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Rheumatology 2001; 40: 683-686
© 2001 British Society for Rheumatology
Original Papers |
Antibodies to Th/To ribonucleoprotein in patients with localized scleroderma
Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo and
1 Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan
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Abstract |
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Objective. Antibodies to Th/To ribonucleoprotein (anti-Th/To) have been detected almost exclusively in patients with systemic sclerosis. Therefore, we aimed to determine the prevalence of anti-Th/To in patients with localized scleroderma.
Methods. Seventy serum samples from patients with localized scleroderma were examined by RNA immunoprecipitation and indirect immunofluorescence analysis using HEp-2 cells as substrate.
Results. Three (4%) of 70 sera from patients with localized scleroderma immunoprecipitated 7-2 (Th) RNA and 8-2 (To) RNA. Indirect immunofluorescence analysis demonstrated that all the sera positive for anti-Th/To showed mainly nucleolar staining. In one patient, the coexistence of anti-histone antibody with anti-Th/To was detected by enzyme-linked immunosorbent assay for anti-histone antibody and confirmed using an absorption test with histones followed by indirect immunofluorescence analysis. Moreover, the localized scleroderma patients with anti-Th/To tended to have significantly fewer sclerotic lesions than those without.
Conclusion. Anti-Th/To is one of the serological abnormalities in localized scleroderma, and the presence of anti-Th/To may be a serological indicator of a mild form of cutaneous involvement.
KEY WORDS: Antinucleolar antibodies, Immunoprecipitation, Humoral immunity.
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Introduction |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Localized scleroderma (LSc) is a connective tissue disorder usually limited to the skin and subcutaneous tissue, but it sometimes involves the muscle beneath the cutaneous lesions. The absence of Raynaud's phenomenon, acrosclerosis and internal organ involvement differentiates LSc from systemic sclerosis (SSc) [1]. LSc has been accompanied by a variety of abnormal immune reactions, such as the presence of antinuclear antibody (ANA), rheumatoid factor (RF), anti-single-stranded DNA antibody (anti-ssDNA) and anti-histone antibody (AHA) [2–6].
Autoantibodies directed against intracellular antigens, such as DNA, RNA, proteins and RNA–protein complexes, are frequently found in various rheumatic diseases [7]. Some of these antibodies are characteristic of certain symptoms and are of diagnostic value. The presence of antibodies to Th/To ribonucleoprotein (anti-Th/To) was detected almost exclusively in patients with SSc and primary Raynaud's phenomenon [8]. The presence of anti-Th/To might be a serological indicator of limited cutaneous involvement, hypothyroidism and small bowel involvement. Sera from patients with anti-Th/To immunoprecipitate two small RNAs, identified as RNase MRP (Th or 7-2 RNA) and RNase P (To or 8-2 RNA), from HeLa cell extracts, and recognize a 40-kDa protein subunit common to both RNAs, which is referred to as Th40 [9]. However, the cloning of a cDNA encoding Th40 has not been accomplished.
The presence and clinical significance of anti-Th/To in patients with LSc has not been described previously. In the present study, we investigated the prevalence of anti-Th/To using RNA immunoprecipitation, and characterized the clinical and laboratory features of LSc patients with anti-Th/To.
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Patients and methods |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Patients and samples
Serum samples were obtained from 70 patients with LSc, 30 patients with SSc and 40 healthy control subjects. The patients with LSc were classified into three subgroups: (i) those with generalized morphea (n=22), (ii) those with linear scleroderma (n=28), and (iii) those with morphea (n=20), as described previously [5, 6]. All patients with SSc met the American College of Rheumatology criteria for the diagnosis of SSc [10].
RNA immunoprecipitation
Anti-Th/To was detected by RNA immunoprecipitation, as described previously [11]. Briefly, 10 µl of patient serum was incubated with 10 µl of protein A–Sepharose in 500 µl of IPP buffer overnight at 4°C. The IgG-coated Sepharose was washed five times with IPP buffer, and was resuspended in 500 µl of NET-2 buffer. This suspension was incubated with 200 µl of HeLa cell extracts for 2 h at 4°C. The Sepharose-bound antigen was washed five times with NET-2 buffer, and resuspended in 300 µl of NET-2 buffer. To extract bound RNAs, 30 µl of 3 M sodium acetate, 30 µl of 10% sodium dodecyl sulphate (SDS), 2 µl of glycogen and 300 µl of phenol–chloroform–isoamyl alcohol (50:50:1, containing 0.1% 8-hydroxyquinolone) were added to the Sepharose beads. RNAs were precipitated with ethanol and dissolved in 20 µl of electrophoresis sample buffer. The RNA samples were denatured at 65°C for 3 min and resolved in a 7M urea–10% polyacrylamide gel, and stained with silver (Bio-Rad Laboratories, Hercules, CA, USA).
