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Expression of autoantibodies to recombinant (U1) RNP-associated 70K antigen in systemic lupus erythematosus
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
54, 266-280 (19%)
Expression of Autoantibodies to Recombinant RNP-Associated 70K Antigen in Systemic Lupus Erythematosus’
(Ul)
E. WILLIAM ST. CLAIR,* CHARLES C. QUERY,? REX BENTLEY,i’ JACK D. KEEN&*9 RICHARD P. POLLSSON,* NANCY B. ALLEN,* DAVID S. CALDWELL,* JOHN R. RICE,* CHRISTINE Cox,* AND DAVID. S. PIsETsKY*‘t,$ *Department Microbiology
of Medicine, Division of Rheumatology and Immunology and fDepartment of and Immunology, Duke University Medical Center and the #Medical Research Service, Durham Veterans Hospital, Durham, North Carolina 27710
To determine the specificity of antibodies to the (Ul) ribonucleoprotein antigen in systemic lupus erythematosus (SLE), patient sera were tested for binding to a recombinant human 70K antigen. By solid-phase immunoassay, we detected anti-70K reactivity in sera from 31 of 96 patients with systemic lupus erythematosus (SLE), demonstrating that anti-70K antibodies may occur in patients with SLE as well as other clinical diagnoses. In sequential sera from 2 of these patients, we found that anti-70K binding varied dramatically over the course of disease. The changes in anti-70K antibody levels did not correlate with clinical events nor evolving antibody reactivity with the Smspecific antigens. 0 1990 Academic Press, Inc.
INTRODUCTION
Antinuclear antibodies (ANAs) serve as markers of underlying immunological disturbances and occur prominently in patients with systemic lupus erythematosus (SLE). These autoantibodies differ in their diagnostic and prognostic significance as well as their presumed role in disease pathogenesis (1, 2). ANA specificities reacting with small nuclear ribonucleoprotein (snRNP) antigens may be pathologically important because of their relationship to clinical features of disease. For example, antibodies to the (Ul)RNP antigen were initially described in association with mixed connective tissue disease (MCTD), a rheumatic syndrome characterized by high titers of anti-(Ul)RNP antibodies and a combination of clinical features similar to SLE, progressive systemic sclerosis (PSS), and polymyositis (3). Despite their striking association with MCTD, anti-(Ul)RNP antibodies are not diagnostic of this syndrome, because they also occur frequently in patients with SLE and other connective tissue diseases (4, 5). Unlike anti(Ul)RNP antibodies, anti-Sm antibodies are present almost exclusively in SLE (6) and, thus, serve as diagnostic markers of a specific disease entity. The frequent coexistence of anti-Sm and anti-(Ul)RNP antibodies is notable ’ This work has been supported in part by General Clinical Research Center Grant RROOO30-26S2 from the Division of Research Resources, National Institutes of Health, Maryland, National Institutes of Health Grant AI-23308, and the Arthritis Foundation’s Devil’s Bag Award. 266 0090-1229190 $1.50 Copyright All rights
0 1990 by Academic Press. Inc. of reproduction in any form reserved.
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because the antigens recognized by these two different autoantibody specificities are present on the same snRNP complex (7). The RNA-protein structures bound by anti-(Ul)RNP and anti-Sm antibodies consist of a set of proteins in association with a small RNA molecule (8). The RNA components are members of the uridine-rich, low molecular weight RNAs found in the nuclear fraction of eukaryotic cells (9). In immunoprecipitation experiments, anti-(Ul)RNP antibodies precipitate Ul snRNP, whereas, anti-Sm antibodies precipitate U1 as well as the less abundant U2, U4, US, and U6 snRNPs (8, 10-12). Using purified snRNP fractions as substrates on immunoblots, the three proteins unique to the Ul snRNP, 70K (M, 70,000), A (M,. 33,000), and C (M, 22,000), have been shown to carry the antigenic determinants recognized by anti-(U l)RNP antibodies (13, 14). In contrast, anti-Sm antibodies react with a different family of antigenic proteins common to the Ul, U2, U4, U5, and U6 snRNP fractions; the proteins reactive with the Sm specificity are B’ (M, 29,000), B (M, 28,000). D (M, 16,000), and sometimes E (M, 13,000) (13, 14). It has been suggested that the specificity of autoantibodies to various snRNP proteins distinguishes certain subsets of patients with connective tissue disease (7). Pettersson and colleagues reported that antibodies to the 70K protein were prominent in sera from patients with MCTD but were rarely present in sera from SLE patients (15). Other studies, however, have suggested that most anti(Ul)RNP sera react with the 70K protein, although a few sera have been described that do not bind the 70K protein but react only to the A and C proteins (14). To investigate further the relationship between anti-70K reactivity and SLE, we quantified the autoantibody response to this polypeptide component of the (Ul)RNP antigen using an enzyme-linked immunosorbent assay (ELISA) with recombinant human 70K fusion protein as antigen. In the studies reported below, we show that 31 of 96 SLE sera contain antibodies to recombinant 70K antigen. Also, in a longitudinal analysis of two SLE patients, we find that anti-70K levels varied markedly during the course of disease. The changes in anti-70K levels were not related to clinical events and were independent of evolving antibody reactivity with the Sm proteins. METHODS Patient sera. To evaluate the operational characteristics of the ELISA, 21 sera containing anti-RNP precipitins were selected without regard to diagnosis from the Clinical Immunology Laboratory, Duke University Medical Center (DUMC). For the cross-sectional analysis, additional serum samples were obtained from unselected patients with SLE and other connective tissue diseases. After informed consent, these sera were collected by venipuncture and frozen at - 20°C. This group included patients with the following diagnoses: SLE, 96; rheumatoid arthritis (RA), 29; progressive systemic sclerosis (PSS), 15; polymyositisl dermatomyositis (PM/DM). 15; and MCTD, 9. The clinical information on these patients was obtained retrospectively from medical records. Patients with SLE satisfied at least four of the 1982 revised American Rheumatism Association criteria for the diagnosis of SLE (6); patients in other diagnostic categories met standard criteria for definite or classic RA (16), PM/DM (17), and PSS (18). The
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9 patients with MCTD met proposed diagnostic MCTD as defined by Sharp (19).
criteria for definite or probable
Expression, purification, and analysis of recombinant fusion protein. Complementary DNAs (cDNAs) encoding 70K protein were isolated from a A gtl 1 recombinant cDNA library derived from human placental tissue and expressed in Escherichia coli Y1089 lysogens as a p-galactosidase fusion protein (20). The experiments presented herein utilized a fusion protein encompassing 383 amino acids located at the carboxyl end of the 70K antigen and were confirmed utilizing a fusion protein containing the full length of the 70K sequence (437 amino acid residues). An E. coli BNN103 lysogen expressing an unrelated B-galactosidase fusion protein was obtained from Barbara Hamilton and Arno Greenleaf, DUMC, and was utilized as a control antigen. The genomic DNA insert expressing the control fusion protein encodes an l&kDa polypeptide from RNA polymerase II of Drosophila melanogaster (Hamilton and Greenleaf, personal communication, 1989). The E. coli Y1089 lysogens were grown to midlog phase at 30°C in 5% Bactotryptone (Difco Laboratories, Detroit, MI), 0.1% Casamino acids (Difco), 0.085 M NaCl, 8 mM MgSO,, pH 7.5, and 100 p,g/ml ampicillin and stimulated to produce fusion protein by a temperature shift to 42°C for 30 min and by addition of isopropyl thio-D-B-galactoside (Bethesda Research Laboratories, Gaithersburg, MD) to a concentration of 6 mM. The BNN103 strain was grown in culture by the same method and stimulated to overproduce fusion protein by incubation for 30 min at 42°C. After culture at 38°C for an additional 3 hr, the bacteria were harvested by centrifugation, resuspended in 0.1 M NaCI, 16 mM MgSO,, 0.05 M Tris, pH 7.5, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.01% gelatin, and frozen at -70°C. The recombinant fusion protein was purified from cells by a previously described centrifugation method (21-23). Briefly, bacteria were lysed by two cycles of freeze-thawing and incubated for 10 min with 10 l&ml of DNase I (Sigma, St. Louis, MO). Next, the cells were incubated for 30 min with 60 pg/ml of lysozyme followed by the addition of Nonidet-P40 (Sigma) to 1% and Zwittergent 3-14 (Calbiochem, San Diego, CA) to 0.5% to solubilize the lysates. The resultant suspension was sonicated, layered on a 40% sucrose cushion, and centrifuged at 10,OOOg for 30 min. The pellet containing the insoluble fusion protein was resuspended in 10 kg/ml of DNase I, recovered again by centrifugation, dissolved in 4.5 M urea, 10 mM Tris, pH 7.5, and 1 mM PMSF, and stored at -70°C. The recombinant fusion protein preparations were analyzed on 6% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting (23). Proteins were identified on gels by staining with Coomassie blue; their antigenicity on nitrocellulose paper was determined by the peroxidase reaction product. In the Western blotting experiments, recombinant fusion protein was identified using a mouse anti-B-galactosidase monoclonal antibody (Promega, Madison, WI). The mouse anti-(Ul)RNP antibody (2.73) was a gift from Dr. Sallie 0. Hoch, Agauron Institute, LaJolla, California. The mouse antiSm monoclonal antibody (Y12) was kindly provided by Dr. Joan A. Steitz, Yale University School of Medicine, New Haven, Connecticut. SDS-PAGE and Western blotting using HeLa cell extracts. HeLa cells were
