Autoimmune response to the spliceosome

Objective. To assess the significance of autoantibodies to RA33, the A2 protein of the heterogeneous nuclear ribonucleoproteins (hnRNP), and to the related hnRNP proteins A1, B1, and B2 in rheumatic diseases. Methods. Using a partially purified preparation of hnRNP-A and hnRNP-B proteins, sera from 303 patients with various rheumatic diseases were investigated by immunoblotting. For the analysis of crossreactivities, autoantibodies were affinity purified by blot elution. Results. Anti-A2/RA33 was found in 35% of rheumatoid arthritis (RA) patients, 38% of mixed connective tissue disease (MCTD) patients, 23% of systemic lupus erythematosus (SLE) patients, and, apart from single exceptions, not in patients with other rheumatic diseases. All anti-A2/RA33-positive sera were also reactive with B1 and B2, and anti-A2/RA33 antibodies cross-reacted with both proteins. Antibodies to hnRNP-A1 were found less frequently; moreover, the majority of anti-A1-positive sera also contained anti-A2/RA33 antibodies. In anti-A1, anti-A2/RA33 doublepositive sera, cross-reactivity between the 2 antibodies was generally observed. In SLE patients, the presence of anti-A2/RA33 was correlated with the presence of anti-(U1) small nuclear RNP (snRNP) and anti-Sm (P < 0.0001 and P < 0.005, respectively), but there was no evidence for cross-reactivity between antibodies to hnRNP and antibodies to snRNP antigens. Conclusion. Since both hnRNPs and snRNPs are essential components of the spliceosome, the data show that the immune systems of patients with RA, SLE, and MCTD react to this functional complex. However, compared with MCTD and SLE patients, RA patients have a more restricted immune response to the spliceosome: they react to hnRNP proteins, particularly to hnRNP-A2/RA33, but not to snRNPs.     

Anticyclic citrullinated peptide antibodies in patients with mixed connective tissue disease

Abstract

The clinical significance of anticyclic citrullinated peptide (CCP) antibodies in patients with mixed connective tissue disease (MCTD) was assessed. Altogether, 86 sera from MCTD patients, 96 from rheumatoid arthritis (RA) patients, 42 from systemic lupus erythematosus (SLE) patients, 23 from systemic sclerosis (SSc) patients, 21 from poymyositis/dermatomyositis (PM/DM) patients, and 17 from those with Sjögren’s syndrome (SjS) were tested for anti-CCP antibodies using an enzyme-lined immunosorbent assay. Among the 96 RA patients, anti-CCP antibodies were detected in 85%, with the frequency being significantly higher than in MCTD, SLE, SSc, PM/DM, and SjS patients (9%, 14%, 13%, 14%, and 18%, respectively; P < 0.001). Among eight MCTD patients who fulfilled the diagnostic criteria for RA, only 50% had anti-CCP antibodies, and the prevalence was significantly lower than for all RA patients (p < 0.01). All eight patients who fulfilled the criteria for RA had overlap of SLE and SSc, except one patient, whereas the four anti-CCP-positive patients who did not fulfill the criteria for RA had SjS without overlapping features of SLE and SSc; moreover, most of their antibody titers were low. These results suggested that anti-CCP antibodies are associated with RA in MCTD patients, but careful diagnosis of RA is required if patients with low titers of anti-CCP antibodies lack overlapping SLE and SSc.     

