In more than 30% of patients with SSc, the autoantigens to which antinuclear antibodies are directed are unknown. In most of these instances, indirect immunofluorescence testing shows nuclear or nucleolar staining, or both. The purpose of our study was to identify one or more of the autoantigens to which these antibodies are directed and to determine the clinical associations and significance of these antibodies.

Methods

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Patients and Clinical Features

We studied 266 consecutive new patients with SSc who had been initially evaluated in our division during the period 1 January 1986 through 31 December 1988. Serum specimens were available for 252 patients (95%). Two hundred twenty-two of the 252 patients (88%) fulfilled the American College of Rheumatology criteria for definite SSc [12]. These criteria were used to assure certainty of diagnosis and did not serve as diagnostic criteria for individual cases [12]. It is well recognized that approximately 10% of patients with SSc, all having limited cutaneous involvement, do not satisfy these classification criteria [13].

The patients were divided into three subsets (dcSSc, lcSSc, or SSc overlap syndrome) as previously described [1, 11] and as defined in the Appendix. One hundred eleven patients had dcSSc, 114 had lcSSc, and 27 had SSc overlap syndrome. A complete history, physical examination, and laboratory evaluation were done on each patient at the first visit, and more limited evaluations were completed during follow-up visits. Records from private physicians were obtained, and survival status was determined during 1992 with 98% ascertainment. Criteria for organ system involvement were based on those of Medsger and Masi [14] with some modifications [11], as discussed in the Appendix. Organ system involvement was attributed to SSc only if findings could not be attributed to any other disease or condition.

Serum Samples

A serum specimen was obtained from each patient with SSc at the time of his or her first visit. Specimens were also obtained from 170 controls, including 20 normal volunteers and 150 patients diagnosed as having systemic lupus erythematosus (n = 55), polymyositis-dermatomyositis (n = 55), or primary Raynaud phenomenon (n = 40). These latter patients were chosen for comparison because they had diseases that are often difficult to distinguish clinically from SSc and because, relatedly, data on these disorders were used to develop classification criteria for SSc [12]. The control patients were consecutive new or returning patients who had been evaluated by us during 1987. The normal control sera were obtained during 1990 from healthy office and laboratory staff members of our division. No sera from patients with other unrelated rheumatic diseases or with nonrheumatic diseases were studied.

Antinuclear Antibody Analysis

We did immunofluorescence tests, Ouchterlony double-immunodiffusion tests, and immunoprecipitation assays using (Phosphorus-32)orthophosphate- or (Sulfur-35)methionine-labeled HeLa cell extracts to identify and characterize antibody in the patients' sera, as previously described [11].

We used serum obtained from a patient with SSc, as a reference serum with anti-RNA polymerase I antibody. This serum autoantibody was shown to be identical to that in the prototype serum described by Reimer and colleagues [15].

Several well-known types of antinuclear antibodies were identified in our study. Immunofluorescence testing for anticentromere antibodies was considered positive if serum antibody produced a centromeric staining pattern. Anti-topoisomerase I and anti-PM-Scl antibodies were detected by Ouchterlony double-immunodiffusion testing with reference sera. Anti-Th ribonucleoprotein, antifibrillarin, anti-nuclear U sn ribonucleoprotein (U₁, U₂, U_4/U6, and U₅), and anti-La (SS-B) antibodies were detected by immunoprecipitation assays using (Phosphorus-32)orthophosphate-labeled cell extracts. We also did immunoprecipitation assays using (Sulfur-35)methionine-labeled cell extracts to confirm the presence of antifibrillarin and anti-PM-Scl antibodies [16, 17].

We used partially purified RNA polymerase III as a substrate reagent in immunoblotting [18]. The reagent was separated on 10% polyacrylamide-sodium dodecyl sulfate (SDS) gel and transferred electrophoretically onto nitrocellulose sheets [19]. Each lane contained 100 µg of HeLa cell extract, in which RNA polymerase III was enriched 1500 times, obtained from 5 x 10⁹ cells. Probing of the subunit proteins with antisera was carried out as previously described [20].