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grown in monolayer cultures with minimal essential media (GIBCO, Grand island, NY) supplemented with penicillin/streptomycin, L-glutamine, nonessential amino acids, vitamins, and 5% calf serum. Cells were pelleted by centrifugation, washed twice with phosphate-buffered saline (PBS), and resuspended in 10 n-&f Tris, pH 7.5, 140 mM NaCI, 1.5 mM MgCl,, and 0.05% Nonidet P40. Extracts of cells were prepared by three cycles of freeze-thawing followed by centrifugation to remove cellular debris. The extract from 5 x 10’ cells was applied to 10% gels: protein separation was achieved according to the method of Laemmli (24). Western blotting was performed as described by Towbin et al. (25). After the electroblotting step, nonspecific binding sites on the nitrocellulose paper were blocked with Blotto (0.01 M Tris, pH 7.5, 5% nonfat dry milk, and 0.05% Tween 20). Patient serum was diluted 1: 100 in Blotto and incubated for 2 hr with nitrocellulose strips. Antibody binding on the strips was detected using “SI-protein A (ICN, Costa Mesa, CA). ELZSA. Polystyrene 96-well microtiter plates (Dynatech, Chantilly. VA) were coated with I kg/ml of purified recombinant 70K fusion protein or with 2 kg/ml of the control fusion protein in 8 M urea, 0.01 M Tris, pH 7.5, and incubated overnight at 4°C. For standardization of the two antigens, the coating concentration of the control fusion protein was adjusted to achieve a level of reactivity with a mouse monoclonal anti-B-galactosidase antibody that was comparable to the binding measured by this monoclonal to the 70K fusion protein. Nonspecific binding sites were blocked for 1 hr with 1% bovine serum albumin in PBS (1% BSA-PBS). After washing with PBS-0.05% Tween 20, antigen-coated wells were incubated for 1 hr with sequential two-fold dilutions of patient sera in 1% BSA-PBS-0.05%J Tween 20. Following a washing step, bound immunoglobulin (Ig) was detected by incubating the wells for 45 min with peroxidase-conjugated goat anti-human IgG (y) (Bionetics Organon Teknika, Oklahoma City, OK). Subsequently. the wells were washed and incubated for 40 min with the peroxidase substrate, 1: 100 dilution of 3.3’,5,5’-tetramethylbenzidine dihydrocholoride (Bionetics Organon Teknika) and a I:3000 dilution of 30% hydrogen peroxide in 0.1 M citric acid, pH 4.0. The peroxidase reaction product was measured by determining the optical density at 380 nm (OD,,,) using a Titertek Multiskan Plus Plate Reader, (Flow Laboratories, McLean, VA). Statistical analysis. Data were managed and analyzed using the CLINFO Data Analysis System of Duke University (RR-30 General Clinical Research Center) supported by the Division of Research Resources, National Institutes of Health. A Wilcoxon rank sum test was used to correlate anti-70K levels in SLE and MCTD sera. RESULTS
The recombinant (Ul)RNP-associated 70K antigen used in these determinations was derived by standard techniques (see Materials and Methods). The authenticity of the cDNA encoding the 70K protein was demonstrated previously (20). We have recently shown that the full-length 70K cDNA encodes a protein with a molecular weight of 52 kDa rather than 70 kDa (26). The in vitro translation product of this cDNA migrates aberrantly on gels at the same position as authentic
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70K protein. For convenience, the protein product is-referred to as 70K protein, despite the observed difference in true molecular weight. Analysis of 70K fusion protein by SDS-PAGE and Western blotting. After purification, the 70K fusion protein preparation was analyzed by SDS-PAGE and Western blotting. Several bands were observed when the 70K fusion protein was analyzed on 6% gels (Fig. 1). By Western blotting (Fig. 2), the 70K fusion protein did not react with normal serum (lane I), but strong reactivity was observed with a serum containing anti-RNP precipitins (lane 2). A murine monoclonal anti-p-galactosidase antibody reacted with the 70K fusion protein as expected (lane 3). In addition, an anti-(U1)RNP monoclonal antibody (2.73) bound strongly to the fusion protein (lane 4), but no reactivity with an anti-Sm monoclonal anti-
FIG. 1. SDS-PAGE analysis of purified recombinant 70K fusion protein. After purification, the 70K fusion protein preparation was analyzed on 6% polyacrylamide gels and protein separation was achieved by the method of Laemmli (22). Gels were stained with Coomassie blue.
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M.W 205Kb
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66KP
LANE
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2
3
4
5
2. Analysis of purified recombinant 70K fusion protein by Western blotting. After separation by SDS-PAGE, the recombinant 70K fusion protein was electroblotted onto nitrocelluiose and reacted with the following sera: a I:1600 dilution of a normal serum, lane 1; a 1:1600 dilution of a serum containing anti-(U1)RNP precipitin, lane 2: a 15000 dilution of a mouse anti-p-galactosidase monoclonal antibody, lane 3; a 1: 100 dilution of a mouse anti-(Ul)RNP monoclonal antibody (2.73). lane 4; and a 1:25 dilution of a mouse anti-Sm monoclonal antibody (Y 12). FIG.
body (Y 12) was observed (lane 5). Partial degradation of the 70K fusion protein was suggested by the presence of several lower molecular weight components that reacted with both the anti-(U l)RNP serum and the anti-l3-galactosidase monoclonal antibody. Analysis of the control fusion protein preparation revealed a band which migrated on gels according to its predicted molecular weight and reacted on Western blots with the murine anti-p-galactosidase monoclonal (data not shown). Several additional lower molecular weight bands were noted in both of the fusion protein preparations. As discussed previously, both of these preparations are presumed to contain small amounts of contaminating E. coli proteins (21). Quantitative ELZSA of anti-70K antibodies. In solid-phase assays, purified recombinant 70K fusion protein was adhered to microtiter wells at 1 l&ml and tested by ELISA for its reactivity with sequential twofold dilutions of patient sera ranging from 1: 100 to 1: 102,400. Initially, 2 1 anti-(U 1)RNP precipitin-positive sera were tested by ELISA to evaluate the operational characteristics of the recombinant 70K fusion protein assay. Figure 3 displays the antibody-binding curves of two anti-(Ul)RNP sera, one containing a high level and the other a low level of binding. A normal serum is included for comparison. The sera containing anti(Ul)RNP precipitins showed strong binding to the recombinant 70K fusion protein, while the normal serum demonstrated only low reactivity. Sera containing anti-R0 and anti-La precipitins, but not anti-(Ul)RNP activity, produced the same level of antibody binding as normal sera (data not shown). To quantify anti-70K antibody levels in patient sera, binding units were defined in these assays as the reciprocal of the serum dilution producing an OD,,, of 1.O absorbance units (AU). In 22 normal sera (diluted 1: 100) the mean OD 380 + standard deviation (SD) was 0.526 +- 0.256 AU; therefore, antibody binding in normal sera by our method
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ST. CLAIR ET AL. + Anti-RNPtSerum +Antl-RNP+ Serum -A- Normal Serum --C
FIG. 3. Measurement of anti-70K antibodies by ELISA. In these solid-phase assays, the purified recombinant 70K fusion protein preparation was adhered to microtiter plates and reacted with sequential two-fold dilutions of two sera containing anti-(U1)RNP precipitins or a normal serum. The amount of antibody binding is expressed as OD,,,.
