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Ig class switch recombination (CSR) and somatic hypermutation serve to diversify antibody responses and are orchestrated by the activity of activation-induced cytidine deaminase and many proteins involved in DNA repair and genome surveillance. Msh5, a gene encoded in the central MHC class III region, and its obligate heterodimerization partner Msh4 have a critical role in regulating meiotic homologous recombination and have not been implicated in CSR. Here, we show that MRL/lpr mice carrying a congenic H-2(b/b) MHC interval exhibit several abnormalities regarding CSR, including a profound deficiency of IgG3 in most mice and long microhomologies at Ig switch (S) joints. We found that Msh5 is expressed at low levels on the H-2(b) haplotype and, importantly, a similar long S joint microhomology phenotype was observed in both Msh5 and Msh4-null mice. We also present evidence that genetic variation in MSH5 is associated with IgA deficiency and common variable immune deficiency (CVID) in humans. One of the human MSH5 alleles identified contains two nonsynonymous polymorphisms, and the variant protein encoded by this allele shows impaired binding to MSH4. Similar to the mice, Ig S joints from CVID and IgA deficiency patients carrying disease-associated MSH5 alleles show increased donor/acceptor microhomology, involving pentameric DNA repeat sequences and lower mutation rates than controls. Our findings suggest that Msh4/5 heterodimers contribute to CSR and support a model whereby Msh4/5 promotes the resolution of DNA breaks with low or no terminal microhomology by a classical nonhomologous end-joining mechanism while possibly suppressing an alternative microhomology-mediated pathway.

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol-anchored membrane protein that restricts complement activation on autologous cells. Previous studies have established a significant protective activity of DAF in the MRL/lpr murine model of human systemic lupus erythematosus. To dissect the mechanism of protection by DAF in this disease model, we evaluated the effect of C3 gene ablation on disease development in MRL/lpr-Daf-1(-/-) mice. We found no significant difference in lymphadenopathy, splenomegaly, or anti-chromatin autoantibody titer between complement-sufficient and complement-deficient MRL/lpr-Daf-1(-/-) mice. On the other hand, complement deficiency strikingly reduced the incidence and severity of dermatitis in MRL/lpr-Daf-1(-/-) mice. To assess the contribution of DAF expression on lymphocytes versus local tissues in suppressing dermatitis, we generated BM chimeric mice between MRL/lpr-Daf-1(-/-) and MRL/lpr-Daf-1(+/+) mice. Compared with MRL/lpr-Daf-1(-/-) --> MRL/lpr-Daf-1(-/-) controls, MRL/lpr-Daf-1(-/-) --> MRL/lpr-Daf-1(+/+) chimeras developed significantly attenuated dermatitis, suggesting that the protective effect of DAF in suppressing dermatitis is primarily attributable to its local expression. We conclude that DAF works as a complement regulator in the skin to protect MRL/lpr mice from skin inflammation, whereas its inhibitory role in the induction phase of MRL/lpr autoimmunity is complement-independent. Together, these results reveal multiple mechanisms of action for DAF in ameliorating systemic autoimmunity.

Formation of reactive nitrogen and oxygen intermediates (RNI and ROI) is an essential part of the innate immune response. Markers of systemic RNI production are increased in the setting of systemic lupus erythematosus (SLE) activity. Several lines of evidence suggest mechanisms through which the activity of inducible nitric oxide synthase (iNOS) is pathogenic in SLE, including the ability of peroxynitrite (ONOO(-), a product of iNOS activity) to modify proteins, lipids, and DNA. These modifications can alter enzyme activity and may increase the immunogenicity of self antigens, leading to a break in immune tolerance. In humans, observational data suggest that overexpression of iNOS and increased production of ONOO(-) lead to glomerular and vascular pathology. Therapies designed to target iNOS activity or scavenge ROI and RNI are in development and may provide the means to reduce the pathogenic consequences of ROI and RNI in SLE.

We previously described a renal protective effect of factor B deficiency in MRL/lpr mice. Factor B is in the MHC cluster; thus, the deficient mice were H2b, the haplotype on which the knockout was derived, whereas the wild-type littermates were H2k, the H2 of MRL/lpr mice. To determine which protective effects were due to H2 vs factor B deficiency, we derived H2b congenic MRL/lpr mice from the 129/Sv (H2b) strain. Autoantibody profiling using autoantigen microarrays revealed that serum anti-Smith and anti-small nuclear ribonucleoprotein complex autoantibodies, while present in the majority of H2k/k MRL/lpr mice, were absent in the H2b/b MRL/lpr mice. Surprisingly, 70% of MRL/lpr H2b/b mice were found to be serum IgG3 deficient (with few to no IgG3-producing B cells). In addition, H2b/b IgG3-deficient MRL/lpr mice had significantly less proteinuria, decreased glomerular immune complex deposition, and absence of glomerular subepithelial deposits compared with MRL/lpr mice of any H2 type with detectable serum IgG3. Despite these differences, total histopathologic renal scores and survival were similar among the groups. These results indicate that genes encoded within or closely linked to the MHC region regulate autoantigen selection and isotype switching to IgG3 but have minimal effect on end-organ damage or survival in MRL/lpr mice.

Ets transcription factors function throughout development in such varied processes as cellular proliferation, apoptosis, differentiation and migration. Many have been implicated to play important roles in hematopoiesis, vasculogenesis/angiogenesis and myogenesis. Fli1 is an Ets family member that is essential for development and increasing evidence suggests modulating Fli1 gene expression impacts lymphocyte function and is important in the autoimmune disease lupus. Presently, it is unknown how Fli1 gene expression is controlled in lymphocytes. Identifying upstream regulators of Fli1 in lymphocytes will be critical for understanding lymphocyte development and the consequences of dysregulation and may be of value in developing future treatments for lupus.

To investigate the role of interferon regulatory factor-1 (IRF-1) in the development of lupus nephritis, IRF-1(-/-) genotype mice were bred onto the MRL/lpJfas(lpr) (MRL/lpr) background. We examined kidney mesangial cell function and disease progression. Endpoints evaluated included inflammatory mediators, autoantibody production, immune complex deposition, renal pathology, T cell subset analysis, and duration of survival. Mesangial cells cultured from IRF-1(-/-) mice produced significantly lower levels of nitric oxide and IL-12 but not TNF-alpha when stimulated with LPS + IFN-gamma. IRF-1(-/-) mice showed less aggravated dermatitis compared to the wild-type mice. Anti-double-stranded DNA production and proteinuria were significantly decreased in IRF-1(-/-) mice compared to IRF-1(+/+) mice. IgG and C3 deposition as well as glomerulonephritis were decreased in IRF-1(-/-) mice at 26 wk of age compared to the IRF-1(+/+) mice. Splenic CD4- CD8- CD44+ T cells were decreased while CD4+ CD25+ T cells were increased in the IRF-1(-/-) mice when compared to IRF-1(+/+) mice. Survival rates (ED50) were 22 wk for IRF-1(+/+) mice and 45 wk for IRF-1(-/-) mice. These findings suggest an important role of IRF-1 in mediating renal disease in MRL/lpr mice.

