1016

ANTIBODIES IN RHEUMATOID SYNOVIAL FLUIDS BIND TO A RESTRICTED SERIES O F PROTEIN ANTIGENS IN RHEUMATOID SYNOVIAL TISSUE ROBERT LAFYATIS, RENE M. FLIPO, BERNARD DUQUESNOY, and ANDRE CAPRON Objective. By searching the synovial fluid of patients with rheumatoid arthritis (RA) for antibodies that react to protein antigens in synovial tissue, we sought to identify putative antigens present in RA synovial tissue that might drive the pathologic immune response believed to be responsible for the joint inflammation. Methods. Synovial tissue was homogenized in sodium dodecyl sulfate polyacrylamide gel buffer, electrophoresed, and analyzed by immunoblotting. Results. Antibodies from synovial fluids of patients with RA bound to several proteins in rheumatoid synovial tissues, including a series of low (27.5-,29-, and 30-kd), middle (43- and 53-kd), and high (140-, la-, and 182-kd) molecular weight proteins. Most of these antigens were also detected in normal synovial tissue, and the high molecular weight proteins were also present in normal dermal, muscle, and liver tissues. The low and middle molecular weight proteins were detected in some, but not all, of the other normal tissues and in Jurkat cell lysates. Antibodies to the low and high series of proteins were present in all rheumatoid synovial From the Centre d’lmmunologie et de Biologie Parasitaire, Unite Mixte INSERM U167-CNRS U624, Institut Pasteur. and the Centre Hospitalier et Universitaire de Lille, Hbpital de la Charite, Lille, France. Robert Lafyatis, MD: Centre d’lmmunologie et de Biologie Parasitaire, Unite Mixte INSERM U 167-CNRS U624, lnstitut Pasteur; Rene M. Flipo, MD: Centre Hospitalier et Universitaire de Lille, Hbpital de la Charite; Bernard Duquesnoy, MD: Centre Hospitalier et Universitaire de Lille, Hbpital de la Charite; Andre Capron, MD: Centre d’lmmunologie et de Biologie Parasitaire, Unite Mixte INSERM U 167-CNRS U624, lnstitut Pasteur. Address reprint requests to Robert Lafyatis, MD, Centre d’lmmunologie et de Biologie Parasitaire, Institut Pasteur, BP 245, I , rue du P. Calmette, 59019 Lille Cedex, France. Submitted for publication October 3, 1991; accepted in revised form March 10. 1992. Arthritis and Rheumatism, Vol. 35, No. 9 (September 1992)

fluids tested, but were generally absent from synovial fluids from patients with other arthritic diseases. Conclusion. These results show that antibodies in synovial fluids consistently react to several proteins in RA and normal synovial tissues. These antigens are possibly the same antigens provoking the T cell response in RA; therefore, understanding the mechanism of the immune response against these proteins will likely lead to important insight into the etiology of RA.

One of the major impediments to understanding the pathophysiology of rheumatoid arthritis (RA) is that the primary stimulus of inflammation has not been identified. Several lines of evidence strongly suggest that T cell activation is a critical target of this stimulus; this evidence includes the prevalence of T cells in the pannus, the association of RA with certain major histocompatibility complex (MHC) class I1 isotypes ( I ) , and the dramatic clinical response to immunosuppressive agents including cyclosporin A (2,3), which appears to act through inhibition of T cell activation. These and several other observations, including the similarities between RA and chronic Lyme arthritis (4) and several animal models of antigen-induced and infectious arthritis, suggest that an unidentified exogenous or endogenous antigen(s) is responsible for stimulating T cell activation (5-9). In some of these animal models, antigen-stimulated T cells and antigenspecific T cell clones from animals with arthritis are able to induce a similar illness in naive animals (9,lO). Additionally, the presence of somatic mutations in at least some rheumatoid factors (RFs) from patients with RA suggests antigen stimulation (1 1); perhaps stimulation of somatic mutation in R F + B cells occurs after antigen-antibody complexes, due to an unidenti-