Anticyclic citrullinated peptide antibodies in patients with mixed connective tissue disease

The clinical significance of anticyclic citrullinated peptide (CCP) antibodies in patients with mixed connective tissue disease (MCTD) was assessed. Altogether, 86 sera from MCTD patients, 96 from rheumatoid arthritis (RA) patients, 42 from systemic lupus erythematosus (SLE) patients, 23 from systemic sclerosis (SSc) patients, 21 from poymyositis/dermatomyositis (PM/DM) patients, and 17 from those with Sjögrens syndrome (SjS) were tested for anti-CCP antibodies using an enzyme-lined immunosorbent assay. Among the 96 RA patients, anti-CCP antibodies were detected in 85%, with the frequency being significantly higher than in MCTD, SLE, SSc, PM/DM, and SjS patients (9%, 14%, 13%, 14%, and 18%, respectively; P < 0.001). Among eight MCTD patients who fulfilled the diagnostic criteria for RA, only 50% had anti-CCP antibodies, and the prevalence was significantly lower than for all RA patients (p < 0.01). All eight patients who fulfilled the criteria for RA had overlap of SLE and SSc, except one patient, whereas the four anti-CCP-positive patients who did not fulfill the criteria for RA had SjS without overlapping features of SLE and SSc; moreover, most of their antibody titers were low. These results suggested that anti-CCP antibodies are associated with RA in MCTD patients, but careful diagnosis of RA is required if patients with low titers of anti-CCP antibodies lack overlapping SLE and SSc.     

PREDOMINANCE OF IgM ANTI-U1RNP ANTIBODIES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Anti-UIRNP antibodies occur in patients with mixed connectivetissue disease (MCTD), systemic sclerosis (SSc), systemic lupuserythematosus (SLE) and other ill-defined connective tissuediseases. To associate the isotypes of anti-UlRNP antibodieswith the diagnosis of the disease, namely SLE or MCTD, sequentialsera of patients positive for anti-UlRNP antibodies by counterimmunoelectrophorcsis(CIE) (32 with SLE, 35 with MCTD) were tested for IgG and IgManti-UlRNP antibodies by enzyme-linked immunosorbent assay (ELISA)using affinity-purified UlsnRNP complexes. Results from ELISAwere confirmed by RNA precipitation. IgG RNA precipitation ofHeLa cellular extracts was performed using the bulk of the IgGfraction removed from each serum after binding to protein A-Sepharosebeads. IgM RNA precipitation was carried out on the IgM fractionof the serum bound to protein A-Scpharose-rabbit anti-humanIgM immune complexes. RNAs were electrophoresed in 10.5% acrylamide-7m urea gels and detected with the silver stain. ELISA showedthat all sera were positive to IgG anti-UlRNP, while 12 of the35 MCTD and 21 of the 32 SLE patients possessed IgM anti-UlRNP(P<0.025). IgM anti-UlRNP reactivity was found during thefollow-up in 20% of 44 sera from 17 MCTD patients and 68% of112 sera from 23 SLE patients (P<0.0001). IgG from all thesera precipitated UlRNPs. Eight of the MCTD sera also precipitatedU2RNPs and 14 of the SLE sera U2 and/or U4/U6, U5 RNPs. IgMfrom MCTD sera did not precipitate URNPs, while IgM from SLEsera precipitated predominantly UlRNPs. These data suggest thatIgM anti-UlRNP antibodies occur predominantly in patients withSLE. The occurrence of IgG anti-UlRNP without IgM is more frequentin MCTD. KEY WORDS: Anti-UIRNP, Anti-Sm, Autoanubodies, Isotypes, SLE, MCTD     

Heterogenous nuclear RNP C1 and C2 core proteins are targets for an autoantibody found in the serum of a patient with systemic sclerosis and psoriatic arthritis

Objective.To determine a target recognized by anti-Bh autoantibody, found in the serum of a patient with the unusual coexistence of systemic sclerosis (SSc) and psoriatic arthritis (PsA). Methods. Antigens recognized by the anti-Bh serum were characterized by indirect immunofluorescence on HeLa cells, by conventional immunoblotting using nuclear extract or partially purified preparation of heterogenous nuclear RNP (hnRNP) proteins, and by 2-dimensional immunoblotting. For the analysis of cross-reactivity and immunofluorescence patterns, autoantibodies were affinity-purified by blot elution and then retested. Results.Comparison of the reactivity of the anti-Bh antibody with the monoclonal antibody 4F4 against both the hnRNP C proteins, together with the determination of biochemical properties of the autoantigens, led to the identification of C1 and C2 core proteins as the targets for the anti-Bh autoantibody. Conclusion. Several essential components of the spliceosome are targeted by autoantibodies that are present in the sera of patients with systemic rheumatic diseases. We also found that the hnRNP core proteins C1 and C2 are recognized by the autoantibody present in the serum of a patient with SSc and PsA. C1 and C2 hnRNP proteins should be added to the several intracellular autoantigens recently shown to be cleaved by interleukin-1β- converting enzyme-like enzymes during apoptosis.     