MRLlpr mice develop spontaneous systemic autoimmunity with many hallmarks of the human disease systemic lupus erythematosus. Although a variety of genes have been implicated in this model, disease pathogenesis is still poorly understood. In an effort to identify novel genes and pathways, we performed genome-wide mRNA expression analysis in the spleens and kidneys of MRLlpr mice throughout the disease course. Samples were collected from cohorts of C57BL/6, MRL+/+ and MRLlpr mice, and profiled by flow cytometry and gene expression microarrays. Serum autoantibodies and renal pathology were studied in parallel. We identified 236 genes in MRLlpr spleen that showed significant threefold or greater changes in expression between 6 and 20 weeks. Of interest, a number of interferon-responsive genes were expressed early, and remained dysregulated throughout the disease course. Many chemokines, cell surface proteins, transcription factors and cytokines, including IFN-gamma, also showed altered expression as disease progressed. Analysis of kidneys indicated the presence of severe inflammation that coincided with evidence for changes in kidney function and elevated expression of IFN-inducible genes, complement components and antigen presentation genes. These data provide a unique genomic view of the progression to fatal autoimmunity in MRLlpr mice, and provide new candidate genes and pathways to explore.

Nitric oxide (NO) production increases with age in the lupus-prone MRL/lpr mouse, paralleling disease activity. One mechanism for excess NO production in MRL/lpr mice may be a defect in down-regulatory mechanisms of the iNOS pathway. A potential modulator of NO is the nuclear hormone receptor peroxisome proliferation activated receptor gamma (PPARgamma). We demonstrate that renal PPARgamma protein expression was altered as disease progressed in MRL/lpr mice, which paralleled increased iNOS protein expression. Additionally, MRL/lpr-derived primary mesangial cells expressed less PPARgamma than BALB/c mesangial cells and produced more NO in response to LPS and IFNgamma. Furthermore, PPARgamma activity was reduced in mesangial cells following exposure to inflammatory mediators. This activity was restored with the addition of a NOS enzyme inhibitor. These results indicate that the activation of inflammatory pathways may lead to reduced activity and expression of PPARgamma, further exacerbating the disease state.

Evidence from animal models and prospective studies of RA, multiple sclerosis, and type-1 diabetes suggest an important role for vitamin D as a modifiable environmental factor in autoimmune disease. This role has not been well studied in human SLE. We compared serum 25-hydroxyvitamin D (25(OH)D) levels between recently diagnosed SLE cases and matched controls, and examined disease characteristics in relationship to 25(OH)D among cases. Data from a population-based cohort of 123 recently diagnosed SLE patients and 240 controls were used. We found a trend toward lower 25(OH)D levels in cases compared to controls, which was statistically significant in Caucasians (p=0.04), controlling for age, sex, season, and smoking. Overall, 67% of the subjects were vitamin D deficient, with mean levels significantly lower among African Americans (15.9 ng/ml) compared to Caucasians (31.3 ng/ml). Critically low vitamin D levels (<10 ng/ml) were found in 22 of the SLE cases, with presence of renal disease being the strongest predictor (OR 13.3, p<0.01) followed by photosensitivity (OR 12.9, p<0.01). These results suggest vitamin D deficiency as a possible risk factor for SLE and provide guidance for future studies looking at a potential role of vitamin D in the prevention and/or treatment of SLE.

This study assessed the developmental immunotoxicity of trichloroethylene (TCE) in B6C3F1 mice exposed via drinking water (0, 1,400, 14,000 ppb) from gestation day 0 (GD0) to either 3 or 8 weeks of age. Lymphocyte proliferation, NK cell activity, SRBC-specific IgM production (PFC response), splenic B220+ cells, and thymic and splenic T-cell immunophenotypes were assessed at 3 and 8 weeks of age. Delayed type hypersensitivity (DTH) and autoantibodies to ds-DNA were assessed in 8-week old animals only. Proliferation and NK cell activity were not affected at either age. Decreased PFC responses were noted in male offspring at both ages and both TCE treatment levels. PFC responses in female offspring were suppressed by treatment with 14,000 ppb TCE at both ages assessed and at 1,400 ppb TCE at 8 weeks of age. Splenic numbers of B220 cells were only decreased in 3-week old pups exposed to 14,000 ppb TCE. The most pronounced alteration in T-cell subpopulations were increases in all thymic (CD4+, CD8+, CD4+/CD8+, and CD4-/CD8-) T-cell types in 8-week old animals. DTH was increased in females at both TCE levels and in males at the high dose only. These results indicate that TCE may be an effective developmental immunotoxicant and suggests that additional studies are required to determine the health risks associated with developmental exposure to TCE.

The nuclear hormone receptor peroxisome proliferation activated receptor gamma (PPARgamma) is a modulator of inflammation including down-regulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. PPARgamma agonists reduce iNOS expression and NO production in a dose-dependent manner in macrophages, mesangial cells and other inflammatory cells. However, the mechanisms involved in the inhibition of iNOS expression by PPARgamma and its agonists are not fully understood. Here we show that the PPARgamma agonist ciglitazone dose-dependently inhibited a murine iNOS-luciferase reporter construct by up to 50% in transfected mesangial cells. Blocking de novo protein synthesis in mesangial cells had no effect on PPARgamma agonist activity, indicating that ciglitazone acts directly to inhibit iNOS transcription. We identified a novel PPAR response element (PPRE) in the murine iNOS promoter that is homologous to the PPRE consensus sequence. In binding assays PPARgamma directly binds to this response element in vitro and can function as a positive element in response to PPARgamma agonists when placed in front of a reporter gene. Site-directed mutagenesis of this PPRE in a murine iNOS promoter/reporter construct did not block the inhibitory activity of a synthetic PPARgamma agonist on the iNOS promoter/reporter construct in transfected mesangial cells. However, the mutated construct exhibited lower basal expression, and higher expression in response to inflammatory stimuli compared to the intact construct. These data suggest that the iNOS PPRE contributes to positive basal expression and negative expression of iNOS in response to inflammatory stimuli. The PPRE is not necessary, however, for synthetic PPARgamma agonists to inhibit iNOS expression.

The New Zealand Black (NZB) Lbw2 locus (lupus NZB x New Zealand White (NZW) 2 locus) was previously linked to mortality and glomerulonephritis, but not to IgG autoantibodies, suggesting that it played a role in a later disease stage. To define its contribution, (NZB x NZW)F1 hybrids (BWF1) containing two, one, or no copies of this locus were generated. Lack of the NZB Lbw2 indeed reduced mortality and glomerulonephritis, but not serum levels of total and anti-DNA IgG Abs. There were, however, significant reductions in the B cell response to LPS, total and anti-DNA IgM and IgG Ab-forming cells, IgM Ab levels, and glomerular Ig deposits. Furthermore, although serum IgG autoantibody levels correlated poorly with kidney IgG deposits, the number of spontaneous IgG Ab-forming cells had a significant correlation. Genome-wide mapping of IgM anti-chromatin levels identified only Lbw2, and analysis of subinterval congenics tentatively reduced Lbw2 to approximately 5 Mb. Because no known genes associated with B cell activation and lupus are in this interval, Lbw2 probably represents a novel B cell activation gene. These findings establish the importance of Lbw2 in the BWF1 hybrid and indicate that Lbw2, by enhancing B cell hyperactivity, promotes the early polyclonal activation of B cells and subsequent production of autoantibodies.