of quantitation is less than 100 binding units of reactivity. The 21 anti-(UI)RNP precipitin positive sera produced anti-70K binding levels ranging from 450 to 90,000 binding units. Anti-70K levels in SLE and other connective tissue disease. To determine the prevalence of anti-70K reactivity in SLE, we tested sera from 96 patients with SLE for binding to the recombinant 70K antigen. None of these patients met diagnostic criteria for MCTD. Furthermore, myositis was noted only infrequently in this SLE group (5 of 96 patients), and no patient exhibited any clinical features of PSS. For comparison, sera from patients with RA, PSS, PM/DM, and MCTD also were evaluated. Each serum was diluted I:100 and assayed in duplicate on three separate occasions for anti-70K binding; those sera demonstrating OD,,, values greater than 1.0 were subsequently titered to quantify antibody levels. A level of approximately 100 binding units, 2 SD above the mean level for 22 normal sera, was the lower limit for a positive result. In the SLE group, anti-70K antibodies were detected by ELISA in 3 1 of 96 sera with levels ranging from 110 to 75,000 binding units (Fig. 4). Antibodies to the 70K fusion protein also were demonstrated in 1 of 29 patients with RA, 1 of 15 patients with PSS, 3 of 15 patients with PM/DM, and 8 of 9 patients with MCTD. All of the RA, PSS, and PM/DM sera with anti-70K antibodies by ELISA contained anti-RNP precipitins, whereas those without anti-70K antibodies were uniformly precipitin negative. Although the anti-70K levels of MCTD sera (mean = 27,650) were higher than those of SLE sera (mean = 9640), the difference was not statistically significant (P = 0.098) and many SLE sera contained high levels of anti-7OK antibodies. We conclude from these results that anti-70K antibodies can be found in sera from patients with SLE as well as in other connective tissue diseases. To determine whether the measured reactivity to the fusion protein in the ELISA might have resulted from binding to the p-galactosidase component of the
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FIG. 4. Anti-70K antibody levels in patients with SLE and other connective tissue diseases. Antibody binding was determined by ELBA and expressed as binding units. Those sera with antibody levels below 100 binding units were not further quantified. The values listed below the closed circles (below the solid line) refer to the total number of negative sera in that diagnostic group.
fusion protein or to contaminating E. coli proteins, 149 sera from the SLE, RA, and PSS group were tested by ELISA for binding to an 18-kDa polypeptide from RNA polymerase II of Drosophila that had been expressed in a P-galactosidase fusion construction. Like the 70K fusion protein, the control antigen was obtained by overproduction in E. co/i, purified from cell lysates by centrifugation, and solubilized in urea. By solid-phase ELISA, only low levels of reactivity were observed between these patient sera and the control fusion protein. The mean OD,,, ? 2 SD of binding was 0.102 +- 0.144 AU with a range of 0.023-0.525 AU. These data indicate that the antibodies bound by the 70K fusion protein in ELISA are directed specifically toward the antigenic sites present on the 70K structure. Comparison of quantitative ELISA results with immunodiffusion and immunoblotting. To assess the relationship in SLE between the anti-70K antibody response and reactivity with the other (Ul)RNP antigens and the Sm antigens, sera were examined by immunodiffusion and immunoblotting techniques for comparison with the results of recombinant protein assays. Anti-(U1)RNP precipitins were found in 26 of the 96 SLE sera; 17 of these 26 sera also contained anti-Sm precipitins. Anti-70K antibodies were detected by the recombinant protein assay in 22 of 26 anti-(Ul)RNP precipitin-positive sera; thus, antibodies to the 70K protein are present very frequently in SLE as a component of the anti-(Ul)RNP response. The four anti-RNP precipitin-positive sera with anti-70K levels below 100 units of reactivity were characterized by immunoblotting of HeLa cell extracts to assess reactivity with the A and C proteins. In all four instances, the immunoblotting method revealed strong binding to the A protein, while two of the sera reacted in addition with the C protein (data not shown). Contrary to the results of the recombinant protein ELISA, two of these four sera also reacted weakly on blots with a 70-kDa protein. Discrepancies between the two methods in detection of anti-70K antibodies could reflect differences in the presentation of 70K protein
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determinants on nitrocellulose paper and the display of these structures adhered to microtiter plates. Alternatively, those sera with anti-Sm as well as anti(Ul)RNP precipitins may react with a different 70-kDa protein that is bound by antibodies with anti-Sm specificity (27). These results indicate that most SLE sera with anti-(U1)RNP precipitins react with the 70K protein and not just to the A or C proteins. The clinical characteristics of the patients whose sera contained anti(U1)RNP precipitins but no anti-70K reactivity were not distinctive in this SLE population. Although relatively few patient sera with this pattern have been analyzed, our observations suggest that the absence of anti-70K binding in anti(U1)RNP positive sera does not distinguish an unusual subset of SLE patients. In nine SLE sera, slightly increased anti-7OK reactivity was detected by ELISA in the absence of anti-(Ul)RNP precipitins, presumably reflecting the greater sensitivity of the recombinant protein assay. To determine whether anti-70K antibodies could be detected in these sera by an independent method, seven were tested for anti-7OK reactivity by immunoblotting using HeLa cell extracts. Six of these seven sera bound weakly on blots to a 70-kDa protein (data not shown). The results from Western blotting support those obtained by ELISA using the recombinant 70K fusion protein as antigen. Comparison of anti-7OK levels with clinical course and the evolution of anti-Sm reactivity. Since an ELISA allows quantitative assessment of antibodies, we analyzed the variation in anti-70K antibody levels as a function of time, clinical disease activity, and the antibody response to other snRNP antigens. Sequential sera from two SLE patients were chosen for this analysis because of the availability of multiple serum samples obtained over many years of their disease course. These sera also were characterized by immunoblotting to interpret anti70K antibody levels in comparison with responses to the other snRNP autoantigens. The results shown in Figs. 5 and 6 are from a 33-year-old woman who
.ARTHRITIS MYOSITIS
ARTHRITIS VASCULITIS
PSYCHOSIS VASCULITIS
ARTHRITIS RENAL
IOOJ 1982
-1983
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1986
1987
FIG. 5. Sequential determination of anti-70K antibody levels in a patient with SLE. Anti-70K binding levels were determined by ELISA (see text for detailed description of the patient’s clinical course). The arrows mark clinical events associated with an increase in disease activity.
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FIG. 6. Analysis of sequential sera using Western blots of HeLa cell extracts. The letters in the right-hand column designate the (UI)RNP antigens (70K. A. and C proteins) and the Sm antigens (B’iB and D proteins). This analysis is from the same patient whose anti-70K levels are shown in Fig. 5.
developed SLE in September of 1981, presenting with polyarthritis, lymphadenopathy, generalized weakness, and mildly elevated muscle enzymes. During the course of her illness, she suffered from other disease manifestations including mononeuritis and retinal vasculitis in late 1983; psychosis, oral ulcers, and digital vasculitis in December of 1985; and renal disease in the latter part of 1986. Except for 2 months of her course, she received daily doses of prednisone, ranging from 1 mg/kg/day when her disease was active to 5-10 mg/day during periods of disease inactivity. By ELISA, the anti-70K antibody levels were highest at the onset of her disease, tending to decline in subsequent years. In comparison, the earliest serum analyzed by immunoblotting revealed strong bands of reactivity with 70K, A, and C proteins. As anti-(Ul)RNP binding decreased, immunoblot reactivity with the B’/B proteins, noted initially in 9/81, increased in lo/83 and was associated with antibodies to the D protein. Subsequent serum samples showed waning reactivity on blots with the 70K, A, and C proteins; serum binding to the A and C proteins eventually disappeared with persistence of anti-B/B’ antibodies and low levels of anti-70K binding. Another case exhibited a strikingly different temporal pattern of antibody binding. This patient was a 28-year-old women with SLE who presented in 1976 with nephrotic syndrome and a positive fluorescent antinuclear antibody test at a titer of 1:2560 (homogeneous pattern). In 1980 she was hospitalized with fever, weight loss, alopecia, polyarthritis, pericarditis, and worsening nephritis, leading to initiation of prednisone therapy. Periodic flares of her disease were controlled by corticosteroid therapy. In late 1987 she died of refractory congestive heart failure
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secondary to ischemic heart disease. As demonstrated in Figs. 7 and 8, her serum remained negative for anti-(Ul)RNP and anti-Sm antibodies from 12/79 until 3/84. In 3/84 she developed serum anti-(U1)RNP and anti-Sm binding, as evidenced by the reactivity on blots with B’/B and D proteins, and a weaker response to the A and C proteins. Anti-70K activity was undectable in the 3/84 serum despite the presence of antibodies to the other (Ul)RNP-specific antigens. The appearance of anti-70K antibodies in serum was delayed until the 9/84 sample after which the levels remained relatively stable up to the time of her death. In both patients, clinical events were not correlated with changes in anti-70K levels. Moreover, in these sera, the magnitude of the anti-70K response did not parallel temporal changes in reactivity with the Sm antigens. DISCUSSION
A difference among patients in their antibody response to antigenic proteins of the (Ul)RNP complex has been suggested to be clinically and pathogenically significant. Although results have been conflicting, some studies have indicated that antibodies to the 70K antigen are primarily associated with MCTD and occur infrequently in sera from patients with SLE (4, 28-31). Other results suggest that anti-70K antibodies are not specific for MCTD. In an analysis using individual snRNP polypeptides isolated by preparative SDS-gel electrophoresis, Yakeda et al. (32) found that antibodies to a 70-kDa protein could be demonstrated in patients with SLE and MCTD, but that the levels of anti-70K antibodies in patients with active MCTD were higher than those observed in association with SLE. Netter and colleagues (33) testing sera for reactivity with a molecularly cloned
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; 1980 1981 1982 1983 1984 1985 1966 1987 7. Sequential determination of anti-70K antibody levels in a patient with SLE. Anti-70K levels were determined by ELBA (see text for description of the patient’s clinical course). The demonstrate the emergence of anti-70K reactivity during the course of SLE. The arrows make events characterized by an increase in disease activity.