Induction of anti-RA33 hnRNP autoantibodies and transient spread to U1-A snRNP complex of spliceosome by idiotypic manipulation with anti-RA33 antibody preparation in mice

OBJECTIVE: Anti-RA33 antibodies occur in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD) and target the A2/B1 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex 4 which forms part of the spliceosome. The aim of the present study was to evaluate the immune response and pathological features induced in mice immunized with anti-RA33 antibodies or patient-derived recombinant single-chain variable fragments (scFv) of anti-RA33 antibodies. METHODS: In the first set of the experiment, two strains of mice (C57BL/6J and BALB/c) were immunized with IgG preparations obtained from two patients with RA and one normal donor. One of the patients had high titer anti-RA33 antibodies; the other one showed weak borderline reactivity. In the second set of the experiment three groups of C57BL/6J mice were immunized, respectively, with affinity-purified (AP) anti-RA33 antibodies, scFv of anti-RA33 antibodies and normal human IgG. The immunological response induced in immunized mice was studied by immunoblotting and line immunoassay (LIA). The presence of arthritis, serositis or myositis was assessed six-months following initial immunization. RESULTS: While anti-RA33 antibodies developed in only two of the mice immunized with different human IgG fractions, anti-RA33 antibodies were clearly detected in 7 sera of 13 mice immunized with AP anti-RA33 antibodies three months after the boost immunization and, moreover, also in 2 sera of 13 mice immunized with scFv of anti-RA33 antibodies. In contrast, mice immunized with normal human IgG did not develop anti-RA33 antibodies. Interestingly, transient autoantibody production against another nuclear autoantigen, U1 snRNP, was observed in 3 C57BL/6J mice immunized with scFv and in 1 mouse immunized with AP autoantibodies. However, these immunological responses were not associated with pathological findings. CONCLUSIONS: Active immunization of naive mice with AP anti-RA33 antibodies and scFv of anti-RA33 antibodies resulted on the one hand in the production of murine anti-RA33 antibodies and led, on the other hand, to transient "autoantibody spread" to snRNP component of the spliceosome and other nuclear autoantigens. This "autoantibody spread" probably reflected disregulation of the idiotypic anti-idiotypic cascade.     

Anti—topoisomerase i antibodies in silica-associated systemic sclerosis. A model for autoimmunity

Objective. To determine the frequency and type of autoantibodies present in patients with systemic sclerosis (SSc) associated with an established environmental toxin. Methods. Clinical data and sera were available from 14 men with silica-associated SSc who had developed SSc after at least 2 years of exposure to silica at work. Controls included 27 men with silicosis without SSc. Autoantibodies were measured by immunodiffusion, immunoblotting, and functional inhibition of topoisomerase I (topo I). Results. Nine of the 14 patients with silica-associated SSc had anti—topo I antibodies. All anti—topo I antibodies in the patients with silica-associated SSc and in 14 anti—topo I—positive patients with idiopathic SSc were directed at an active site of topo I, or at least sterically inhibited its function. One patient with silica-associated SSc had anticentromere antibodies. Unexpectedly, 2 patients with silicosis who had no symptoms of a connective tissue disease had autoantibodies to Ro/SS-A and La/SS-B autoantigens. Conclusion. Anti—topo I antibodies are the predominant autoantibodies present in silica-associated SSc. The generation of anti—topo I antibodies in genetically susceptible individuals may depend partly on the patient's sex and on the site of organ involvement, and may be triggered by silica particles acting as an immune adjuvant.     