Peroxisome proliferation-activated receptor (PPAR)gamma agonists inhibit inducible nitric-oxide synthase (iNOS), tumor necrosis factor-alpha, and interleukin-6. Because of these effects, synthetic PPARgamma agonists, including thiazolidinediones, are being studied for their impact on inflammatory disease. The anti-inflammatory concentrations of synthetic PPARgamma agonists range from 10 to 50 microM, whereas their binding affinity for PPARgamma is in the nanomolar range. The specificity of synthetic PPARgamma agonists for PPARgamma at the concentrations necessary for anti-inflammatory effects is thus in question. We report that PPARgamma is not necessary for the inhibition of iNOS by synthetic PPARgamma agonists. RAW 264.7 macrophages possess little PPARgamma, yet lipopolysaccharide (LPS)/interferon (IFN)gamma-induced iNOS was inhibited by synthetic PPARgamma agonists at 20 microM. Endogenous PPARgamma was inhibited by the transfection of a dominant-negative PPARgamma construct into murine mesangial cells. In the transfected cells, synthetic PPARgamma agonists inhibited iNOS production at 10 microM, similar to nontransfected cells. Using cells from PPARgamma Cre/lox conditional knockout mice, baseline and LPS/IFNgamma-induced nitric oxide levels were higher in macrophages lacking PPARgamma versus controls. However, synthetic PPARgamma agonists inhibited iNOS at 10 microM in the PPARgamma-deficient cells, similar to macrophages from wild-type mice. These results indicate that PPARgamma is not necessary for inhibition of iNOS expression by synthetic PPARgamma agonists at concentrations over 10 microM. Intrinsic PPARgamma function, in the absence of synthetic agonists, however, may play a role in inflammatory modulation.

Objective: There have been few studies of occupational exposures and systemic lupus erythematosus (SLE). We examined the association between the risk of SLE and occupational exposures (mercury, solvents, and pesticides), specific jobs (ever worked in teaching, healthcare, and cosmetology), and working night or rotating shifts., Methods: Patients with recently diagnosed SLE (n = 265) were recruited through 4 university based and 30 community based rheumatology practices in North Carolina and South Carolina, USA. Controls (n = 355) were identified through driver's license records and were frequency matched to patients by age, sex, and state. Data collection included an in-person interview with detailed farming and work histories., Results: Associations were seen with self-reported occupational exposure to mercury (OR 3.6, 95% CI 1.3, 10.0), mixing pesticides for agricultural work (OR 7.4, 95% CI 1.4, 40.0), and among dental workers (OR 7.1, 95% CI 2.2, 23.4). Although these associations were fairly strong and statistically significant, the prevalence of these exposures was very low and thus these estimates are based on a small number of exposed cases and controls. Weaker associations were seen between SLE and shift work (OR 1.6, 95% CI 0.99, 2.7) and among healthcare workers with patient contact (OR 1.7, 95% CI 0.99, 2.9). There was no association of SLE with use of solvents or among teachers or cosmetologists., Conclusion: This study reveals the potential contribution of occupational exposures to the development of SLE, and highlights some exposures and experiences that should be examined in other studies using more extensive exposure assessment techniques and in experimental studies of autoimmunity.

Epigenetic regulation of gene expression is involved in the development of many diseases. Histone acetylation is a posttranslational modification of the nucleosomal histone tails that is regulated by the balance of histone deacetylases and histone acetyltransferases. Alterations in the balance of histone acetylation have been shown to cause aberrant expression of genes that are a hallmark of many diseases, including systemic lupus erythematosus. In this study, we determined whether suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor: 1) inhibits inflammatory mediator production in vitro and 2) modulates lupus progression in vivo. Mesangial cells isolated from 10-wk-old MRL/lpr mice were stimulated with LPS/IFN-gamma and incubated with SAHA. TNF-alpha, IL-6, NO, and inducible NO synthase expression were inhibited by SAHA. We then treated MRL/lpr mice with daily injections of SAHA from age 10 to 20 wk. The animals treated with SAHA had decreased spleen size and a concomitant decrease in CD4-CD8- (double-negative) T cells compared with controls. Serum autoantibody levels and glomerular IgG and C3 deposition in SAHA-treated mice were similar to controls. In contrast, proteinuria and pathologic renal disease were significantly inhibited in the mice receiving SAHA. These data indicate that SAHA blocks mesangial cell inflammatory mediator production in vitro and disease progression in vivo in MRL/lpr mice., (Copyright 2004 The American Association of Immunologists, Inc.)

Tumor necrosis factor (TNF) is involved in the pathogenesis of systemic lupus erythematosus (SLE), but the role of TNF polymorphisms in SLE susceptibility remains unclear. Previous studies in different populations report an inconsistent association of the TNF-alpha -308A allele with SLE, sometimes depending on the presence of HLA-DR3. We examined the association of polymorphisms in TNF-alpha (-308G/A, -238G/A) and TNFbeta (+252A/G) in a population-based study of SLE in the southeastern United States and considered TNF-SLE associations with respect to HLA-DR3 and DR2 and the interleukin (IL)-1alpha -889C/T polymorphism, previously linked to SLE in this population. Genotypes were analyzed for 230 recently diagnosed SLE patients who met American College of Rheumatology classification criteria and 276 age- and sex-matched controls, randomly selected from driver's license registries. Carriage of the TNF-alpha -308A allele was significantly associated with SLE in Caucasians (OR = 2.3; 95% CI 1.4, 3.9), but not African Americans. Analyses stratified by IL-1alpha -889 genotypes (C/C vs C/T or T/T) revealed independent associations of SLE with TNF-alpha -308A or HLA-DR2 and DR3. This reflected a significant interaction of TNF and IL-1 genotypes in Caucasians, and yielded a strong association (OR = 8.0, p < 0.00001) for the combined "HLA-DR3, TNF-alpha -308A, IL-1alpha -889C/C" genotype. These findings provide evidence of cytokine gene epistasis in SLE susceptibility.

Lupus nephritis is characterized by immune complex deposition and infiltration of inflammatory cells into the kidney including Ab-producing cells (AbPCs). Although AbPCs play a central role in the pathogenesis of immune complex glomerulonephritis in lupus, the specificity and pathogenic role of AbPCs infiltrating into the kidneys in lupus are poorly understood. To characterize AbPCs present in lupus kidneys, we isolated AbPCs from diseased MRL/MpJ-Faslpr (MRL/lpr) mouse kidneys. ELISPOT assays, using glomerular Ag (GA) extracts as Ag, demonstrated significant enhancement of anti-GA AbPCs in the kidneys as compared in peripheral blood or spleen of the same mouse. We isolated hybridomas with anti-GA specificity from MRL/lpr mouse kidneys. All the anti-GA mAbs had polyreactive binding to ssDNA, dsDNA, and IgG (i.e., rheumatoid factor), but not to histones or Sm. Sequence analysis of anti-GA Abs suggested the occurrence of somatic mutations and amino acid replacement in complementarity-determining regions with a high replacement to silent ratio resulting in charged amino acids. Intravenous administration of the monoclonal anti-GA Abs into BALB/c mice resulted in graded deposition in glomeruli paralleling their ELISA anti-GA reactivity. These results suggest that AbPCs infiltrating the kidneys in MRL/lpr mice accumulate as a result of Ag selection and likely play a pathologic role in lupus nephritis.