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FIG. 8. Analysis of sequential sera using Western blots of HeLa cell extracts. The letters in the right-hand column designate the (Ul)RNP antigens (70K. A, and C proteins) and the Sm antigens (B’IB and D proteins). This analysis is from the same patient whose anti-70K levels are shown in FIG. 7.
70K antigen, also identified anti-70K antibodies in patients with SLE who were selected from a small sample of anti-(Ul)RNP precipitin-positive individuals. Our study design differs from that of Netter et al. in that a large group of unselected SLE sera was examined. Using recombinant 70K protein as antigen, we detected anti-70K reactivity in 31 of 96 SLE sera and in 22 of 26 SLE sera containing anti-(Ul)RNP precipitins. While sera from patients with MCTD demonstrated a higher mean level of anti-70K antibodies than sera from patients with SLE, the difference was not statistically significant. Together, these results show that anti70K antibodies are frequently a component of the anti-(U1)RNP response in SLE. The specificity of the cloned 70K fusion protein for detecting antibodies to the (Ul)RNP-associated ‘IO-kDa protein has been experimentally verified. Affinitypurified antibodies to the recombinant 70K fusion protein immunoprecipitate only (Ul)snRNP particles and react on a Western blot of HeLa cell extracts with a 70-kDa protein (26). Not all antibodies to the 70K protein, however, may be bound by the cloned antigen. Furthermore, significant reactivity with the P-galactosidase moiety or contaminating E. coli proteins has not been found. An RNA polymerase II fusion protein was chosen as a control in these experiments because it contained the identical @galactosidase component and a polypeptide from D. melanogaster that was not known to be immunoreactive with sera from patients with autoimmune disease. In addition, the control fusion protein was derived by the same procedure as the 70K fusion protein, so both of these preparations would be expected to contain similar amounts of E. cofi proteins. Since patient sera demonstrated only minimal reactivity with this control fusion protein,
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we conclude that the antibodies bound by the recombinant 70K fusion protein are primarily directed to epitopes located within the region defined by the 70K protein sequence. In only 4 of 26 SLE sera containing anti-(UI)RNP precipitin were we unable to demonstrate reactivity by ELISA to the recombinant human 70K antigen. By Western blotting of HeLa cell extracts these 4 sera also failed to react with a ‘IO-kDa cellular protein. Instead, they reacted with bands migrating at the expected location of the A protein, and in two cases with the C protein. The patient with MCTD whose serum failed to react with the 70K fusion protein bound strongly on immunoblots to A, B’, and B proteins, but only weakly to a 70-kDa protein. A previous study has indicated that anti-RNP and anti-Sm antibody determinations by ELISA and immunoblotting do not strictly correlate (34), so occasional discrepancies between these two assay procedures may relate to methodological differences. To gain insights into possible mechanisms of anti-70K antibody production, we studied the evolution of this response in sequential serum samples obtained from four patients during the course of SLE. We observed in two SLE patients marked variations of anti-70K levels over time that were unrelated to clinical flares of disease. These fluctuations in anti-70K levels also were not related to variations in the response to the anti-Sm-specific proteins. In this regard, Fisher el al. (35) have described two SLE patients who shifted temporally from an anti-Sm to an anti-(U1)RNP pattern. Less dramatic shifts in anti-70K antibody binding have been noted in several of our other patients (unpublished observations) as well as other cases where immunoblotting was used to quantify anti-70K reactivity (30). We infer from these results that the antibody response to the (Ul)RNP-associated 70K antigen may vary during the course of disease and may be controlled, at least in part, by mechanisms operating independently from those governing the anti-Sm response. The finding that anti-70K responses vary over time also has implications in the design and interpretation of studies which attempt to correlate serological parameters with other features of autoimmune disease. Since antibody levels may, in fact, change significantly in certain patients during their clinical course, a serological profile obtained at any single point in time may be insufficient to investigate correlations between autoantibodies (and their levels) and the presence of specific genetic, diagnostic, or prognostic variables of disease. Accurate assessment of these responses would seem to require multiple sampling over the course of the illness, preferably with reagents such as we describe which allow quantitative measurement of antibodies to individual components of complex antigens. Further studies using molecularly cloned autoantigens should help to elucidate the dynamics of these autoantibody responses and their relationship to clinical and pathogenic events. ACKNOWLEDGMENTS The authors thank Mr. James Burch for his expert technical assistance and Mrs. Judy Gentry for typing the manuscript.
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REFERENCES 1. Pisetsky, D. S.. Systemic lupus erythematosus. Med. C/in. North Amer. 70, 337-353, 1986. 2. Smith, H. R., and Steinberg, A. D.. Autoimmunity: A perspective. Atmu. Rev. Immunol. 1, 175-210, 1983. 3. Sharp, G. C., Irvin. W. S., Tan, E. M., Gould, R. G., and Holman. H. R.. Mixed connective tissue disease: An apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Amer. J. Med. 52, 148-159. 1972. 4. Munves, E. F., and Schur, P. H., Antibodies to Sm and RNP. prognosticators of disease involvement. Arthritis Rheum. 26, 848-853. 1983. 5. Tan, E. M., Autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine. Adr. Immunol.
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6. Tan, E. M.. Cohen, A. S.. Fries, J. F.. et al.. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25, 1271-1277, 1982. 7. Hardin. J. A., and Mimori. T., Autoantibodies to ribonucleoproteins. C/in. Rheum. L)t.c. 11, 485-505, 1985. 8. Lerner, M. R., and Steitz, J. A., Antibodies to small nuclear RNAs complexed with protems are produced by patients with systemic lupus erythematosus. Proc. Narl. Acad. Sci. USA 76, 549% 5499, 1979. 9. McNeilage, L. J.. Whittingham. S., and Mackay. I. R., Autoantibodies reactive with small ribonucleoprotein antigens: A convergence of molecular biology and clinical immunology. J. C/in. Luh. Immunol. 15, I-17. 1984. IO. Lerner. M. R.. Boyle, J. A.. Hardin, J. A.. and Steitz, J. A., Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus. Science 211, 4WO2. 1981. Il. Lerner, M. R.. and Steitz. J. A., Snurps and scryps. Cell 25, 298-300. 1981. 12. Mather, L., Schopfer, K.. Wilhelm, J. A.. Nyffenegger. T.. Parisot. R. F., and DeRobertis. E. M.. Molecular characterization of ribonucleoprotein antigens bound by antinuclear antibodies. Arthritis Rheum. 25, 1278-1283. 1982. 13. Billings, P. B., and Hoch. S. O., Characterization of U small nuclear RNA-associated proteins, J. Biol. Chem. 259, 12850-12856. 1984. 14. Pettersson. I., Hinterberger. M., Mimori. T.. Gottlieb, E.. and Steitz. J. A.. The structure of mammalian small nuclear ribonucleoproteins: Identification of multiple protein components reactive with antiribonucleoprotein and anti-Sm autoantibodies. J. Biol. Chem. 259, 5907-5914. 1984. 15. Pettersson, 1.. Wang. F., Smith, E. 1.. Wigzell. H.. Hedfors, E.. Horn. J.. and Sharp, G. C ., i‘he use of immunoblotting and immunoprecipitation of (U) small nuclear ribonucleoproteins in the analysis of sera of patients with mixed connective tissue disease and systemic lupus erythematosus. Arthritis Rheum. 29, 986-996, 1986. 16. Ropes. M. W.. Bennett. G. A., Cobb, S., Jacox. R.. and Jessar, R. A.. 1958 revision of diagnostic criteria for rheumatoid arthritis. Bull. Rheum. Dis. 9, 175-176. 1958. 17. Bohan. A.. and Peter. J. B.. Polymyositis and dermatomyositis. N. Eng1. .I. Med. 292. W-347. 1975. 18. Masi, A. T., Rodnan, G. P., and Medsger, T. A., Jr.. ef (I/., Preliminary criteria for the classification of systemic sclerosis fscleroderma). Arthritis Rheum. 23, 581-591. 1980. 19. Sharp, G. C., Diagnostic criteria for classifications of MCTD. In “Excerpta Medica” tR. Kasukawa and G. Sharp, Eds.1, pp. 23-32, Elsevier, Amsterdam, 1987. 20. Query, C. C., and Keene, J. D.. A human autoimmune protein associated with U I RNA contains a region of homology that is cross-reactive with retroviral p3oP”a antigen. Cell 51, 21 l-220, 1987. 21. St. Clair, E. W., Pisetsky, D. S.. Reich, C. F., Chambers, J. C., and Keene, J. D., Quantitative immunoassay of anti-La antibodies using purified recombinant La antigen. Arrhritis Rheum. 31, 506-514, 1988. 22. Adam, S. A., Nakagawa. T., Swanson. M. S.. Woodruff, T. K., and Dreyfuss. G., mRNA polyadenylate-binding protein: Gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol. Cell. Biol. 6, 2932-2943, 1986.