Identification of an immunodominant epitope on RNA polymerase III recognized by systemic sclerosis sera: Application to enzyme‐linked immunosorbent assay

Objective

To characterize an immunodominant epitope on RNA polymerase III (RNAP III) recognized by systemic sclerosis (SSc) sera and to develop an enzyme‐linked immunosorbent assay (ELISA) for the detection of serum anti–RNAP I/III antibodies.

Methods

RNAP III–specific subunits RPC62 and RPC155 were generated in a bacterial expression system as a series of recombinant fragments. Reactivities to these recombinant fragments were examined by immunoblots and/or ELISA in 16 SSc sera containing anti–RNAP I/III antibodies, 89 SSc sera lacking anti–RNAP I/III antibodies, 61 systemic lupus erythematosus (SLE) sera, and 61 healthy control sera.

Results

Anti–RNAP I/III–positive SSc sera recognized several distinct epitopes on RPC62 and RPC155 in various combinations, but the fragment encoding amino acids at positions 732–1166 of RPC155 was recognized by all 11 anti–RNAP I/III–positive SSc sera tested. Carboxyl‐ and amino‐terminal deletion studies showed that at least 130 amino acids at positions 891–1020 of RPC155 were necessary for the antibody binding, but strong reactivity required an additional amino‐terminal extension. When a purified recombinant fragment containing the immunodominant epitope was used as the antigen source in an ELISA, elevated antibody reactivity was detected in all 16 anti–RNAP I/III–positive SSc sera, but in no anti–RNAP I/III–negative SSc, SLE, or healthy control sera, representing a sensitivity of 100% and a specificity of 100%.

Conclusion

A major epitope commonly recognized by SSc sera containing anti–RNAP I/III antibodies was identified on RPC155. The ELISA using a recombinant fragment expressing the immunodominant epitope should be a valuable tool for routine screening for anti–RNAP I/III antibodies in clinical diagnostic laboratories.

     

Ribosomal P protein P0 as a candidate for the target antigen of anti-endothelial cell antibodies in mixed connective tissue disease

OBJECTIVE: To evaluate the presence of anti-endothelial cell antibodies (AECA) in patients with mixed connective tissue disease (MCTD) compared to those with systemic sclerosis (SSc) and to determine the candidates for the endothelial auto-antigen that reacts with AECA in patients with MCTD using a molecular cloning strategy. METHODS: AECA were measured by a cellular enzyme-linked immunosorbent assay (ELISA) using fixed human umbilical vein endothelial cells (HUVEC) in 47 MCTD patients, 68 SSc patients, and 52 normal controls. A HUVEC cDNA expression library was immunoscreened with pooled sera from 6 patients with high AECA levels determined by cellular ELISA to explore the endothelial autoantigens in MCTD. An ELISA assay for anti-ribosomal protein P0 antibodies was used to assess the correlation with AECA levels. RESULTS: The candidate target proteins recognized by AECA in MCTD included: (i) ribosomal protein P0; (ii) a putative oncogene derived from dek mRNA; (iii) SS-B/La protein; (iv) U1 RNA-associated 70K protein; and (v) DNA-binding protein B. A significant correlation between the levels of AECA and anti-ribosomal protein P0 antibodies was demon-strated in MCTD, but not in systemic sclerosis. The sera containing high levels of AECA from patients with MCTD frequently cross-reacted with ribosomal protein P0. On the other hand, sera without AECA activity from patients with MCTD never reacted with ribosomal protein P0. CONCLUSION: AECA were more frequently seen in patients with MCTD than in patients with SSc. Ribosomal protein P0 may be one of the major target antigens of AECA in patients with MCTD.     

