Autoantibodies to RNA polymerases recognize multiple subunits and demonstrate cross-reactivity with RNA polymerase complexes.
OBJECTIVE: To determine the subunit specificity of autoantibody directed to RNA polymerases (RNAP) I, II, and III, which is one of the major autoantibody responses in patients with systemic sclerosis (SSc). METHODS: Thirty-two SSc sera with anti-RNAP antibodies (23 with anti-RNAP I/III, 5 with anti-RNAP I/III and II, and 4 with anti-RNAP II alone) were analyzed by immunoblotting using affinity-purified RNAP and by immunoprecipitation using 35S-labeled cell extracts in which RNAP complexes were dissociated. Antibodies bound to individual RNAP subunits were eluted from preparative immunoblots and were further analyzed by immunoblotting and immunoprecipitation. RESULTS: At least 15 different proteins were bound by antibodies in anti-RNAP-positive SSc sera in various combinations. All 9 sera immunoprecipitating RNAP II and all 28 sera immunoprecipitating RNAP I/III recognized the large subunit proteins of RNAP II and III, respectively. Reactivity to RNAP I large subunits was strongly associated with bright nucleolar staining by indirect immunofluorescence. Affinity-purified antibodies that recognized a 62-kd subunit protein cross-reacted with a 43-kd subunit protein and immunoprecipitated both RNAP I and RNAP III. Antibodies that recognized a 21-kd subunit protein obtained from sera that were positive for anti-RNAP I/III and II antibodies immunoprecipitated both RNAP II and RNAP III. CONCLUSION: Anti-RNAP antibodies recognize multiple subunits of RNAP I, II, and III. Moreover, the results of this study provide the first direct evidence that antibodies that recognize shared subunits of human RNAPs or epitopes present on different human RNAP subunits are responsible for the recognition of multiple RNAPs by SSc sera. (+info)
Estrogen enhancement of anti-double-stranded DNA antibody and immunoglobulin G production in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.
OBJECTIVE: To study the in vitro effect of estrogen on IgG anti-double-stranded DNA (anti-dsDNA) antibody and total IgG production in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE), in order to elucidate its regulatory role in SLE. METHODS: PBMC from SLE patients and normal donors were cultured with 17beta-estradiol (E2). IgG anti-dsDNA antibodies, total IgG, and cytokine activity in the culture supernatants were measured by enzyme-linked immunosorbent assay. RESULTS: E2 enhanced production of IgG anti-dsDNA antibodies as well as total IgG in PBMC from SLE patients. Anti-dsDNA production in patients with inactive disease was less responsive to E2 than that in patients with active disease. E2 also enhanced total IgG, but not anti-dsDNA, production in the PBMC of normal donors. Antibody production was increased by E2 to a lesser extent in patients' B cells than in their PBMC. Anti-interleukin-10 (anti-IL-10) antibodies partially blocked the E2-induced increase in antibody production in patients' PBMC, but anti-IL-10 had no effect on B cells. E2 increased IL-10 production by patients' monocytes. Exogenous IL-10 acted additively with E2 in increasing antibody production in patients' B cells. CONCLUSION: These results suggest that E2 may polyclonally increase the production of IgG, including IgG anti-dsDNA, in SLE patients' PBMC by enhancing B cell activity and by promoting IL-10 production in monocytes. These findings support the involvement of E2 in the pathogenesis of SLE. (+info)
Up-regulation of glomerular extracellular matrix and transforming growth factor-beta expression in RF/J mice.
BACKGROUND: RF/J mice were first reported as a murine model of spontaneous glomerulosclerosis by Gude and Lupton in 1960, but the precise histologic characteristics and immunopathological background of this mouse have not been investigated further. METHODS: Measurements of serum levels of immunoglobulins, anti-single strand DNA (anti-ss-DNA) antibody, complement (C3), and circulating immune complex (IC) were performed. Analyses of glomerular histological and immunopathological lesions in association with the detection of mRNA expression of collagen IV, TGF-beta, matrix protein turnover related enzymes, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2) and platelet-derived growth factor (PDGF) were also performed in young (10-week-old) and elderly (60-week-old) RF/J mice with age-matched BALB/C mice as the controls. RESULTS: High levels of serum IgA and IgG from as early as 20 weeks of age were noted in the RF/J mice. Serum anti-ss-DNA antibody of aged RF/J mice increased up to 23% of that of aged MRL-lpr/lpr mice, and serum C3 concentration significantly decreased with age, reaching lower levels than that of BALB/c mice. IgA-IC levels were significantly high compared to BALB/C mice both in the early and late stages of life, whereas IgG-IC levels were high only in mice younger than 20 weeks. Semiquantitative and quantitative analyzes of renal histopathological findings revealed significantly marked and age-related mesangial matrix expansion in RF/J mice, with increasing frequency of global glomerular sclerosis and tubulointerstitial damage. On the other hand, although precise measurements of glomerular cell numbers also showed an apparent augmentation in both young and old RF/J mice compared to BALB/C mice, glomerular cellularity decreased with age in RF/J mice. Immunohistochemical study revealed massive immunoglobulin deposition from a young age in association with significantly higher accumulation of matrix proteins, such as types I and IV collagen and laminin from the early stage of life. In addition, in these glomeruli, transforming growth factor-beta1 (TGF-beta1) was highly expressed both in young and old mice. The mRNA expression of MMP-2 was up-regulated only in the early stage of life. Although PDGF mRNA of RF/J mice was significantly up-regulated in the early stage of life, the differences between the mice disappeared in the late stage of life. CONCLUSIONS: These findings suggest that in RF/J mice, an immunopathological background inducing high serum immunoglobulin and IC levels from the early stage of life is closely related to mesangioproliferative glomerular lesions mediated by PDGF, and that development of massive extracellular matrix accumulation in glomeruli was induced by up-regulated expression of TGF-beta with inappropriate regulation of protein turnover-related enzyme production. (+info)
Cryoglobulinaemia and rheumatic manifestations in patients with hepatitis C virus infection.