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23. St. Clair, E. W., Pisetsky, D. S., Reich, C. F., and Keene, J. D., Analysis of autoantibody binding to different regions of the human La antigen expressed in recombinant fusion proteins. .I. Immuno/. 141, 4173-4180, 1988. 24. Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685, 1970. 25. Towbin, H., Staehelin, T., and Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proc. Nat/. Acad. Sci. USA 76, 43504354, 1979. 26. Query, C. C., Bentley, R. C., and Keene, J. D., A common RNA recognition motif identified within a defined Ul RNA binding domain of the 70 K Ul snRNP protein. Cell 57, 89-101, 1989. 27. Gerke, V., and Steitz, J. A., A protein associated with small nuclear ribonucleoprotein particles recognizes the 3’ splice site of premessenger RNA. Cell 47, 973-984, 1986. 28. Habets, W. J., De Rooij, D. J., and Salden, M. H.. er al., Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease. C/in. Exp. Immunol. 54, 265-276, 1983. 29. Van Venrooij, W. J., and Habets, W. J., Detection of nuclear antibodies recognized by human autoantibodies. Stand. .I. Rheumatol. Suppl. 56, 32-41, 1985. 30. Habets, W. G.. De Rooij, D. J., Hoet, M. H., Van De Putte, L. B., and Van Venrooij, W. J., Quantitation of anti-RNP and anti-Sm antibodies in MCTD and SLE patients by immunoblotting. Clin. Exp.
Immunol.
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31. Williams, D. G., Charles, P. J., and Maini, R. N., Preparative isolation of ~67, A, B, B’ and D from nRNP/Sm antigens by reverse phase chromatography. .I. Immunol. Methods 113, 25-35, 1988. 32. Yakeda, Y., Wang, G. S., Wang, R. J., Anderson, S. K., Pettersson, I., Amaki, S., and Sharp, G. C., Enzyme-linked immunosorbent assay using isolated (U) small nuclear ribonucleoprotein polypeptides as antigens to investigate the clinical significance of autoantibodies to these polypeptides. Clin. Immunol. Immunopathol. 50, 213-220, 1989. 33. Netter, H. J., Gulder, H. H., Szostecki, C., Lakomek, H. J., and Will, H., A recombinant autoantigen derived from the human (Ul) small nuclear RNP-specific 69-kd protein. Arthritis Rheum. 31, 616-622, 1988. 34. Fronek, Z., Valle, R. R., Eaton, R. B., and Schur, P. H., The relationship between Sm and RNP antigens. C/in. Immunol. Immunopathol. 41, 91-107, 1986. 35. Fisher, D. E., Reeves, W. H., Wisniewolski, R., Lahita, R. G., and Chiorazzi, N., Temporal shifts from Sm to ribonucleoprotein reactivity in systemic lupus erythematosus. Arthritis Rheum. 28, 1348-1355, 1985. Received April 28, 1989; accepted with revision September 19, 1989
IMMUNOLOGY
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IMMUNOPATHOLOGY
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Expression of Autoantibodies to Recombinant RNP-Associated 70K Antigen in Systemic Lupus Erythematosus’
(Ul)
E. WILLIAM ST. CLAIR,* CHARLES C. QUERY,? REX BENTLEY,i’ JACK D. KEEN&*9 RICHARD P. POLLSSON,* NANCY B. ALLEN,* DAVID S. CALDWELL,* JOHN R. RICE,* CHRISTINE Cox,* AND DAVID. S. PIsETsKY*‘t,$ *Department Microbiology
of Medicine, Division of Rheumatology and Immunology and fDepartment of and Immunology, Duke University Medical Center and the #Medical Research Service, Durham Veterans Hospital, Durham, North Carolina 27710
To determine the specificity of antibodies to the (Ul) ribonucleoprotein antigen in systemic lupus erythematosus (SLE), patient sera were tested for binding to a recombinant human 70K antigen. By solid-phase immunoassay, we detected anti-70K reactivity in sera from 31 of 96 patients with systemic lupus erythematosus (SLE), demonstrating that anti-70K antibodies may occur in patients with SLE as well as other clinical diagnoses. In sequential sera from 2 of these patients, we found that anti-70K binding varied dramatically over the course of disease. The changes in anti-70K antibody levels did not correlate with clinical events nor evolving antibody reactivity with the Smspecific antigens. 0 1990 Academic Press, Inc.
INTRODUCTION
Antinuclear antibodies (ANAs) serve as markers of underlying immunological disturbances and occur prominently in patients with systemic lupus erythematosus (SLE). These autoantibodies differ in their diagnostic and prognostic significance as well as their presumed role in disease pathogenesis (1, 2). ANA specificities reacting with small nuclear ribonucleoprotein (snRNP) antigens may be pathologically important because of their relationship to clinical features of disease. For example, antibodies to the (Ul)RNP antigen were initially described in association with mixed connective tissue disease (MCTD), a rheumatic syndrome characterized by high titers of anti-(Ul)RNP antibodies and a combination of clinical features similar to SLE, progressive systemic sclerosis (PSS), and polymyositis (3). Despite their striking association with MCTD, anti-(Ul)RNP antibodies are not diagnostic of this syndrome, because they also occur frequently in patients with SLE and other connective tissue diseases (4, 5). Unlike anti(Ul)RNP antibodies, anti-Sm antibodies are present almost exclusively in SLE (6) and, thus, serve as diagnostic markers of a specific disease entity. The frequent coexistence of anti-Sm and anti-(Ul)RNP antibodies is notable ’ This work has been supported in part by General Clinical Research Center Grant RROOO30-26S2 from the Division of Research Resources, National Institutes of Health, Maryland, National Institutes of Health Grant AI-23308, and the Arthritis Foundation’s Devil’s Bag Award. 266 0090-1229190 $1.50 Copyright All rights
0 1990 by Academic Press. Inc. of reproduction in any form reserved.
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because the antigens recognized by these two different autoantibody specificities are present on the same snRNP complex (7). The RNA-protein structures bound by anti-(Ul)RNP and anti-Sm antibodies consist of a set of proteins in association with a small RNA molecule (8). The RNA components are members of the uridine-rich, low molecular weight RNAs found in the nuclear fraction of eukaryotic cells (9). In immunoprecipitation experiments, anti-(Ul)RNP antibodies precipitate Ul snRNP, whereas, anti-Sm antibodies precipitate U1 as well as the less abundant U2, U4, US, and U6 snRNPs (8, 10-12). Using purified snRNP fractions as substrates on immunoblots, the three proteins unique to the Ul snRNP, 70K (M, 70,000), A (M,. 33,000), and C (M, 22,000), have been shown to carry the antigenic determinants recognized by anti-(U l)RNP antibodies (13, 14). In contrast, anti-Sm antibodies react with a different family of antigenic proteins common to the Ul, U2, U4, U5, and U6 snRNP fractions; the proteins reactive with the Sm specificity are B’ (M, 29,000), B (M, 28,000). D (M, 16,000), and sometimes E (M, 13,000) (13, 14). It has been suggested that the specificity of autoantibodies to various snRNP proteins distinguishes certain subsets of patients with connective tissue disease (7). Pettersson and colleagues reported that antibodies to the 70K protein were prominent in sera from patients with MCTD but were rarely present in sera from SLE patients (15). Other studies, however, have suggested that most anti(Ul)RNP sera react with the 70K protein, although a few sera have been described that do not bind the 70K protein but react only to the A and C proteins (14). To investigate further the relationship between anti-70K reactivity and SLE, we quantified the autoantibody response to this polypeptide component of the (Ul)RNP antigen using an enzyme-linked immunosorbent assay (ELISA) with recombinant human 70K fusion protein as antigen. In the studies reported below, we show that 31 of 96 SLE sera contain antibodies to recombinant 70K antigen. Also, in a longitudinal analysis of two SLE patients, we find that anti-70K levels varied markedly during the course of disease. The changes in anti-70K levels were not related to clinical events and were independent of evolving antibody reactivity with the Sm proteins. METHODS Patient sera. To evaluate the operational characteristics of the ELISA, 21 sera containing anti-RNP precipitins were selected without regard to diagnosis from the Clinical Immunology Laboratory, Duke University Medical Center (DUMC). For the cross-sectional analysis, additional serum samples were obtained from unselected patients with SLE and other connective tissue diseases. After informed consent, these sera were collected by venipuncture and frozen at - 20°C. This group included patients with the following diagnoses: SLE, 96; rheumatoid arthritis (RA), 29; progressive systemic sclerosis (PSS), 15; polymyositisl dermatomyositis (PM/DM). 15; and MCTD, 9. The clinical information on these patients was obtained retrospectively from medical records. Patients with SLE satisfied at least four of the 1982 revised American Rheumatism Association criteria for the diagnosis of SLE (6); patients in other diagnostic categories met standard criteria for definite or classic RA (16), PM/DM (17), and PSS (18). The
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9 patients with MCTD met proposed diagnostic MCTD as defined by Sharp (19).