Presence of antinucleosome autoantibodies in a restricted set of connective tissue diseases: antinucleosome antibodies of the IgG3 subclass are markers of renal pathogenicity in systemic lupus erythematosus

OBJECTIVE: To study the frequency and disease specificity of antinucleosome antibody reactivity in diverse connective tissue diseases (CTD), and to determine factors, such as antibody subclass, that may influence the pathogenicity of these antibodies in relation to disease activity. METHODS: IgG and IgM antinucleosome activities on nucleosome core particles from 496 patients with 13 different CTD and 100 patients with hepatitis C were measured by enzyme-linked immunosorbent assay (ELISA). Of the patients with CTD, 120 had systemic lupus erythematosus (SLE), 37 had scleroderma (systemic sclerosis; SSc), 20 had mixed connective tissue disease (MCTD), and 319 had other CTD, including Sj?gren's syndrome, inflammatory myopathy, rheumatoid arthritis, primary antiphospholipid syndrome, Wegener's granulomatosis, Takayasu arteritis, giant cell arteritis, relapsing polychondritis, Beh?et's syndrome, and sarcoidosis. Antinucleosome-positive sera were further analyzed, by isotype-specific ELISA, for antinucleosome and anti-double-stranded DNA (anti-dsDNA) IgG subclasses. RESULTS: SLE, SSc, and MCTD were the only 3 CTD in which antinucleosome IgG were detected (71.7%, 45.9%, and 45.0% of patients, respectively). Antinucleosomes of the IgG3 subclass were present at high levels in patients with active SLE and were virtually absent in those with SSc, MCTD, or inactive SLE, and their levels showed a positive correlation with SLE disease activity. Of note, an increase in levels of antinucleosome of the IgG3 isotype was observed during SLE flares, and this increase was found to be closely associated with active nephritis. Levels of antinucleosome of the IgG1 subclass showed a trend toward an inverse correlation with SLE disease activity. No significant fluctuation in the anti-dsDNA isotype profile was observed in relation to SLE severity or clinical signs. CONCLUSION: Our data suggest that IgG antinucleosome is a new marker that may help in the differential diagnosis of CTD; antinucleosome of the IgG3 isotype might constitute a selective biologic marker of active SLE, in particular, of lupus nephritis.     

The centromere kinesin-like protein,CENP-E. An autoantigen in systemic sclerosis

Objective. Autoantibodies directed against centromere proteins (CENPs) are a serologic feature in some patients with systemic sclerosis (SSc). Previous studies have focused on autoantibodies to CENPs A, B, and C. CENP-E is a recently described 312-kd protein that also localizes to the centromere. Therefore, we studied the presence of autoantibodies to recombinant CENP-E in patients with SSc. Methods. Sixty sera from patients with the SSc spectrum of diseases were screened for the presence of autoantibodies against CENP-E, by indirect immunofluorescence and immunoblotting using recombinant CENPE protein. HLA class II alleles were determined by DNA oligotyping. Results. Among the SSc sera, 15 of 60 (25%) demonstrated antibody reactivity with recombinant CENP-E, and 14 of these 15 sera (93%) had antibodies directed against another CENP. Anti–CENP-E was seen in 13 of 30 sera with anti-CENP (43%). All patients with anti–CENP-E had a limited form of SSc, known as the CREST variant (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). When patients with anti-CENPs A, B, or C were compared with patients with anti–CENP-E, no unique clinical features in the anti–CENP-E positive group were identified. Ninety-three percent of the patients with anti–CENP-E had HLA–DQB1 alleles that had polar amino acids at position 26 (primarily DQB1*05), similar to patients with other CENP autoantibodies. Conclusion. Antibodies to CENP-E are common in patients with SSc, and are seen in higher frequency in sera from patients with a limited form, or CREST variant, of the disease.     

Frequency and clinical associations of anti-chromo antibodies in connective tissue disease.