Pyridostigmine bromide (PYR) is an anticholinesterase drug indicated for the treatment of myasthenia gravis and neuromuscular blockade reversal. It acts as a reversible cholinesterase inhibitor and was used as a pretreatment for soldiers during Operation Desert Storm to protect against possible nerve gas attacks. Since that time, PYR has been implicated as a possible causative agent contributing to Gulf War Illness. PYR's mechanism of action has been well-delineated with regards to its effects on the nervous system, yet little is known regarding potential effects on immunological function. To evaluate the effects of PYR on immunological function, adult female B6C3F1 mice were gavaged daily for 14 days with PYR (0, 1, 5, 10, or 20 mg/kg/day). Immune parameters assessed were lymphoproliferation, natural killer cell activity, the SRBC-specific antibody plaque-forming cell (PFC) response, thymus and spleen weight and cellularity, and thymic and splenic CD4/CD8 lymphocyte subpopulations. Exposure to PYR did not alter splenic and thymus weight or splenic cellularity. However, 20 mg PYR/kg/day decreased thymic cellularity with decreases in both CD4+/CD8+ (20 mg/kg/day) and CD4-/CD8- (10 and 20 mg/kg/day) cell types. Functional immune assays indicated that lymphocyte proliferative responses and natural killer cell activity were normal; whereas exposure to PYR significantly decreased primary IgM antibody responses to a T-cell dependent antigen at the 1, 5, 10 and 20 mg/kg treatment levels for 14 days. This is the first study to examine the immunotoxicological effects of PYR and demonstrate that this compound selectively suppresses humoral antibody responses.

Objective: To examine the association between N-acetyl transferase (NAT) genotype (NAT1 and NAT2) and risk of developing systemic lupus erythematosus (SLE)., Methods: DNA samples were collected from 243 recently diagnosed cases and 298 controls enrolled in a population based case-control study conducted in 60 counties in North Carolina and South Carolina, USA., Results: There was no association between SLE and NAT1 genotype (OR 0.96, 95% CI 0.65, 1.4 for the presence of a *10 allele) or NAT2 genotype (OR 1.1, 95% CI 0.73, 1.6 for the slow- compared with fast-acetylation genotype). We saw some evidence of interaction between NAT genotypes and use of hair dyes (a source of arylamines), with higher risk seen among hair dye users who had both the *10 NAT1 allele and the NAT2 slow-acetylation genotype (OR 2.9, 95% CI 1.2, 6.9 in this subgroup compared with all others)., Conclusion: Our results suggest that although there is little overall association between NAT genotypes and risk of developing SLE, the interaction between NAT1 and NAT2 and specific exposures such as hair dyes may be important. This finding highlights the need to consider exposure when assessing genetic susceptibility.

Cytotoxic lymphocyte antigen-4 (CTLA-4) plays an important role in regulating T cell activation, and may help to limit T cell response under conditions of inflammation. Genetic variability in CTLA-4 has been implicated in the development of several autoimmune diseases. Some studies have described associations between CTLA-4 polymorphisms and systemic lupus erythematosus (SLE), but findings have been inconsistent. We examined polymorphisms in the CTLA-4 gene promoter region (-1722T/C, -1661 A/G, -318C/T) and exon I (+49G/A) with respect to SLE in a population-based case-control study in the southeastern US. Genotypes from 230 recently diagnosed cases and 276 controls were examined separately for African-Americans and whites. We observed no overall associations between SLE and the four CTLA-4 polymorphisms examined. Subgroup analyses revealed effect modification by age for the presence of the -1661G allele, yielding a significant positive association with SLE in younger (<35 years) African-Americans (OR = 3.3). CTLA-4 genotypes also interacted with HLA-DR2 and GM allotype to contribute to risk of SLE. These findings suggest allelic variation in this region of CTLA4 is not a major independent risk factor for SLE, but may contribute to risk of disease in younger African-Americans or in the presence of certain immunogenetic markers.

Background: The alternative complement pathway (AP) is activated in individuals with lupus nephritis and in murine models of systemic lupus erythematosus, including MRL/lpr mice. A previous study from our laboratory evaluated the development of renal disease in MRL/lpr mice genetically deficient in factor B (Bf-/-), a protein necessary for AP activation. MRL/lpr Bf-/- mice developed less renal disease and had improved survival; however, these mice were also a different major histocompatibility complex (MHC) haplotype (H-2b) than their wild-type littermates (H-2k) due to the gene for Bf being located in the MHC gene complex. We undertook the current study to determine if the decreased renal disease in MRL/lpr Bf-/- mice was due to the lack of AP activation or the H-2b haplotype by studying the effects of factor D (Df) deficiency, a critical protein for AP activation, on disease development in MRL/lpr mice., Methods: Df-deficient mice were backcrossed with MRL/lpr mice for four to nine generations. MRL/lpr H-2k Df-/-, Df+/-, and Df+/+ littermates were evaluated for disease development. Lack of AP activation in MRL/lpr Df-/- mice was determined by the zymosan assay. Serum creatinine levels were measured using a creatinine kit. Proteinuria and autoantibody levels were determined by enzyme-linked immunosorbent assay (ELISA). Sections from one kidney were stained with fluorescein isothiocyanate (FITC) alpha-murine C3 or alpha-murine IgG to detect C3 and IgG deposition. The remaining kidney was cut in half with one half fixed, sectioned, and stained with hematoxylin and eosin and periodic acid-Schiff (PAS) to evaluate pathology and another half fixed in glutaraldehyde and examined via electron microscopy., Results: MRL/lpr Df-/- mice had similar glomerular IgG deposition, proteinuria and autoantibody levels, as Df+/+ and Df+/- littermates. However, glomerular C3 deposition, serum creatinine levels, and pathologic renal disease were significantly reduced in Df-/- mice. Despite the lack of renal disease in Df-/- mice, life span was not impacted by factor D deficiency., Conclusion: The absence of Df and AP activation is protective against the development of proliferative renal disease in MRL/lpr mice suggesting the similar effect of Bf deficiency in MRL/lpr mice was also due to the lack of AP activation.