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ANTIGENS IN RA

fied antigen, stimulate T cell-dependent B cell activation through surface (RF) immunoglobulin (12). The ability to stimulate R F production through this mechanism has been recently demonstrated in vitro (13). Several series of studies have suggested possible antigen(s) responsible for initiating the immune response in RA. Immunization of rats with native type I1 collagen induces arthritis (9), and several studies have demonstrated both an antibody and cellular reactivity to type I1 collagen in patients with RA (14-17). However, antibodies to type I1 collagen have also been found in patients with other forms of arthritis (IS), and the development of these antibodies in RA apparently follows the onset of the arthritis (18). This suggests that a reaction against this antigen is not the primary immune stimulus in RA, but rather a secondary response, which perhaps contributes significantly to cartilage degradation (19). Elevated levels of RA-associated nuclear antigen (RANA), an antigen associated with Epstein-Barr virus (EBVrinfected cells, have also been described in patients with RA (20,21). Several groups have studied the potential association of elevated titers of antibodies to various EBV antigens, RANA, and rheumatoid arthritis; antibodies to both RANA and EpsteinBan- nuclear antigen-l (EBNA-I) appear in patients early after EBV infection and in patients with RA (22-24). The presence of these antibodies in patients with RA is possibly due to the increased numbers of circulating B cells infected with EBV, which is apparently due to a T cell defect (25). The finding that EBV antigens are not elevated early in the course of RA suggested that EBV did not directly cause RA (26); however, other studies showing that an antigen which cross-reacts with antibodies to EBNA-I is present in the synovial tissues of patients with RA, and that there is a shared epitope between the HLA-DR4 region associated with RA and the EBV glycoprotein gpl10, suggest alternative roles for EBV in the pathogenesis of RA (27,28). Recently, heat-shock proteins have received attention as possible antigens in RA, following the demonstration that arthritogenic T lymphocyte clones from rats with adjuvant arthritis react to the 65-kd heat-shock protein of Mycobacterium bovis and that pretreatment with this protein prevents subsequent attempts to induce both adjuvant and streptococcal cell wall arthritis in rats (29,30). Further studies have shown that antibodies to this protein are present in patients with RA, but are also present in patients with other forms of arthritis (31). Synovial fluid T cell

reactivity to this antigen has also been reported (31,32); however, the importance of this or a crossreactive antigen in RA remains controversial (33,34). Finally, the presence of RFs implies that immunoglobulin might be stimulating the primary immune response in RA. Several observations, however, suggest that production of RFs is a secondary response: They are present in many illnesses characterized by prolonged immune system stimulation; they are produced in response to aggregated IgG, immune complexes (see above), and other stimuli of polyclonal B cell activation; and they appear to have a physiologic role, enhancing antibody affinity through cross-linking (for review, see refs. 12 and 35). Although each of these antigens may play a role in the pathophysiology of RA, none has been convincingly shown to be directly responsible for initiating the immune response. The localized nature of the immune response in RA indicates that some aspect of the biology of synovial tissue provides a tropism for its initiation. One possible explanation is that synovial tissue expresses an antigen(s) which is either not expressed in other tissues, o r is more immunogenic in rheumatoid synovial tissues than elsewhere in the body. This antigen could be either exogenous (for example, come into the synovium by an infectious organism), o r endogenous (potentially provoking inflammation through mechanisms such a s crossreactivity to an infectious organism, o r result from some other defect in immune regulation). Antibodies in synovial fluid are likely to react to these putative antigens, which are possibly the same antigens that stimulate the T cell response. We have therefore searched for and characterized the presence of proteins in rheumatoid synovial tissues which are recognized by immunoglobulins in synovial fluids of patients with RA.

PATIENTS AND METHODS Patients. Synovial fluid samples were obtained from 5 patients with chronic ( > 2 years duration), erosive, definite or classic RA (36), 3 patients with osteoarthritis (OA), 2

patients with psoriatic arthritis (PA), 2 patients with calcium pyrophosphate deposition disease (CPDD), 1 patient with palindromic rheumatism (PR), and 1 patient with systemic lupus erythematosus (SLE). Synovial fluid samples were centrifuged at high speed in a microfuge and stored frozen. Synovial tissue was obtained from patients with RA at the time of elective joint replacement. Normal synovial tissue was obtained from the ankle of a patient who underwent amputation because of vascular insufficiency, and normal muscle, liver, and skin tissues were obtained from