OBJECTIVES--To assess the frequency and clinical associations of anti-chromo antibodies in connective tissue disease and to study their relationship to anti-centromere autoreactivity. METHODS--Anti-chromo and anti-centromere antibodies (ACA) were measured by immunoblotting in 50 patients with systemic sclerosis (SSc) and 58 connective tissue disease controls. Common epitopes on centromere and chromo antigens were sought using affinity-purified antibodies from immunoblots. RESULTS--Anti-chromo antibodies were detected in three of 32 sera with ACA and no controls. The three patients with anti-chromo antibodies had limited cutaneous SSc, and two had erosive arthritis. No cross-reactivity between chromo or centromere-related proteins was demonstrated. CONCLUSIONS--Anti-chromo antibodies form a rare autoantibody specificity that may identify an overlap group of patients with SSc and arthritis. Chromo and centromere antigens are associated but immunologically distinct autoimmune targets. Whether they localise to the same chromosomal site remains unknown.     

Identification of hnRNP C1/C2 as an Autoantigen in Patients with Behcet’s Disease

Background: Ribonucleoproteins particles that form the spliceosomes are among the most frequently targeted molecules of the autoimmune response. In the last few years, autoantibodies against all A/B hnRNP proteins have been found in the sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), and serve as diagnostic markers for several rheumatic diseases. However, the functional role of hnRNP C1/C2 in autoimmune diseases is still not clearly understood. Objective: To identify hnRNP C1/C2 as an autoantigen in patients with Behcet’s Disease (BD). Methods: First, HaCaT and EA.hy926 cells were cultured and RNA was extracted. Second, amplification of the corresponding gene by RT-PCR, cloning, and purification techniques was applied to acquire the recombinant protein hnRNP C1/C2. Third, the target protein band was excised from gel electrophoresis, digested with trypsin, and analyzed by (MALDI-TOF/). Finally, Western blotting and ELISA were performed to verify the immunoreactivity of BD serum with recombinant hnRNPC1/C2. Results: Results demonstrated that the reactivity of BD serum against recombinant hnRNP C1/C2 protein was significantly higher as compared to healthy control (P<0.001). Conclusion: hnRNP C1/C2 can be considered as a self antigen which might be involved in BD pathology in Hans Chinese population.     

Lack of natural antibodies in rheumatic diseases

Summary The prevalence and Ig class of natural antibodies in the sera of healthy individuals and of patients with rheumatic diseases were studied. The presence, even in high concentrations, of natural antibodies in normals was confirmed. In the rheumatic diseases tested, however, a discordance in the levels and Ig class of anti-actin, anti-myosin and anti-ssDNA antibodies was noted. IgM anti-actin antibodies occur infrequently, whereas IgM anti-myosin and anti-ssDNA are increased in these diseases. IgG anti-actin antibodies are increased in the sera of patients with RA, but not in patients with SLE, polymyositis or mixed connective tissue disease (MCTD). IgG anti-myosin as well as anti-ssDNA antibodies were increased in patients with RA and SLE, but not in patients with polymyositis or MCTD. These findings suggest that these natural antibodies are unlike the multispecific autoantibodies produced by lymphocytes from the CD5+B cell lineage and that they may have undergone affinity maturation. Perhaps natural antibodies are a feature of health rather than of disease.     

Autoantigenic epitopes on dna topoisomerase i

Objective. To elucidate the clinical and immunogenetic associations with reactivity to autoantigenic epitopes on DNA topoisomerase I (topo I) recognized by sera from patients with systemic sclerosis (SSc). Methods. Autoantigenic epitopes on topo I were identified by screening an epitope library constructed from topo I complementary DNA restriction fragments using autoimmune anti–topo I–positive sera as a probe. Epitope reactivities of sera from 43 anti–topo I–positive SSc patients were surveyed by immunoblotting, and associations with clinical symptoms and HLA–DR types were examined. Results. Four different epitope regions were identified on the topo I molecule. Immunoreactivity to the region encompassing amino acid residues 658–700, termed ER4, was found to be associated with diffuse cutaneous SSc, progressive pulmonary interstitial fibrosis, and poor prognosis for 15-year survival. SSc patients with ER4 reactivity frequently displayed the DR2/DRw52 phenotype. Conclusion. Molecular analysis of precise antigenic epitopes on topo I is helpful in classifying clinical subsets of SSc.     

