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The Association of Anti-Ro52 Autoantibodies with Myositis and Scleroderma Autoantibodies
Article No. jaut.1998.0265, available online at http://www.idealibrary.com on
Journal of Autoimmunity (1999) 12, 137–142
The Association of Anti-Ro52 Autoantibodies with Myositis and Scleroderma Autoantibodies Mark Barton Frank1, Victoria McCubbin1, Edward Trieu1, Yajaun Wu1, David A. Isenberg2 and Ira N. Targoff3 1 Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA 2 Centre for Rheumatology/Bloomsbury Rheumatology Unit, Department of Medicine, University College London, London W1P 9PG UK 3 Veterans Affairs Medical Center, Oklahoma City, OK, USA
Received 9 October 1998 Accepted 19 October 1998 Key words: Ro52, Ro/SS-A, myositis, autoantibodies
The presence of autoantibodies to the Ro52 protein in sera from patients with idiopathic inflammatory myopathies has recently been reported. These antibodies were found predominately in sera with the myositis-specific autoantibody anti-histidyl-tRNA synthetase (anti-Jo-1). In this report, we analysed sera from 216 patients to determine whether anti-Ro52 antibodies are associated with myositis autoantibodies other than anti-Jo-1. These included sera containing antibodies that recognize threonyl- or alanyl-tRNA synthetases, Mi-2, PM-Scl, signal recognition particle (SRP), as well as the systemic sclerosis-related antibodies anti-topoisomerase I (Scl-70) and anticentromere. A high proportion of sera that contain anti-aminoacyl-tRNA synthetase antibodies, anti-SRP, or anti-PM-Scl antibodies were found to contain antibodies to the Ro52 protein. In contrast, in sera containing antiMi-2, anti-Scl-70 or anti-centromere antibodies, anti-Ro52 antibodies were absent or occurred infrequently. In addition, only one serum from 41 rheumatoid arthritis patients was positive for anti-Ro52 autoantibodies. These data indicate that anti-Ro52 antibodies are produced in particular subsets of myositis patients, and are not limited to sera with anti-Jo-1 antibodies. © 1999 Academic Press
Introduction
idiopathic inflammatory myopathies. Anti-Ro52 was coexpressed in the majority of sera that also contained antibodies to histidyl-tRNA synthetase, also known as anti-Jo-1 [7]. Anti-Ro52 autoantibodies were detected in similar proportions of patients with polymyositis and dermatomyositis, and the epitopes that they recognized appeared to be similar, if not identical to those recognized by anti-Ro52 antibodies in Sjg¨ren’s syndrome and lupus sera. Although anti-Jo-1 is the most common myositis antibody, several other autoantibodies are either associated with or specific to myositis. Among these are autoantibodies directed at other aminoacyl-tRNA synthetases. These include anti-threonyl-tRNA synthetase (also known as anti-PL-7), anti-alanyl-tRNA synthetase (anti-PL-12), anti-glycyl-tRNA synthetase (anti-EJ), anti-isoleucyl-tRNA synthetase (anti-OJ) and anti-asparaginyl-tRNA synthetase (anti-KS). Antibodies to these enzymes are not cross-reactive, and occur independently of one another (i.e. in different sera). Clinically, patients with any of these antibodies tend to have similar features collectively termed ‘antisynthetase syndrome’ consisting of polymyositis or dermatomyositis, interstitial lung disease, arthritis, Raynaud’s phenomenon, and other clinical problems. A number of features make it difficult to understand a mechanism that may couple the antibody responses to Jo-1 and Ro52. First, the antibodies to these proteins
The autoimmune response by patients with lupus and Sjo¨gren’s syndrome is directed, in part, against a variety of intracellular molecules, including the 60 kDa Ro/SS-A (Ro60) and 52 kDa Ro/SS-A (Ro52) molecules. These non-homologous proteins are encoded by distinct genes on different chromosomes [1–3]. The role of autoantibodies that bind to these proteins in the pathogenesis of these diseases is not clear, although considerable proportions of IgG in patient sera can be directed against them. The Ro52 protein sequence contains both zinc finger and leucine zipper motifs, suggesting that it functions as a nucleic acid binding molecule [1, 2]. Direct support for this hypothesis was recently obtained [4]. The function of the Ro60 molecule is also unknown, but one report indicates that it may play a role in the degradation of small, aberrant RNA [5]. While antibodies to Ro52 and Ro60/SS-A occur primarily in patients with systemic lupus erythematosus and Sjo¨gren’s syndrome, a recent paper by Rutjes et al. [6] identified antibodies to Ro52 in patients with Correspondence to: Mark Barton Frank, Arthritis and Immunology Program, Oklahoma Medical Research Foundation 825 NE 13th Street, Oklahoma City, OK 73104, USA. Fax: 405–271–4110. E-mail: [email protected] 137 0896–8411/99/020137+06 $30.00/0
© 1999 Academic Press
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were shown not to be cross-reactive [6]. Second, while tRNA synthetases charge their cognate tRNAs in the cytoplasm, Ro52 resides primarily in the nucleus [8]. The role of molecules in the nucleus that are antigenically similar to Jo-1 is unknown [9]. Furthermore, anti-Jo-1 antibodies are rarely, if ever, found in patients with SLE or primary Sjo¨gren’s syndrome without myositis. Finally, anti-Ro52 antibodies in the latter two diseases are frequently detected with antibodies to the Ro60/SS-A and La/SS-B molecules. Among sera from patients with myositis, a higher proportion of anti-Jo-1-positive sera has anti-Ro60 than do anti-Jo-1-negative myositis sera [10]. However, in the sera studied by Rutjes et al. [6], antibodies to Ro60/SS-A were rare relative to anti-Ro52. Thus, if the anti-Jo-1 and anti-Ro52 antibodies are coupled, the mechanism may be unique to myositis. In order to better understand this association, we have examined sera from patients that contain anti-Jo-1 and other antibodies that have been shown to be associated with myositis, as well as with myositis–scleroderma overlap syndrome.