Background: MRL/MpJ-Tnfrsf6lpr(MRL/lpr) mice, a murine model of systemic lupus erythematosus (SLE), have defective expression of Fas, substantially reducing signaling for apoptosis via this mechanism. However, it is known that MRL/lpr mice have increased spontaneous apoptosis of leukocytes. These conflicting observations have stimulated interest in apoptosis in this SLE model. MRL/lpr mice overproduce nitric oxide (NO) as autoimmune disease progresses. In vitro administration of NO may induce or decrease apoptosis depending on the cell type. Therefore, we hypothesized that NO induces MRL/lpr spleen lymphocyte apoptosis independent of Fas receptor engagement., Methods: Percentages of apoptotic spleen lymphocytes from MRL/lpr and BALB/cJ mice were determined ex vivo after in vivo treatment with NG-monomethyl-L-arginine (NMMA), a nitric oxide synthase (NOS) inhibitor. After culture in varying concentrations of a slow-acting NO donor, the following were determined in spleen lymphocytes: (1) levels of apoptosis, (2) the effect of phorbol myristate acid (PMA) on levels of NO-induced apoptosis, and (3) protein kinase C (PKC) activity., Results: Spleen lymphocytes from MRL/lpr mice with active disease had increased levels of ex vivo apoptosis when compared with BALB/cJ controls. This increase was reduced by pharmacologic inhibition of NOS in MRL/lpr but not in BALB/cJ mice. Exogenous administration of NO in vitro reduced PKC activity and induced apoptosis in MRL/lpr spleen lymphocytes, an effect that could be reduced via coadministration of PMA in vitro., Conclusion: These results suggest that NO plays a role in spleen lymphocyte apoptosis in MRL/lpr mice, possibly via inhibition of PKC, despite a Fas defect.

Terminal deoxynucleotidyl transferase (TdT) places non-template-coded nucleotides (N additions) in the VH CDR3 of T cell receptors and immunoglobulins. Amino acids coded for by N additions are important in autoantibody binding of dsDNA in lupus. We hypothesized that a genetic lack of TdT would modulate disease in lupus-prone mice. To test this hypothesis, we derived TdT-deficient MRL/lpr mice. Serum levels of anti-dsDNA antibodies and anti-dsDNA producing splenocytes were significantly lower in the TdT(-) versus TdT(+) littermates. Albuminuria, glomerular IgG deposition, and pathologic renal disease were significantly reduced in the TdT(-) mice. Sequence analysis of anti-dsDNA hybridomas derived from TdT(-) mice revealed a lack of N additions, short VH CDR3 segments, yet the presence of VH CDR3 arginines. Thus, the genetic absence of TdT reduces autoantibody production and clinical disease in MRL/lpr mice, confirming the importance of N additions in the autoimmune response in these mice.

Objective: Multiple genetic factors modulate predisposition to systemic lupus erythematosus (SLE). The glutathione S-transferase (GST) genes GSTM1, GSTT1, and GSTP1 catalyze metabolic pathways for the excretion of reactive oxygen species that may be generated by cellular oxidative stress induced by ultraviolet radiation in sunlight. We hypothesized that risk of SLE associated with occupational sun exposure is modulated by GSTM1, GSTT1, and GSTP1 genotypes., Methods: DNA samples and occupational history were collected from 243 cases and 298 controls in the Carolina Lupus Study, a population based case-control study of patients with recently diagnosed SLE., Results: There was no independent association between SLE and presence of the homozygous null GSTM1 or GSTT1 genotype, the homozygous Val/Val or heterozygous Val/Ile GSTP1 genotype, or occupational sunlight exposure. The prevalence of Ro autoantibodies was significantly increased among Caucasians with the GSTM1 null genotype (OR 2.6, 95% CI 1.0, 6.8), but was somewhat weaker among African-Americans (OR 1.5, 95% CI 0.7, 3.5). In the combined analysis of occupational sunlight exposure and GSTM1 genotype, the effect of sun exposure among Caucasians varied depending on GSTM1 genotype. There was a 3-fold increased risk (OR 3.1, 95% CI 0.9, 10.8) of SLE associated with 24 or more months' occupational sun exposure among Caucasians with the GSTM1 null genotype, but sun exposure was not associated with risk among GSTM1 positive Caucasians (OR 0.6, 95% CI 0.3, 1.5). The interaction was statistically significant (p = 0.028)., Conclusion: Our results suggest that GSTM1 homozygous null genotype may modify the effect of occupational sun exposure on the risk of SLE in caucasians.

Studies in human systemic lupus erythematosus (SLE) suggest a possible role for histone deacetylases (HDACs) in skewed gene expression and disease pathogenesis. We used the MRL-lpr/lpr murine model of lupus to demonstrate that HDACs play a key role in the heightened levels of both Th1 and Th2 cytokine expression that contribute to disease. The availability of specific HDAC inhibitors (HDIs) such as trichostatin A (TSA) and suberonylanilide hydroxamic acid (SAHA) permits the study of the role of HDACs in gene regulation. Our results indicate that HDIs downregulate IL-12, IFN-gamma, IL-6, and IL-10 mRNA and protein levels in MRL-lpr/lpr splenocytes. This effect on gene transcription is associated with an increased accumulation of acetylated histones H3 and H4 in total cellular chromatin. To elucidate the in vivo effects of TSA on lupuslike disease, we treated MRL-lpr/lpr mice with TSA (0.5 mg/kg/d) for 5 weeks. Compared with vehicle-treated control mice, TSA-treated mice exhibited a significant reduction in proteinuria, glomerulonephritis, and spleen weight. Taken together, these findings suggest that increased expression of HDACs leading to an altered state of histone acetylation may be of pathologic significance in MRL-lpr/lpr mice. In addition, TSA or other HDIs may have therapeutic benefit in the treatment of SLE.

Ischemia/reperfusion (I/R) injury of the kidney is a common cause of acute renal failure (ARF) and is associated with high morbidity and mortality in the intensive care unit. The mechanisms underlying I/R injury are complex. Studies have shown that complement activation contributes to the pathogenesis of I/R injury in the kidney, but the exact mechanisms of complement activation have not been defined. We hypothesized that complement activation in this setting occurs via the alternative pathway and that mice deficient in complement factor B, an essential component of the alternative pathway, would be protected from ischemic ARF. Wild-type mice suffered from a decline in renal function and had significant tubular injury, particularly in the outer medulla, after I/R. We found that factor B-deficient mice (fB(-/-)) developed substantially less functional and morphologic renal injury after I/R. Furthermore, control wild-type mice had an increase in tubulointerstitial complement C3 deposition and neutrophil infiltration in the outer medulla after I/R, whereas fB(-/-) mice demonstrated virtually no C3 deposition or neutrophil infiltration. Our results demonstrate that complement activation in the kidney after I/R occurs exclusively via the alternative pathway, and that selective inhibition of this pathway provides protection to the kidneys from ischemic ARF.

Objective: Systemic lupus erythematosus (SLE) is an autoimmune disease in which morbidity and mortality are higher in African-Americans. The etiology of this racial disparity is unknown. A genetic predisposition to enhanced nitric oxide (NO) production may predispose African-Americans to develop SLE and may increase disease severity. We have demonstrated a correlation between NO production and disease activity in SLE. Two polymorphisms in the inducible NO synthase (NOS2) promoter region (G-954C and CCTTT microsatellite repeat polymorphisms) are associated with improved outcome in some African patients with malaria. This study was designed to determine if these polymorphisms are associated with SLE., Methods: We assessed the frequency of both the G-954C and CCTTT microsatellite repeat NOS2 promoter polymorphisms in a cohort of patients with SLE and age, sex, and race matched controls in North Carolina and South Carolina., Results: Both polymorphisms were more frequent among African-American female SLE patients when compared with controls (p = 0.04 for the G-954C polymorphism and p = 0.03 for the CCTTT-8 repeat polymorphism). Further, the G-954C and CCTTT-8 repeat polymorphisms were in linkage disequilibrium (D cent = 0.89, p = 0.0001) among African-American female SLE patients., Conclusion: Altered genetic control of NOS2 transcription may be a risk factor for SLE among African-American females. The extent of linkage disequilibrium between the G-954C and CCTTT-8 repeat NOS2 promoter polymorphisms suggests that they were co-inherited.