LAFYATIS ET AL

another patient during surgery for resection of a colon tumor. Cell culture. The Jurkat cell line was cultured in RPMI and the HeLa cell line was cultured in Iscove's modified Dulbecco's medium. In both cases, the media were supplemented with 10% fetal bovine serum and antibiotics (50 unitslml penicillin, 50 pg/ml streptomycin). Cells were grown in a humidified incubator at 37°C in 5% CO,. Sample preparation for protein blotting and analysis. Tissues were prepared for electrophoresis by mincing finely, adding 5 volumes of I .2 x polyacrylamide gel electrophoresis (PAGE) sample buffer (37), and then homogenizing with an Ultra-turrax homogenizer (Janrke and Kunkle, Staufen) at top speed for 20-30 seconds. Jurkat cells were pelleted, washed with phosphate buffered saline, 5 volumes of 1 . 2 ~ PAGE sample buffer was added, and samples were vortexed for 5 minutes. HeLa cell nuclear extract was prepared from cultured HeLa cells as described by Hassfeld et al (38). and the resulting protein extract was diluted 1 : l in 2x PAGE sample buffer. After protein solubilization, all samples were centrifuged at high speed in a microfuge for 5 minutes, and the supernatant was removed for analysis. Prior to electrophoresis, dithiothreitol was added to 0. I M , and the samples were heated in boiling water for 5 minutes. Electrophoresis and blotting. Sodium dodecyl sulfate (SDS)-PAGE gel electrophoresis was carried out on 10% acrylamide gels as has been described (37). Due to the difficulty of determining protein concentration in the presence of SDS, loading of the gel was determined empirically, and the maximum amount of tissue homogenate giving distinct bands was run (probably -50-100 pdlane after cutting the protein blot). Molecular weights were determined by the migration of prestained high molecular weight markers (Gibco/BRL, Bethesda, MD) in an adjacent lane. Following electrophoresis, the proteins were transferred to nitrocellulose utilizing a Transphor apparatus (Hoeffler Scientific Instruments, San Francisco, CA) in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol) for 8-12 hours at 6OV in a refrigerator. Detection of immunoreactive proteins. Following transfer, blots were placed in TNT buffer (I0 mM Tris, pH 8.0, 0. ISM NaCI, 0.05% Tween 20) plus 5% low-fat milk for 5-12 hours at 4°C. The blot was then cut into strips, incubated with the primary antibody (generally a 1:100 dilution of synovial fluid) diluted in TNT buffer plus 0.5% low-fat milk for 18 hours at 4°C. and washed with TNT buffer 5 times for 5 minutes. The blot was then incubated with the secondary antibody diluted in TNT buffer plus 0.5% low-fat milk for 30 minutes at room temperature. The secondary antibody consisted of either goat anti-human IgG (y chain-specific)/alkaline phosphatase, or goat anti-human IgM ( p chain-specific)/alkaline phosphatase (both from Zymed, San Francisco, CA). The blot was toludeveloped using 5-bromo-4-chloro-3-indolyl-phosphate idine saltlp-nitroblue tetrazolium chloride (BCIP/NBT) in developing buffer (100 mM NaCI, 5 mM MgCI2, 100 mM Tris, pH 9.5) as described elsewhere (37). RF, antinuclear antibody (ANA), antineutrophil cytoplasmic antibody (ANCA), and anticollagen antibody determinations. Rheumatoid factors were measured by latex agglutination utilizing the Arthri-Slidex kit (bioMerieux. Lyon.

France); a titer of 1:20 was considered positive. Enzymelinked immunosorbent assay (ELISA) for RFs was performed as described (39). Briefly, heat-aggregated (63°C. 20 minutes) human IgG (Sigma, Poole, UK) in 0.5M carbonate, pH 8.5, was allowed to bind overnight to microtiter wells (20 pglwell). Wells were then washed 5 times with TNT buffer, and synovial fluids (diluted in TNT plus 0.5% low-fat milk) were added. After 1 hour of incubation, wells were washed 5 times with TNT buffer and incubated another hour with goat anti-human IgM ( p chain-specific)/alkaline phosphatase at a dilution of 1:5,OOO in TNT plus 0.5% low-fat milk. Wells were developed using BCIP/NBT as above. ANA were detected using indirect immunofluorescence on HEp-2 cells. ANCA were detected using indirect immunofluorescence on human neutrophils as described (40). Anti-type I1 collagen antibodies were assayed by ELISA as described (41).

RESULTS Identification of antigens in rheumatoid synovial tissue. Synovial tissue proteins from a patient with RA (patient RA2) were homogenized in electrophoresis buffer and probed for antigens binding to synovial fluid antibodies by protein blotting and immunochemical staining. Synovial fluids used in initial experiments were from several patients with RA and a patient with OA. Binding of antibodies to antigens was detected utilizing a goat anti-human IgG/alkaline phosphataseconjugated antibody. In addition to binding to antibodies from synovial fluid, which were bound to antigens on the protein blot, this antibody also bound directly to blotted IgG from the synovial tissue (see lanes 4 and 13 of Figures la and Ic, respectively). The bands seen in these lanes correspond to the approximate molecular weights of IgG heavy chain (dark band) and light chain (light band; seen better in lane 13) and represent control binding by the secondary antibody alone. Antibodies from synovial fluid of patients with RA reacted against several proteins in the diseased synovial tissue, including a series of low molecular weight proteins, 27.5 kd, 29 kd, and 30 kd, 2 middle molecular weight proteins, 43 kd and 53 kd, and a series of high molecular weight proteins, 140 kd and 164 kd (appearing as a doublet), and 182 kd (Figures la-c, lanes 2, 3, 6, 7, and 1 I ) . Staining of the high and low molecular weight proteins was found in all RA synovial fluids. Staining of the 2 middle molecular weight proteins, however, was quite variable (Figures la and b, lanes 2, 3 , 6, and 7). The antibodies in the synovial fluid from the patient with OA did not react with any of these proteins at the same dilution (Figure la, lane 1).