Materials and Methods
sample buffer, electrophoresed in 10% polyacrylamide gels, and visualized by autoradiography. The human Ro52 protein was expressed in E. coli from its full-length cDNA, and antibodies to it were detected by ELISA [1, 12]. Briefly, inclusion bodies containing the Ro52 protein were solubilized in 7 M urea, and then diluted in a carbonate buffer to coat microtiter plates (Costar, Cambridge, MA, USA). After washing and blocking the plates, dilutions of patient sera were added, and bound antibodies were detected using an alkaline phosphatase-conjugated goat antihuman ã chain specific antiserum (Sigma, St. Louis, MO, USA) and p-nitrophenyl phosphate substrate. Optical densities (OD) were quantified in a Dynatech MRX microplate reader (Dynex Technologies, Inc., Chantilly, VA, USA) at 405 nm. Controls included OD from wells with no antibody, as well as extracts from bacteria transformed with pUC19 that lacked the human Ro52 cDNA. These control values were subtracted from the OD of patients’ sera reacting with the Ro52 protein to obtain a net OD. Antibodies to Ro60 and La/SS-B were detected similarly by Ouchterlony immunodiffusion and immunoprecipitation analysis. Anti-centromere antibodies were detected by indirect immunofluorescence of Hep-2 cells.
Sera Sera were obtained from patients of the authors (INT and DAT), or from the Myositis Serum Bank or the Clinical Immunology Laboratory at the Oklahoma Medical Research Foundation (Oklahoma City, OK, USA). The latter sera were referred either for clinical testing or as part of other studies. Sera were stored at −20°C prior to use. Two hundred and sixteen sera were selected for analysis based on the presence of specific autoantibodies that are associated with myositis, scleroderma, or myositis–scleroderma overlap syndrome. Because the sera used here were selected on the basis of their autoantibody profiles, the results may not represent a random sampling of myositis patients. An additional 41 sera from patients with rheumatoid arthritis were also analysed for the presence of anti-Ro52 antibodies.
Detection of patient autoantibodies The specificities of autoantibodies related to myositis were determined using Ouchterlony immunodiffusion with calf thymus extract as antigen, and by immunoprecipitation of nucleic acids and proteins. These methods have been previously described in detail [11]. Briefly, for nucleic acid analysis, patient antibodies were bound to protein A-Sepharose beads. The beads were washed and then reacted with extract from sonicated HeLa cells. Immunoprecipitates were phenol-extracted and then subjected to electrophoresis in 8 M urea, 10% polyacrylamide gels. RNA was detected by silver staining. For protein analysis, immunoprecipitates of 35S-labeled HeLa cells were recovered from the beads by heat denaturation in
Statistics Fisher’s Exact test was used to test for differences between serologic groups. Calculated P-values were corrected for the multiple comparisons made.
Results The autoimmune response to the Ro52 protein in anti-Jo-1-positive sera We analysed the response to the Ro52 protein in 216 sera that contained antibodies associated with, or specific for, patients with polymyositis, dermatomyositis, or scleroderma. Ninety-two (43%) of these sera contained IgG antibodies directed to the Ro52 protein, and 40 sera (19%) contained anti-Jo-1 antibodies. The concurrence of anti-Ro52 and myositis-specific antibodies (MSA) was analysed. Among the 40 antiJo-1-positive sera, 70% had antibodies to the Ro52 protein. This percentage is similar to the 58% of sera reported by Rutjes et al. [6]. The proportion of sera with anti-Ro52 antibodies was significantly higher (P=8×10 −7) in the anti-Jo-1-positive sera than in antiJo-1-negative sera (37%), consistent with the findings of Rutjes et al. [6]. Antibodies to Ro52 in SLE and Sjo¨gren’s syndrome patients frequently occur in sera that also contain antibodies to the Ro60/SS-A and La/SS-B proteins. Accordingly, we tested the hypothesis that the anti-Ro52 antibodies in anti-Jo-1-positive patients predominantly occur in the latter sera. The results
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Table 1. Anti-Ro52 antibodies in anti-Jo-1-positive myositis patients Anti-Ro60
Anti-La
Anti-Ro52-positive
Anti-Ro52-negative
Total
% Ro52 +
Negative Positive Positive Total
Negative Negative Positive
12 2 14 28
9 0 3 11
21 2 17 40
57 100 82 70
Table 2. Anti-Ro52 antibodies in myositis sera with anti-threonyl-tRNA synthetase (PL-7) antibodies Anti-Ro60
Anti-La
Anti-Ro52-positive
Anti-Ro52-negative
Total
% Ro52 +
Negative Positive Total
Negative Negative
4 7 11
5 0 5
9 7 16
44 100 69
Table 3. Anti-Ro52 antibodies in myositis sera with anti-alanyl-tRNA synthetase (PL-12) antibodies Anti-Ro60
Anti-La
Anti-Ro52-positive
Anti-Ro52-negative
Total
% Ro52 +
Negative Positive Positive Total
Negative Negative Positive
4 2 4 10*
5 0 0 5
9 2 4 15
44 100 100 67
*Testing for anti-La/SS-B antibodies was equivocal in one serum that was positive for both anti-Ro52 and anti-Ro60/SS-A antibodies. This serum is omitted from the table.
suggest that the autoimmune response to the Ro52 protein by anti-Jo-1-positive patients is statistically independent of the presence of anti-Ro60/SS-A or anti-La/SS-B antibodies (Table 1, P=0.24).
antibodies to Ro52. Therefore, the immune response to the Ro52 protein in anti-tRNA synthetase-positive patients is not limited to those containing anti-Jo-1 autoantibodies.