In assays for RNA polymerase III transcription in vitro using HeLa S100 extracts, we prepared transcription extracts containing 30 µL of HeLa S100 extract and 60 µL of dithiothreitol (0.5 mmol/L), ethylenediaminetetraacetic acid (0.2 mmol/L), KCl (70 mmol/L), MgCl (₂) (5 mmol/L), and HEPES (8 mmol/L) (pH 7.9). This extract was reacted for 1 hour at 4 °C with 6 mg of protein-A sepharose that had been incubated with 30 µL of serum and washed. The immunoprecipitate was removed from the extract by centrifugation in an Eppendorf microcentrifuge for 10 seconds. The supernatant was recovered and subjected to two more 10-second centrifugations. We added to the supernatant adenosine triphosphate, cytidine triphosphate, and uridine triphosphate (each in a concentration of 2 mmol/L); guanosine triphosphate (0.1 mmol/L) with 1 microcuries of (a- Phosphorus-32)guanosine triphosphate (3000 Ci/mmol; ICN Radiochemicals, Costa Mesa, California), and 1 µg of adenovirus pVA I [21]. The reaction was continued for 90 minutes at 30 °C and stopped by the addition of 100 µL of RNA extraction buffer (1% SDS, ethylenediaminetetraacetic acid [20 mmol/L], NaCl [200 mmol/L], and 25 µg/mL carrier of transfer RNA). The mixture was extracted with phenol and precipitated with ethanol; RNA was resolved by electrophoresis on 8% polyacrylamide-urea (7 mol/L) gels and visualized by autoradiography.

Statistical Analysis

Comparisons of proportions were done using chi-square tests and the Fisher exact test where appropriate. Differences between mean values were determined using the Student t-test. All P values were corrected for multiple comparisons. Cumulative survival rates were calculated using life-Table methods, and comparisons were made using log-rank tests.

Results

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Identification of Antinuclear Antibody

We examined sera from 252 consecutive new patients with SSc. Only one serum specimen, that obtained at the first visit, was studied for each patient. Two hundred forty-four patients (97%) had a positive antinuclear antibody test result. Overall, sera from 153 patients had one or more antibodies that were directed to previously known nuclear antigens. Sera from 110 patients had antibody to one of the following three SSc-specific antigens: topoisomerase I (n = 50), centromere (n = 52), and PM-Scl (n = 8). None of the serum specimens contained two or more of these three antibodies. We also examined all 252 serum specimens by immunoprecipitation assay using (Phosphorus-32)orthophosphate-labeled HeLa cell extract to identify antibody to nuclear or cytoplasmic ribonucleoprotein. Two additional SSc-specific antibodies, anti-Th ribonucleoprotein and antifibrillarin, were each found in 11 patients, 1 of whom had both antibodies; thus, a total of 21 patients had at least one of these antibodies. In addition to these five SSc-specific antibodies, antibody to U sn ribonucleoprotein or La, or both, were detected in sera from 28 patients. In 6 of these patients, these two antibodies coexisted with one of the other SSc-specific antibodies. Specimens from 8 patients were negative by these tests. Sera from the remaining 91 patients produced positive nuclear or nucleolar staining, or both, but the antigen to which these antibodies were directed was unidentified.

Immunoprecipitation Studies in Patients with Unidentified Antinuclear Antibody

We tested serum specimens from the 91 patients with unidentified antinuclear antibody by an immunoprecipitation assay using [Sulfur-35]methionine-labeled HeLa cell extract. Thirteen specimens precipitated 12 proteins with molecular weights of 220, 197, 155, 145, 138, 126, 63, 44, 41, 33, 23, and 16 kd. Representative immunoprecipitates are shown in lanes 2 and 3 of Figure 1. The 220-kd band frequently appeared as a broad band or a doublet. Forty-four specimens precipitated many but not all of these 12 proteins Figure 1, lanes 4, 5, 6, and 7). Twenty-seven specimens precipitated 10 proteins but not the 2 proteins with molecular weights of 220 and 145 kd Figure 1, lanes 4 and 5], and 17 specimens precipitated 9 proteins but not the 3 with molecular weights of 220, 145, and 126 kd Figure 1, lanes 6 and 7). The 126-kd protein appeared faintly in some experiments. We classified these 57 patients into three groups based on their reactivity (Table 1). All six proteins with the lowest molecular weights were uniformly precipitated by sera from the three groups.