Direct binding of anti-DNA topoisomerase I autoantibodies to the cell surface of fibroblasts in patients with systemic sclerosis

OBJECTIVE: Fibroblasts play a crucial role in the development of systemic sclerosis (SSc), and antifibroblast antibodies (AFAs) capable of inducing a proinflammatory phenotype in fibroblasts have been detected in the sera of SSc patients. This study examined the prevalence of AFAs in SSc and other diseases and the possible correlation between AFAs and known antinuclear antibody specificities in SSc patients. METHODS: Sera from 99 patients with SSc, 123 patients with other autoimmune and nonautoimmune diseases, and 30 age- and sex-matched healthy controls were examined. AFA prevalence was assessed by flow cytometry and further characterized by indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Anti-topoisomerase I (anti-topo I) from SSc sera were purified by affinity chromatography on topo I. RESULTS: AFAs were more common in SSc patients (26.3%) than in any other disease groups studied. The presence of AFA was significantly associated with pulmonary involvement and death. AFA-positive sera from SSc patients bound to all human and rodent fibroblasts tested, but not to human primary endothelial cells or smooth muscle cells. All SSc AFAs strongly reacted with topo I by ELISA and immunoblotting. The binding intensity of SSc AFAs correlated strongly with reactivity against topo I on immunoblots of fibroblast extracts and with the immunofluorescence pattern typical of anti-topo I on permeabilized cells. Total IgG and affinity-purified anti-topo I from AFA-positive SSc sera were found to react with the surface of unpermeabilized fibroblasts by flow cytometry as well as by immunofluorescence and confocal microscopy. CONCLUSION: This is the first report establishing that AFAs in SSc are strongly correlated with anti-topo I and, furthermore, that anti-topo I antibodies themselves display AFA activity by reacting with determinants at the fibroblast surface.     

Direct binding of anti–DNA topoisomerase I autoantibodies to the cell surface of fibroblasts in patients with systemic sclerosis

Objective

Fibroblasts play a crucial role in the development of systemic sclerosis (SSc), and antifibroblast antibodies (AFAs) capable of inducing a proinflammatory phenotype in fibroblasts have been detected in the sera of SSc patients. This study examined the prevalence of AFAs in SSc and other diseases and the possible correlation between AFAs and known antinuclear antibody specificities in SSc patients.

Methods

Sera from 99 patients with SSc, 123 patients with other autoimmune and nonautoimmune diseases, and 30 age‐ and sex‐matched healthy controls were examined. AFA prevalence was assessed by flow cytometry and further characterized by indirect immunofluorescence, enzyme‐linked immunosorbent assay (ELISA), and immunoblotting. Anti–topoisomerase I (anti–topo I) from SSc sera were purified by affinity chromatography on topo I.

Results

AFAs were more common in SSc patients (26.3%) than in any other disease groups studied. The presence of AFA was significantly associated with pulmonary involvement and death. AFA‐positive sera from SSc patients bound to all human and rodent fibroblasts tested, but not to human primary endothelial cells or smooth muscle cells. All SSc AFAs strongly reacted with topo I by ELISA and immunoblotting. The binding intensity of SSc AFAs correlated strongly with reactivity against topo I on immunoblots of fibroblast extracts and with the immunofluorescence pattern typical of anti–topo I on permeabilized cells. Total IgG and affinity‐purified anti–topo I from AFA‐positive SSc sera were found to react with the surface of unpermeabilized fibroblasts by flow cytometry as well as by immunofluorescence and confocal microscopy.

Conclusion

This is the first report establishing that AFAs in SSc are strongly correlated with anti–topo I and, furthermore, that anti–topo I antibodies themselves display AFA activity by reacting with determinants at the fibroblast surface.