The autoimmune response to the Ro52 protein in other anti-aminoacyl-tRNA synthetase sera
The frequency of anti-Ro52 antibodies in sera with other MSAs
Because of the common clinical spectrum among patients who produce antibodies to different aminoacyl-tRNA synthetases, the response to Ro52 in sera containing antibodies to two other synthetases was also analysed. The proportion of myositis patients with non-Jo-1 anti-tRNA synthetase antibodies is low (approximately 2% of myositis patients for each antibody); therefore, the number of sera available for study is relatively small. Sera from 16 patients with anti-PL-7, and another 16 patients with anti-PL-12 antibodies were examined. Among these sera without anti-Ro60 antibodies, 44% had antibodies to the Ro52 protein. As was the case with sera containing anti-Jo-1 antibodies, the immune response to Ro52 was statistically independent of the response to the Ro60 particle, although anti-Ro52 antibodies were present in all of the sera that contained anti-Ro60 antibodies (Tables 2 and 3). Collectively, of the 72 sera that were positive for any anti-tRNA synthetase antibodies, 50 (69%) contained
Although anti-synthetase antibodies are the most common, other MSAs have also been described. These include antibodies to Mi-2, produced primarily by dermatomyositis patients. Antibodies to Ro52 were not detected in any of 16 anti-Mi-2-positive sera that were studied, including 10 that contained antibodies to the Ro60/SS-A molecule. Thus, the autoimmune response to Ro52 is not equally represented among serologically distinct groups of myositis patients. Autoantibodies to the signal recognition particle (SRP) are produced primarily by polymyositis patients. This particle is involved in the transport of newly synthesized proteins into the endoplasmic reticulum. Anti-SRP antibodies occur in about 4% of myositis patients and are associated with HLA-DR5 [11]. Of the 21 anti-SRP-positive sera that were analysed, nine (43%) had antibodies to the Ro52 protein. All of these anti-SRP-positive sera were negative for antibodies to the Ro60 and La/SS-B molecules. These data demonstrate that anti-Ro52 autoantibodies
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Table 4. Antibodies to Ro52 in anti-PM-Scl-positive sera Anti-Ro60
Anti-La
Anti-Ro52-positive
Anti-Ro52-negative
Total
% Ro52-positive
Negative Positive Positive Total*
Negative Negative Positive
13 9 1 23
22 2 2 26
35 11 3 49
37 82 33 47
*The presence of anti-La/SS-B antibodies could not definitively be determined in one anti-Ro52-positive and one anti-Ro52-negative. These sera are not included in this table.
in myositis patients are not limited to sera that contain anti-tRNA synthetase antibodies.
low titer. None of the ANA-negative rheumatoid arthritis sera had detectable anti-Ro52 antibodies.
The autoimmune response to the Ro52 protein in sera from patients with anti-PM-Scl antibodies
Discussion
Antibodies to the PM-Scl antigen are present in approximately 25% of sera from patients with polymyositis and scleroderma overlap [13]. The antigen consists of at least 11 proteins in the nucleus and nucleolus whose function(s) is unknown [13]. Sera from 51 patients with anti-PM-Scl antibodies were studied to determine if antibodies to the Ro52 protein were present. Twenty-four of the sera (47%) contained antibodies to Ro52 (Table 4). As was the case with the anti-aminoacyl-tRNA synthetase-positive sera, antiRo52 antibodies were present in a higher proportion of sera that also contained anti-Ro60 antibodies. However, the association between antibodies to the two Ro antigens did not reach statistical significance (P=0.12). The presence of anti-Ro52 antibodies was also not associated with the titer of anti-PM-Scl antibodies in these sera (data not shown) as determined by a recombinant ELISA [14].
The autoimmune response to Ro52 in sera from patients with other autoimmune disorders Given the findings from the anti-PM-Scl-positive sera, we tested the hypothesis that patients with scleroderma who do not have polymyositis also produce anti-Ro52 autoantibodies. Scleroderma sera containing autoantibodies to either topoisomerase I (Scl-70) or anti-centromere antibodies were tested. Antitopoisomerase I antibodies are often associated with diffuse cutaneous systemic sclerosis with an increased frequency of lung involvement, while anti-centromere antibodies are strongly associated with limited cutaneous systemic sclerosis. Anti-Ro52 antibodies were detected in only three of 19 anti-Scl-70-positive sera, and one of nine anti-centromere sera that were screened. All 28 of these sera were negative for anti-Ro60 and anti-La/SSB antibodies. Forty-one sera from rheumatoid arthritis patients were also screened for anti-Ro52 antibodies. Twentyfour of these sera were ANA-positive. Only one serum, which was weakly positive for ANA, contained anti-Ro52 antibodies, which were present at
In this study, sera from 216 patients known to have autoantibodies associated with myositis or scleroderma were screened for the presence of IgG autoantibodies to the Ro52 protein. A significant proportion of these patients produced antibodies to this protein. These include patients with anti-PM-Scl antibodies, which are associated with an overlap syndrome of myositis and scleroderma. However, antiRo52 antibodies were rarely present in sera from patients with scleroderma-specific antibodies. Similarly, anti-Ro52 autoantibodies were infrequently found in rheumatoid arthritis sera. The quantitative levels of these antibodies varied in each of the serologic groups and were similar to those in lupus patients. With few exceptions, very little work has been reported on the presence of anti-Ro52 antibodies in patients other than lupus or Sjo¨gren’s syndrome. Ricchiuti et al. [15] described anti-Ro52 antibodies in 24% of rheumatoid and 19% of juvenile rheumatoid arthritis sera, and in 19% of sera from patients with mixed connective tissue disease. No anti-Ro52 autoantibodies were detected in scleroderma sera. Rutjes et al. [6] were the first to identify these antibodies in patients with idiopathic inflammatory myopathies. Their data indicated these antibodies occurred primarily in sera containing antibodies to histidyltRNA synthetase (Jo-1). Because the authors used a secondary antiserum that detected human IgM, IgG, and IgA antibodies, the specific anti-Ro52 antibody isotypes were not discussed. The data reported here extend these findings of Rutjes et al. [6] in a number of ways. We confirm that antibodies to Ro52 frequently occur in sera with anti-Jo-1 antibodies. However, anti-Ro52 antibodies also occur in similar proportions of sera containing other MSAs, in particular other anti-tRNA-synthetase, anti-SRP, and anti-PM-Scl antibodies. Because the majority of sera used in this study were selected for particular myositis-associated antibody patterns, the associations of the anti-Ro52 response to MSAs in myositis clinical groups is unknown. A separate study is underway to analyse the association of anti-Ro52 antibodies with disease.