ANTIGENS IN RA

1019

Figure 1. Identification of antigens in rheumatoid arthritis (RA) synovial tissue. a, b, and c, Three experiments utilizing the same protein blot. Synovial tissue proteins from a patient with RA were separated on a 10% polyacrylamide gel, transferred to nitrocellulose, and probed for immunoreactive proteins, using patient’s synovial fluid as primary antibody ( I : 100 dilution unless otherwise indicated). Secondary antibody was alkaline phosphatase-conjugated goat anti-human IgG (unless otherwise indicated). Lane 1, Osteoarthritis patient I ( O A l ) , lane 2. RAI, lane 3, RA2, lane 4. no primary antibody. lane 5. stained total proteins. lane 6, RA3, lane 7, RA4, lane 8, O A l ( 1 : I O dilution). lane 9. OAI, lane 10. RAI (goat anti-human IgM a s secondary antibody). lane I I , RAI, lane 12, no primary antibody (rabbit anti-human IgM as secondary antibody). lane 13. no primary antibody (anti-human IgG as secondary antibody). Molecular weight markers are shown to the left of lane I ; antigens identified by synovial fluids from patients with RA are marked with solid circles to the left of lane 2. (See also Table I . )

To determine whether the lack of staining of these antigens by antibodies in OA synovial fluid was due to differences in antibody concentration, the synovial fluid sample from the patient with OA was also tested at a 10-fold lower dilution ( l : l O ) , and the immunochemical staining at this dilution was compared with staining by RA synovial fluids at the higher dilution. At this lower dilution, the OA sample revealed light staining of the series of low molecular weight proteins; however, there was no staining of either the middle or the high molecular weight proteins (Figure Ib, lane 8). We, therefore, observed in four

consecutive synovial fluids from patients with RA, antibodies binding to several antigens in inflamed synovial tissue: 1 of the synovial fluid samples was from the same patient as the blotted synovial tissue proteins (patient RA2). We further characterized the antibodies to these antigens, showing that antibodies to the series of middle and low molecular weight sets of antigens include both IgM and IgG isotypes (Figure Ic, lane 10). IgM antibody to the series of high molecular weight proteins was not detected (Figure Ic, compare lane 10 with anti-IgG staining in lane I I).

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Table 1. Titers of rheumatoid factor, antinuclear antibody (ANA), antineutrophil cytoplasmic antibody (ANCA), and anti-type 11 collagen antibodies, as well as the presence of antibodies to synovial tissue proteins in the 14 patients studied

Rheumatoid factor* Diagnosis, patient number Rheumatoid arthritis RAI RA2 RA3 RA4 RA5 Osteoarthritis OA 1 OA2 OA3 Psoriatic arthritis PA I PA2 Chondrocalcinosis CPDDI CPDD2 Systemic lupus erythematosus SLE I Palindromic rheumatism PR I

Latex ELISA titer positivity

1:20

1 :40

-

+ +

Synovial tissue proteins (MW)t 27.5

29

+ ++ + +++ +

++ +++ + +++ +

30

43

53

++ f + + +++ +++ f TR + +++ +

1:20

+

-

-

TR

-

-

-

-

-

-

-

-

-

140

164

Other

++ +++

f

++ +++ + +++

TR 8 5 k d , + TR 85 kd, + TR 8 5 k d , + + + + 85 kd, +

+ +

++ +

f

-

-

TR

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

TR

182

-

-

-

-

-

-

-

-

-

-

18, 21, 37, 42 kd, +++ 37. 42 kd, TR

-

-

-

-

-

+ + + +++

-

-

TR

+

TR -

-

-

-

-

-

-

T

R

-

-

1 :80 -

-

-

-

1:160 1:320

95 kd, TR

-

-

-

-

-

Antitype I1 ANA ANCA collagen titer$ titer titer

-

18, 21, 37, 42

kd,

+++

* Rheumatoid factor was determined by latex agglutination and by enzyme-linked immunosorbent assay (ELISA; positive at >4-fold optical density over background, at a dilution of 1:lOO). t Data were compiled from values shown in Figures I , 2, 3, and 4, and represent band intensity, where - = no staining, TR = trace, and + + + = greatest intensity. $ ANA were present in both a speckled pattern (at 1:160 in patient RA2 and 1:320 in patient RA3) and a homogeneous pattern (at 1:20 in both patients RA2 and RA3).