     

Detection of autoantibodies to extractable nuclear antigens in autoimmune diseases of rheumatic origin using immunoblotting--comparison with counter-electrophoresis

Sera from 82 patients with rheumatic autoimmune disease were tested for anti-ENA antibodies by immunoblotting and counterimmunoelectrophoresis (CIE), using HeLa cell extract and rabbit thymus extract, respectively, as antigens. Anti-ENA antibodies were more frequently detected and could be better differentiated by immunoblotting rather than by CIE. There was especially an increase in anti-Sm antibodies (23, in contrast to four positive results), which was only detectable in serum samples for the 59 SLE patients. The sera of the six patients with MCTD all reacted with the 68 U1-RNP antigen. The sera of the five patients with Sj?gren's syndrome only recognized the 50k La antigen, while other anti-ENA antibodies were not observed. In SLE anti-La antibodies were often associated with other anti-ENA antibodies. Seven out of eight SLE patients showing a combined detection of antibodies against Sm, U1-RNP and La by immunoblotting demonstrated severe organ involvements, especially lupus nephritis. Therefore, the characterization of anti-ENA antibodies by immunoblotting may contribute to improve the differentiation of connective tissue diseases.     

Antibodies to retroviral proteins in autoimmune connective tissue disease. Relation to clinical manifestations and ribonucleoprotein autoantibodies

Objective. To study the relationship between antibodies that recognize human retroviral proteins and the presence of clinical features and ribonucleoprotein antibodies in patients with autoimmune connective tissue diseases (CTDs). Methods. Antibodies against native human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia/lymphoma virus type I, recombinant HIV-1 Nef protein, and ribonucleoprotein antigens were determined by immunoblot of sera from 65 prospectively studied patients with definite or suspected CTDs of autoimmune type. Results. Antibodies to retroviral proteins (ARP), most frequently to HIV Gag proteins p55 and p24, were found in 64% of 22 patients with systemic lupus erythematosus (SLE), in 63% of 8 patients with discoid LE (DLE), in 75% of 8 patients with mixed connective tissue disease (MCTD), and in 26% of 19 individuals with chronic biologically false-positive (CBFP) seroreactions, but not in 8 patients with subacute cutaneous lupus erythematosus. No clear correlation of ARP with antibodies to any specific small nuclear RNP antigen was observed. The most striking finding was that recurrent infections, both in LE patients and in those with CBFP reactions and widespread, acral discoid skin lesions, occurred significantly more often in ARP-positive patients. Conclusion. The occurrence of antibodies reacting with human retroviral proteins is associated with severe skin lesions and recurrent infections in SLE, DLE, and MCTD patients, and with a disposition toward developing systemic disease in CBFP reactors.     

Antibodies to type IV collagen in rheumatic diseases

Rheumatic disease sera were examined by a sensitive and specific enzyme linked immunosorbent assay (ELISA) for antibodies to native and to denatured type IV collagen from basement membranes of bovine anterior lens capsules or human placenta. The frequency of antitype IV collagen antibodies depended on the antigen and the disease studied. No controls had human type IV collagen antibodies and only 5.6% had antibody to bovine type IV collagen. Antibody to one or more of our 4 antigens was seen in 20% of children with juvenile rheumatoid arthritis (JRA), 35% of patients with mixed connective tissue disease (MCTD), 40% of those with juvenile dermatomyositis (DM), 52% of adults with rheumatoid arthritis (RA), 56% of patients with scleroderma and 60% of patients with systemic lupus erythematosus (SLE). Antibodies to native human type IV collagen were rare (0-10%) except in SLE (45%). Antibodies to denatured human type IV collagen were commoner in RA, scleroderma and SLE. Antibodies to native bovine type IV collagen occurred in 8-20% of patient sera, and to denatured antigen in 25-26% of scleroderma, MCTD and DM patients. Antibovine type IV collagen activity measured by ELISA could be absorbed from positive sera by preincubation of the sera with bovine type IV collagen but not bovine type I collagen or native human placental type IV collagen, indicating that the antibodies were specific for bovine type IV collagen.