criteria for definite or probable
Expression, purification, and analysis of recombinant fusion protein. Complementary DNAs (cDNAs) encoding 70K protein were isolated from a A gtl 1 recombinant cDNA library derived from human placental tissue and expressed in Escherichia coli Y1089 lysogens as a p-galactosidase fusion protein (20). The experiments presented herein utilized a fusion protein encompassing 383 amino acids located at the carboxyl end of the 70K antigen and were confirmed utilizing a fusion protein containing the full length of the 70K sequence (437 amino acid residues). An E. coli BNN103 lysogen expressing an unrelated B-galactosidase fusion protein was obtained from Barbara Hamilton and Arno Greenleaf, DUMC, and was utilized as a control antigen. The genomic DNA insert expressing the control fusion protein encodes an l&kDa polypeptide from RNA polymerase II of Drosophila melanogaster (Hamilton and Greenleaf, personal communication, 1989). The E. coli Y1089 lysogens were grown to midlog phase at 30°C in 5% Bactotryptone (Difco Laboratories, Detroit, MI), 0.1% Casamino acids (Difco), 0.085 M NaCl, 8 mM MgSO,, pH 7.5, and 100 p,g/ml ampicillin and stimulated to produce fusion protein by a temperature shift to 42°C for 30 min and by addition of isopropyl thio-D-B-galactoside (Bethesda Research Laboratories, Gaithersburg, MD) to a concentration of 6 mM. The BNN103 strain was grown in culture by the same method and stimulated to overproduce fusion protein by incubation for 30 min at 42°C. After culture at 38°C for an additional 3 hr, the bacteria were harvested by centrifugation, resuspended in 0.1 M NaCI, 16 mM MgSO,, 0.05 M Tris, pH 7.5, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.01% gelatin, and frozen at -70°C. The recombinant fusion protein was purified from cells by a previously described centrifugation method (21-23). Briefly, bacteria were lysed by two cycles of freeze-thawing and incubated for 10 min with 10 l&ml of DNase I (Sigma, St. Louis, MO). Next, the cells were incubated for 30 min with 60 pg/ml of lysozyme followed by the addition of Nonidet-P40 (Sigma) to 1% and Zwittergent 3-14 (Calbiochem, San Diego, CA) to 0.5% to solubilize the lysates. The resultant suspension was sonicated, layered on a 40% sucrose cushion, and centrifuged at 10,OOOg for 30 min. The pellet containing the insoluble fusion protein was resuspended in 10 kg/ml of DNase I, recovered again by centrifugation, dissolved in 4.5 M urea, 10 mM Tris, pH 7.5, and 1 mM PMSF, and stored at -70°C. The recombinant fusion protein preparations were analyzed on 6% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting (23). Proteins were identified on gels by staining with Coomassie blue; their antigenicity on nitrocellulose paper was determined by the peroxidase reaction product. In the Western blotting experiments, recombinant fusion protein was identified using a mouse anti-B-galactosidase monoclonal antibody (Promega, Madison, WI). The mouse anti-(Ul)RNP antibody (2.73) was a gift from Dr. Sallie 0. Hoch, Agauron Institute, LaJolla, California. The mouse antiSm monoclonal antibody (Y12) was kindly provided by Dr. Joan A. Steitz, Yale University School of Medicine, New Haven, Connecticut. SDS-PAGE and Western blotting using HeLa cell extracts. HeLa cells were
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grown in monolayer cultures with minimal essential media (GIBCO, Grand island, NY) supplemented with penicillin/streptomycin, L-glutamine, nonessential amino acids, vitamins, and 5% calf serum. Cells were pelleted by centrifugation, washed twice with phosphate-buffered saline (PBS), and resuspended in 10 n-&f Tris, pH 7.5, 140 mM NaCI, 1.5 mM MgCl,, and 0.05% Nonidet P40. Extracts of cells were prepared by three cycles of freeze-thawing followed by centrifugation to remove cellular debris. The extract from 5 x 10’ cells was applied to 10% gels: protein separation was achieved according to the method of Laemmli (24). Western blotting was performed as described by Towbin et al. (25). After the electroblotting step, nonspecific binding sites on the nitrocellulose paper were blocked with Blotto (0.01 M Tris, pH 7.5, 5% nonfat dry milk, and 0.05% Tween 20). Patient serum was diluted 1: 100 in Blotto and incubated for 2 hr with nitrocellulose strips. Antibody binding on the strips was detected using “SI-protein A (ICN, Costa Mesa, CA). ELZSA. Polystyrene 96-well microtiter plates (Dynatech, Chantilly. VA) were coated with I kg/ml of purified recombinant 70K fusion protein or with 2 kg/ml of the control fusion protein in 8 M urea, 0.01 M Tris, pH 7.5, and incubated overnight at 4°C. For standardization of the two antigens, the coating concentration of the control fusion protein was adjusted to achieve a level of reactivity with a mouse monoclonal anti-B-galactosidase antibody that was comparable to the binding measured by this monoclonal to the 70K fusion protein. Nonspecific binding sites were blocked for 1 hr with 1% bovine serum albumin in PBS (1% BSA-PBS). After washing with PBS-0.05% Tween 20, antigen-coated wells were incubated for 1 hr with sequential two-fold dilutions of patient sera in 1% BSA-PBS-0.05%J Tween 20. Following a washing step, bound immunoglobulin (Ig) was detected by incubating the wells for 45 min with peroxidase-conjugated goat anti-human IgG (y) (Bionetics Organon Teknika, Oklahoma City, OK). Subsequently. the wells were washed and incubated for 40 min with the peroxidase substrate, 1: 100 dilution of 3.3’,5,5’-tetramethylbenzidine dihydrocholoride (Bionetics Organon Teknika) and a I:3000 dilution of 30% hydrogen peroxide in 0.1 M citric acid, pH 4.0. The peroxidase reaction product was measured by determining the optical density at 380 nm (OD,,,) using a Titertek Multiskan Plus Plate Reader, (Flow Laboratories, McLean, VA). Statistical analysis. Data were managed and analyzed using the CLINFO Data Analysis System of Duke University (RR-30 General Clinical Research Center) supported by the Division of Research Resources, National Institutes of Health. A Wilcoxon rank sum test was used to correlate anti-70K levels in SLE and MCTD sera. RESULTS
The recombinant (Ul)RNP-associated 70K antigen used in these determinations was derived by standard techniques (see Materials and Methods). The authenticity of the cDNA encoding the 70K protein was demonstrated previously (20). We have recently shown that the full-length 70K cDNA encodes a protein with a molecular weight of 52 kDa rather than 70 kDa (26). The in vitro translation product of this cDNA migrates aberrantly on gels at the same position as authentic
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70K protein. For convenience, the protein product is-referred to as 70K protein, despite the observed difference in true molecular weight. Analysis of 70K fusion protein by SDS-PAGE and Western blotting. After purification, the 70K fusion protein preparation was analyzed by SDS-PAGE and Western blotting. Several bands were observed when the 70K fusion protein was analyzed on 6% gels (Fig. 1). By Western blotting (Fig. 2), the 70K fusion protein did not react with normal serum (lane I), but strong reactivity was observed with a serum containing anti-RNP precipitins (lane 2). A murine monoclonal anti-p-galactosidase antibody reacted with the 70K fusion protein as expected (lane 3). In addition, an anti-(U1)RNP monoclonal antibody (2.73) bound strongly to the fusion protein (lane 4), but no reactivity with an anti-Sm monoclonal anti-
FIG. 1. SDS-PAGE analysis of purified recombinant 70K fusion protein. After purification, the 70K fusion protein preparation was analyzed on 6% polyacrylamide gels and protein separation was achieved by the method of Laemmli (22). Gels were stained with Coomassie blue.
