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Josef S. Smolen - One of the best experts on this subject based on the ideXlab platform.

  • immunodominant t cell epitopes of hnrnp a2 associated with disease activity in patients with rheumatoid arthritis
    European Journal of Immunology, 2010
    Co-Authors: Sylvie Trembleau, Markus H Hoffmann, Brigitte Meyer, Valerie Nell, Helga Radner, Wolfgang Zauner, Juergen Hammer, Gerald Aichinger, Gottfried Fischer, Josef S. Smolen
    Abstract:

    The heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) has been described as an important autoantigen in rheumatoid arthritis (RA) since it is targeted by autoantibodies, autoreactive T cells, and is aberrantly expressed in synovial cells in patients. To identify hnRNP-A2-specific T-cell epitopes possibly associated with pathogenicity, we used an innovative approach. We first scanned 280 overlapping hnRNP-A2 peptides for binding to the RA-associated class II molecules HLA-DR4 and HLA-DR1, leading to a comprehensive selection of binders. The selected peptides were tested in IFN-γ-specific ELISPOT assay: PBMC from 18% of RA patients showed a significant IFN-γ response to hnRNP-A2 peptides, 15% to the overlapping sequences 117–133 and/or 120–133, whereas PBMC from healthy individuals tested negative. We measured proliferative responses to these two peptides in another cohort of patients with RA or osteoarthritis: positive responses were found in 28% of RA, but also in 11% of osteoarthritis patients and these responses could be blocked by anti-MHC class II Ab. Remarkably, the presence of 117/120–133-specific T cells was significantly associated with active disease in RA patients, and bone erosion appeared to be more common in T-cell positive patients. These data suggest involvement of hnRNP-A2 specific cellular autoimmune responses in RA pathogenesis.

  • the rheumatoid arthritis associated autoantigen hnrnp a2 ra33 is a major stimulator of autoimmunity in rats with pristane induced arthritis
    Journal of Immunology, 2007
    Co-Authors: Jonatan Tuncel, Makiyeh Tohidastakrad, Birgit Turk, Serafin Pinolroma, Markus Hoffmann, Karl Skriner, Georg Schett, Guy Serre, Josef S. Smolen
    Abstract:

    A single intradermal injection of the mineral oil pristane in susceptible DA.1F rats induces erosive arthritis closely mimicking rheumatoid arthritis (RA). Pristane-induced arthritis (PIA) is driven by autoreactive T cells but no autoantigen has been identified to date. We therefore analyzed B and T cell responses to autoantigens potentially involved in the pathogenesis of RA, including IgG, citrullinated proteins, stress proteins, glucose-6-phosphate isomerase, and heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (RA33). IgG and IgM autoantibodies to hnRNP-A2 were detectable in sera of pristane-primed DA.1F rats already 1 wk before disease onset, reached maximum levels during the acute phase, and correlated with arthritis severity. Apart from rheumatoid factor, autoantibodies to other Ags were not observed. CD4 + lymph node cells isolated 10 days after pristane injection produced IFN-γ but not IL-4 in response to stimulation with hnRNP-A2, whereas none of the other candidate Ags elicited cytokine secretion. Surprisingly, hnRNP-A2 also stimulated lymph node cells of naive animals to produce inflammatory cytokines in a MyD88-dependent manner. Furthermore, hnRNP-A2 was highly overexpressed in the joints of rats injected with pristane. Overexpression coincided with the appearance of anti-RA33 Abs and preceded the onset of clinical symptoms of PIA by several days. Taken together, these data suggest hnRNP-A2 to be among the primary inducers of autoimmunity in PIA. Therefore, this Ag might play a pivotal role in the pathogenesis of PIA and possibly also human RA.

  • anti ra33 antibodies antibodies to the heterogeneous nuclear ribonucleoprotein a2
    Autoantibodies (Second Edition), 2007
    Co-Authors: Günter Steiner, Josef S. Smolen
    Abstract:

    ABSTRACT Anti-RA33 antibodies (Ab) are directed to the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, an abundant nuclear protein that is involved in processing, transport and translation of mRNA. Although hnRNP-A2 appears to be constitutively expressed in most organs and tissues, aberrant overexpression has been observed in certain cancers and in inflamed synovial tissue of patients with rheumatoid arthritis (RA). Anti-RA33 Ab occur in approximately one-third of patients with RA but are rarely detected in other rheumatic diseases, with the exception of systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) where they are as prevalent as in RA. Since anti-RA33 Ab are already detectable in the earliest stages of RA they are useful diagnostic markers showing a positive predictive value (PPV) for RA of approximately 74%. In the absence of rheumatoid factor (RF) and anti-citrullinated protein Ab (ACPA), anti-RA33 Ab appear to be associated with a relatively favourable prognosis. Similar to RF and ACPA, their role in the pathogenesis of RA is unclear. However, autoreactive T cells directed to hnRNP-A2 appear to be commonly present in blood and synovial fluid of RA patients. Their Th1-like phenotype is suggestive of pathogenic involvement of anti-RA33 autoimmunity.