OBJECTIVES: To investigate the association of cryoglobulinaemia and rheumatic manifestations in Korean patients with hepatitis C virus (HCV) infection. METHODS: Forty nine Korean patients with HCV infection were recruited. The prevalence, concentration, and type of cryoglobulin (by immunofixation), rheumatoid factor (RF), antinuclear antibody (ANA), and various rheumatological symptoms were investigated and HCV genotype was determined by polymerase chain reaction with genotype specific primer. RESULTS: The prevalence of cryoglobulin was 59% in Korean HCV patients and the concentration of cryoglobulin was 9.8 (7.9) g/l (mean (SD)). The type of cryoglobulinaemia was identified in 23 (80%) of 29 HCV patients with cryoglobulinaemia and they were all type III. There were no differences in age, sex, history of operation and transfusion, proportion of liver cirrhosis between the patients with cryoglobulinaemia and those without cryoglobulinaemia. The frequencies of RF and ANA were 14% and 3.4% respectively in HCV patients with cryoglobulinaemia. There was no difference in HCV genotype between the patients with cryoglobulinaemia and those without cryoglobulinaemia. Clinical features of HCV patients were as follows: arthralgia/arthritis (35%), cutaneous manifestation (37%), Raynaud's phenomenon (8%), paresthesia (44%), dry eyes (22%), dry mouth (10%), oral ulcer (33%), and abdominal pain (14%). However, these rheumatological symptoms did not differ between the two groups. CONCLUSION: Although the rheumatological symptoms were not different between HCV patients with and without cryoglobulinaemia, HCV patients showed various rheumatological manifestations. These result suggests that HCV infection could be included as one of the causes in patients with unexplained rheumatological symptoms. (+info)
A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of defined specificities. I. Precision, sensitivity, and specificity.
OBJECTIVE: To determine the performance characteristics of enzyme-based immunoassay (EIA) kits for the detection of antinuclear and other autoantibodies of defined specificities. METHODS: Nine manufacturers of EIA kits to detect antibodies of defined specificities participated in a study in which they received coded sera from the Centers for Disease Control and Prevention. These coded sera contained different dilutions of antibody of one specificity mixed with sera containing antibodies of other specificities. The manufacturers were asked to use their standard technology to determine antibody content and send the data to a committee of the International Union of Immunological Societies for analysis. The data were analyzed for sensitivity and specificity in the detection of anti-double-stranded DNA (anti-dsDNA), anti-single-stranded DNA, antihistone, anti-Sm, anti-U1 RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70 (DNA topoisomerase I), anticentromere, and anti-Jo-1 antibodies. In addition, replicate samples were included in the coded sera to evaluate the precision of each EIA method. RESULTS: Lack of sensitivity and specificity was most evident in the anti-dsDNA and anti-Sm kits, although 2 kits for anti-dsDNA achieved acceptable sensitivity and specificity. Generally, anti-SSA/Ro, anti-SSB/La, anti-Scl-70, anticentromere, and anti-Jo-1 kits performed well. Many false-positive results were obtained with a multiple myeloma serum containing cryoprecipitates, but multiple myeloma sera without cryoprecipitates presented no problem in the EIA system. Precision, based on evaluation of replicate samples, varied from very good to poor. CONCLUSION: No single manufacturer was clearly superior to others in terms of their products' overall sensitivity, specificity, and precision. Areas that needed improvement were in kits for the detection of antibodies to dsDNA and to Sm antigen. Some EIA kits achieved good sensitivity and specificity. Individual manufacturers were informed of the performance of their respective kits so they could take measures to correct perceived deficiencies and thus improve the reliability of a group of important diagnostic assays used in the evaluation of systemic rheumatic diseases. (+info)
Influence of ethnic background on clinical and serologic features in patients with systemic sclerosis and anti-DNA topoisomerase I antibody.