View larger version (99K): [in this window] [in a new window]   Figure 1. Precipitated proteins of (Sulfur-35)methionine-labeled HeLa cell extract obtained using serum specimens and fractionated on 7.5% (left) and 12% (right) polyacrylamide-SDS gel. Lane Mr shows molecular weight markers; lane 1 shows nonspecific proteins precipitated by normal human serum; lanes 2, 3, 4, 5, 6, and 7 show specific proteins precipitated in serum specimens from six patients with systemic sclerosis. The positions of molecular weight standards (in kilodaltons) are shown to the left of lane Mr. Two panels are included to show the separation of proteins with molecular weights of more than 30 kd and less than 40 kd, respectively.

The 12 proteins shown by immunoprecipitation in 13 patients (group 1) were almost identical in molecular weight to the 13 proteins in the anti-RNA polymerase I reference serum, except 1 protein with a molecular weight of 27 kd previously described [16]. We confirmed this identity by parallel electrophoresis using our anti-RNA polymerase I reference serum (not shown). Because sera from certain patients with SSc were identical in several respects to rabbit polyclonal antisera induced by immunization with biochemically purified RNA polymerase I [16], we concluded that the 12 proteins precipitated by these sera were constituent subunits of RNA polymerase I. However, when we reviewed structures of RNA polymerases I, II, and III obtained by biochemical purification in previous studies [22, 23], certain differences were apparent. The 6 proteins with the highest molecular weights (>100 kd) appeared to occur in pairs characterizing each of the RNA polymerase I, II, and III enzymes; these three sets of "couplets" had molecular weights of 197 and 126 kd, 220 and 145 kd, and 155 and 138 kd, corresponding to the two proteins with the highest molecular weights in RNA polymerases I, II, and III, respectively. Therefore, we believe that group 1 sera reacted with all three RNA polymerases, group 2 sera reacted with RNA polymerases I and III, and group 3 sera reacted with RNA polymerase III only. Thus, all 57 patients had antibody to RNA polymerase III, with some also showing reactivity to RNA polymerase I or to RNA polymerases I and II.

Immunoblotting Studies

To test the hypothesis that sera from all three groups had antibody directed against RNA polymerase III, we did immunoblotting using partially purified RNA polymerase III (Figure 2). This reagent was confirmed by polyacrylamide-SDS gel fractionation to contain all 10 subunit proteins [molecular weights of 155, 138, 84, 63, 44, 34, 33, 27, 23, and 16 kd] of the enzyme. The enzyme also contained additional proteins, probably contaminants, with molecular weights of 50, 28, 25, and 24 kd. We selected four representative serum specimens, two from group 1 Figure 2, lanes 2 and 3), one from group 2 (lane 4), and one from group 3 (lane 5). All four specimens reacted with two proteins having molecular weights of 138 and 23 kd. In addition, three specimens reacted with a 63-kd protein (lanes 3, 4, and 5), and two reacted with three proteins having molecular weights of 44, 25, and 16 kd (lanes 4 and 5). These observations suggest that all four serum specimens reacted with the subunit proteins of RNA polymerase III, except for a 25-kd protein that showed slightly different electrophoretic mobility from the 27-kd subunit protein.

