected sibpairs. Familiality of the age at SLE diagnosis in relative pairs suggests that shared genes and/or shared environmental exposures impact disease susceptibility. Shared immediate environmental triggers appear less compelling, because the average time between dates of diagnosis was 11 years in parent–offspring pairs and 7.5 years in affected sibpairs. The significantly earlier age at disease diagnosis in offspring compared with their parents suggests that some forms of anticipation might play a role in susceptibility to SLE. Stratifying families by subphenotypes that are familial may reduce heterogeneity and facilitate identification of genetic risk factors for SLE.

Objective To test the hypothesis that an artificial peptide, based on an algorithm describing T cell stimulatory sequences from the VH regions of murine IgG antibodies to DNA, is an effective tolerogen in vivo in the (NZB/NZW)F1 (BWF1) mouse model of systemic lupus erythematosus. Methods Using proliferative T cell responses to 439 Ig peptides, an algorithm was constructed that describes the amino acid sequences likely to stimulate BWF1 T cells. Stimulatory (pConsensus [pCONS]) or nonstimulatory (pNegative [pNEG]) peptides were synthesized. Groups of 10-week-old (healthy) or 20-week-old (diseased) BWF1 mice received monthly intravenous injections of 1,000 μg of peptide or saline. Ex vivo splenic T cell responses and in vivo clinical effects were measured. Results Tolerance was induced by pCONS, but not by pNEG, with respect to ex vivo T cell proliferation and T cell help for antibodies to DNA. T cell help for IgG anti-DNA was impaired not only after T cell stimulation by pCONS but also after stimulation by some peptides from nucleosomal and Ro antigens. Treatment with pCONS significantly delayed the onset of nephritis and inhibited increases in the plasma levels of total IgG, IgG antibodies to DNA, nucleosome, cardiolipin, interferon-γ, and interleukin-4. In contrast, antibody responses to an exogenous antigen were not impaired. Survival was significantly prolonged in both healthy and diseased mice treated with pCONS. Conclusion Induction of immune tolerance in response to treatment with pCONS in autoreactive T cell helper populations is highly effective in delaying the appearance of multiple autoantibodies, cytokine increases, and nephritis in BWF1 mice, and dramatically prolongs survival. A striking effect is the ability of the peptide to tolerize T cell help for anti-DNA that is induced by multiple, structurally unrelated self antigens.

Objective Signaling molecules from the T cell receptor zeta/Fcepsilon receptor gamma (TCRzeta/FcRgamma) family play a critical role in the function of Fcgamma receptors and the TCR and are located on human chromosome 1, where lupus susceptibility genes are located. This study was undertaken to investigate the possibility of polymorphisms and/or mutations of TCRzeta in systemic lupus erythematosus (SLE). Methods We amplified the whole coding region of TCRzeta by reverse transcriptase-polymerase chain reaction (PCR) and directly sequenced the PCR products with a dye primer technique to facilitate heterozygote detection. Results An alternative splicing form of TCRzeta, with a CAG codon (glutamine) inserted at the splice junction of exons 4 and 5, was found both in SLE and in non-SLE subjects. Both splice isoforms of TCRzeta occurred in human mixed peripheral blood mononuclear cells, natural killer cells, and Jurkat T cells. In TCRzeta, 2 silent and 2 missense mutations were found, but neither coding change occurred in the immunoreceptor tyrosine-activation motif. No unique mutations were found in Caucasian, African American, Hispanic, Chinese, or Japanese SLE patients living in North America. Conclusion The uncommon and equal occurrence of novel single-nucleotide polymorphisms in both SLE patients and normal subjects makes it improbable that they play important roles in genetic susceptibility to SLE.

Objective Abnormal immune complex clearance is a feature of systemic lupus erythematosus (SLE). Polymorphisms of the Fc gamma receptor type IIa (Fc gammaRIIa) genes (the receptor binds IgG2 and IgG3) are important disease susceptibility factors in some populations. This study sought to determine the effects of these polymorphisms among Korean patients with SLE. Methods Polymerase chain reaction of genomic DNA and allele-specific oligonucleotide hybridization were used to determine Fc gammaRIIa genotypes in Korean patients with SLE and healthy control subjects. Clinical manifestations were analyzed in each patient and correlated with the genotypes. Results Among the 73 SLE patients, there was an abnormal distribution of Fc gammaRIIa alleles when compared with 64 controls: 11.0% of the SLE patients were homozygous for Fc gammaRIIa-H131 compared with 34.4% of the controls (odds ratio [OR] 0.20, 95% confidence interval [95% CI] 0.04-0.95, chi2 = 5.7, P = 0.01699). The allelic frequency of Fc gammaRIIa-H131 was significantly lower in the SLE patients than in the controls (49.3% versus 63.3%; P = 0.02019), and it was also significantly lower in lupus patients with nephritis compared with the normal population (OR 0.53, 95% CI 0.29-0.95, chi2 = 5.15, P = 0.02330), but was not significantly lower in lupus patients without nephritis (P = 0.13663 versus controls). Clinically, the level of proteinuria was significantly higher in the lupus nephritis patients who had R/R131 than in those who had H/H131 or R/H131. Conclusion An abnormal distribution of Fc gammaRIIa polymorphisms was associated with SLE in Korean patients. There was a significant decrease in the Fc gammaRIIa-H/H131 genotype and H131 allelic frequency in SLE patients, particularly in those with nephritis. This suggests that the H131 allele confers some protection from SLE in this population.