OBJECTIVE: To investigate the effect of ethnicity on clinical and serologic expression in patients with systemic sclerosis (SSc) and anti-DNA topoisomerase I (anti-topo I) antibody. METHODS: Clinical and serologic features, as well as HLA class II allele frequencies, were compared among 47 North American white, 15 North American black, 43 Japanese, and 12 Choctaw Native American SSc patients with anti-topo I antibody. RESULTS: The frequency of progressive pulmonary interstitial fibrosis was lower, and cumulative survival rates were better in white compared with black and Japanese patients. Sera of white and black patients frequently recognized the portion adjacent to the carboxyl terminus of topo I, sera of Japanese patients preferentially recognized the portion adjacent to the amino terminus of topo I, and sera of Choctaw patients recognized both portions of topo I. Anti-RNA polymerase II and anti-SSA/Ro antibodies were present together with anti-topo I antibody more frequently in sera of Japanese patients than in sera of white patients. The HLA-DRB1 alleles associated with anti-topo I antibody differed; i.e., DRB1*1101-*1104 in whites and blacks, DRB1*1502 in Japanese, and DRB1*1602 in Choctaws. Multivariate analysis showed that ethnic background was an independent determinant affecting development of severe lung disease as well as survival. CONCLUSION: Clinical and serologic features in SSc patients were strongly influenced by ethnic background. The variability of disease expression in the 4 ethnic groups suggests that multiple factors linked to ethnicity, including genetic and environmental factors, modulate clinical manifestations, disease course, and autoantibody status in SSc. (+info)
Disparate T cell requirements of two subsets of lupus-specific autoantibodies in pristane-treated mice.
Intraperitoneal injection of pristane induces a lupus-like disease in BALB/c and other non-autoimmune mice characterized by autoantibody production and the development of immune complex disease closely resembling lupus nephritis. Two subsets of autoantibodies are induced by pristane: IgG anti-DNA DNA and -chromatin autoantibodies are strongly IL-6-dependent, whereas IgG anti-nRNP/Sm and -Su antibodies are not. The present studies were carried out to examine the role of T cells in establishing this dichotomy between the production of anti-nRNP/Sm/Su versus anti-DNA/chromatin autoantibodies. Autoantibody production and renal disease were evaluated in athymic (nude) mice treated with pristane. BALB/c nu/nu mice spontaneously developed IgM and IgG anti-single-stranded (ss)DNA and -chromatin, but not anti-nRNP/Sm or -Su, autoantibodies. Pristane treatment increased the levels of IgG anti-chromatin antibodies in nu/nu mice, but did not induce production of anti-nRNP/Sm or -Su antibodies. In contrast, BALB/c nu/+ and +/+ control mice did not spontaneously produce autoantibodies, whereas anti-nRNP/Sm and -Su autoantibodies were induced by pristane in approx. 50% of nu/+ and +/+ mice and anti-DNA/chromatin antibodies at lower frequencies. Nude mice spontaneously developed mild renal lesions that were marginally affected by pristane, but were generally milder than the lesions developing in pristane-treated nu/+ and +/+ mice. The data provide further evidence that two distinct pathways with different cytokine and T cell requirements are involved in autoantibody formation in pristane-induced lupus. This dichotomy may be relevant to understanding differences in the regulation of anti-DNA versus anti-nRNP/Sm autoantibodies in systemic lupus erythematosus, as well as the association of anti-DNA, but not anti-nRNP/Sm, with lupus nephritis. (+info)
Electrocardiographic abnormalities in a murine model injected with IgG from mothers of children with congenital heart block.
BACKGROUND: It is a widely held view that congenital heart block (CHB) is caused by the transplacental transfer of maternal autoantibodies (anti-SSA/Ro and/or anti-SSB/La) into the fetal circulation. To test this hypothesis and to reproduce human CHB, an experimental mouse model (BALB/c) was developed by passive transfer of human autoantibodies into pregnant mice. METHODS AND RESULTS: Timed pregnant mice (n=54) were injected with a single intravenous bolus of purified IgG containing human anti-SSA/Ro and anti-SSB/La antibodies from mothers of children with CHB. To parallel the "window period" of susceptibility to CHB in humans, 3 groups of mice were used: 8, 11, and 16 days of gestation. Within each group, we tested 10, 25, 50, and 100 microg of IgG. At delivery, ECGs were recorded and analyzed for conduction abnormalities. Bradycardia and PR interval were significantly increased in 8-, 11-, and 16-day gestational groups when compared with controls (P<0.05). QRS duration was not significantly different between all groups. Antibody levels measured by ELISA in both mothers and their offspring confirmed the transplacental transfer of the human antibodies to the pups. CONCLUSIONS: The passive transfer model demonstrated bradycardia, first-degree but not complete atrioventricular block in pups. The greater percentage and degree of bradycardia and PR prolongation in the 11-day mouse group correlates with the "window period" of susceptibility observed in humans. The high incidence of bradycardia suggests possible sinoatrial node involvement. All together, these data provide relevant insights into the pathogenesis of CHB. (+info)