Synovial fluid R F levels were determined in all patients, and we found that there was no relationship between R F titer and reactivity to synovial tissue proteins (Table 1). RF-negative synovial fluids also reacted against synovial tissue antigens. In other studies, synovial fluid RFs (from RF-positive patient RAS) were absorbed using IgG bound to agarose beads; these absorbed synovial fluids gave the same patterns of reactivity against synovial tissue antigens as did the unabsorbed synovial fluids (data not shown), indicating that RFs are not the antibody directed against the synovial tissue antigens. In order to further confirm the specific antibodyantigen nature of the observed bands, experimental controls using human IgG and heat-aggregated IgG were performed. These controls gave only background staining, similar to that in lane 1 of Figure 1 (data not shown). Also, for several of the synovial fluids, immunoglobulin was purified on a protein G-agarose col-

umn and used as the primary antibody against synovial tissues. Results were the same as those shown utilizing unpurified immunoglobulins from synovial fluid. Identification of antigens in normal synovial tissue. The immune response in RA might potentially be directed against normally expressed endogenous proteins, pathologically expressed endogenous proteins, or exogenous antigens (an infectious organism, for example). We therefore determined whether the antigens identified in inflamed synovial tissue by synovial fluid antibodies from patients with RA were also present in normal synovial tissue. A series of high molecular weight proteins with the same molecular weight as the proteins identified in inflamed synovial tissue were stained in normal synovial tissue (Figure 2, lanes 1-5). Similar to the staining seen in inflamed tissue (Figure I), antibodies to these antigens were stained by synovial fluids from patients RAI-RA4, as well as a fifth patient, RAS. Given the markedly similar

ANTIGENS IN RA

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RAI and RA2 in rheumatoid synovium are actually different proteins (again comparing lanes 2 and 3 in Figure I with lanes I and 2 in Figure 2). In addition, a new 85-kd protein was stained most intensely by synovial fluid RA4, but was also stained lightly with RA2, RA3, and RA5 synovial fluids. Staining of the low molecular weight proteins was not seen in this experiment, which was developed a relatively short time; however, these proteins were observed to be present, apparently at a relatively low concentration, in normal synovial tissue in other experiments developed longer (see Figure 3, lane 1). Staining was also noted at a molecular weight of approximately 65 kd; however, this appeared to represent an artifact. This staining (also seen in Figure 3 ) . which was generally irregular and not confined to the lane of the gel, was also seen in control experiments using heat-aggregated human IgG as a control for

Figure 2. Identification of antigens in normal synovial tissue. Synovial tissue proteins were electrophoresed and transferred to nitrocellulose as in Figure I . Patient's synovial fluid was used as primary antibody ( I : 100 dilution); alkaline phosphatase-conjugated goat anti-human IgG was used as secondary antibody. Lane I . Rheumatoid arthritis patient I (RAI). lane 2, RA2. lane 3. RA3. lane 4, RA4. lane 5 , RA5. Molecular weight markers are shown to the left. (See also Table I .)

pattern of staining, it seems likely that these represent the same proteins present in inflamed tissues. One of the middle molecular weight proteins identifiedby R A 1 synovial fluid and the ldar weightprotein identifiedby RA2 (53-kd and 43-kd bands, lanes 2 and 3, respectively, Figure I ) were also present in normal svnovial tissue (lanes I and 2.. Figure 2; i.e., bands just at;ove [lane 11 andjust below [lane 21 background Ig heavy-chain Stainingin norsynovial tissue of the 43-kd protein by RA2* but not R A I , indicates that the 43-kd proteins stained by

Figure 3. Identification of antigens in dermal, muscle, and liver tissues. Normal tissue proteins were electrophoresed in adjacent lanes and transferred to nitrocellulose a s in Figure I . Synovial fluid from rheumatoid arthritis patient 2 (RA2) was used a s primary antibody (I:100 dilution): alkaline phosphatase-conjugated goat anti-human IgG was used as secondary antibody. Lanes I , 2. 3. and 4. Staining of normal synovial. dermal. liver. and muscle tissues. respectively; lanes 5 . 6 . 7 . and 8. total transferred proteins from normal svnovial. dermal. liver. and muscle tissues. resDectivelv. Molecular weight markers are shown to the left. (See also Table 1 . 1