Anti-Ro52 antibodies with myositis sera
No sera studied here with the dermatomyositisspecific anti-Mi-2 antibodies contained anti-Ro52 antibodies. This contrasts with the finding of Rutjes et al. [6] who reported similar proportions of anti-Ro52 antibody-positive sera in sera from patients with polymyositis and dermatomyositis. This distinction may reflect different proportions of dermatomyositis patients with non-Jo-1 anti-aminoacyl-tRNA synthetase antibodies, different anti-Ro52 isotypes (only IgG was detected here), or racial differences between these cohorts. The response to Mi2 is associated with HLA-DR53 and HLA-DR7, in contrast to HLA-DR52 and HLA-DR3 specificities that predominate in patients who produce anti-synthetase antibodies [10]. HLA typing of patients in the future may be useful in testing the hypothesis that the Mi-2-associated HLA proteins are incapable of presenting or inefficiently presenting Ro52 to T cells. No clear HLA association with anti-Ro52 has been described in lupus or Sjo¨gren’s patients. The findings reported here show that the response to Ro52 is rare in scleroderma patients, in support of data by Ricchiuti et al. [15]. However, this immune response is clearly present in patients with anti-PMScl antibodies, which are found in some patients with polymyositis overlap syndrome and other disorders. Detection of anti-Ro52 antibodies may prove to be a useful addition in discriminating between patients in these groups. While tRNA synthetases charge their nucleic acids in the cytoplasm, and Ro52 occurs primarily in the nucleus, two recent reports provide data to indicate the possibility for interactions between these proteins. Vazques-Abad et al. [9] reported that anti-Jo-1 antibodies bound to antigen in both the cytoplasm and nucleus of a laryngeal epithelial carcinoma cell line. Scmitz et al. found evidence for Ro52 in the nucleus with patient and monoclonal antibodies, but also in the cytoplasm of a bladder carcinoma cell line [8]. Venables has speculated that an interaction between these proteins may occur in abnormal cells or cells undergoing apoptosis [16]. A striking feature of the data reported here was the independent response to the Ro52 and Ro60 molecules in myositis sera. The initial description of anti-Ro52 antibodies in Sjo¨gren’s Syndrome patients reported these antibodies in >80% of sera with anti-Ro60 antibodies [17]. A later report by the same group found that they co-occurred in about half of lupus and Sjo¨gren’s syndrome sera, while anti-Ro52 antibodies alone were detected only in Sjo¨gren’s sera, and antiRo60 antibodies alone occurred only in SLE [18]. Reports by others also indicate that certain SLE patients produce antibodies to Ro52 in the absence of those to Ro60 [19, 20]. The analyses of lupus sera in our laboratory [12, 21, 22] are more consistent with those of Ben-Chetrit et al. [17, 18]. The frequent cooccurrence of antibodies to both forms of Ro has led to investigations supporting models of antigenic spreading between Ro52, Ro60 and La/SS-B proteins [23–25]. Interestingly, in many of the sera described here, the frequency of antibodies to Ro60 and La/SS-B is lower than the frequency of anti-Ro52 antibodies. This may
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suggest either that intermolecular spreading may not occur in these patients [16], or that the anti-Ro52 antibodies predate the immune response to these other molecules. The high proportions of patients with myositis and only anti-Ro52 antibodies may serve as an interesting group of patients for follow-up study on this issue.
Acknowledgements The authors thank the following individuals for their valuable referral of patient sera: Morris Reichlin, MD (Oklahoma City, USA); Frank C. Arnett, MD (Houston, USA); Frederic W. Miller, MD, PhD, (Bethesda, USA); Paul H. Plotz, MD, (Bethesda USA); and Chester Oddis, MD, (Pittsburgh, USA). We also thank Ms Lori Lewis for technical assistance with ELISA. This work was supported in part by Public Health Service Grant AR41919 to MBF from the National Institute of Arthritis and Musculoskeletal and Skin Diseases.