  • characterization of autoreactive t cells to the autoantigens hnrnp a2 ra33 and filaggrin in patients with rheumatoid arthritis and controls
    Arthritis Research & Therapy, 2001
    Co-Authors: Ruth Fritsch, Karl Skriner, Josef S. Smolen, Daniela Eselbock, Beatrice Jahnschmid, Clemens Scheinecker, Barbara Bohle, Josef Neumuller, G Steiner
    Abstract:

    In an attempt elucidate the role of autoimmune processes in the pathogenesis of rheumatoid arthritis (RA) we investigated the T cell responses to two autoantigens targeted by autoantibodies of patients with RA, (i) the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/RA33 and (ii) filaggrin which is one of the target structures recognized by anti-citrulline antibodies. Stimulation assays were performed with peripheral blood mononuclear cells of 50 RA patients, 20 patients with osteoarthritis and 21 healthy control individuals using recombinant hnRNP-A2/RA33 as well as some fragments thereof and recombinant filaggrin both in unmodified and citrullinated form. Antigen-specific T cell clones (TCC) were obtained by cultivating T cell lines in the presence of antigen and IL-2 followed by limiting-dilution cloning. Proliferative responses to hnRNP-A2/RA33 were seen in 60% of the RA patients with a mean stimulation index (SI) of 3.5 ± 2.8 and were significantly higher than those observed in the control group (mean SI=1.7 ± 1, P < 0.00005). There was no correlation with the presence of anti-A2/RA33 autoantibodies nor with MHC genes, although more than 60% of the responsive patients carried the shared epitope. Results obtained with recombinant fragments indicated a major T cell epitope to be located in the N-terminal first RNA binding domain of the protein. Anti-A2/RA33 specific TCC (n = 16) derived from RA patients were almost exclusively CD4?, whereas only 7 of 12 TCC derived from controls showed this phenotype, and secreted high amounts of IFNg upon antigen stimulation as did all TCC derived from controls. Proliferative responses to filaggrin in either form were seen in only 25% of the RA patients tested and did not differ from those observed in the control group indicating that filaggrin-reactive T cells do presumably not drive the autoantibody response to citrullinated antigens. Taken together, a Th1 type autoimmune response to hnRNP-A2/RA33 was commonly observed in RA patients suggesting this nuclear protein to constitute a major T cell autoantigen which might be fuelling one of the pathological autoimmune reactions that drive the destructive processes effective in RA.

  • anti a2 ra33 autoantibodies are directed to the rna binding region of the a2 protein of the heterogeneous nuclear ribonucleoprotein complex differential epitope recognition in rheumatoid arthritis systemic lupus erythematosus and mixed connective tis
    Journal of Clinical Investigation, 1997
    Co-Authors: Karl Skriner, Josef S. Smolen, W H Sommergruber, Andrea Barta, V Tremmel, I Fischer, Günter Steiner
    Abstract:

    The recently described anti-A2/RA33 autoantibodies occur in 20-40% of patients with RA, SLE, and mixed connective tissue disease (MCTD). They are directed to the A2 protein of the heterogeneous nuclear ribonucleoprotein complex (hnRNP-A2), an abundant nuclear protein associated with the spliceosome. The NH2-terminal half of the antigen contains two conserved RNA binding domains whereas its COOH-terminal part is extremely glycine-rich. The aim of this study was to characterize the autoepitopes of hnRNP-A2 and to investigate the effects of anti-A2/RA33 autoantibodies on possible functions of the antigen. Using bacterially expressed fragments, two major discontinuous epitopes were identified. One containing the complete second RNA binding domain was recognized by the majority of patients with RA and SLE but not by patients with MCTD. The second epitope contained sequences of both RNA binding domains and was preferentially targeted by patients with MCTD. When the RNA binding properties of the antigen were investigated, oligoribonucleotides containing the sequence motif r(UUAG) were found to bind to a site closely adjacent or overlapping with the epitope targeted by autoantibodies from patients with RA and SLE. Moreover, anti-A2/RA33 autoantibodies from patients with RA or SLE, but not from patients with MCTD, inhibited binding of RNA. Thus, anti-A2/RA33 autoantibodies recognize conformation-dependent epitopes located in a functionally important region of the antigen. Furthermore, the specific recognition of an epitope by MCTD patients may be used as another argument in favor of considering MCTD a distinct connective tissue disease.

Karl Skriner - One of the best experts on this subject based on the ideXlab platform.

  • the rheumatoid arthritis associated autoantigen hnrnp a2 ra33 is a major stimulator of autoimmunity in rats with pristane induced arthritis
    Journal of Immunology, 2007
    Co-Authors: Jonatan Tuncel, Makiyeh Tohidastakrad, Birgit Turk, Serafin Pinolroma, Markus Hoffmann, Karl Skriner, Georg Schett, Guy Serre, Josef S. Smolen
    Abstract:

    A single intradermal injection of the mineral oil pristane in susceptible DA.1F rats induces erosive arthritis closely mimicking rheumatoid arthritis (RA). Pristane-induced arthritis (PIA) is driven by autoreactive T cells but no autoantigen has been identified to date. We therefore analyzed B and T cell responses to autoantigens potentially involved in the pathogenesis of RA, including IgG, citrullinated proteins, stress proteins, glucose-6-phosphate isomerase, and heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (RA33). IgG and IgM autoantibodies to hnRNP-A2 were detectable in sera of pristane-primed DA.1F rats already 1 wk before disease onset, reached maximum levels during the acute phase, and correlated with arthritis severity. Apart from rheumatoid factor, autoantibodies to other Ags were not observed. CD4 + lymph node cells isolated 10 days after pristane injection produced IFN-γ but not IL-4 in response to stimulation with hnRNP-A2, whereas none of the other candidate Ags elicited cytokine secretion. Surprisingly, hnRNP-A2 also stimulated lymph node cells of naive animals to produce inflammatory cytokines in a MyD88-dependent manner. Furthermore, hnRNP-A2 was highly overexpressed in the joints of rats injected with pristane. Overexpression coincided with the appearance of anti-RA33 Abs and preceded the onset of clinical symptoms of PIA by several days. Taken together, these data suggest hnRNP-A2 to be among the primary inducers of autoimmunity in PIA. Therefore, this Ag might play a pivotal role in the pathogenesis of PIA and possibly also human RA.

