Distribution and antigen specificity of anti-U1RNP antibodies in patients with systemic sclerosis

Abstract

Systemic sclerosis (SSc) is a generalized connective tissue disease which is characterized by the presence of several autoantibodies. To determine the prevalence and antigen specificity of anti-U1RNP antibodies (anti-U1RNP) in patients with SSc, serum samples from 223 patients with SSc, 117 patients with systemic lupus erythematosus (SLE), 18 patients with mixed connective tissue disease (MCTD) and 40 healthy control subjects were examined by indirect immunofluorescent analysis (IIF), double immunodiffusion, and immunoblotting using nuclear extract of HeLa cells. Eighteen of the 223 (8%) serum samples from patients with SSc were shown to be positive for anti-U1RNP. The frequency of anti-U1RNP positivity in limited cutaneous SSc (14%) was significantly higher than that in those with diffuse cutaneous SSc (3%). Anti-Sm antibodies were detected in patients with SLE positive for anti-U1RNP, but not in those with SSc positive for anti-U1RNP or those with MCTD. Immunoblotting demonstrated that anti-70-kD antibodies were detected more often in patients with SSc positive for anti-U1RNP and in those with MCTD than in those with SLE. Furthermore, anti-U1RNP was closely correlated with pulmonary fibrosis and joint involvement in patients with SSc. These results suggest that anti-70-kD antibodies are useful in the classification of patients with anti-U1RNP.

INTRODUCTION

Scleroderma or systemic sclerosis (SSc) is a generalized connective tissue disease which involves sclerotic changes in the skin and many other organ systems [1,2].The pathogenesis and aetiology are unknown, but the presence of several autoantibodies is recognized to be one of its prominent features [3]. Anti-centromere antibody (ACA) and anti-topoisomerase I antibody (anti-topo I) are considered to be highly specific for SSc.

Anti-U1RNP antibody (anti-U1RNP) is generally known to be a serological marker for mixed connective tissue disease (MCTD) [4], but can be detected also in patients with definite SSc or systemic lupus erythematosus (SLE) [5,6]. Studies have revealed that the presence of each antibody closely correlates with clinical features, organ involvement, and prognosis, respectively [7,8].

The autoantigens are often nucleic acid-associated proteins, such as small nuclear RNP (snRNP) [9]. Small nuclear RNP are important components in pre-messenger RNA processing [10,11]. They consist of at least 11 polypeptides (termed 70K, A, A′, B, B′, B′′, C, D, E, F, and G) over a molecular weight range of 11–70 kD, and five snRNA molecules (U1, U2, U4, U5, and U6) [12–15]. Anti-Sm antibodies precipitate U1, U2, U4, U5, U6 and recognize primarily B/B′ and D. Anti-Sm antibodies are strongly associated with SLE. Anti-(U1, U2) snRNP autoantibodies precipitate both U1 and U2 and recognize U2-specific polypeptides A′ and B′′ [13].

Previous analyses revealed that the RNP autoantigens are located on small RNP particles containing uridine-rich (U1) small nuclear RNA, which is known to play a major role in splicing of pre-messenger RNA [16]. Further investigations by immunoblot analyses with nuclear extracts identified the antigenic epitopes for anti-U1RNP reside on the p70 protein (70 kD), the A protein (33 kD), and occasionally on the C protein (22 kD) [17,18]. Antibodies reacting to p70 protein were reported to be associated with anti-RNP specificity in MCTD, but rarely occurred in SLE patients' sera [17,19]. However, the prevalence or antigen specificity of U1RNP in patients with SSc, to our knowledge, has not been described previously. In this study, we investigated the prevalence and antigen specificity of anti-U1RNP in patients with SSc.