View larger version (134K): [in this window] [in a new window]   Figure 2. Serum antibodies from patients with SSc shown by immunoblotting to be reactive with HeLa cell RNA polymerase III. Each lane contained partially purified RNA polymerase III obtained from 5 x 109 HeLa cells by biochemical procedures. The substrate was fractionated on a 11% polyacrylamide-SDS gel and electrophoretically transferred to nitrocellulose sheets. The sheets were incubated with normal serum (lane 1) and four representative serum specimens from groups 1, 2, and 3 (lanes 2-5); the groups are defined in Table 1. The IgG bound to proteins was visualized using enzyme-linked immunoassay. Dots beside lanes indicate the positive protein bands. The nitrocellulose strip was also stained with amido-black (lane 6). An arrow to the right of lane 6 indicates the position of each subunit protein, according to the conformity of its molecular weight, as previously described [21]. Four arrowheads to the right of lane 6 indicate the positions of four additional proteins, which are likely contaminants. The positions of molecular weight standards (in kilodaltons) are given to the right of the panel.

Immunodepletion Studies

Next, we used serum specimens from the three reactivity groups in immunodepletion studies of RNA polymerase III enzymatic activity in HeLa S100 extracts. We selected six test samples, two each from groups 1, 2, and 3 Figure 3, lanes 4, 5, 6, 7, 8, and 9) and five control specimens, including one sample from a normal human control and specimens from four patients with various types of antinuclear antibody (lanes 1, 2, 3, 10, and 12). Serum IgG was bound to protein-A sepharose and used in immunoprecipitation from a transcription extract. The transcription extract, in which the antigen had been depleted by immunoprecipitation using all six test sera, failed to yield the expected adenovirus VA I RNA product (lanes 4 through 9), whereas no inhibition was observed in the control sera. Thus, immunoprecipitation using sera from all three groups completely eliminated RNA polymerase III transcription activity from the S100 extract. Taken together with the results of the immunoprecipitation assay and immunoblotting, these data offer convincing evidence that RNA polymerase III was recognized by sera from patients with SSc.

View larger version (62K): [in this window] [in a new window]   Figure 3. Depletion of RNA polymerase III activity from HeLa S100 extracts by immunoprecipitation. Antibodies were coupled to protein-A sepharose and used in depletion experiments. The HeLa S100 extract was preincubated with IgG-coupled protein-A sepharose, obtained using normal human serum (lane 1), four control sera with various antinuclear antibodies (the specificities are marked at the top of lanes), and six representative serum specimens from the three groups [lanes 4-9]; see Table 2 for group classification). We also carried out mock immunoprecipitation (no antibodies coupled to the protein-A sepharose). After removal of the sepharose by brief centrifugation, the supernatant was subjected to the RNA polymerase III transcription reactions using adenovirus pVA I as the DNA template. The (Phosphorus-32)orthophosphate-labeled adenovirus VA I RNA products by the in vitro transcription reactions were determined by 8% polyacrylamide-urea (7 mol/L) gels and visualized by autoradiography. Anti-topo I = anti-topoisomerase I antibody I; anti-U3 RNP = anti-U3 ribonucleoprotein antibody (fibrillarin).

We examined all 57 serum specimens by immunofluorescence testing. The results in two representative specimens are shown in Figure 4. Characteristic anti-RNA polymerase I staining [16] was observed in sera from 17 patients. In all these cases, serum specimens showed both nucleolar and nuclear staining when diluted at 1:40 Figure 4, top]. Such staining was observed in 7 serum specimens from group 1, 10 from group 2, and none from group 3. However, serum specimens from the remaining 40 patients, including all patients from group 3, showed only nuclear speckled staining Figure 4, bottom). The relation between the immunoprecipitation results and the immunofluorescence staining pattern is summarized in Table 1.

View larger version (75K): [in this window] [in a new window]   Figure 4. Indirect immunofluorescence test for antinuclear antibody using HEp-2 cells as substrate. Serum specimens from patients with systemic sclerosis were diluted 1:40 with phosphate-buffered saline and used as the first antibody. Top. Immunofluorescence shows nucleolar staining, suggesting that serum contains antinuclear antibody that is apparently identical to the anti-RNA polymerase I antibody shown with antinucleolar staining by Reimer and colleagues [16]. Bottom. Immunofluorescence shows nuclear speckled staining without nucleolar staining.