References 1. Itoh K., Itoh Y., Frank M.B. 1991. Protein heterogeneity in the human Ro/SSA ribonucleoproteins: the 52-and 60-kD Ro/SSA autoantigens are encoded by separate genes. J. Clin. Invest. 87: 177–186 2. Chan E.K.L., Hamel J.C., Buyon J.P., Tan E.M. 1991. Molecular definition and sequence motifs of the 52-kD component of human SS-A/Ro autoantigen. J. Clin. Invest. 87: 68–76 3. Frank M.B., Mattei M.G. 1994. Mapping of the human 60,000 Mr Ro/SSA locus: the genes for three Ro/SSA autoantigens are located on separate chromosomes. Immunogenetics 39: 428–431 4. Frank M.B., McCubbin V., Heldermon C. 1995. Expression and DNA binding of the human 52 kD Ro/SSA autoantigen. Biochem. J. 305: 359–362 5. O’Brien C.A., Wolin S.L. 1994. A possible role for the 60-kD Ro autoantigen in a discard pathway for defective 5S rRNA precursors. Genes Dev. 8: 2891–2903 6. Rutjes S.A., Vree Egberts W.T.M., Jongen P., van den Hoogen F., Pruijn G.J.M., van Venrooij W.J. 1997. Anti-Ro52 antibodies frequently co-occur with anti-Jo-1 antibodies in sera from patients with idiopathic inflammatory myopathy. Clin. Exper. Immunol. 109: 32–40 7. Mathews M.B., Bernstein R.M. 1983. Myositis antibody inhibits histidyl-tRNA synthetase: a model for autoimmunity. Nature 304: 177–179 8. Schmitz M., Bachmann M., Laubinger J., Thijssen J.P.H., Pruijn G.J.M. 1997. Characterization of murine monoclonal antibodies against the Ro52 autoantigen. Clin. Exper. Immunol. 110: 53–62 9. Vazques-Abad D., Carson J.H., Rothfield N. 1996. Localization of histidyl-tRNA synthetase (Jo-1) in the laryngeal epithelial carcinoma cell line (Hep-2 cells). Cell Tissue Res. 286: 487–491 10. Love L.A., Leff R.L., Fraser D.D., Targoff I.N., Dalakas M., Plotz P.H., Miller F.W. 1991. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups. Medicine 70: 360–374
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Journal of Autoimmunity (1999) 12, 137–142
The Association of Anti-Ro52 Autoantibodies with Myositis and Scleroderma Autoantibodies Mark Barton Frank1, Victoria McCubbin1, Edward Trieu1, Yajaun Wu1, David A. Isenberg2 and Ira N. Targoff3 1 Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA 2 Centre for Rheumatology/Bloomsbury Rheumatology Unit, Department of Medicine, University College London, London W1P 9PG UK 3 Veterans Affairs Medical Center, Oklahoma City, OK, USA
Received 9 October 1998 Accepted 19 October 1998 Key words: Ro52, Ro/SS-A, myositis, autoantibodies
The presence of autoantibodies to the Ro52 protein in sera from patients with idiopathic inflammatory myopathies has recently been reported. These antibodies were found predominately in sera with the myositis-specific autoantibody anti-histidyl-tRNA synthetase (anti-Jo-1). In this report, we analysed sera from 216 patients to determine whether anti-Ro52 antibodies are associated with myositis autoantibodies other than anti-Jo-1. These included sera containing antibodies that recognize threonyl- or alanyl-tRNA synthetases, Mi-2, PM-Scl, signal recognition particle (SRP), as well as the systemic sclerosis-related antibodies anti-topoisomerase I (Scl-70) and anticentromere. A high proportion of sera that contain anti-aminoacyl-tRNA synthetase antibodies, anti-SRP, or anti-PM-Scl antibodies were found to contain antibodies to the Ro52 protein. In contrast, in sera containing antiMi-2, anti-Scl-70 or anti-centromere antibodies, anti-Ro52 antibodies were absent or occurred infrequently. In addition, only one serum from 41 rheumatoid arthritis patients was positive for anti-Ro52 autoantibodies. These data indicate that anti-Ro52 antibodies are produced in particular subsets of myositis patients, and are not limited to sera with anti-Jo-1 antibodies. © 1999 Academic Press
Introduction
idiopathic inflammatory myopathies. Anti-Ro52 was coexpressed in the majority of sera that also contained antibodies to histidyl-tRNA synthetase, also known as anti-Jo-1 [7]. Anti-Ro52 autoantibodies were detected in similar proportions of patients with polymyositis and dermatomyositis, and the epitopes that they recognized appeared to be similar, if not identical to those recognized by anti-Ro52 antibodies in Sjg¨ren’s syndrome and lupus sera. Although anti-Jo-1 is the most common myositis antibody, several other autoantibodies are either associated with or specific to myositis. Among these are autoantibodies directed at other aminoacyl-tRNA synthetases. These include anti-threonyl-tRNA synthetase (also known as anti-PL-7), anti-alanyl-tRNA synthetase (anti-PL-12), anti-glycyl-tRNA synthetase (anti-EJ), anti-isoleucyl-tRNA synthetase (anti-OJ) and anti-asparaginyl-tRNA synthetase (anti-KS). Antibodies to these enzymes are not cross-reactive, and occur independently of one another (i.e. in different sera). Clinically, patients with any of these antibodies tend to have similar features collectively termed ‘antisynthetase syndrome’ consisting of polymyositis or dermatomyositis, interstitial lung disease, arthritis, Raynaud’s phenomenon, and other clinical problems. A number of features make it difficult to understand a mechanism that may couple the antibody responses to Jo-1 and Ro52. First, the antibodies to these proteins
The autoimmune response by patients with lupus and Sjo¨gren’s syndrome is directed, in part, against a variety of intracellular molecules, including the 60 kDa Ro/SS-A (Ro60) and 52 kDa Ro/SS-A (Ro52) molecules. These non-homologous proteins are encoded by distinct genes on different chromosomes [1–3]. The role of autoantibodies that bind to these proteins in the pathogenesis of these diseases is not clear, although considerable proportions of IgG in patient sera can be directed against them. The Ro52 protein sequence contains both zinc finger and leucine zipper motifs, suggesting that it functions as a nucleic acid binding molecule [1, 2]. Direct support for this hypothesis was recently obtained [4]. The function of the Ro60 molecule is also unknown, but one report indicates that it may play a role in the degradation of small, aberrant RNA [5]. While antibodies to Ro52 and Ro60/SS-A occur primarily in patients with systemic lupus erythematosus and Sjo¨gren’s syndrome, a recent paper by Rutjes et al. [6] identified antibodies to Ro52 in patients with Correspondence to: Mark Barton Frank, Arthritis and Immunology Program, Oklahoma Medical Research Foundation 825 NE 13th Street, Oklahoma City, OK 73104, USA. Fax: 405–271–4110. E-mail: [email protected] 137 0896–8411/99/020137+06 $30.00/0
© 1999 Academic Press
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were shown not to be cross-reactive [6]. Second, while tRNA synthetases charge their cognate tRNAs in the cytoplasm, Ro52 resides primarily in the nucleus [8]. The role of molecules in the nucleus that are antigenically similar to Jo-1 is unknown [9]. Furthermore, anti-Jo-1 antibodies are rarely, if ever, found in patients with SLE or primary Sjo¨gren’s syndrome without myositis. Finally, anti-Ro52 antibodies in the latter two diseases are frequently detected with antibodies to the Ro60/SS-A and La/SS-B molecules. Among sera from patients with myositis, a higher proportion of anti-Jo-1-positive sera has anti-Ro60 than do anti-Jo-1-negative myositis sera [10]. However, in the sera studied by Rutjes et al. [6], antibodies to Ro60/SS-A were rare relative to anti-Ro52. Thus, if the anti-Jo-1 and anti-Ro52 antibodies are coupled, the mechanism may be unique to myositis. In order to better understand this association, we have examined sera from patients that contain anti-Jo-1 and other antibodies that have been shown to be associated with myositis, as well as with myositis–scleroderma overlap syndrome.