B lymphocytes in individuals with systemic lupus erythematosus (SLE) secrete pathogenic autoantibodies to DNA which cause clinical nephritis. (NZB X NZW) F1 (BW) female mice also secrete pathogenic anti-DNA autoantibodies, and therefore are considered to be an animal model of SLE. The rearranged immunoglobulin (Ig) genes that encode an anti-DNA antibody from a diseased BW mouse have been cloned, and transgenic (Tg) mice have been created by microinjection of these constructs into fertilized eggs from normal mice. As we reported previously, when the construct contains the C gamma 2a heavy chain constant (CH) region, the mice spontaneously secrete anti-DNA IgG and they develop mild nephritis. This demonstrated that the Ig encoded by the transgene is pathogenic. In contrast, here we report that when the construct contains the same anti-DNA Ig variable (V) regions used previously, along with the C mu region, the autoreactive B cells are rendered tolerant. Most B cells in the Tg mice express the mu transgene product on their surface, and rearrangement of endogenous light chain genes is partially suppressed. Furthermore, most hybridomas made from Tg B cells secrete IgM anti-DNA. Despite this, the Tg mice have reduced levels of total serum Ig and they do not secrete anti-DNA IgM either spontaneously or following immunization with DNA. We conclude that most B cells in the Tg mice have been rendered anergic. Anergy is however reversible in vitro; lipopolysaccharide stimulation of Tg B cells leads to the production of a significant amount of IgM anti-DNA antibody. The studies demonstrate that in this line of Tg mice on a normal mouse genetic background potentially pathogenic B cells that express a high-affinity Ig specific for a natural autoantigen are subject to tolerance by induction of anergy.

Objective. To assess the ability of peptides derived from anti-DNA to stimulate syngeneic T lymphocytes and influence lupus in (NZB × NZW)F1 (NZB/W) mice. Methods. We synthesized (by Geysen pin method) overlapping 12-mer peptides recapitulating the amino acid sequence of the VH region of a nephritogenic monoclonal IgG2a anti-DNA antibody (A6.1) from an NZB/W mouse. Splenic T cells were cultured with the peptides; multiple experiments assayed 12-mer and 16-mer peptides which contained a triple-base motif (KFKGK). We immunized 20-week-old NZB/W mice with the 12-mer and evaluated them for evidence of nephritis and for quantities of antibodies in plasma and glomeruli. Results. Three clusters of peptides caused proliferation of spleen cells from young, nonimmunized mice. Both the 12-mer FYNQKEKGKATL and the 16-mer GDTFYNQKEKGKATLT peptides stimulated purified T cells. The KXKXK motif occurs in 15% of murine IgVH (NBRF protein database), compared with 100% (6 of 6) of NZB/W anti-DNA monoclonal antibody. Immunization with the 12-mer peptide increased plasma levels of IgG, anti-DNA, and immune complexes, and levels of anti-DNA in glomeruli; nephritis was accelerated. Conclusion. NZB/W anti-DNA contain peptide sequences in their VH regions that stimulate self—T cells. At least one motif is frequent in NZB/W anti-DNA. If some activated T cells provide help, this mechanism may contribute to sustained up-regulation of autoantibodies in murine lupus.

Several genome-wide scans for systemic lupus ery-thematosus (SLE) susceptibility genes have shown linkage to aregion on human chromosome lq41-42 (1–3). Poly(ADP-ribose) polymerase (PARP) is one of the candidates in thisregion implicated by a transmission disequilibrium study of 124multiethnic (primarily Caucasian) SLE families, suggestingthat PARP alleles may be (or be close to) the SLE suscepti-bility gene within lq41-q42 (4). Therefore, we read withinterest the article by Delrieu et al (5) reporting a lack ofassociation of PARP alleles with SLE among French Cauca-sians. Our data described below suggest that PARP alleles maybe in linkage disequilibrium with an SLE susceptibility gene.The incidence of SLE in African Americans is 2–4times greater than that in Caucasians (6). Our study usedbanked DNA obtained from consecutive African AmericanSLE patients (n 5 91) at the Division of Rheumatologyand Immunogenetics, University of Texas–Houston MedicalSchool and from normal controls (n 5 53) recruited fromamong medical center personnel. All SLE patients fulfilledthe American College of Rheumatology criteria for thedisease (7).Consistent with the findings reported by Delrieu et al(5), no specific PARP promoter allele was found to besignificantly associated with SLE or with any subset of SLE(patients stratified by the presence of lupus nephritis, anti–double-stranded DNA [by