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M.W 205Kb
L
66KP
LANE
I
2
3
4
5
2. Analysis of purified recombinant 70K fusion protein by Western blotting. After separation by SDS-PAGE, the recombinant 70K fusion protein was electroblotted onto nitrocelluiose and reacted with the following sera: a I:1600 dilution of a normal serum, lane 1; a 1:1600 dilution of a serum containing anti-(U1)RNP precipitin, lane 2: a 15000 dilution of a mouse anti-p-galactosidase monoclonal antibody, lane 3; a 1: 100 dilution of a mouse anti-(Ul)RNP monoclonal antibody (2.73). lane 4; and a 1:25 dilution of a mouse anti-Sm monoclonal antibody (Y 12). FIG.
body (Y 12) was observed (lane 5). Partial degradation of the 70K fusion protein was suggested by the presence of several lower molecular weight components that reacted with both the anti-(U l)RNP serum and the anti-l3-galactosidase monoclonal antibody. Analysis of the control fusion protein preparation revealed a band which migrated on gels according to its predicted molecular weight and reacted on Western blots with the murine anti-p-galactosidase monoclonal (data not shown). Several additional lower molecular weight bands were noted in both of the fusion protein preparations. As discussed previously, both of these preparations are presumed to contain small amounts of contaminating E. coli proteins (21). Quantitative ELZSA of anti-70K antibodies. In solid-phase assays, purified recombinant 70K fusion protein was adhered to microtiter wells at 1 l&ml and tested by ELISA for its reactivity with sequential twofold dilutions of patient sera ranging from 1: 100 to 1: 102,400. Initially, 2 1 anti-(U 1)RNP precipitin-positive sera were tested by ELISA to evaluate the operational characteristics of the recombinant 70K fusion protein assay. Figure 3 displays the antibody-binding curves of two anti-(Ul)RNP sera, one containing a high level and the other a low level of binding. A normal serum is included for comparison. The sera containing anti(Ul)RNP precipitins showed strong binding to the recombinant 70K fusion protein, while the normal serum demonstrated only low reactivity. Sera containing anti-R0 and anti-La precipitins, but not anti-(Ul)RNP activity, produced the same level of antibody binding as normal sera (data not shown). To quantify anti-70K antibody levels in patient sera, binding units were defined in these assays as the reciprocal of the serum dilution producing an OD,,, of 1.O absorbance units (AU). In 22 normal sera (diluted 1: 100) the mean OD 380 + standard deviation (SD) was 0.526 +- 0.256 AU; therefore, antibody binding in normal sera by our method
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FIG. 3. Measurement of anti-70K antibodies by ELISA. In these solid-phase assays, the purified recombinant 70K fusion protein preparation was adhered to microtiter plates and reacted with sequential two-fold dilutions of two sera containing anti-(U1)RNP precipitins or a normal serum. The amount of antibody binding is expressed as OD,,,.
of quantitation is less than 100 binding units of reactivity. The 21 anti-(UI)RNP precipitin positive sera produced anti-70K binding levels ranging from 450 to 90,000 binding units. Anti-70K levels in SLE and other connective tissue disease. To determine the prevalence of anti-70K reactivity in SLE, we tested sera from 96 patients with SLE for binding to the recombinant 70K antigen. None of these patients met diagnostic criteria for MCTD. Furthermore, myositis was noted only infrequently in this SLE group (5 of 96 patients), and no patient exhibited any clinical features of PSS. For comparison, sera from patients with RA, PSS, PM/DM, and MCTD also were evaluated. Each serum was diluted I:100 and assayed in duplicate on three separate occasions for anti-70K binding; those sera demonstrating OD,,, values greater than 1.0 were subsequently titered to quantify antibody levels. A level of approximately 100 binding units, 2 SD above the mean level for 22 normal sera, was the lower limit for a positive result. In the SLE group, anti-70K antibodies were detected by ELISA in 3 1 of 96 sera with levels ranging from 110 to 75,000 binding units (Fig. 4). Antibodies to the 70K fusion protein also were demonstrated in 1 of 29 patients with RA, 1 of 15 patients with PSS, 3 of 15 patients with PM/DM, and 8 of 9 patients with MCTD. All of the RA, PSS, and PM/DM sera with anti-70K antibodies by ELISA contained anti-RNP precipitins, whereas those without anti-70K antibodies were uniformly precipitin negative. Although the anti-70K levels of MCTD sera (mean = 27,650) were higher than those of SLE sera (mean = 9640), the difference was not statistically significant (P = 0.098) and many SLE sera contained high levels of anti-7OK antibodies. We conclude from these results that anti-70K antibodies can be found in sera from patients with SLE as well as in other connective tissue diseases. To determine whether the measured reactivity to the fusion protein in the ELISA might have resulted from binding to the p-galactosidase component of the
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l
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----&-.a se
65 SLE (N = 96)
.p.
l*
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v
4::. .
.
20
14
12
1
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PSS (N = 15)
PMlDM (N = 15)
MCTD (N = 9)
FIG. 4. Anti-70K antibody levels in patients with SLE and other connective tissue diseases. Antibody binding was determined by ELBA and expressed as binding units. Those sera with antibody levels below 100 binding units were not further quantified. The values listed below the closed circles (below the solid line) refer to the total number of negative sera in that diagnostic group.
fusion protein or to contaminating E. coli proteins, 149 sera from the SLE, RA, and PSS group were tested by ELISA for binding to an 18-kDa polypeptide from RNA polymerase II of Drosophila that had been expressed in a P-galactosidase fusion construction. Like the 70K fusion protein, the control antigen was obtained by overproduction in E. co/i, purified from cell lysates by centrifugation, and solubilized in urea. By solid-phase ELISA, only low levels of reactivity were observed between these patient sera and the control fusion protein. The mean OD,,, ? 2 SD of binding was 0.102 +- 0.144 AU with a range of 0.023-0.525 AU. These data indicate that the antibodies bound by the 70K fusion protein in ELISA are directed specifically toward the antigenic sites present on the 70K structure. Comparison of quantitative ELISA results with immunodiffusion and immunoblotting. To assess the relationship in SLE between the anti-70K antibody response and reactivity with the other (Ul)RNP antigens and the Sm antigens, sera were examined by immunodiffusion and immunoblotting techniques for comparison with the results of recombinant protein assays. Anti-(U1)RNP precipitins were found in 26 of the 96 SLE sera; 17 of these 26 sera also contained anti-Sm precipitins. Anti-70K antibodies were detected by the recombinant protein assay in 22 of 26 anti-(Ul)RNP precipitin-positive sera; thus, antibodies to the 70K protein are present very frequently in SLE as a component of the anti-(Ul)RNP response. The four anti-RNP precipitin-positive sera with anti-70K levels below 100 units of reactivity were characterized by immunoblotting of HeLa cell extracts to assess reactivity with the A and C proteins. In all four instances, the immunoblotting method revealed strong binding to the A protein, while two of the sera reacted in addition with the C protein (data not shown). Contrary to the results of the recombinant protein ELISA, two of these four sera also reacted weakly on blots with a 70-kDa protein. Discrepancies between the two methods in detection of anti-70K antibodies could reflect differences in the presentation of 70K protein
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determinants on nitrocellulose paper and the display of these structures adhered to microtiter plates. Alternatively, those sera with anti-Sm as well as anti(Ul)RNP precipitins may react with a different 70-kDa protein that is bound by antibodies with anti-Sm specificity (27). These results indicate that most SLE sera with anti-(U1)RNP precipitins react with the 70K protein and not just to the A or C proteins. The clinical characteristics of the patients whose sera contained anti(U1)RNP precipitins but no anti-70K reactivity were not distinctive in this SLE population. Although relatively few patient sera with this pattern have been analyzed, our observations suggest that the absence of anti-70K binding in anti(U1)RNP positive sera does not distinguish an unusual subset of SLE patients. In nine SLE sera, slightly increased anti-7OK reactivity was detected by ELISA in the absence of anti-(Ul)RNP precipitins, presumably reflecting the greater sensitivity of the recombinant protein assay. To determine whether anti-70K antibodies could be detected in these sera by an independent method, seven were tested for anti-7OK reactivity by immunoblotting using HeLa cell extracts. Six of these seven sera bound weakly on blots to a 70-kDa protein (data not shown). The results from Western blotting support those obtained by ELISA using the recombinant 70K fusion protein as antigen. Comparison of anti-7OK levels with clinical course and the evolution of anti-Sm reactivity. Since an ELISA allows quantitative assessment of antibodies, we analyzed the variation in anti-70K antibody levels as a function of time, clinical disease activity, and the antibody response to other snRNP antigens. Sequential sera from two SLE patients were chosen for this analysis because of the availability of multiple serum samples obtained over many years of their disease course. These sera also were characterized by immunoblotting to interpret anti70K antibody levels in comparison with responses to the other snRNP autoantigens. The results shown in Figs. 5 and 6 are from a 33-year-old woman who
.ARTHRITIS MYOSITIS
ARTHRITIS VASCULITIS
PSYCHOSIS VASCULITIS
ARTHRITIS RENAL
IOOJ 1982
-1983
I984
1985
1986
1987
FIG. 5. Sequential determination of anti-70K antibody levels in a patient with SLE. Anti-70K binding levels were determined by ELISA (see text for detailed description of the patient’s clinical course). The arrows mark clinical events associated with an increase in disease activity.