Materials and Methods
sample buffer, electrophoresed in 10% polyacrylamide gels, and visualized by autoradiography. The human Ro52 protein was expressed in E. coli from its full-length cDNA, and antibodies to it were detected by ELISA [1, 12]. Briefly, inclusion bodies containing the Ro52 protein were solubilized in 7 M urea, and then diluted in a carbonate buffer to coat microtiter plates (Costar, Cambridge, MA, USA). After washing and blocking the plates, dilutions of patient sera were added, and bound antibodies were detected using an alkaline phosphatase-conjugated goat antihuman ã chain specific antiserum (Sigma, St. Louis, MO, USA) and p-nitrophenyl phosphate substrate. Optical densities (OD) were quantified in a Dynatech MRX microplate reader (Dynex Technologies, Inc., Chantilly, VA, USA) at 405 nm. Controls included OD from wells with no antibody, as well as extracts from bacteria transformed with pUC19 that lacked the human Ro52 cDNA. These control values were subtracted from the OD of patients’ sera reacting with the Ro52 protein to obtain a net OD. Antibodies to Ro60 and La/SS-B were detected similarly by Ouchterlony immunodiffusion and immunoprecipitation analysis. Anti-centromere antibodies were detected by indirect immunofluorescence of Hep-2 cells.
Sera Sera were obtained from patients of the authors (INT and DAT), or from the Myositis Serum Bank or the Clinical Immunology Laboratory at the Oklahoma Medical Research Foundation (Oklahoma City, OK, USA). The latter sera were referred either for clinical testing or as part of other studies. Sera were stored at −20°C prior to use. Two hundred and sixteen sera were selected for analysis based on the presence of specific autoantibodies that are associated with myositis, scleroderma, or myositis–scleroderma overlap syndrome. Because the sera used here were selected on the basis of their autoantibody profiles, the results may not represent a random sampling of myositis patients. An additional 41 sera from patients with rheumatoid arthritis were also analysed for the presence of anti-Ro52 antibodies.
Detection of patient autoantibodies The specificities of autoantibodies related to myositis were determined using Ouchterlony immunodiffusion with calf thymus extract as antigen, and by immunoprecipitation of nucleic acids and proteins. These methods have been previously described in detail [11]. Briefly, for nucleic acid analysis, patient antibodies were bound to protein A-Sepharose beads. The beads were washed and then reacted with extract from sonicated HeLa cells. Immunoprecipitates were phenol-extracted and then subjected to electrophoresis in 8 M urea, 10% polyacrylamide gels. RNA was detected by silver staining. For protein analysis, immunoprecipitates of 35S-labeled HeLa cells were recovered from the beads by heat denaturation in
Statistics Fisher’s Exact test was used to test for differences between serologic groups. Calculated P-values were corrected for the multiple comparisons made.
Results The autoimmune response to the Ro52 protein in anti-Jo-1-positive sera We analysed the response to the Ro52 protein in 216 sera that contained antibodies associated with, or specific for, patients with polymyositis, dermatomyositis, or scleroderma. Ninety-two (43%) of these sera contained IgG antibodies directed to the Ro52 protein, and 40 sera (19%) contained anti-Jo-1 antibodies. The concurrence of anti-Ro52 and myositis-specific antibodies (MSA) was analysed. Among the 40 antiJo-1-positive sera, 70% had antibodies to the Ro52 protein. This percentage is similar to the 58% of sera reported by Rutjes et al. [6]. The proportion of sera with anti-Ro52 antibodies was significantly higher (P=8×10 −7) in the anti-Jo-1-positive sera than in antiJo-1-negative sera (37%), consistent with the findings of Rutjes et al. [6]. Antibodies to Ro52 in SLE and Sjo¨gren’s syndrome patients frequently occur in sera that also contain antibodies to the Ro60/SS-A and La/SS-B proteins. Accordingly, we tested the hypothesis that the anti-Ro52 antibodies in anti-Jo-1-positive patients predominantly occur in the latter sera. The results
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Table 1. Anti-Ro52 antibodies in anti-Jo-1-positive myositis patients Anti-Ro60
Anti-La
Anti-Ro52-positive
Anti-Ro52-negative
Total
% Ro52 +
Negative Positive Positive Total
Negative Negative Positive
12 2 14 28
9 0 3 11
21 2 17 40
57 100 82 70
Table 2. Anti-Ro52 antibodies in myositis sera with anti-threonyl-tRNA synthetase (PL-7) antibodies Anti-Ro60
Anti-La
Anti-Ro52-positive
Anti-Ro52-negative
Total
% Ro52 +
Negative Positive Total
Negative Negative
4 7 11
5 0 5
9 7 16
44 100 69
Table 3. Anti-Ro52 antibodies in myositis sera with anti-alanyl-tRNA synthetase (PL-12) antibodies Anti-Ro60
Anti-La
Anti-Ro52-positive
Anti-Ro52-negative
Total
% Ro52 +
Negative Positive Positive Total
Negative Negative Positive
4 2 4 10*
5 0 0 5
9 2 4 15
44 100 100 67
*Testing for anti-La/SS-B antibodies was equivocal in one serum that was positive for both anti-Ro52 and anti-Ro60/SS-A antibodies. This serum is omitted from the table.