  • aberrant expression of the autoantigen heterogeneous nuclear ribonucleoprotein a2 ra33 and spontaneous formation of rheumatoid arthritis associated anti ra33 autoantibodies in tnf α transgenic mice
    Journal of Immunology, 2005
    Co-Authors: Silvia Hayer, Makiyeh Tohidastakrad, Serafin Pinolroma, Karl Skriner, Hélène Dumortier, Beatrice Jahnschmid, Silva Haralambous, Sylvie Trembleau, Kurt Redlich, Georg Schett
    Abstract:

    Human TNF-α transgenic (hTNFtg) mice develop erosive arthritis closely resembling rheumatoid arthritis (RA). To investigate mechanisms leading to pathological autoimmune reactions in RA, we examined hTNFtg animals for the presence of RA-associated autoantibodies including Abs to citrullinated epitopes (anti-cyclic citrullinated peptide), heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (anti-RA33), and heat shock proteins (hsp) (anti-hsp). Although IgM anti-hsp Abs were detected in 40% of hTNFtg and control mice, IgG anti-hsp Abs were rarely seen, and anti-cyclic citrullinated peptide Abs were not seen at all. In contrast, >50% of hTNFtg mice showed IgG anti-RA33 autoantibodies, which became detectable shortly after the onset of arthritis. These Abs were predominantly directed to a short epitope, which was identical with an epitope previously described in MRL/ lpr mice. Incidence of anti-RA33 was significantly decreased in mice treated with the osteoclast inhibitor osteoprotegerin and also in c- fos -deficient mice lacking osteoclasts. Pronounced expression of hnRNP-A2 and a smaller splice variant was seen in joints of hTNFtg mice, whereas expression was low in control animals. Although the closely related hnRNP-A1 was also overexpressed, autoantibodies to this protein were infrequently detected. Because expression of hnRNP-A2 in thymus, spleen, brain, and lung was similar in hTNFtg and control mice, aberrant expression appeared to be restricted to the inflamed joint. Finally, immunization of hTNFtg mice with recombinant hnRNP-A2 or a peptide harboring the major B cell epitope aggravated arthritis. These findings suggest that overproduction of TNF-α leads to aberrant expression of hnRNP-A2 in the rheumatoid joint and subsequently to autoimmune reactions, which may enhance the inflammatory and destructive process.

  • 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
    Clinical and Experimental Rheumatology, 2002
    Co-Authors: G Steiner, Karl Skriner, Ora Shovman, Boris Gilburd, Pnina Langevitz, M Miholits, R Hoet, Y Levy, Gisele Zandmangoddard, Elisabeth Hoefler
    Abstract:

    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.

  • characterization of autoreactive t cells to the autoantigens hnrnp a2 ra33 and filaggrin in patients with rheumatoid arthritis and controls
    Arthritis Research & Therapy, 2001
    Co-Authors: Ruth Fritsch, Karl Skriner, Josef S. Smolen, Daniela Eselbock, Beatrice Jahnschmid, Clemens Scheinecker, Barbara Bohle, Josef Neumuller, G Steiner
    Abstract:

    In an attempt elucidate the role of autoimmune processes in the pathogenesis of rheumatoid arthritis (RA) we investigated the T cell responses to two autoantigens targeted by autoantibodies of patients with RA, (i) the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/RA33 and (ii) filaggrin which is one of the target structures recognized by anti-citrulline antibodies. Stimulation assays were performed with peripheral blood mononuclear cells of 50 RA patients, 20 patients with osteoarthritis and 21 healthy control individuals using recombinant hnRNP-A2/RA33 as well as some fragments thereof and recombinant filaggrin both in unmodified and citrullinated form. Antigen-specific T cell clones (TCC) were obtained by cultivating T cell lines in the presence of antigen and IL-2 followed by limiting-dilution cloning. Proliferative responses to hnRNP-A2/RA33 were seen in 60% of the RA patients with a mean stimulation index (SI) of 3.5 ± 2.8 and were significantly higher than those observed in the control group (mean SI=1.7 ± 1, P < 0.00005). There was no correlation with the presence of anti-A2/RA33 autoantibodies nor with MHC genes, although more than 60% of the responsive patients carried the shared epitope. Results obtained with recombinant fragments indicated a major T cell epitope to be located in the N-terminal first RNA binding domain of the protein. Anti-A2/RA33 specific TCC (n = 16) derived from RA patients were almost exclusively CD4?, whereas only 7 of 12 TCC derived from controls showed this phenotype, and secreted high amounts of IFNg upon antigen stimulation as did all TCC derived from controls. Proliferative responses to filaggrin in either form were seen in only 25% of the RA patients tested and did not differ from those observed in the control group indicating that filaggrin-reactive T cells do presumably not drive the autoantibody response to citrullinated antigens. Taken together, a Th1 type autoimmune response to hnRNP-A2/RA33 was commonly observed in RA patients suggesting this nuclear protein to constitute a major T cell autoantigen which might be fuelling one of the pathological autoimmune reactions that drive the destructive processes effective in RA.

  • anti a2 ra33 autoantibodies are directed to the rna binding region of the a2 protein of the heterogeneous nuclear ribonucleoprotein complex differential epitope recognition in rheumatoid arthritis systemic lupus erythematosus and mixed connective tis
    Journal of Clinical Investigation, 1997
    Co-Authors: Karl Skriner, Josef S. Smolen, W H Sommergruber, Andrea Barta, V Tremmel, I Fischer, Günter Steiner
    Abstract:

    The recently described anti-A2/RA33 autoantibodies occur in 20-40% of patients with RA, SLE, and mixed connective tissue disease (MCTD). They are directed to the A2 protein of the heterogeneous nuclear ribonucleoprotein complex (hnRNP-A2), an abundant nuclear protein associated with the spliceosome. The NH2-terminal half of the antigen contains two conserved RNA binding domains whereas its COOH-terminal part is extremely glycine-rich. The aim of this study was to characterize the autoepitopes of hnRNP-A2 and to investigate the effects of anti-A2/RA33 autoantibodies on possible functions of the antigen. Using bacterially expressed fragments, two major discontinuous epitopes were identified. One containing the complete second RNA binding domain was recognized by the majority of patients with RA and SLE but not by patients with MCTD. The second epitope contained sequences of both RNA binding domains and was preferentially targeted by patients with MCTD. When the RNA binding properties of the antigen were investigated, oligoribonucleotides containing the sequence motif r(UUAG) were found to bind to a site closely adjacent or overlapping with the epitope targeted by autoantibodies from patients with RA and SLE. Moreover, anti-A2/RA33 autoantibodies from patients with RA or SLE, but not from patients with MCTD, inhibited binding of RNA. Thus, anti-A2/RA33 autoantibodies recognize conformation-dependent epitopes located in a functionally important region of the antigen. Furthermore, the specific recognition of an epitope by MCTD patients may be used as another argument in favor of considering MCTD a distinct connective tissue disease.