ANTIBODIES
TO
97K
RECOMBINANT
70K
ANTIGEN
IN
SLt
275
*
70K 55K* 43K* 36K* 29Kw
A e'/e
FIG. 6. Analysis of sequential sera using Western blots of HeLa cell extracts. The letters in the right-hand column designate the (UI)RNP antigens (70K. A. and C proteins) and the Sm antigens (B’iB and D proteins). This analysis is from the same patient whose anti-70K levels are shown in Fig. 5.
developed SLE in September of 1981, presenting with polyarthritis, lymphadenopathy, generalized weakness, and mildly elevated muscle enzymes. During the course of her illness, she suffered from other disease manifestations including mononeuritis and retinal vasculitis in late 1983; psychosis, oral ulcers, and digital vasculitis in December of 1985; and renal disease in the latter part of 1986. Except for 2 months of her course, she received daily doses of prednisone, ranging from 1 mg/kg/day when her disease was active to 5-10 mg/day during periods of disease inactivity. By ELISA, the anti-70K antibody levels were highest at the onset of her disease, tending to decline in subsequent years. In comparison, the earliest serum analyzed by immunoblotting revealed strong bands of reactivity with 70K, A, and C proteins. As anti-(Ul)RNP binding decreased, immunoblot reactivity with the B’/B proteins, noted initially in 9/81, increased in lo/83 and was associated with antibodies to the D protein. Subsequent serum samples showed waning reactivity on blots with the 70K, A, and C proteins; serum binding to the A and C proteins eventually disappeared with persistence of anti-B/B’ antibodies and low levels of anti-70K binding. Another case exhibited a strikingly different temporal pattern of antibody binding. This patient was a 28-year-old women with SLE who presented in 1976 with nephrotic syndrome and a positive fluorescent antinuclear antibody test at a titer of 1:2560 (homogeneous pattern). In 1980 she was hospitalized with fever, weight loss, alopecia, polyarthritis, pericarditis, and worsening nephritis, leading to initiation of prednisone therapy. Periodic flares of her disease were controlled by corticosteroid therapy. In late 1987 she died of refractory congestive heart failure
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secondary to ischemic heart disease. As demonstrated in Figs. 7 and 8, her serum remained negative for anti-(Ul)RNP and anti-Sm antibodies from 12/79 until 3/84. In 3/84 she developed serum anti-(U1)RNP and anti-Sm binding, as evidenced by the reactivity on blots with B’/B and D proteins, and a weaker response to the A and C proteins. Anti-70K activity was undectable in the 3/84 serum despite the presence of antibodies to the other (Ul)RNP-specific antigens. The appearance of anti-70K antibodies in serum was delayed until the 9/84 sample after which the levels remained relatively stable up to the time of her death. In both patients, clinical events were not correlated with changes in anti-70K levels. Moreover, in these sera, the magnitude of the anti-70K response did not parallel temporal changes in reactivity with the Sm antigens. DISCUSSION
A difference among patients in their antibody response to antigenic proteins of the (Ul)RNP complex has been suggested to be clinically and pathogenically significant. Although results have been conflicting, some studies have indicated that antibodies to the 70K antigen are primarily associated with MCTD and occur infrequently in sera from patients with SLE (4, 28-31). Other results suggest that anti-70K antibodies are not specific for MCTD. In an analysis using individual snRNP polypeptides isolated by preparative SDS-gel electrophoresis, Yakeda et al. (32) found that antibodies to a 70-kDa protein could be demonstrated in patients with SLE and MCTD, but that the levels of anti-70K antibodies in patients with active MCTD were higher than those observed in association with SLE. Netter and colleagues (33) testing sera for reactivity with a molecularly cloned
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FIG. binding results clinical
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RASH ORAL ULCERS, PLEURltlS,ARTHRALGIAS, RENAL
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; 1980 1981 1982 1983 1984 1985 1966 1987 7. Sequential determination of anti-70K antibody levels in a patient with SLE. Anti-70K levels were determined by ELBA (see text for description of the patient’s clinical course). The demonstrate the emergence of anti-70K reactivity during the course of SLE. The arrows make events characterized by an increase in disease activity.
ANTIBODlES
TO RECOMBINANT
70K ANTIGEN
IN
S1.E
777
MW 97K *
70K
43K t
2.
36K 29K w
A B'/El
/ '-
C
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FIG. 8. Analysis of sequential sera using Western blots of HeLa cell extracts. The letters in the right-hand column designate the (Ul)RNP antigens (70K. A, and C proteins) and the Sm antigens (B’IB and D proteins). This analysis is from the same patient whose anti-70K levels are shown in FIG. 7.
70K antigen, also identified anti-70K antibodies in patients with SLE who were selected from a small sample of anti-(Ul)RNP precipitin-positive individuals. Our study design differs from that of Netter et al. in that a large group of unselected SLE sera was examined. Using recombinant 70K protein as antigen, we detected anti-70K reactivity in 31 of 96 SLE sera and in 22 of 26 SLE sera containing anti-(Ul)RNP precipitins. While sera from patients with MCTD demonstrated a higher mean level of anti-70K antibodies than sera from patients with SLE, the difference was not statistically significant. Together, these results show that anti70K antibodies are frequently a component of the anti-(U1)RNP response in SLE. The specificity of the cloned 70K fusion protein for detecting antibodies to the (Ul)RNP-associated ‘IO-kDa protein has been experimentally verified. Affinitypurified antibodies to the recombinant 70K fusion protein immunoprecipitate only (Ul)snRNP particles and react on a Western blot of HeLa cell extracts with a 70-kDa protein (26). Not all antibodies to the 70K protein, however, may be bound by the cloned antigen. Furthermore, significant reactivity with the P-galactosidase moiety or contaminating E. coli proteins has not been found. An RNA polymerase II fusion protein was chosen as a control in these experiments because it contained the identical @galactosidase component and a polypeptide from D. melanogaster that was not known to be immunoreactive with sera from patients with autoimmune disease. In addition, the control fusion protein was derived by the same procedure as the 70K fusion protein, so both of these preparations would be expected to contain similar amounts of E. cofi proteins. Since patient sera demonstrated only minimal reactivity with this control fusion protein,
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we conclude that the antibodies bound by the recombinant 70K fusion protein are primarily directed to epitopes located within the region defined by the 70K protein sequence. In only 4 of 26 SLE sera containing anti-(UI)RNP precipitin were we unable to demonstrate reactivity by ELISA to the recombinant human 70K antigen. By Western blotting of HeLa cell extracts these 4 sera also failed to react with a ‘IO-kDa cellular protein. Instead, they reacted with bands migrating at the expected location of the A protein, and in two cases with the C protein. The patient with MCTD whose serum failed to react with the 70K fusion protein bound strongly on immunoblots to A, B’, and B proteins, but only weakly to a 70-kDa protein. A previous study has indicated that anti-RNP and anti-Sm antibody determinations by ELISA and immunoblotting do not strictly correlate (34), so occasional discrepancies between these two assay procedures may relate to methodological differences. To gain insights into possible mechanisms of anti-70K antibody production, we studied the evolution of this response in sequential serum samples obtained from four patients during the course of SLE. We observed in two SLE patients marked variations of anti-70K levels over time that were unrelated to clinical flares of disease. These fluctuations in anti-70K levels also were not related to variations in the response to the anti-Sm-specific proteins. In this regard, Fisher el al. (35) have described two SLE patients who shifted temporally from an anti-Sm to an anti-(U1)RNP pattern. Less dramatic shifts in anti-70K antibody binding have been noted in several of our other patients (unpublished observations) as well as other cases where immunoblotting was used to quantify anti-70K reactivity (30). We infer from these results that the antibody response to the (Ul)RNP-associated 70K antigen may vary during the course of disease and may be controlled, at least in part, by mechanisms operating independently from those governing the anti-Sm response. The finding that anti-70K responses vary over time also has implications in the design and interpretation of studies which attempt to correlate serological parameters with other features of autoimmune disease. Since antibody levels may, in fact, change significantly in certain patients during their clinical course, a serological profile obtained at any single point in time may be insufficient to investigate correlations between autoantibodies (and their levels) and the presence of specific genetic, diagnostic, or prognostic variables of disease. Accurate assessment of these responses would seem to require multiple sampling over the course of the illness, preferably with reagents such as we describe which allow quantitative measurement of antibodies to individual components of complex antigens. Further studies using molecularly cloned autoantigens should help to elucidate the dynamics of these autoantibody responses and their relationship to clinical and pathogenic events. ACKNOWLEDGMENTS The authors thank Mr. James Burch for his expert technical assistance and Mrs. Judy Gentry for typing the manuscript.
ANTIBODIES
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RECOMBINANT
70K
ANTIGEN
IN
SLE
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