suggest that the autoimmune response to the Ro52 protein by anti-Jo-1-positive patients is statistically independent of the presence of anti-Ro60/SS-A or anti-La/SS-B antibodies (Table 1, P=0.24).
antibodies to Ro52. Therefore, the immune response to the Ro52 protein in anti-tRNA synthetase-positive patients is not limited to those containing anti-Jo-1 autoantibodies.
The autoimmune response to the Ro52 protein in other anti-aminoacyl-tRNA synthetase sera
The frequency of anti-Ro52 antibodies in sera with other MSAs
Because of the common clinical spectrum among patients who produce antibodies to different aminoacyl-tRNA synthetases, the response to Ro52 in sera containing antibodies to two other synthetases was also analysed. The proportion of myositis patients with non-Jo-1 anti-tRNA synthetase antibodies is low (approximately 2% of myositis patients for each antibody); therefore, the number of sera available for study is relatively small. Sera from 16 patients with anti-PL-7, and another 16 patients with anti-PL-12 antibodies were examined. Among these sera without anti-Ro60 antibodies, 44% had antibodies to the Ro52 protein. As was the case with sera containing anti-Jo-1 antibodies, the immune response to Ro52 was statistically independent of the response to the Ro60 particle, although anti-Ro52 antibodies were present in all of the sera that contained anti-Ro60 antibodies (Tables 2 and 3). Collectively, of the 72 sera that were positive for any anti-tRNA synthetase antibodies, 50 (69%) contained
Although anti-synthetase antibodies are the most common, other MSAs have also been described. These include antibodies to Mi-2, produced primarily by dermatomyositis patients. Antibodies to Ro52 were not detected in any of 16 anti-Mi-2-positive sera that were studied, including 10 that contained antibodies to the Ro60/SS-A molecule. Thus, the autoimmune response to Ro52 is not equally represented among serologically distinct groups of myositis patients. Autoantibodies to the signal recognition particle (SRP) are produced primarily by polymyositis patients. This particle is involved in the transport of newly synthesized proteins into the endoplasmic reticulum. Anti-SRP antibodies occur in about 4% of myositis patients and are associated with HLA-DR5 [11]. Of the 21 anti-SRP-positive sera that were analysed, nine (43%) had antibodies to the Ro52 protein. All of these anti-SRP-positive sera were negative for antibodies to the Ro60 and La/SS-B molecules. These data demonstrate that anti-Ro52 autoantibodies
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Table 4. Antibodies to Ro52 in anti-PM-Scl-positive sera Anti-Ro60
Anti-La
Anti-Ro52-positive
Anti-Ro52-negative
Total
% Ro52-positive
Negative Positive Positive Total*
Negative Negative Positive
13 9 1 23
22 2 2 26
35 11 3 49
37 82 33 47
*The presence of anti-La/SS-B antibodies could not definitively be determined in one anti-Ro52-positive and one anti-Ro52-negative. These sera are not included in this table.
in myositis patients are not limited to sera that contain anti-tRNA synthetase antibodies.
low titer. None of the ANA-negative rheumatoid arthritis sera had detectable anti-Ro52 antibodies.
The autoimmune response to the Ro52 protein in sera from patients with anti-PM-Scl antibodies
Discussion
Antibodies to the PM-Scl antigen are present in approximately 25% of sera from patients with polymyositis and scleroderma overlap [13]. The antigen consists of at least 11 proteins in the nucleus and nucleolus whose function(s) is unknown [13]. Sera from 51 patients with anti-PM-Scl antibodies were studied to determine if antibodies to the Ro52 protein were present. Twenty-four of the sera (47%) contained antibodies to Ro52 (Table 4). As was the case with the anti-aminoacyl-tRNA synthetase-positive sera, antiRo52 antibodies were present in a higher proportion of sera that also contained anti-Ro60 antibodies. However, the association between antibodies to the two Ro antigens did not reach statistical significance (P=0.12). The presence of anti-Ro52 antibodies was also not associated with the titer of anti-PM-Scl antibodies in these sera (data not shown) as determined by a recombinant ELISA [14].