Coexistence of Anti-RNA Polymerase III Antibody and Other SSc-specific Antibodies

We examined serum specimens from 95 of the 153 patients with SSc who had known antibodies to determine whether they also had anti-RNA polymerase III antibody. The 95 patients included 22 of the 50 patients with anti-topoisomerase I antibody, 22 of the 52 with anticentromere antibody, and all 51 with anti-PM-Scl, antifibrillarin, anti-Th ribonucleoprotein, anti-nuclear U sn ribonucleoprotein, or anti-La antibody. Anti-RNA polymerase III antibody was not detected in sera from any of these 95 patients by immunoprecipitation assays of (Sulfur-35)methionine-labeled cell extracts. Thus, no single patient had both anti-RNA polymerase III and either anti-topoisomerase I or anticentromere antibody. Furthermore, anti-RNA polymerase antibody was not detected in any of the sera from the 8 patients with SSc who had negative nuclear staining on immunofluorescence testing.

Clinical Features

Using the classification system outlined in the Appendix, we determined that 111 patients with SSc had diffuse cutaneous involvement, 114 had limited skin thickening, and 27 had SSc in overlap. Consistent with previous findings, statistically significant demographic differences were found among these subsets. The patients with an SSc overlap syndrome were youngest at the time of both disease onset (mean age, 31.7 years) and first visit (mean age, 39.2 years); patients with lcSSc were oldest (51.9 years) at the time of the first visit. The proportion of women was 93% in the overlap group, 82% in the dcSSc group, and 77% in the lcSSc group. The proportion of blacks was highest in the dcSSc and overlap groups.

The medications received by the three groups of patients were reviewed. Corticosteroids were administered to 70% of patients with the SSc overlap syndrome, to 52% of patients with dcSSc, and to 39% of patients with lcSSc at some time during their illness. In contrast, D-penicillamine was received by 79% of patients with dcSSc, by 33% of patients with the overlap syndrome, and by 24% of patients with lcSSc, and colchicine was received by 25% of patients with dcSSc and by fewer than 10% of patients in each of the other groups. Methotrexate was given to 25% of patients with the overlap syndrome and to fewer than 10% of patients in each of the other groups; azathioprine was given to 15% of patients with the overlap syndrome and to fewer than 5% of patients in each of the other groups; and cyclophosphamide was given to fewer than 5% of patients in all groups.

Anti-RNA polymerase III antibody was present in 57 (23%) of the 252 patients with SSc and in none of the 170 controls (P < 0.0001 by Fisher exact test). Thus, anti-RNA polymerase III antibody is highly specific to SSc.

When the 252 patients with SSc were analyzed according to disease classification, anti-RNA polymerase III antibody was detected in 50 of the 111 (45%) patients with dcSSc, 7 of the 114 (6%) patients with lcSSc, and none of the 27 patients with SSc in overlap. The frequency of anti-RNA polymerase III antibody in patients with dcSSc differed significantly from that in the other subsets (P < 0.001).

Because of the strong association between anti-RNA polymerase III antibody and dcSSc, we limited our comparison of clinical features to patients with dcSSc. We compared the 50 patients with dcSSc who had anti-RNA polymerase III antibody and the 30 patients with dcSSc who had anti-topoisomerase I antibody (Table 2). Age at disease onset, age at first diagnosis by a physician, disease duration at first visit, race, sex, and medication use were not significantly different in these two groups. However, these patient groups did differ regarding certain clinical features. (Comparison of organ system involvement and clinical features was done using the definitions in the Appendix.) As shown in Table 2, patients with anti-RNA polymerase III less frequently had telangiectasias and had significantly more extensive skin involvement, as judged by the total skin thickness score (mean maximum score, 38.4 compared with 30.3, P < 0.022).