Minoru Satoh - One of the best experts on this subject based on the ideXlab platform.

  • sat0267 serum anti heterogeneous nuclear ribonucleoprotein antibody characterizes systemic sclerosis with anti centromere antibody and severe raynaud s phenomenon with digital ulcers
    Annals of the Rheumatic Diseases, 2019
    Co-Authors: Natasa Isailovic, Minoru Satoh, Angela Ceribelli, Carolina Gorlino, Elena Generali, Maria De Santis, Marta Caprioli, Giacomo Maria Guidelli, Piercarlo Sarziputtini, Carlo Selmi
    Abstract:

    Background: Systemic Sclerosis (SSc) is characterized by the presence of serum autoantibodies (autoAbs) which may be crucial in the diagnosis, prediction of organ involvement, follow-up, and treatment choices. However, SSc sera can be negative for autoAbs identified using commercially available tests, and several new and rare autoAbs have been identified in the last decade with immunoprecipitation (IP) techniques. We recently reported a new IP pattern corresponding to anti-heterogeneous nuclar ribonucleoprotein (hnRNP) antibodies in a cohort of SSc patients. Objectives: To analyze the IP pattern of anti-hnRNP antibodies in SSc sera and determine their clinical and laboratory correlations. Methods: We investigated sera from 63 consecutive patients with SSc attending our Unit between 2014 and 2018, using protein-IP of 35S-methionine-labeled K562 cell extract followed by SDS-PAGE and autoradiography, and IP-Western Blot for hnRNP-L and C1+C2 following established protocols. Clinical charts were used to analyze clinical and laboratory data for possible statistical correlations, performed by Prism (GraphPad Software 4.0, Fisher exact test) with statistical significance set at p values Results: We identified a new protein-IP pattern characterized by a set of several proteins of 140/40-25kD in 8/63 (13%) SSc sera (Figure panel A). This IP pattern corresponds to the hnRNP complex which is composed of several proteins and RNAs involved in RNA processing and splicing. These proteins have molecular weights ranging between 32 and 45kD as the set of proteins we identified in our experiments. To define the 140/40-25kD pattern identified in 8 SSc cases, we performed IP-WB using two anti-hnRNP antibodies selected by the molecular weight of the target antigen. We tested anti-hnRNP C1+C2 and anti-hnRNP L monoclonal antibodies in these 8 samples after preparation by crosslinking of IgG to PAS beads and protein-IP protocol, followed by WB (Figure panel B). Results show that 5/8 SSc anti-140/40-25kD samples are positive for hnRNP L, while no reactivity was observed with hnRNP C1+C2 monoclonal antibody. We further observed that anti- hnRNP antibodies are significantly associated with serum ACApositivity (p=0.008), severe Raynaud’s phenomenon with digital ulcers requiring IV prostacyclin (75%, 6/8 vs 7/54 ACA+, 13%; p=0.0006), and esophageal involvement in 4 cases. Conclusion: We identified anti-hnRNP antibodies as new target antigen of a subset of SSc patients with ACA positivity and severe peripheral vascular involvement. Additional analysis is necessary to study the several components of anti-hnRNP antibodies, and longitudinal data will provide evidence on the predictive value of this biomarker. Reference [1] Ceribelli A, et al. J Rheumatol. 2010 Oct;37(10):2071-5 Disclosure of Interests: Natasa Isailovic: None declared, Angela Ceribelli: None declared, Carolina Gorlino: None declared, Elena Generali: None declared, Maria De Santis: None declared, Marta Caprioli: None declared, Giacomo Maria Guidelli: None declared, Piercarlo Sarzi-Puttini: None declared, Minoru Satoh: None declared, Carlo Selmi Grant/research support from: AbbVie, Janssen, MSD, Novartis, Pfizer, Consultant for: AbbVie, Alfa-Sigma, Biogen, Bristol-Myrs Squibb, Celgene, Eli-Lilly, Janssen, Merck Sharp and Dohme, Novartis, Pfizer, Roche, Sanofi-Genzyme, UCB, Speakers bureau: AbbVie, Alfa-Sigma, Biogen, Bristol-Myrs Squibb, Celgene, Eli-Lilly, Janssen, Merck Sharp and Dohme, Novartis, Pfizer, Roche, Sanofi-Genzyme, UCB

  • Brief Definitive Report Interleukin 6 Dependence of Anti-DNA Antibody Production: Evidence for Two Pathways of Autoantibody Formation in Pristane-induced Lupus
    2013
    Co-Authors: B. Richards, Melody Shaw, Claude Libert, Valeria Poli, Minoru Satoh, Westley H. Reeves
    Abstract:

    Pristane induces a lupus-like syndrome in nonautoimmune mice characterized by the development of glomerulonephritis and lupus-associated autoantibodies. This is accompanied by overproduction of interleukin (IL)-6, a cytokine linked with autoimmune phenomena. The goal of this study was to evaluate the role of IL-6 in autoantibody production in pristane-induced lupus. BALB/cAn IL-6–deficient (���) and –intact (���) mice were treated with pristane or phosphate-buffered saline, and autoantibody production was evaluated. Pristane induced high levels of immunoglobulin (Ig)G anti-single-stranded DNA, –double-stranded (ds)DNA, and-chromatin antibodies in IL-6�� � , but not IL-6�� � mice by enzyme-linked immunosorbent assay. High titer IgG anti-dsDNA antibodies also were detected in sera from ���, but not ���, mice by Crithidia luciliae kinetoplast staining. The onset of IgG anti-dsDNA antibody production in �� � mice occurred �5 mo after pristane treatment, well after the onset of nephritis, suggesting that these antibodies are not directly responsible for inducing renal disease. In contrast to anti-DNA, the frequencies of Anti-nRNP/Sm and anti-Su antibodies were similar in pristane-treated IL-6�� � and IL-6�� � mice. However, levels were higher in the �� � group. These results suggest that IgG anti-DNA and chromatin antibodies in pristane-treated mice are strictly IL-6 dependent, whereas induction of Anti-nRNP/Sm and Su autoantibodies is IL-

  • pristane induced autoimmunity in germ free mice
    Clinical Immunology, 2005
    Co-Authors: Akiei Mizutani, Jun Akaogi, Yoshioki Yamasaki, Yoshiki Kuroda, Michito Hirakata, Victoria M. Shaheen, Westley H. Reeves, Dina C. Nacionales, Hideo Yoshida, Minoru Satoh
    Abstract:

    Hypergammaglobulinemia and autoantibodies are reduced in pristane-treated specific pathogen-free mice vs. conventionally housed controls, consistent with the role of microbial stimulation in this model. To determine whether microbial stimulation is required, BALB/c mice housed under germ-free conditions were treated i.p. with sterile PBS or pristane and examined 6 months later. As in conventional mice, pristane-treated germ-free mice developed peritoneal granulomas and hypergammaglobulinemia with increased IgG2a/IgG1 ratios. LPS stimulation induced more IL-6, IL-12, and TNF-α, and anti-CD3 induced more IFN-γ and IL-4 by peritoneal cells from pristane-treated mice vs. control. Anti-nRNP/Sm and -Su autoantibodies were found in 40% and 43%, respectively, of pristane-treated germ-free mice by immunoprecipitation. Thus, bacterial stimulation was not required for lupus autoantibodies, peritoneal granuloma formation, hypergammaglobulinemia, or cytokine overproduction. Although microbial stimulation acts synergistically with pristane, these results clearly indicate that pristane does not act merely by increasing exposure to microbial products such as LPS.

  • x linked immunodeficient mice spontaneously produce lupus related anti rna helicase a autoantibodies but are resistant to pristane induced lupus
    International Immunology, 2003
    Co-Authors: Minoru Satoh, Akiei Mizutani, Krista M Behney, Jun Akaogi, Yoshiki Kuroda, Michito Hirakata, Dina C. Nacionales, Hideo Yoshida, Westley H. Reeves
    Abstract:

    Murine lupus can occur spontaneously or be induced by hydrocarbons, such as pristane. Spontaneous disease in MRL and NZB/W F1 mice is suppressed by the xid (X-linked immunodeficiency) mutation, which greatly diminishes T cell-independent type 2 responses as well as the number of peritoneal B1 cells. The present study asked whether lupus induced by i.p. injection of pristane likewise is inhibited by the xid defect. Male CBA/N (xid) mice were refractory to the induction of autoantibodies by pristane, whereas 23% of pristane-treated male CBA/CaJ controls produced Anti-nRNP/Sm, -Su and/or -OJ (isoleucyl tRNA synthetase) antibodies. Unexpectedly, 43% (12 of 28) of the xid mice spontaneously produced anti-nuclear antibodies that proved highly specific for the lupus antigen RNA helicase A (RHA). Strikingly, this specificity was absent in CBA/CaJ mice (none of 51). Moreover, pristane treatment suppressed the production of anti-RHA antibodies when administered prior to the onset of autoantibody production, but enhanced anti-RHA levels when given after the onset of autoantibody production, suggesting that pristane interferes with anti-RHA production at an early stage. Large amounts of IgG1 anti-RHA autoantibodies were detected in the sera of xid mice, whereas pristane-induced Anti-nRNP/Sm and -Su autoantibodies were almost exclusively IgG2a. Cytokine production within the peritoneal cavity reflected the predominant isotypes: IL-12 and IFN-g predominated in pristane-treated mice, whereas IL-4 and IL-6 were more predominant in untreated xid mice. The spontaneous production of antiRHA by xid mice and its suppression by pristane treatment at the level of autoantibody induction supports the idea that lupus autoantibodies may be generated through a variety of mechanisms.

  • interferon γ is required for lupus nephritis in mice treated with the hydrocarbon oil pristane
    Kidney International, 2001
    Co-Authors: Byron P. Croker, Hanno B. Richards, Minoru Satoh, Hideo Yoshida, Charles J Jennette
    Abstract:

    Interferon-γ is required for lupus nephritis in mice treated with the hydrocarbon oil pristane. Background Although the precise mechanisms leading to lupus nephritis remain obscure, both T H 1 and T H 2 cytokines have been implicated. The present study examined the roles of interleukin (IL)-4 and interferon-γ (IFN-γ) in a novel inducible form of lupus that develops in non-autoimmune mice treated with the hydrocarbon oil pristane. Methods BALB/c IL-4 or IFN-γ deficient mice (IL-4 -/-, IFNγ -/-) and wild type controls (+/+) received either pristane or phosphate-buffered saline (PBS) IP. Serial sera were analyzed for anti-DNA/chromatin, anti-RNP/Sm, and total immunoglobulin levels. Proteinuria was measured and kidneys were examined by direct immunofluorescence and light microscopy. Results Renal disease did not develop in pristane-treated IFN-γ -/- mice, as assessed by the absence of capillary immune deposits, glomerular pathology and proteinuria whereas IL-4 -/- mice developed renal disease similar to +/+ mice. Production of IgG anti-single stranded DNA and anti-chromatin antibodies was abrogated in IFN-γ -/- mice. In contrast, these autoantibodies were produced at similar or higher frequencies and levels by IL-4 -/- versus wild-type mice. The frequency of Anti-nRNP/Sm was markedly reduced in IFN-γ -/- mice. IL-4 deficiency had little effect on the production of anti-DNA/chromatin and Anti-nRNP/Sm. Conclusions IFN-γ is essential for the induction of nephritis and anti-DNA/chromatin following pristane exposure in BALB/c mice, suggesting that genetic or environmental factors influencing T H 1-T H 2 balance could be an important determinant of renal disease in lupus.