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binding by immune complexes (data not shown). It has been described previously as a relatively common artifact on Western blots (discussed in ref. 42). Identification of antigens in normal skin, liver, and muscle tissues. Since the antigens identified in inflamed synovial tissue were also present in normal synovial tissue, we next investigated whether expression of these antigens was limited to synovial tissue or was more widespread, occurring in other tissues. For these studies we used normal subcutaneous tissue (representing a tissue similar to synovial tissue, composed primarily of fibroblasts and connective tissue), muscle tissue (representing a tissue of similar, mesodermal origin), and liver tissue (representing a more distantly related tissue, composed primarily of cells of entodermal origin). These tissues were homogenized in PAGE buffer and analyzed in the same manner as the synovial tissues. Using synovial fluid from patient RAI as the source of primary antibody, we found that the high molecular weight antigens that stained in normal and diseased synovium were also present in dermal, muscle, and liver tissues (Figure 3). Although the 182-kd band is not clearly seen in any of the tissues, this appears to be due to overloading and distortion at the top of the gel. The 53-kd protein was more intensely stained in the normal synovial tissue than were any of the other tissues, and the series of low molecular weight proteins were present in both synovial and dermal tissues, but not the liver. The presence of the low molecular weight antigens in muscle tissue was obscured by staining of some new proteins in muscle tissue, which were not present in normal synovial tissues. The significance of antibodies to these proteins, which, of the tissues examined, were expressed only in muscle, is unclear. In other studies, synovial fluid was analyzed by immunoblotting; however, we were not able to detect antibodies in synovial fluid against proteins present in RA synovial fluid (data not shown). Identification of antigens by use of synovial fluids from patients with other rheumatic conditions. The primary aim of the current studies was to identify antigens involved in the immune response of RA; therefore, to further characterize the specificity of the antigens for RA, synovial fluids from patients with several other arthritic conditions were studied. For these studies, normal synovial tissue was used as the source of blotted proteins. Two patients each with OA. PA, and CPDD, as well as 1 patient with SLE and 1 with PR. were studied. The antibodies in synovial

LAFYATIS ET AL

Figure 4. Reactivity of synovial fluids from patients with other arthritic conditions toward synovial tissue proteins. Synovial tissue proteins were electrophoresed and transferred to nitrocellulose a s in Figure I . Patient’s synovial fluid was used as primary antibody ( I : 100 dilution); alkaline phosphatase-conjugated goat anti-human IgG was used as secondary antibody. Lane I , Osteoarthritis patient 2 (OA2). lane 2. OA3. lane 3. psoriatic arthritis patient I (PAI), lane 4. PA2. lane 5 . calcium pyrophosphate deposition disease patient I (CPDDI). lane 6. CPDDZ. lane 7, systemic lupus erythematosus patient I (SLEI). lane 8. palindromic rheumatism patient I (PRI). lane 9. rheumatoid arthritis patient 2 (RA2). Molecular weight markers are shown to the left. (See also Table I . )

fluids from these patients revealed several patterns of reactivity . Synovial fluid from the 2 patients with OA did not react against any of the previously identified antigens except for faint reactivity against a 95-kd protein by sample OA3 (Figure 4, lanes I and 2). One of the patients with PA and the patient with PR showed strikingly similar reactivities to several proteins, none of which stained with rheumatoid synovial fluids (Figure 4, lanes 3 and 8). Samples from the other

ANTIGENS IN RA

Figure 5. Reactivity of synovial fluids from patients with rheumatoid arthritis (RA) toward cellular antigens. Jurkat cell proteins were electrophoresed and transferred to nitrocellulose as in Figure I . Patient's synovial fluid was used as primary antibody ( I : 100 dilution); alkaline phosphatase-conjugated goat anti-human IgG was used as secondary antibody. Lane I , RA2. lane 2. RA3. lane 3. RA5, lane 4. RAI. lane 5 , RA4. lane 6, anti-Ro antiserum, lane 7. stained total proteins. Proteins identified by the anti-Rolanti-La antiserum are marked with solid circles to the left of lane 6. (See also Table 1 .)

patient with PA showed a similar, but much weaker, pattern of reactivity (Figure 4, lane 4). Samples from neither of the 2 patients with CPDD reacted against any synovial tissue proteins (Figure 4, lanes 5 and 6). Synovial fluid antibodies from the patient with SLE reacted strongly to the low molecular weight antigens and very weakly to the high molecular weight proteins (Figure 4. lane 7). This pattern of reactivity was somewhat similar to that for the patients with RA.