Skeletal muscle involvement, defined by the presence of inflammatory myopathy, was absent in patients with anti-RNA polymerase III (odds ratio, 0.00; 95% CI, 0.00 to 0.86). Eleven patients (22%) with anti-RNA polymerase antibody had mild proximal weakness, but none had elevated serum creatine kinase levels or other objective evidence of myopathy. Corticosteroids were infrequently administered in these patients, but if so, they were given in low doses. Thus, it is unlikely that inadvertently treated polymyositis was missed. A lower proportion of patients with anti-RNA polymerase III antibody had a forced vital capacity less than 60% of predicted normal when compared with patients with anti-topoisomerase I antibody (7% and 33%, respectively; odds ratio = 0.15; 95% CI, 0.02 to 0.95). Patients who had anti-RNA polymerase III antibody had an increased frequency of renal crisis compared with patients who had anti-topoisomerase I (24% and 10%, respectively; P > 0.05).

The mean maximum Westergren erythrocyte sedimentation rate was significantly lower in patients with anti-RNA polymerase III antibody than in those with anti-topoisomerase I (26.8 mm/h compared with 41.0 mm/h, P = 0.01). No differences (P > 0.2) were observed in the frequency of anemia (hematocrit < 0.36), leukopenia (leukocyte count <4 x 10⁹/L), or elevated serum gammaglobulin levels in the two patient subsets. The most abnormal values recorded during the course of illness and considered attributable to SSc were used for these calculations.

Because our patients were first evaluated during the period 1986 to 1988, the maximum available follow-up time was 4 to 6 years, limiting comparisons of survival. The 5-year cumulative survival rate from the time of our initial evaluation was 90% for patients with anti-RNA polymerase III antibody and 74% for patients with anti-topoisomerase I antibody (P > 0.2). Thus, in our short-term follow-up, the presence of anti-RNA polymerase III antibody did not correlate significantly with improved survival.

It is difficult to determine the clinical significance of anti-RNA polymerase I or II antibody because we found that they always occurred in conjunction with anti-RNA polymerase III antibody. No demographic differences and no statistically significant clinical differences were observed among the three reactivity groups. Small numbers of patients may be an important limiting aspect of these comparisons.

Discussion

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We have described a novel serum autoantibody directed against RNA polymerase III. Identification of this antibody was based on several lines of evidence: 1) Serum specimens from certain patients with SSc precipitated proteins with molecular weights similar to those of RNA polymerase III subunit proteins; 2) these sera reacted with several subunit proteins of the partially purified enzyme; and 3) they depleted the enzymatic activity of RNA polymerase III from transcription extracts by immunoprecipitation.

Analysis of eukaryotic RNA polymerases purified from various organisms has revealed structural complexity [22, 23]. The three classes of RNA polymerase are each multisubunit enzymes composed of two large distinct proteins with molecular weights of more than 100 kd and a collection of several smaller proteins shared by either all three or two classes of the enzymes. In a previous report, Reimer and colleagues [16] described the identification of anti-RNA polymerase I antibody in sera from patients with SSc [16]. One finding supporting this conclusion was that patient serum and specific rabbit antisera, obtained by immunization with purified RNA polymerase I, shared identity with 13 precipitated proteins with molecular weights ranging from 210 to 14 kd. Therefore, it was suggested that all 13 proteins were subunit constituents of this enzyme. Our anti-RNA polymerase I reference serum, as well as serum specimens from 13 patients with SSc, precipitated 12 of the 13 proteins but not a 27-kd protein.

However, we have concluded that these 13 proteins contain subunits of RNA polymerases II and III as well as those of RNA polymerase I. First, we have identified anti-RNA polymerase III antibody as noted above. Second, we have identified anti-RNA polymerase II antibody in immunoabsorption experiments [24] using a specific monoclonal antibody (8WG16) to the carboxyl terminal domain with heptapeptide repeats of the 220-kd protein of RNA polymerase II [25]. Finally, the rabbit antisera used by Reimer and colleagues [15] were induced by immunization with a whole purified molecule of RNA polymerase I. Because this enzyme molecule contains the shared low molecular weight proteins, the rabbit antisera may also contain antibody reactive with RNA polymerase II and III molecules.