PATIENTS AND METHODS

Patients

Serum samples were obtained from 223 patients with SSc, 117 patients with SLE, 18 patients with MCTD and 40 healthy control subjects. For a diagnosis of MCTD the criteria proposed by Alarcon-Segovia [20] were used. In this study, patients with MCTD had clinical features of SLE, SSc and polymyositis and anti-U1RNP antibodies [4], and did not satisfy the criteria for other connective tissue diseases, such as SSc, SLE, or polymyositis/dermatomyositis (PM/DM) [21,22,23]. Patients with SLE met the American Rheumatism Association criteria for definite SLE [21] and did not have features diagnostic of other connective tissue disorders [22,23]. Patients with SSc met the American College of Rheumatology (formerly American Rheumatism Association) criteria for the diagnosis of definite scleroderma (systemic sclerosis) [22], and did not have features diagnostic of other connective tissue diseases [21,23]. The patients with SSc included in this study did not satisfy the criteria for MCTD [20], and did not have cytopenias or PM. Two of them had scleroderma kidney. Patients with SSc were grouped according to the classification system proposed by LeRoy et al. [24]: 102 patients had limited cutaneous SSc (lcSSc) and 121 patients had diffuse cutaneous SSc (dcSSc). Patients who met one or more of the criteria of connective tissue disease and had features diagnostic of other connective tissue diseases [21–23] were excluded from this study. Aliquots of serum were frozen at −80°C until assayed.

Clinical assessment

The clinical and laboratory data reported in this study were obtained at the time the blood samples were drawn. The patients were evaluated for the presence of gastrointestinal, pulmonary, cardiac, renal or joint involvement as described previously [25,26].

Analysis of antinuclear antibodies

Antinuclear antibodies (ANA) were detected by immunofluorescence using HEp-2 cells, as described [25,27].

Double immunodiffusion

Antibodies against U1RNP, Ro/SS-A, La/SS-B, and Sm antigens were detected using double immunodiffusion in 0.5% agarose as described [28].

Preparation of nuclear extract for immunoblotting

Nuclear extract was prepared following a procedure described previously [19,29]. HeLa S3 cells were harvested and washed in PBS and scraped into 1 ml of cold buffer A (10 mm HEPES–KOH pH 7.9, 1.5 mm MgCl₂, 10 mm KCl, 1 mm dithiothreitol, 0.2 mm PMSF). The cells were allowed to swell on ice for 10 min and then vortexed for 10 s. After centrifugation for 3 min, the supernatant was discarded. The pellet was resuspended in 100 μl of cold buffer C (20 mm HEPES–KOH pH 7.9, 25% glycerol, 420 mm NaCl, 1.5 mm MgCl₂, 0.2 mm EDTA, 1 mm dithiothreitol, 0.7 mm PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation for 2 min at 4°C and the supernatant fraction was stored at −80°C and used as substrate for immunoblotting.

Immunoblotting

Immunoblotting was performed as described [26,30]. Briefly, nuclear antigens from HeLa cells were subjected to electrophoresis on 15% SDS–polyacrylamide slab gels, and then electrotransferred from the gels on to nitrocellulose sheets. The nitrocellulose sheets were then incubated overnight with serum samples diluted 1:50. Bound antibodies were detected with alkaline phosphatase-conjugated goat anti-human IgG antibody, and colour was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphatase. Immunoblotting analyses using 7.5% gels were also performed and anti-70-kD antibodies were distinguished from anti-70-kD ACA.

Statistical analysis

Statistical analysis was carried out with Fisher's exact probability test for the analysis of frequency. Two-tailed P values < 0.05 were considered significant.

RESULTS

Prevalence of anti-U1RNP

The frequency of anti-U1RNP positivity by double immunodiffusion is shown in Table 1. Eighteen (8%) of the 223 serum samples from patients with SSc were shown to be positive for anti-U1RNP. The frequency of anti-U1RNP positivity was 14% in patients with lcSSc, significantly higher than that in those with dcSSc (3%, P < 0.01). The frequency of anti-U1RNP positivity was 24% in patients with SLE, significantly higher than in those with SSc (P < 0.01).

Table 1.

Frequencies of anti-U1RNP antibodies (anti-U1RNP) in patients with systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD), detected by double immunodiffusions

Unless indicated otherwise, values are numbers.

lcSSc, limited cutaneous SSc; dcSSc, diffuse cutaneous SSc.