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Identification of antigens in Jurkat cell lysates. Since previous studies of protein antigens in autoimmune disease have identified primarily cell-associated antigens, we wanted to determine whether the observed antigens in synovial tissues could be identified utilizing a cell lysate. A cell lysate from Jurkat cells was therefore probed for antigens utilizing synovial fluids from patients with RA or serum from an antiRo/anti-La positive patient with SLE as a positive control. Sera from the patient with SLE reacted against 2 proteins of approximate molecular weight of 60 kd, consistent with the molecular weight of 2 of the 3 cloned Ro proteins, and a third protein of 45 kd, likely representing La protein (Figure 5, lane 6). Synovial fluid RA2 reacted weakly against a 43-kd protein (Figure 5, lane I), the same molecular weight as I of the middle molecular weight proteins previously identified (Figure I , lane 3). The low molecular weight proteins were also stained lightly (best seen in lane I ) . Notably, the high molecular weight proteins identified in synovial tissues were not stained. These high molecular weight proteins were not stained even after overdevelopment of blotted Jurkat cell proteins (probed as above) (data not shown) and were also not stained in cell extract prepared from a cultured rat synovial cell lysate (data not shown). These findings suggest that these proteins are secreted extracellular proteins that do not appreciably accumulate intracellularly. Comparison of autoantibodies identified against synovial tissue proteins with other autoantibodies described in patients with RA. Previous studies of autoantibodies in patients with RA have identified several autoantibodies occasionally associated with this disease, including ANA (43), ANCA (44), and antibodies against 33-kd protein, called RA33. which was identified in HeLa cell nuclear extracts by immunoblotting (38). We therefore wanted to determine whether the antigens observed in synovial tissues could be related to the antigens associated with these various antibodies. The 5 RA synovial fluids used in the experiments above were tested for ANA, ANCA, and antitype I1 collagen antibodies. Two of the synovial fluids, RA2 and RA3, were positive for ANA. both with homogeneous and speckled patterns. None were positive for ANCA, and only I (RA4) was positive for anti-type I1 collagen antibodies (summarized in Table I ) . None of the bands seen by immunoblotting (Figure I ) appeared to correlate with the positive ANA or anti-type 11 collagen antibodies, since all of the bands

1024

LAFYATIS ET AL

were present at similar or greater intensity in synovial fluids negative for ANA or anti-type I1 collagen antibodies. It therefore does not appear that the antigens identified in synovial tissues are related specificities determined by ANA, ANCA, or anti-type I1 collagen antibodies. In order to study the relationship between the antigens identified in synovial tissues and the nuclear antigen described by Hassfeld et al (38). a nuclear extract was prepared from HeLa cells according to the protocol described by those authors, and synovial fluids RA2 and RA3 were analyzed for antibodies against this extract. As a control, synovial tissue was analyzed in an adjacent lane of the same gel. Synovial fluids RA2 and RA3 intensely stained proteins in the nuclear extract of the same molecular weight as the series of low molecular weight proteins we described in synovial tissue: these were the only proteins stained (Figure 6). It therefore appears likely that these proteins represent the same antigen described by Hassfeld et al, although there are some discrepancies. That group described a single protein with a molecular weight of 33 kd, whereas we note several proteins. The 2 most intensely stained were of molecular weights 29 kd and 30 kd. Several differences in experimental protocol might account for these discrepancies. Hassfeld and coworkers apparently separated the extracted proteins on a gradient gel (although this is not specifically described), rather than a 10% acrylamide gel, and used '%labeled protein A for identification of the bound primary antibody (rather than an alkaline phosphataseconjugated secondary antibody). Either of these differences in protocol could account for the resolution of 2 proteins rather than I . Further, the molecular weight standards we used were prestained, and therefore, a discrepancy of several kilodaltons would not be surprising. Overall, however, the intensity of staining of the antigen in this extract, along with the similarity of molecular weight, provide strong evidence in favor of these being the same, or closely related, antigens.