In addition to the 13 patients whose sera precipitated the 12 proteins, two other groups of patients were identified whose sera precipitated some of the 12 proteins but not 2 (group 2) or 3 (group 3) of the 6 proteins with the highest molecular weights (see Table 1). We suspected that sera from group 2 would react with RNA polymerases I and III and those from group 3 would react with only RNA polymerase III, based on their reactivity with the large specific proteins of the enzymes in immunoprecipitation. Thus, all three groups probably had antibody to RNA polymerase III, but group 1 also had antibody to RNA polymerases I and II and group 2 had antibody to RNA polymerase I.

In immunoblotting studies using partially purified RNA polymerase III, four representative serum specimens from the three groups showed reactivity to two or more subunit proteins of the enzyme. This result was, in part, unexpected because serum specimens from groups 2 and 3 showed reactivity with the 23-kd protein (see Figure 2). The latter protein is likely to be shared in all three enzyme classes and has been shown to be antigenically cross-reactive with subunits of similar molecular weight in RNA polymerases I and II [22, 23]. However, serum specimens from these two groups appeared to react with only RNA polymerases I and III or RNA polymerase III alone, respectively, in immunoprecipitation. The reason for this discrepancy is unclear, but it is possible that some antigenic determinants on a 23-kd protein of RNA polymerase III enzyme are unique or that only a 23-kd protein of RNA polymerases I and III, or of RNA polymerase III alone, was accessible to the sera from groups 2 or 3, respectively, in their native condition. Further investigation will focus on characterization of the antigenic determinants on subunit proteins of RNA polymerases I, II, and III.

The observed immunofluorescence staining patterns, especially the presence of nucleolar staining, were not consistent with the immunoprecipitation results (see Table 1). RNA polymerase I resides in nucleoli, whereas RNA polymerases II and III are located in nuclei [22]. Although serum specimens from groups 1 and 2 appeared to react with RNA polymerase I in immunoprecipitation studies, not all specimens in these two groups produced the characteristic anti-RNA polymerase I nucleolar staining in immunofluorescence testing. It is possible that antigenic determinants in the nucleolus either are present in insufficient copy number or are altered by cell fixation techniques. No serum specimens from group 3 showed nucleolar staining, supporting the observation that these sera did not precipitate RNA polymerase I.

Anti-RNA polymerase III antibody was found in 57 of 252 (22%) patients with SSc and was highly specific to SSc, similar to the previously described anti-RNA polymerase I antibody [7, 15]. The frequency of this autoantibody is equal to that of anti-topoisomerase I antibody or anticentromere antibody among all patients with SSc, thus establishing anti-RNA polymerase III antibody as an important marker serum antibody for SSc.

After we first reported our initial observations on anti-RNA polymerase III antibodies [26], Kuwana and colleagues [27] confirmed the findings. They identified autoantibodies that precipitated either 14 or 12 proteins with molecular weights similar to those of RNA polymerases I, II, and III and that inhibited all three enzymes in vitro. These two types of antibody appear to correspond to those detected in our groups 1 and 2 because of the similar reactivities to the 6 largest proteins identified in immunoprecipitation studies. Kuwana and colleagues concluded that all three enzymes, including RNA polymerase II, were targets for the sera that precipitated the 12 proteins, based on the inhibition assay for in vitro transcription alone. We do not believe this conclusion was warranted because these sera did not immunoprecipitate two proteins with 220/200 kd and 140 kd, which correspond to the two largest subunits of RNA polymerase II. Furthermore, their immunoblots did not show substrate purity for the enzymes or the protein bands in SDS-polyacrylamide electrophoresis.

In the study by Kuwana and colleagues [27], the antibodies were highly specific for SSc, particularly the diffuse cutaneous variant, but occurred in a much lower proportion of Japanese patients with dcSSc (18%) than did anti-RNA polymerase antibodies. Those having the antibody were significantly more often male, were older at disease onset, had higher frequencies of renal and cardiac involvement, had lower frequencies of peripheral vascular disease and pulmonary involvement, and had a reduced 5-year cumulative survival. Except for the frequency of cardiac involvement, termed "serious" by our definition in the U.S. patients, these associations of autoantibody and clinical features are strikingly similar.