*P < 0.01; **P < 0.02.

Association of anti-U1RNP with anti-Ro/SS-A, anti-La/SS-B, or anti-Sm

The frequency of anti-Ro/SS-A, anti-La/SS-B, or anti-Sm in the sera positive for anti-U1RNP was also studied. Anti-Ro/SS-A were detected in four (22%) of the 18 patients with SSc positive for anti-U1RNP, in eight (29%) of the 28 patients with SLE positive for anti-U1RNP and in six (33%) of the 18 patients with MCTD. There was no significant difference in the frequencies of anti-Ro/SS-A between patients with SSc positive for anti-U1RNP and those with SLE positive for anti-U1RNP, between those with SSc positive for anti-U1RNP and those with MCTD, or between those with SLE positive for anti-U1RNP or those with MCTD. Anti-La/SS-B were detected in one (6%) of the 18 patients with SSc positive for anti-U1RNP, in four (14%) of the 28 patients with SLE positive for anti-U1RNP and in none (0%) of the 18 patients with MCTD. The frequency of the coexistence of anti-U1RNP and anti-La/SS-B in patients with SLE was higher than that in SSc or that in MCTD, but there was no significant difference in the frequencies of coexistence of anti-La/SS-B and anti-U1RNP between patients with SSc and those with SLE, between those with SSc and those with MCTD, or between those with SLE and those with MCTD. Anti-Sm were detected in eight (29%) of the 28 patients with SLE positive for U1RNP, but not detected in patients with SSc positive for anti-U1RNP or in those with MCTD. There were significant differences in the frequencies of anti-Sm between the patients with SLE positive for anti-U1RNP and those with SSc positive for anti-U1RNP (P < 0.01), as well as between those with SLE positive for anti-U1RNP and those with MCTD (P < 0.01).

Immunoblotting for anti-U1RNP

To determine the antigen specificities of anti-U1RNP, the serum samples positive for anti-U1RNP in double immunodiffusion were assessed. These sera were reacted with nuclear antigens derived from HeLa cells. Figure 1 shows representative results of immunoblotting and these results are summarized in Table 2. Anti-70-kD antibodies were detected in 14 (78%) of the 18 patients with SSc positive for anti-U1RNP, in eight (29%) of the 28 patients with SLE positive for anti-U1RNP and in 11 (61%) of the 18 patients with MCTD. Anti-70-kD antibodies were detected more often in patients with SSc positive for anti-U1RNP than in those with MCTD, but there was no significant difference in the frequencies between these two groups. There were significant differences in the frequencies of anti-70-kD between patients with SSc positive for anti-U1RNP and those with SLE positive for anti-U1RNP (P < 0.01), and between those with SLE positive for anti-U1RNP and MCTD (P < 0.02).

Fig. 1.

Immunoblot analysis of sera from patients with systemic sclerosis (SSc) (a), those with mixed connective tissue disease (MCTD) (b), and those with systemic lupus erythematosus (SLE) (c). Sera from healthy control subjects were also included (lane 19 (a); lane 19 (b); lane 29 (c)). The nitrocellulose blot strips contained HeLa nuclear extracts transferred after separation on 15% SDS–polyacrylamide gels. Each strip was incubated with patient serum at 1:50 dilution and bound antibodies were detected with alkaline phosphatase-conjugated goat anti-human IgG antibody, and colour was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphatase.

Table 2.

Antigen specificity of anti-U1RNP antibodies (anti-U1RNP) in patients with systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) investigated by immunoblotting

Unless indicated otherwise, values are numbers.

*P < 0.01, compared with SSc or MCTD patients.