We examined risk factors for systemic lupus erythematosus (SLE) in 265 recently diagnosed patients in North Carolina and South Carolina and 355 control subjects identified through driver's license records and frequency matched to patients by age, sex, and state. Analyses were limited to exposures before diagnosis (cases) or reference year (control subjects). SLE patients were more likely than control subjects to report a history of allergy to medications (odds ratio [OR] 3.1, 95% confidence interval [CI], 2.1-4.5), particularly to antibiotics. SLE risk increased with history of shingles (OR 2.5, 95% CI 1.1-5.9) and with frequent (more than once per year) cold sores in the 3 years before diagnosis (OR 2.8, 95% CI 1.4-5.4). There was little association with history of mononucleosis, a marker of late infection with Epstein-Barr virus, implanted medical devices, or hepatitis B vaccination. History of lupus in parents or siblings was associated with an increased risk (OR 3.3, 95% CI 1.2-8.6). Further research is needed to clarify whether medication allergies and specific infectious agents are involved in the etiology of SLE. Published by Elsevier Science Inc.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of autoantibodies and the development of immune complex glomerulonephritis. Lupus nephritis (LN) remains a leading cause of morbidity and mortality in SLE. As a result, defining pathogenetic mediators in LN remains a major research effort. Progression to LN in SLE is dependent on the host breaking immune tolerance and forming autoantibodies that deposit in the kidney. A variety of predisposing factors in the host must then be present for this event to result in renal pathology. In this article, the authors review recent reports that advance our understanding of LN disease mediators, from autoantibody production and immune complex deposition to end stage fibrosis.

Objective: Estrogen and prolactin may accelerate the progression of murine systemic lupus erythematosus (SLE). In humans, 85% of lupus patients are women, which also suggests the importance of hormonal factors in disease pathogenesis. The purpose of this study was to examine hormonal and reproductive risk factors for lupus among women., Methods: This population-based, case-control study included 240 female SLE patients diagnosed between January 1, 1995 and July 31, 1999 who fulfilled the American College of Rheumatology classification criteria. Female controls (n = 321) were identified through driver's license records. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) as measures of association, adjusting for age, state, race, and education. Analyses were limited to exposures before diagnosis., Results: Breast-feeding was associated with a decreased risk of developing SLE (OR 0.6, 95% CI 0.4-0.9), with a statistically significant trend for number of babies breast-fed and total weeks of breast-feeding. There were no associations with number of pregnancies or live births. Natural menopause occurred earlier in women with subsequent development of SLE compared with controls (P < 0.001). There was little association between SLE and current use or duration of use of hormone replacement therapy or oral contraceptives, and no association with previous use of fertility drugs., Conclusion: We found little evidence that estrogen- or prolactin-related exposures are associated with an increased risk of lupus. The reduced risk observed among women who had breast-fed one or more babies should be examined in other studies. Early natural menopause, rather than decreasing risk of SLE because of reduced estrogen exposure, may be a marker of susceptibility to development of SLE.

Objective: Crystalline silica may act as an immune adjuvant to increase inflammation and antibody production, and findings of occupational cohort studies suggest that silica exposure may be a risk factor for systemic lupus erythematosus (SLE). We undertook this population-based study to examine the association between occupational silica exposure and SLE in the southeastern US., Methods: SLE patients (n = 265; diagnosed between January 1, 1995 and July 31, 1999) were recruited from 4 university rheumatology practices and 30 community-based rheumatologists in 60 contiguous counties. Controls (n = 355), frequency-matched to patients by age, sex, and state of residence, were randomly selected from driver's license registries. The mean age of the patients at diagnosis was 39 years; 91% were women and 60% were African American. Detailed occupational and farming histories were collected by in-person interviews. Silica exposure was determined through blinded assessment of job histories by 3 industrial hygienists, and potential medium- or high-level exposures were confirmed through followup telephone interviews. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated by logistic regression., Results: More patients (19%) than controls (8%) had a history of medium- or high-level silica exposure from farming or trades. We observed an association between silica and SLE (medium exposure OR 2.1 [95% CI 1.1-4.0], high exposure OR 4.6 [95% CI 1.4-15.4]) that was seen in separate analyses by sex, race, and at different levels of education., Conclusion: These results suggest that crystalline silica exposure may promote the development of SLE in some individuals. Additional research is recommended in other populations, using study designs that minimize potential selection bias and maximize the quality of exposure assessment.

Background: MRL-MPJFaslpr (MRL/lpr) mice spontaneously develop lupus-like disease characterized by immune complex glomerulonephritis and overproduction of nitric oxide (NO). Blocking NO production pharmacologically by a non-specific nitric oxide synthase (NOS) inhibitor ameliorated renal disease in MRL/lpr mice while genetically deficient inducible NOS (iNOS) mice developed proliferative glomerulonephritis similar to wild-type controls., Methods: To clarify the role of iNOS in the pathogenesis of nephritis in MRL/lpr mice, we treated mice with two different NOS inhibitors. Either NG-monomethyl-l-arginine (L-NMMA), a nonspecific NOS inhibitor, or l-N6-(1-iminoethyl)lysine (L-NIL), an iNOS specific inhibitor, was administered in the drinking water from 10 through 22 weeks of age with disease progression monitored over time. Control mice received water alone., Results: Both L-NMMA and L-NIL blocked NO production effectively in MRL/lpr mice. As expected, neither L-NNMA nor L-NIL had an effect on antibody production, immune complex deposition or complement activation. Although both NOS inhibitors decreased protein excretion, L-NMMA was more effective than L-NIL. Pathologic renal disease was significantly decreased at 19 weeks in both treatment groups. At 22 weeks the L-NIL treated mice, but not the L-NMMA mice, had significantly reduced renal disease scores compared to controls., Conclusion: These results indicate that specific inhibition of iNOS blocks the development of pathologic renal disease in MRL/lpr mice.

MRL-MPJ Fas(lpr) (MRL/lpr) mice are a prototypic murine model for lupus characterized by an accelerated autoimmune syndrome. Disease begins as early as 8-wk-of-age in these animals with polyclonal B cell activation and elevated levels of serum IgM. By 12 to 16-wk-of-age MRL/lpr mice begin to produce a variety of autoantibodies including anti-dsDNA and anti-ss-DNA antibodies. From 16 to 24 wk, MRL/lpr mice develop proliferative immune complex mediated glomerulonephritis, vasculitis, arthritis, and massive lymphadenopathy that results in renal failure and death in 50% of the mice by 24-wk-of-age. This review will discuss several different genetic knockout experimental approaches used to study disease expression in MRL/lpr mice including various approaches in our laboratory aimed at autoantibody (Ab) production and inflammatory mediators.