Westley H. Reeves - One of the best experts on this subject based on the ideXlab platform.

  • Brief Definitive Report Interleukin 6 Dependence of Anti-DNA Antibody Production: Evidence for Two Pathways of Autoantibody Formation in Pristane-induced Lupus
    2013
    Co-Authors: B. Richards, Melody Shaw, Claude Libert, Valeria Poli, Minoru Satoh, Westley H. Reeves
    Abstract:

    Pristane induces a lupus-like syndrome in nonautoimmune mice characterized by the development of glomerulonephritis and lupus-associated autoantibodies. This is accompanied by overproduction of interleukin (IL)-6, a cytokine linked with autoimmune phenomena. The goal of this study was to evaluate the role of IL-6 in autoantibody production in pristane-induced lupus. BALB/cAn IL-6–deficient (���) and –intact (���) mice were treated with pristane or phosphate-buffered saline, and autoantibody production was evaluated. Pristane induced high levels of immunoglobulin (Ig)G anti-single-stranded DNA, –double-stranded (ds)DNA, and-chromatin antibodies in IL-6�� � , but not IL-6�� � mice by enzyme-linked immunosorbent assay. High titer IgG anti-dsDNA antibodies also were detected in sera from ���, but not ���, mice by Crithidia luciliae kinetoplast staining. The onset of IgG anti-dsDNA antibody production in �� � mice occurred �5 mo after pristane treatment, well after the onset of nephritis, suggesting that these antibodies are not directly responsible for inducing renal disease. In contrast to anti-DNA, the frequencies of Anti-nRNP/Sm and anti-Su antibodies were similar in pristane-treated IL-6�� � and IL-6�� � mice. However, levels were higher in the �� � group. These results suggest that IgG anti-DNA and chromatin antibodies in pristane-treated mice are strictly IL-6 dependent, whereas induction of Anti-nRNP/Sm and Su autoantibodies is IL-

  • pristane induced autoimmunity in germ free mice
    Clinical Immunology, 2005
    Co-Authors: Akiei Mizutani, Jun Akaogi, Yoshioki Yamasaki, Yoshiki Kuroda, Michito Hirakata, Victoria M. Shaheen, Westley H. Reeves, Dina C. Nacionales, Hideo Yoshida, Minoru Satoh
    Abstract:

    Hypergammaglobulinemia and autoantibodies are reduced in pristane-treated specific pathogen-free mice vs. conventionally housed controls, consistent with the role of microbial stimulation in this model. To determine whether microbial stimulation is required, BALB/c mice housed under germ-free conditions were treated i.p. with sterile PBS or pristane and examined 6 months later. As in conventional mice, pristane-treated germ-free mice developed peritoneal granulomas and hypergammaglobulinemia with increased IgG2a/IgG1 ratios. LPS stimulation induced more IL-6, IL-12, and TNF-α, and anti-CD3 induced more IFN-γ and IL-4 by peritoneal cells from pristane-treated mice vs. control. Anti-nRNP/Sm and -Su autoantibodies were found in 40% and 43%, respectively, of pristane-treated germ-free mice by immunoprecipitation. Thus, bacterial stimulation was not required for lupus autoantibodies, peritoneal granuloma formation, hypergammaglobulinemia, or cytokine overproduction. Although microbial stimulation acts synergistically with pristane, these results clearly indicate that pristane does not act merely by increasing exposure to microbial products such as LPS.

  • x linked immunodeficient mice spontaneously produce lupus related anti rna helicase a autoantibodies but are resistant to pristane induced lupus
    International Immunology, 2003
    Co-Authors: Minoru Satoh, Akiei Mizutani, Krista M Behney, Jun Akaogi, Yoshiki Kuroda, Michito Hirakata, Dina C. Nacionales, Hideo Yoshida, Westley H. Reeves
    Abstract:

    Murine lupus can occur spontaneously or be induced by hydrocarbons, such as pristane. Spontaneous disease in MRL and NZB/W F1 mice is suppressed by the xid (X-linked immunodeficiency) mutation, which greatly diminishes T cell-independent type 2 responses as well as the number of peritoneal B1 cells. The present study asked whether lupus induced by i.p. injection of pristane likewise is inhibited by the xid defect. Male CBA/N (xid) mice were refractory to the induction of autoantibodies by pristane, whereas 23% of pristane-treated male CBA/CaJ controls produced Anti-nRNP/Sm, -Su and/or -OJ (isoleucyl tRNA synthetase) antibodies. Unexpectedly, 43% (12 of 28) of the xid mice spontaneously produced anti-nuclear antibodies that proved highly specific for the lupus antigen RNA helicase A (RHA). Strikingly, this specificity was absent in CBA/CaJ mice (none of 51). Moreover, pristane treatment suppressed the production of anti-RHA antibodies when administered prior to the onset of autoantibody production, but enhanced anti-RHA levels when given after the onset of autoantibody production, suggesting that pristane interferes with anti-RHA production at an early stage. Large amounts of IgG1 anti-RHA autoantibodies were detected in the sera of xid mice, whereas pristane-induced Anti-nRNP/Sm and -Su autoantibodies were almost exclusively IgG2a. Cytokine production within the peritoneal cavity reflected the predominant isotypes: IL-12 and IFN-g predominated in pristane-treated mice, whereas IL-4 and IL-6 were more predominant in untreated xid mice. The spontaneous production of antiRHA by xid mice and its suppression by pristane treatment at the level of autoantibody induction supports the idea that lupus autoantibodies may be generated through a variety of mechanisms.