The discovery of anti-RNA polymerase III antibody fills a major gap in the identification of specific serum autoantibodies in SSc. With this addition, our laboratory is able to identify the antigen against which serum antinuclear antibodies are directed in more than 85% of U.S. patients with SSc. Almost all of these serum antibodies, including the newly identified anti-RNA polymerase III, are mutually exclusive and never occur in the same serum specimen [4, 11, 16]. These results should be confirmed by other laboratories and in a study including a similar broad-based population of patients with SSc and controls with other closely related connective tissue diseases.

Appendix

Subset Classification

1. Systemic sclerosis with diffuse cutaneous involvement: skin thickness of the extremities both distal and proximal to the elbows or knees or both; may include trunk

2. Systemic sclerosis with limited cutaneous involvement: skin thickness of the extremities distal to, but not proximal to, the elbows or knees and not affecting the trunk; may include face

3. Systemic sclerosis overlap syndrome with either diffuse or limited cutaneous involvement plus clinical and laboratory features of another connective tissue disease (for example, systemic lupus erythematosus, polymyositis-dermatomyositis, or rheumatoid arthritis)

Definitions of Organ System Involvement

1. Blood vessel: a) Raynaud phenomenon [episodic cold-or emotion-induced blanching or cyanosis of the digital tips, or both]; b) evidence of digital tip ischemia (digital pitting scars, digital tip ulcerations, or gangrene)

2. Skin: a) cutaneous thickening in any location, measured using a semiquantitative physical examination measure of the extent and severity of skin thickening [total skin score]; b) the presence of matt-like telangiectasias in any location; c) palpable or radiographic calcinosis

3. Joint: a) inflammatory joint pain (arthralgias) or swelling [arthritis]; b) digital contractures

4. Tendon: one or more palpable tendon friction rubs

5. Skeletal muscle: inflammatory myopathy, with proximal muscle weakness on physical examination plus one or more of the following: elevated serum creatine kinase level; abnormal electromyogram consistent with myopathy; or muscle biopsy showing myofibril necrosis or interstitial inflammation

6. Gastrointestinal tract: a) esophagus: distal esophageal hypomotility on cine-esophagram or manometry; b) small intestine: hypomotility or dilatation of one or more parts of the small intestine [duodenum, jejunum, ileum] on small-bowel radiographic series; c) colon wide-mouthed sacculations on barium enema

7. Lung: a) pulmonary interstitial fibrosis: bibasilar fibrosis on chest radiograph; b) abnormal pulmonary function: forced vital capacity less than 60% of predicted normal or DLco [diffusing capacity of the lung for carbon monoxide] less than 60% of predicted normal on pulmonary function testing; c) pulmonary arterial hypertension: clinical evidence or proved at right heart catheterization

8. Heart: a) serious symptomatic cardiac problems including clinical evidence of left ventricular congestive heart failure; conduction defect or arrhythmia that is symptomatic and requires medical therapy or pacemaker insertion; or clinical evidence of pericarditis, that is, pericardial pain and one of the following findings: pericardial rub on physical examination, electrocardiographic evidence of pericarditis, echocardiographic detection of pericardial effusion, or clinical evidence of pericardial tamponade; b) other cardiac involvement: any objective abnormalities on electrocardiogram, echocardiogram, multiple gated acquisition scan, or Holter monitoring considered secondary to SSc

9. Kidney (renal crisis): acute or subacute development of renal insufficiency often, but not always, associated with accelerated arterial hypertension or microangiopathic hemolytic anemia, or both.

Abbreviations

Ssc = Systemic sclerosis

dcSSc = Systemic sclerosis with diffuse cutaneous involvement

lcSSc = Systemic sclerosis with limited cutaneous involvement