We examined the prevalence of clinical and immunologic features of systemic lupus erythematosus (SLE) by race, sex and age in a population-based study of 265 SLE patients. Patients fulfilled the American College of Rheumatology classification criteria. The median time between diagnosis and study enrollment was 13 months. The clinical and hematologic data were limited to occurrences up to 6 months after the diagnosis date, as documented in medical records. We used sera collected at study enrollment from 244 (92%) patients for serologic testing of autoantibodies. The associations between clinical and immunological features of SLE and age, sex and race were examined using logistic regression. The effect of each of these variables was examined adjusting for the other two demographic factors. Mean age at diagnosis was 6 years younger among African-Americans and other minorities compared with white patients (P < 0.01). Discoid lupus, proteinuria, anti-Sm and anti-RNP autoantibodies were more commonly seen in African-American patients, with odds ratios higher than 3.0. Photosensitivity and mucosal ulcers were noted less often in African-American patients. Proteinuria, leukopenia, lymphopenia and thrombocytopenia were approximately three times more common in men compared with women. The prevalence of oral or nasal ulcers and anti-DNA autoantibodies declined with age. The extent to which the differences we observed reflect genetic or environmental influences on the disease process should be investigated.

Objective: To examine the association between smoking and hair treatments (dyes, permanents) and risk of developing systemic lupus erythematosus (SLE)., Methods: Patients (n = 265) diagnosed between January 1, 1995, and July 31, 1999, were recruited through 4 university based and 30 community based rheumatology practices in eastern North Carolina and South Carolina. Controls (n = 355) were identified through driver's license records and were frequency matched to patients by age, sex, and state. Data collection included a 60 min in-person interview. Analyses were limited to experiences that occurred before age at diagnosis (patients) or reference age (controls). Because the prevalence of use of hair treatments among men was very low, the analyses of those exposures were limited to women., Results: There was no association with smoking history and risk of developing SLE when analyzed as status (current, former, or never-smoker) or measures of dose (duration or pack-years). Use of permanent hair dyes in women was associated with a small increased risk of developing SLE (OR 1.5, 95% CI 1.0, 2.2). This association increased with longer duration of use (compared with nonusers, OR 1.7, 95% CI 1.0, 2.7 for 6 or more years). There was little evidence of an association between SLE and use of temporary dyes or of permanents and straighteners., Conclusion: These results suggest at most a weak association between SLE risk and permanent hair dyes or smoking. Genetic variability in the metabolism of these products may be important to assess in future studies.

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.

The aim of the current study was to test the hypothesis that the induction of an underlying immunologic condition in rabbits may enhance the development of steroid-induced osteonecrosis. Thirty-five adult rabbits were divided into four groups. Group I: 10 rabbits were immunized at 15-day intervals for 2 months by murine antibodies to deoxyribonucleic acid autoantibodies. Four weeks after the end of the immunization, the animals received injections of methylprednisolone for 7 days and then prednisolone per os for 8 months. Group II: 10 animals only received immunizations according to the protocol used in Group I. Group III: 10 animals only were treated with corticosteroids according to the protocol used in Group I. Group IV: five animals were used as controls. Various changes were observed in the proximal metaphysis and diaphysis of the femur in eight of 10 animals in Group I (80%) and in five of 10 animals in Group II (50%) when compared with the animals in Group III and Group IV. The most common feature was evidence of new and old hemorrhage through the sinusoids, exudative reaction and thrombus formation in veins and small arteries. Focal necrotic areas of bone marrow showed an accumulation of cell debris, residue of hemorrhage, and disappearance of marrow elements. These findings suggest that (1) corticosteroids may potentiate the effects of a preexisting condition to increase the risk of osteonecrosis; (2) immunologic reaction may be an important factor in the pathogenesis of necrotic lesions; and (3) repeated intramedullary hemorrhage and thrombus formation may represent early major pathologic findings in bone necrosis.

Mesangial cells from MRL/lpr mice, a model of lupus, overproduce nitric oxide (NO) compared to controls. J series prostaglandins (PG) and thiazolidinediones block LPS stimulation of NO production via the activation of peroxisome proliferator-activator receptor-gamma (PPAR-gamma) in macrophages but utilize an alternative mechanism in microglial cells. We investigated the mechanism by which PGJ(2) inhibits NO production in LPS/IFN-gamma-stimulated MRL/lpr mesangial cells. Our results demonstrated that LPS/IFN-gamma addition to MRL/lpr mesangial cells stimulated iNOS activation, expression of p-38 kinase and p44/42 MAPK, and NF-kappaB translocation to the nucleus. Both pioglitazone, a specific PPAR-gamma agonist, and PGJ(2) blocked NO production, iNOS protein expression, and iNOS mRNA transcription. PGJ(2) failed to inhibit nuclear NF-kappaB translocation or p44/42 MAPK or p-38 kinase induction in stimulated mesangial cells. These data suggest that PGJ(2) blocks iNOS expression and subsequent NO production in mesangial cells via a PPAR-gamma-mediated mechanism either by interfering with NF-kappaB transcriptional activity or by an NF-kappaB-independent mechanism., (Copyright 2001 Academic Press.)

Genetic variation in immunoglobulin gamma (GM) and kappa (KM) chains was associated with systemic lupus erythematosus (SLE) in some studies. However, the data are conflicting, and only one study examined associations in African-Americans. We examined GM and KM allotypes, by race, in a population-based case-control study of SLE. Sera from patients (n = 222) and controls (n = 273) were typed for GM and KM allotypes by a hemagglutination inhibition method. GM phenotypes were not significantly associated with SLE in African-Americans or Caucasians. However, the frequency of KM phenotypes in Caucasian patients was significantly different from that in controls (p = 0.032). KM3,3 was associated with an increased risk, whereas KM1,3 was associated with a lower relative risk of SLE. In African-Americans, however, the pattern of associations with KM phenotypes differed from that in Caucasians, and the overall difference between patients and controls was not statistically significant., (Copyright 2001 S. Karger AG, Basel)