Clinical and serological correlations

The clinical and serological features in patients positive and negative for anti-U1RNP are summarized in Table 3. There were no significant differences between these groups in gender, age or duration of disease. Pulmonary fibrosis was significantly more frequent in the patients positive for anti-U1RNP than in those negative for these antibodies (72% versus 36%, P < 0.01). The frequency of reduced percentage vital capacity (%VC) was also greater in the patients positive for anti-U1RNP (50% versus 16%, P < 0.001). The frequency of joint involvement and of elevated erythrocyte sedimentation rate (ESR) was also greater in the patients positive for anti-U1RNP (50% versus 23%, P < 0.01; and 83% versus 43%, P < 0.001).

Table 3.

Correlation of anti-U1RNP antibodies (anti-U1RNP) with clinical and serological features in patients with systemic sclerosis (SSc)

Values are percentages except where indicated otherwise.

VC, Vital capacity; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate.

**P < 0.01; ***P < 0.001.

DISCUSSION

The present study is a comprehensive analysis of the distribution of anti-U1RNP in patients with SSc. Anti-U1RNP was detected in 8% of patients with SSc. The frequency of anti-U1RNP positivity was 14% in patients with lcSSc, significantly higher than in those with dcSSc (3%, P < 0.01). This result is consistent with a previous report that anti-U1RNP was more often detected in patients with lcSSc than in those with dcSSc [31,32]. Kuwana et al. reported that anti-U1RNP was detected in about 10% of patients with SSc [31]. Bunn et al. also performed detailed analyses of autoantibody specificities in SSc, and reported that anti-U1RNP was detected in 6.4% of their SSc patients [32]. In this study, the frequency of anti-U1RNP positivity was 24% in patients with SLE, significantly higher than in those with SSc (P < 0.01). These results emphasize the fact that anti-U1RNP is also detected in definite SSc, but that the frequency of this antibody in SSc is less than that in SLE. Anti-Sm antibodies are detected in 29% of anti-RNP-positive SLE sera, but not in anti-RNP-positive SSc sera, nor in MCTD sera. This result confirms previous reports that the presence of anti-Sm is a highly specific serological marker for SLE [17].

Since previous studies have reported that the Sm and RNP antigens were associated with snRNP protein particles implicated in the splicing of heterogeneous nuclear RNA [33–36], several laboratories have attempted to fractionate and isolate snRNP and to analyse its protein and RNA composition by biochemical and immunochemical methods. Increasingly, detailed knowledge of the RNA and protein composition of the snRNPs also has allowed investigators to analyse the specificities of anti-RNP and anti-Sm in autoimmune diseases. Consistent with previous reports [17,19], this study revealed that the anti-70-kD antibodies are rarely found in anti-RNP-positive SLE sera. It seems that the anti-RNP specificities in these patients are primarily composed of reactions with the A and C proteins. To our knowledge, the antigen specificity of U1RNP in patients with SSc has not been described previously. This study suggested that anti-70-kD antibodies are detected in patients with SSc most frequently (82%) among these patient populations. These results, including the results reported in previous studies, suggest that anti-U1RNP is detected in patients with definite SSc and that the specificity of the antibody is similar to that seen in patients with MCTD. Therefore, the presence of anti-70-kD antibodies could be regarded as a serological marker for SSc or MCTD after statistical analysis of a large number of sera.

SSc is a heterogeneous condition with a range of visceral complications and diversity in the extent of skin sclerosis [24]. Markers indicating the increased risk of developing significant complications have been sought to assist in patient management. Serological associations with particular features in SSc patients have been reported, such as the association of anti-topoisomerase I antibodies with lung fibrosis [24]. We have also reported the association of anti-histone antibodies with pulmonary fibrosis and that of autoantibodies to cardiolipin β₂-glycoprotein-I complex with pulmonary hypertension [25,26]. In our patients, the presence of anti-U1RNP was closely correlated with the presence of pulmonary fibrosis, joint involvement, and elevated ESR levels. This suggests that anti-U1RNP is a serological indicator of the presence of pulmonary fibrosis and of joint involvement in patients with SSc.

In conclusion, our study implies that anti-70-kD antibodies may be useful in the classification of patients with anti-U1RNP, and anti-U1RNP may be a serological marker of pulmonary fibrosis and joint involvement in patients with SSc.