  • widespread susceptibility among inbred mouse strains to the induction of lupus autoantibodies by pristane
    Clinical and Experimental Immunology, 2000
    Co-Authors: Minoru Satoh, J O Naim, Paul H. Wooley, Hanno B. Richards, Victoria M. Shaheen, Hideo Yoshida, M. Shaw, Westley H. Reeves
    Abstract:

    Unlike other agents associated with drug-induced lupus, the isoprenoid alkane pristane induces autoantibodies pathognomonic of lupus, including anti-Sm, anti-dsDNA, and anti-ribosomal P in BALB/c and SJL/J mice. The susceptibility of other strains of mice to pristane-induced lupus is unknown and is the focus of the present study. Anti-nRNP/Sm, anti-Su, and anti-ribosomal P autoantibodies were produced by most strains of mice surveyed within several months of pristane treatment, although there was marked interstrain variability in their frequencies, levels, and times of onset. In sharp contrast, the production of autoantibodies against the double-stranded RNA binding proteins NF45/NF90/p110 was restricted to B6 and B10.S mice. We conclude that pristane selectively induces lupus-specific autoantibodies in virtually any strain of mouse regardless of its genetic background. However, H-2-linked as well as non-H2 genes influenced the expression of individual autoantibody markers. The widespread susceptibility of pristane-treated mice to lupus autoantibody production and the relatively small effect of MHC are unique features of this chemically induced lupus syndrome, with potential implications for understanding the pathogenesis of autoantibodies in idiopathic human systemic lupus erythematosus.

  • fas and fas ligand mutations inhibit autoantibody production in pristane induced lupus
    Journal of Immunology, 2000
    Co-Authors: Minoru Satoh, Jory P Weintraub, Melody Shaw, Hanno B. Richards, Victoria M. Shaheen, Hideo Yoshida, Westley H. Reeves
    Abstract:

    Mutations of Fas ( lpr ) or Fas ligand ( gld ) cause a limited lupus-like syndrome in B6 mice by interfering with the deletion of autoreactive B and/or T cells. A more generalized lupus syndrome reminiscent of that of MRL mice can be induced in nonautoimmune strains by pristane, which causes a nonspecific inflammatory response in the peritoneal cavity. We hypothesized that, as in MRL mice, the lpr and gld mutations might accelerate lupus in pristane-treated mice. Pristane-treated B6 mice developed Anti-nRNP/Sm, Su, and ribosomal P Abs, but little anti-ssDNA or chromatin. In contrast, B6/ lpr and B6/ gld mice spontaneously developed anti-ssDNA/chromatin Abs, but not Anti-nRNP/Sm/Su/ribosomal P. Unexpectedly, B6/ lpr and B6/ gld mice were highly resistant to the induction by pristane of IgM anti-ssDNA (2 wk) and IgG Anti-nRNP/Sm/Su/ribosomal P autoantibodies (6 mo), suggesting that intact Fas signaling is necessary. Interestingly, pristane did not enhance IgG chromatin Ab production in B6/ lpr or B6/ gld mice, suggesting that it did not influence the production of autoantibodies that develop spontaneously in the setting of Fas deficiency. Pristane treatment also decreased lymphoproliferation in B6/ lpr mice. Increased production of IL-12 was associated consistently with the production of Anti-nRNP/Sm/Su/ribosomal P as well as anti-DNA/chromatin. In contrast, production of anti-DNA/chromatin Abs was associated with IL-6 overproduction in pristane-treated mice, but not in lpr mice. The data strongly support the idea that different subsets of autoantibodies are regulated differentially by cytokine stimulation and/or Fas signaling.

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  • B cell epitopes of the heterogeneous nuclear ribonucleoprotein A2: identification of a new specific antibody marker for active lupus disease.
    Annals of the Rheumatic Diseases, 2009
    Co-Authors: Georg Schett, Hélène Dumortier, Elisabeth Hoefler, Sylviane Muller, Günter Steiner
    Abstract:

    OBJECTIVES: Autoantibody formation and T cell reactivity against the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) has been observed in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Since no differences in epitope recognition were reported and the usefulness of anti-hnRNP-A2 antibodies as diagnostic markers of SLE is unknown, it was our objective to characterise linear B cell epitopes of hnRNP-A2 and to relate the anti-hnRNP-A2 antibody responses to disease activity and clinical features of SLE. METHODS: Sequential serum samples from 15 patients with SLE and sera from patients with other rheumatic diseases and healthy subjects were investigated by ELISA for autoantibody reactivities against a set of 13 overlapping peptides spanning the RNA-binding region of hnRNP-A2. Antibody reactivity against the complete protein was determined by western immunoblotting and ELISA. SLE disease activity was assessed by European Consensus Lupus Activity Measure scores, by SLE Index scores and the British Isles Lupus Assessment index. RESULTS: Anti-peptide antibody reactivities were found in 60% of SLE sera but in only 5% of control samples, and were mainly directed to four peptides, one of which (p155-175) appeared to be immunodominant. Antibodies to p155-175 were exclusively seen in patients with SLE and correlated with clinical disease activity as well as kidney and skin involvement. No correlations were found for the other anti-peptide antibody responses. CONCLUSION: Peptide p155-175 encompasses a disease-specific immunodominant epitope of hnRNP-A2. Since antibodies to p155-175 correlate with disease activity and nephritis, they may be useful as markers for active SLE.