Background: (MRL)-lpr/lpr mice spontaneously develop autoimmune disease characterized by arthritis and glomerulonephritis. Nitric oxide is postulated to play a role in the disease pathogenesis, as mice treated with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMMA) show markedly reduced manifestations of the disease. The purpose of this study was to examine the role of peroxynitrite in disease development in MRL-lpr/lpr mice., Materials and Methods: We examined kidney extracts from control and MRL-lpr/lpr mice for nitrotyrosine by immunoblot with a rabbit polyclonal anti-nitrotyrosine antibody. Catalase activity was determined spectrophotometrically or by activity staining of native polyacrylamide gels. In some experiments, we studied the ability of peroxynitrite and other agents to modify purified catalase in vitro., Results: Kidney extracts from diseased mice had elevated levels of nitrotyrosine, and decreased levels of catalase activity and protein, relative to control mice. MRL-lpr/lpr mice treated with NMMA in vivo had decreased levels of nitrotyrosine, and demonstrated a partial restoration of both catalase activity and protein levels. Treatment of catalase in vitro with peroxynitrite or tetranitromethane at pH 8.0 resulted in protein nitration and a decrease in catalase activity. 1,3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, also decreased the activity of catalase., Conclusions: These observations suggest that peroxynitrite formation, with an associated decrease in catalase activity and general decrease in antioxidant enzyme activity, may result in increased levels of hydrogen peroxide and other oxidants that can contribute to the pathogenesis of disease in MRL-lpr/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice develop immune complex glomerulonephritis similar to human lupus. Glomerular mesangial cells are key modulators of the inflammatory response in lupus nephritis. When activated, these cells secrete inflammatory mediators including NO and products of cyclooxygenase perpetuating the local inflammatory response. PGJ2, a product of cyclooxygenase, is a potent in vitro inhibitor of macrophage inflammatory functions and is postulated to function as an in vivo inhibitor of macrophage-mediated inflammatory responses. We hypothesized that in lupus, a defect in PGJ2 production allows the inflammatory response to continue unchecked. To test this hypothesis, mesangial cells were isolated from MRL/lpr and BALB/c mice and stimulated with IL-1beta or LPS plus IFN-gamma. In contrast to the 2- to 3-fold increase in PGJ2 production by stimulated BALB/c mesangial cells, supernatant PGJ2 did not increase in MRL/lpr mesangial cell cultures. NO production in stimulated MRL/lpr and BALB/c mesangial cells, was blocked by PGJ2 and pioglitazone. These studies suggest that abnormalities in PGJ2 production are present in MRL/lpr mice and may be linked to the heightened activation state of mesangial cells in these mice.

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.

Terminal deoxynucleotidyl transferase (TdT) adds nontemplate coded nucleotides (N additions) between the recombining ends of immunoglobulin and T cell receptor genes. These nucleotides add significant diversity to the Ig and TCR repertoires. Amino acids coded for by these nucleotides play a key role in the binding of self antigens by autoantibodies and autoreactive T cells. To determine the effect of a lack of N additions on autoantibody production, we bred the TdT knockout genotype onto the autoimmune C57BL/6-Fas(lpr) background. TdT-deficient mice had significantly lower sera anti-DNA and rheumatoid factor activity than their TdT-producing littermates. C57BL/6-Fas(lpr) TdT-deficient mice had shorter VH CDR3 regions and fewer VH CDR3 arginines [0.6% versus 4. 7%] than their TdT-producing littermates. These data indicate that the absence of TdT limited the production of anti-DNA antibodies and rheumatoid factors in C57BL/6-Fas(lpr) mice, likely due to constraints on Ig diversity secondary to the lack of TdT-derived N additions., (Copyright 2000 Academic Press.)

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease. Overproduction of nitric oxide (NO) has been implicated in its pathogenesis. Several retrospective studies have indicated a correlation between serum nitrate and nitrite (NOx) and disease activity. This measure of NO production can be falsely elevated by exogenous dietary and medication sources of NOx and variably reduced by serum thiols. These variables can make NOx a less reliable tool for studying the role of NO in SLE. Peroxynitrite, a by-product of NO and superoxide, nitrates tyrosine moieties. The resulting 3-nitrotyrosine (3NT) serves as a long-term indicator of NO-mediated protein modifications that is not affected by exogenous sources of NOx or serum thiols. We hypothesized that for these reasons serum 3NT levels would correlate with lupus disease activity more significantly than serum NOx. To address this hypothesis, we prospectively evaluated lupus disease activity, serum protein 3NT levels, and NOx levels in a cohort of lupus patients at 3-month intervals. Serum 3NT correlated with disease activity among African-Americans, while NOx correlated with disease activity among Caucasians. Subjects with active lupus nephritis had higher levels of serum 3NT than those without renal disease. Immunohistochemical analysis of renal biopsies from subjects with active proliferative lupus nephritis revealed renal expression of inducible NO synthase. The results of this study suggest that overproduction of NO may play a pathogenic role in SLE and lupus nephritis. Serum 3NT may be a useful, new tool for studying the contributions of NO to the pathogenesis of SLE.

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-gamma compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-gamma secreting cells, there was a significant rise in serum IFN-gamma levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-gamma while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.

To determine if soluble CD16 (sCD16) could alter the expression of lupus-like disease, groups of 10 female NZB/NZW mice (age 16-20 weeks) were given sCD16 three times a week for 5 weeks (control; 100 microg; 200 microg/dose) after onset of proteinuria. Results of this study indicate that the administration of sCD16 after onset of disease lowered anti-DNA levels, delayed the development of proteinuria, and significantly prolonged survival while the mice were on treatment. These results indicate that sCD16 alters the expression of autoantibodies and the progression of renal disease in NZB/NZW mice, suggesting that therapies directed at Fc receptors may be useful in the treatment of SLE., (Copyright 1998 Academic Press.)

MRL-lpr/lpr mice spontaneously develop manifestations of autoimmunity including arthritis, vasculitis, and glomerulonephritis. The paramagnetic molecule nitric oxide has been implicated as an effector molecule in initiation and propagation of these inflammatory conditions. In this study, we utilized electron paramagnetic resonance spectroscopy to directly detect nitrosylated protein complexes as products of nitric oxide in whole blood and in kidneys of MRL-lpr/lpr mice. Electron paramagnetic resonance spectra of blood samples from MRL-lpr/lpr mice showed nitrosyl hemoglobin species. Amounts of blood nitrosyl hemoglobin in MRL-lpr/lpr mice were significantly increased as compared to age-matched control mice. Electron paramagnetic resonance spectra of MRL-lpr/lpr kidney tissue exhibited a signal characteristic of a dinitrosyl-iron-dithiolate complex at g approximately 2.04. Formation of nitrosylated nonheme protein in diseased kidneys is associated with development of glomerulonephritis in the autoimmune mice. The presence of nitrosylated nonheme protein indicates the formation of nitric oxide within the kidneys of the diseased mice signifying in situ renal nitric oxide formation.

Anti-DNA induced in BALB/c mice by immunization with bacterial (Escherichia coli) DNA resemble spontaneous anti-DNA from lupus mice in V gene use and cross-reactivity with other nuclear Ags, but lack the high V(H) CDR3 arginine content seen in anti-DNA from lupus mice. Moreover, the induced anti-DNA bind bacterial and mammalian single-stranded (ss) DNA and bacterial double-stranded (ds) DNA, but do not bind mammalian dsDNA. This reactivity profile is in contrast to that of the spontaneously arising anti-DNA of lupus mice, among which mammalian dsDNA reactive Abs are prominent. In this study we demonstrate that the addition of arginine to V(H) CDR3 of an induced anti-DNA confers the mammalian dsDNA binding characteristic of anti-DNA from lupus mice. The ability to confer mammalian dsDNA binding is dependent on both the position of the arginine in V(H) CDR3 and the light chain with which the heavy chain is paired, suggesting the light chain plays a more substantial role in DNA binding by this Ab than has previously been reported for other anti-DNA. Our data support the argument that V(H) CDR3 arginines tend to confer antimammalian dsDNA reactivity, leading to censure of B cells expressing these Abs and provides an explanation for the absence of arginine-rich V(H) CDR3 in the bacterial DNA-induced response.