Enzyme-linked immunosorbent assay for AHA and indirect immunofluorescence study
Enzyme-linked immunosorbent assay (ELISA) for AHA was carried out, as described previously [6]. ANA was detected by indirect immunofluorescence using HEp-2 cells as substrate, as described previously [2]. We carried out an absorption test using histones in indirect immunofluorescence analysis on the sera, which were considered to be positive for IgG AHA by ELISA, as described previously [12].
Statistical analysis
The statistical analysis was carried out with the Mann–Whitney U-test for the comparison of means and Fisher's exact probability test for the analysis of frequency. Two-tailed P values less than 0.05 were considered significant.
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Results |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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RNA immunoprecipitation
Three (4%) of the 70 serum samples from patients with LSc immunoprecipitated 7-2 RNA and 8-2 RNA (Fig. 1
). The frequency of anti-Th/To positivity was 7% in the patients with SSc, and there was no significant difference in the frequency of this antibody between the patients with SSc and those with LSc.
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Anti-La (SS-B) antibody is known to immunoprecipitate 7-2 RNA and 8-2 RNA in addition to precursor tRNAs, pre-5 S and pre-5.8 S ribosomal RNAs, Ro (SS-A) RNAs. In the present study, there was no serum which immunoprecipitated La RNAs. Moreover, antibodies to La were not detected by double immunodiffusion or ELISA for antibodies to La.
ELISA for AHA and indirect immunofluorescence studies
IgG AHA was detected in 24 (34%) of the 70 patients with LSc. Among the three patients with anti-Th/To, IgG AHA was detected in one serum, but the titre was low. In indirect immunofluorescence analysis, all of the three sera positive for anti-Th/To by RNA immunoprecipitation showed mainly nucleolar staining (Fig. 2A). One serum which contained both AHA and anti-Th/To showed nucleolar staining, weak homogeneous staining and chromosome staining (Fig. 2B). Because this serum was positive for AHA by ELISA, we performed the absorption test using histones. After incubation of the serum with 3 mg/ml total histones, the homogeneous staining and chromosome staining were markedly decreased, leaving nucleolar staining (Fig. 2C). These findings suggested that AHA played the major role in the weak homogeneous and chromosome staining pattern in indirect immunofluorescence analysis of this serum.
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Clinical correlations
There was no significant difference between these groups in sex, age or duration of the disease. The number of sclerotic lesions was significantly lower in the patients with anti-Th/To than in those without (2.3±2.3 vs 3.5±3.2, P<0.05).
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Discussion |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Anti-Th/To has been considered to be fairly specific to SSc, because the presence of anti-Th/To is detected in patients with SSc and primary Raynaud's phenomenon but not in those with SLE, polymyositis/dermatomyositis or undifferentiated connective tissue disease [8]. In the present study, anti-Th/To was detected in 4% of patients with LSc by RNA immunoprecipitation. This frequency is similar to that in patients with SSc [8]. The LSc patients with anti-Th/To did not have sclerodactyly or nailfold bleeding. There were no specific autoantibodies, such as anti-topoisomerase I antibody or anti-centromere antibody, in sera from these patients by indirect immunofluorescence or immunodiffusion. Raynaud's phenomenon did not occur at any time in their disease course. Taken together, our findings show that anti-Th/To occurs in patients with LSc as well as those with SSc. In the present study, patients with SSc-associated reactivities may not always have systemic disease and some of the same events leading to anti-Th/To or histology of skin sclerosis in SSc might occur in LSc, which should be elucidated in future studies. Two of the patients with anti-Th/To were diagnosed as having linear scleroderma and the other was diagnosed as having morphea. In addition, the patients with anti-Th/To had significantly fewer sclerotic lesions than those without. Our finding indicates that the LSc patients with anti-Th/To may have a mild form of cutaneous involvement, as SSc patients with anti-Th/To are inclined to have limited cutaneous involvement. However, the notion that anti-Th/To or anti-Ku may be associated with mild skin involvement in SSc has not yet been determined because of a small number of reports. Further cases with anti-Th/To are needed to demonstrate a definite association between anti-Th/To and the clinical and laboratory features of patients with LSc.
Previous studies showed that the coexistence of antibodies to U3RNP, Ro (SS-A) and U1RNP was sometimes found [8]. The coexistence of anti-U1RNP, anti-U3RNP and anti-Ro can be detected by RNA immunoprecipitation, but the coexistence of these autoantibodies with anti-Th/To was not detected in our patients. The coexistence of AHA with anti-Th/To was detected in one of the three patients with anti-Th/To. This was confirmed by the absorption test using histones in indirect immunofluorescence analysis and the ELISA for AHA. These results, together with those of previous studies, suggest that anti-Th/To may coexist with various other autoantibodies.
In conclusion, we showed for the first time that anti-Th/To is found in patients with LSc by RNA immunoprecipitation. In one patient, the coexistence of AHA with anti-Th/To was detected. The LSc patients with anti-Th/To tended to have fewer sclerotic lesions than the patients without. Our study suggests that anti-Th/To is one of the serological abnormalities in LSc, and that the presence of anti-Th/To may be a diagnostic and pathobiological feature of LSc.
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Notes |
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Correspondence to: H. Ihn, Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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