  • anti ra33 antibodies antibodies to the heterogeneous nuclear ribonucleoprotein a2
    Autoantibodies (Second Edition), 2007
    Co-Authors: Günter Steiner, Josef S. Smolen
    Abstract:

    ABSTRACT Anti-RA33 antibodies (Ab) are directed to the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, an abundant nuclear protein that is involved in processing, transport and translation of mRNA. Although hnRNP-A2 appears to be constitutively expressed in most organs and tissues, aberrant overexpression has been observed in certain cancers and in inflamed synovial tissue of patients with rheumatoid arthritis (RA). Anti-RA33 Ab occur in approximately one-third of patients with RA but are rarely detected in other rheumatic diseases, with the exception of systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) where they are as prevalent as in RA. Since anti-RA33 Ab are already detectable in the earliest stages of RA they are useful diagnostic markers showing a positive predictive value (PPV) for RA of approximately 74%. In the absence of rheumatoid factor (RF) and anti-citrullinated protein Ab (ACPA), anti-RA33 Ab appear to be associated with a relatively favourable prognosis. Similar to RF and ACPA, their role in the pathogenesis of RA is unclear. However, autoreactive T cells directed to hnRNP-A2 appear to be commonly present in blood and synovial fluid of RA patients. Their Th1-like phenotype is suggestive of pathogenic involvement of anti-RA33 autoimmunity.

  • molecular definition of heterogeneous nuclear ribonucleoprotein r hnrnp r using autoimmune antibody immunological relationship with hnrnp p
    Nucleic Acids Research, 1998
    Co-Authors: Wolfgang Hassfeld, Günter Steiner, Gideon Dreyfuss, Edward K L Chan, David A Mathison, Douglas S Portman, Eng M Tan
    Abstract:

    Serum from a patient showing symptoms related to autoimmunity was found to contain autoantibodies to the nuclear mitotic apparatus (NuMA) protein and to several novel nuclear antigens with estimated molecular weights of 40, 43, 72, 74 and 82 kDa. Using this serum for screening a human cDNA expression library a 2.5 kb cDNA clone was isolated which encoded the complete sequence of a protein of 633 amino acids. Sequence analysis revealed a modular structure of the protein: an acidic N-terminal region of approximately 150 amino acids was followed by three adjacent consensus sequence RNA binding domains located in the central part of the protein. In the C-terminal portion a nuclear localization signal and an octapeptide (PPPRMPPP) with similarity to a major B cell epitope of the snRNP core protein B were identified. This was followed by a glycine- and arginine-rich section of approximately 120 amino acids forming another type of RNA binding motif, a RGG box. Interestingly, three copies of a tyrosine-rich decapeptide were found interspersed in the RGG box region. The major in vitro translation product of the cDNA co-migrated in SDS-PAGE with the 82 kDa polypeptide that was recognized by autoantibodies. The structural motifs as well as the immunofluorescence pattern generated by anti-82 kDa antibodies suggested that the antigen was one of the proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Subsequently the 82 kDa antigen was identified as hnRNP R protein by its presence in immunoprecipitated hnRNP complexes and co-migration of the recombinant protein with this hitherto uncharacterized hnRNP constituent in two-dimensional gel electrophoresis. The concomitant autoimmune response to a hnRNP component of the pre-mRNA processing machinery and to NuMA, a protein engaged in mitotic events and reported to be associated with mRNA splicing complexes in interphase, may indicate physical and functional association of these antigens. Support for this notion comes from observations that concomitant or coupling of autoantibody responses to proteins which are associated with each other as components of subcellular particles are often found in autoimmune diseases.

  • anti a2 ra33 autoantibodies are directed to the rna binding region of the a2 protein of the heterogeneous nuclear ribonucleoprotein complex differential epitope recognition in rheumatoid arthritis systemic lupus erythematosus and mixed connective tis
    Journal of Clinical Investigation, 1997
    Co-Authors: Karl Skriner, Josef S. Smolen, W H Sommergruber, Andrea Barta, V Tremmel, I Fischer, Günter Steiner
    Abstract:

    The recently described anti-A2/RA33 autoantibodies occur in 20-40% of patients with RA, SLE, and mixed connective tissue disease (MCTD). They are directed to the A2 protein of the heterogeneous nuclear ribonucleoprotein complex (hnRNP-A2), an abundant nuclear protein associated with the spliceosome. The NH2-terminal half of the antigen contains two conserved RNA binding domains whereas its COOH-terminal part is extremely glycine-rich. The aim of this study was to characterize the autoepitopes of hnRNP-A2 and to investigate the effects of anti-A2/RA33 autoantibodies on possible functions of the antigen. Using bacterially expressed fragments, two major discontinuous epitopes were identified. One containing the complete second RNA binding domain was recognized by the majority of patients with RA and SLE but not by patients with MCTD. The second epitope contained sequences of both RNA binding domains and was preferentially targeted by patients with MCTD. When the RNA binding properties of the antigen were investigated, oligoribonucleotides containing the sequence motif r(UUAG) were found to bind to a site closely adjacent or overlapping with the epitope targeted by autoantibodies from patients with RA and SLE. Moreover, anti-A2/RA33 autoantibodies from patients with RA or SLE, but not from patients with MCTD, inhibited binding of RNA. Thus, anti-A2/RA33 autoantibodies recognize conformation-dependent epitopes located in a functionally important region of the antigen. Furthermore, the specific recognition of an epitope by MCTD patients may be used as another argument in favor of considering MCTD a distinct connective tissue disease.

  • autoimmune response to the spliceosome an immunologic link between rheumatoid arthritis mixed connective tissue disease and systemic lupus erythematosus
    Arthritis & Rheumatism, 1995
    Co-Authors: Wolfgang Hassfeld, Karl Skriner, Günter Steiner, Andrea Studnickabenke, Winfried Graninger, Inge Fischer, Josef S. Smolen
    Abstract:

    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.