The autoimmune response to Ro52 in sera from patients with other autoimmune disorders Given the findings from the anti-PM-Scl-positive sera, we tested the hypothesis that patients with scleroderma who do not have polymyositis also produce anti-Ro52 autoantibodies. Scleroderma sera containing autoantibodies to either topoisomerase I (Scl-70) or anti-centromere antibodies were tested. Antitopoisomerase I antibodies are often associated with diffuse cutaneous systemic sclerosis with an increased frequency of lung involvement, while anti-centromere antibodies are strongly associated with limited cutaneous systemic sclerosis. Anti-Ro52 antibodies were detected in only three of 19 anti-Scl-70-positive sera, and one of nine anti-centromere sera that were screened. All 28 of these sera were negative for anti-Ro60 and anti-La/SSB antibodies. Forty-one sera from rheumatoid arthritis patients were also screened for anti-Ro52 antibodies. Twentyfour of these sera were ANA-positive. Only one serum, which was weakly positive for ANA, contained anti-Ro52 antibodies, which were present at
In this study, sera from 216 patients known to have autoantibodies associated with myositis or scleroderma were screened for the presence of IgG autoantibodies to the Ro52 protein. A significant proportion of these patients produced antibodies to this protein. These include patients with anti-PM-Scl antibodies, which are associated with an overlap syndrome of myositis and scleroderma. However, antiRo52 antibodies were rarely present in sera from patients with scleroderma-specific antibodies. Similarly, anti-Ro52 autoantibodies were infrequently found in rheumatoid arthritis sera. The quantitative levels of these antibodies varied in each of the serologic groups and were similar to those in lupus patients. With few exceptions, very little work has been reported on the presence of anti-Ro52 antibodies in patients other than lupus or Sjo¨gren’s syndrome. Ricchiuti et al. [15] described anti-Ro52 antibodies in 24% of rheumatoid and 19% of juvenile rheumatoid arthritis sera, and in 19% of sera from patients with mixed connective tissue disease. No anti-Ro52 autoantibodies were detected in scleroderma sera. Rutjes et al. [6] were the first to identify these antibodies in patients with idiopathic inflammatory myopathies. Their data indicated these antibodies occurred primarily in sera containing antibodies to histidyltRNA synthetase (Jo-1). Because the authors used a secondary antiserum that detected human IgM, IgG, and IgA antibodies, the specific anti-Ro52 antibody isotypes were not discussed. The data reported here extend these findings of Rutjes et al. [6] in a number of ways. We confirm that antibodies to Ro52 frequently occur in sera with anti-Jo-1 antibodies. However, anti-Ro52 antibodies also occur in similar proportions of sera containing other MSAs, in particular other anti-tRNA-synthetase, anti-SRP, and anti-PM-Scl antibodies. Because the majority of sera used in this study were selected for particular myositis-associated antibody patterns, the associations of the anti-Ro52 response to MSAs in myositis clinical groups is unknown. A separate study is underway to analyse the association of anti-Ro52 antibodies with disease.
Anti-Ro52 antibodies with myositis sera
No sera studied here with the dermatomyositisspecific anti-Mi-2 antibodies contained anti-Ro52 antibodies. This contrasts with the finding of Rutjes et al. [6] who reported similar proportions of anti-Ro52 antibody-positive sera in sera from patients with polymyositis and dermatomyositis. This distinction may reflect different proportions of dermatomyositis patients with non-Jo-1 anti-aminoacyl-tRNA synthetase antibodies, different anti-Ro52 isotypes (only IgG was detected here), or racial differences between these cohorts. The response to Mi2 is associated with HLA-DR53 and HLA-DR7, in contrast to HLA-DR52 and HLA-DR3 specificities that predominate in patients who produce anti-synthetase antibodies [10]. HLA typing of patients in the future may be useful in testing the hypothesis that the Mi-2-associated HLA proteins are incapable of presenting or inefficiently presenting Ro52 to T cells. No clear HLA association with anti-Ro52 has been described in lupus or Sjo¨gren’s patients. The findings reported here show that the response to Ro52 is rare in scleroderma patients, in support of data by Ricchiuti et al. [15]. However, this immune response is clearly present in patients with anti-PMScl antibodies, which are found in some patients with polymyositis overlap syndrome and other disorders. Detection of anti-Ro52 antibodies may prove to be a useful addition in discriminating between patients in these groups. While tRNA synthetases charge their nucleic acids in the cytoplasm, and Ro52 occurs primarily in the nucleus, two recent reports provide data to indicate the possibility for interactions between these proteins. Vazques-Abad et al. [9] reported that anti-Jo-1 antibodies bound to antigen in both the cytoplasm and nucleus of a laryngeal epithelial carcinoma cell line. Scmitz et al. found evidence for Ro52 in the nucleus with patient and monoclonal antibodies, but also in the cytoplasm of a bladder carcinoma cell line [8]. Venables has speculated that an interaction between these proteins may occur in abnormal cells or cells undergoing apoptosis [16]. A striking feature of the data reported here was the independent response to the Ro52 and Ro60 molecules in myositis sera. The initial description of anti-Ro52 antibodies in Sjo¨gren’s Syndrome patients reported these antibodies in >80% of sera with anti-Ro60 antibodies [17]. A later report by the same group found that they co-occurred in about half of lupus and Sjo¨gren’s syndrome sera, while anti-Ro52 antibodies alone were detected only in Sjo¨gren’s sera, and antiRo60 antibodies alone occurred only in SLE [18]. Reports by others also indicate that certain SLE patients produce antibodies to Ro52 in the absence of those to Ro60 [19, 20]. The analyses of lupus sera in our laboratory [12, 21, 22] are more consistent with those of Ben-Chetrit et al. [17, 18]. The frequent cooccurrence of antibodies to both forms of Ro has led to investigations supporting models of antigenic spreading between Ro52, Ro60 and La/SS-B proteins [23–25]. Interestingly, in many of the sera described here, the frequency of antibodies to Ro60 and La/SS-B is lower than the frequency of anti-Ro52 antibodies. This may
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suggest either that intermolecular spreading may not occur in these patients [16], or that the anti-Ro52 antibodies predate the immune response to these other molecules. The high proportions of patients with myositis and only anti-Ro52 antibodies may serve as an interesting group of patients for follow-up study on this issue.
Acknowledgements The authors thank the following individuals for their valuable referral of patient sera: Morris Reichlin, MD (Oklahoma City, USA); Frank C. Arnett, MD (Houston, USA); Frederic W. Miller, MD, PhD, (Bethesda, USA); Paul H. Plotz, MD, (Bethesda USA); and Chester Oddis, MD, (Pittsburgh, USA). We also thank Ms Lori Lewis for technical assistance with ELISA. This work was supported in part by Public Health Service Grant AR41919 to MBF from the National Institute of Arthritis and Musculoskeletal and Skin Diseases.
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