DISCUSSION We report here the presence of antigens in rheumatoid synovial tissues that are identified by antibodies from the synovial fluid of patients with RA. Our results show that all patients with RA who were studied have antibodies against the same series of both high and low molecular weight proteins: variable reactivity to 3 middle molecular weight proteins was also

Figure 6. Reactivity of synovial fluids from patients with rheumatoid arthritis (RA) toward nuclear antigens. HeLa cell nuclear proteins (lanes I and 2) and RA synovial tissue proteins (lane 3) were electrophoresed and transferred to nitrocellulose as in Figure I . Patient's synovial fluid was used as primary antibody (1:lOO dilution); alkaline phosphatase-conjugated goat anti-human IgG was used as secondary antibody. Lane I , RA2. lane 2. RA3, lane 3. RA3. Molecular weight markers are shown to the left. (See also Table I . )

seen. These data suggest that the antibody response to synovial tissue proteins in various RA patients is relatively uniform in regard to antigen specificity. The antigens were identified in both normal and diseased synovium, indicating that these antigens are not exogenous, i.e., do not arise de novo in the synovial tissue as the result of an infection, and further, do not arise as the result of an endogenous genetic defect leading to overexpression in synovial tissue of these patients with RA. The high molecular weight antigens were also identified in several other normal tissues, such as muscle and liver, which rarely are affected in RA. The presence of these antigens in dermal, muscle, and liver tissues shows that the specificity of the immune response in RA is not simply due to selective expression of the high molecular weight antigens in synovial tissues. Rather, if the immune response in R A is primarily directed against these antigens, other factors, such as increased accessibility of these antigens

ANTIGENS IN RA

to immune effector cells or altered antigenicity of these synovial tissue proteins, would be required to explain the predisposition of the immune response in RA to synovial tissue. The pattern of expression of the 53-kd protein and the series of low molecular weight proteins in different tissues was more variable, leaving open the possibility that tissue-specific expression of one or more of these antigens could be important in the tissue-specific inflammation that is characteristic of RA. From these initial data, it is impossible to tell if the identified antigens are directly stimulating the immune response in RA, or are involved in a secondary response after inflammation has been initiated via another mechanism. The consistent finding of antibodies to both the high and low molecular weight proteins in patients with RA (7 of 8 additional patients tested since these experiments have also been positive for these antibodies) and the lack of these antibodies in most other patients with arthritis of other etiologies, including such inflammatory arthritides as psoriatic arthritis, suggest that these antigens could be directly involved in stimulating the immune response in RA. Serum from the patient with SLE also reacted against both the high and low molecular weight proteins, possibly suggesting that the same antigens are responsible for initiating synovial tissue inflammation in these 2 diseases. The synovial fluid from patients with PA and the patient with PR reacted to a quite distinct set of proteins. Possibly, these proteins represent important antigens in PA. In this regard, it will be interesting to see if the patient with palindromic rheumatism develops psoriatic arthritis. Studies are under way to determine whether these antibodies are more widely present in other patients with PA. The identity of the antigenic proteins is not known. The observation that the high molecular weight antigens are easily identified in a variety of tissues, but are not identified in cell lysates, suggests that they are primarily extracellular, possibly secreted by fibroblasts, since this cell type is common to the normal tissues studied. These proteins are of similar molecular weight as collagen; however, given the tissue distribution and lack of anti-type I1 collagen antibodies in most of the synovial fluids studied, they are clearly not type I1 collagen. Other collagen types are possible candidates; cellular sensitivity to type I11 collagen has been reported in patients with RA, and lymphocyte transformation in response to type 1 collagen has been reported both in patients with RA and controls (healthy subjects and patients with other arthritic disorders) (14,16). Absorption of synovial fluids

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with type I collagen bound to agarose beads did not inhibit staining, and I of the synovial fluids has been studied and found to be negative for antibodies to type I collagen by ELISA (unpublished observations), which strongly suggests that these antibodies are also not directed against type I collagen. Similarly, the identity of the middle molecular weight antigens is unknown. None of the proteins identified have the same molecular weight as the human analog (reported molecular weight 63 kd) of the 65-kd mycobacterial heat-shock protein implicated in the cause of adjuvant arthritis, or the previously described 62-kd protein having a shared antigenic epitope with EBNA (28). The lack of correlation with a positive ANA or staining of protein antigens associated with a positive ANA in Jurkat cell lysates shows that the middle molecular weight antigens are not the same protein antigens previously identified in patients with SLE. Conceivably, one of these bands is the same as a currently undefined 56-kd protein; antibodies against this protein have been seen occasionally in patients with RA (45). Further, we do not know the relationship between the antibodies identified here and anti-keratin and anti-perinuclear factor antibodies, which have been identified by immunofluorescence against, respectively, buccal mucosa and rat esophageal cells (46,47). It has been suggested that these antibodies may be directed against the same antigen, and that this antigen may not actually be keratin (47). The low molecular weight antigens appear to be nuclear antigens; these proteins are likely the same as or are closely related to the RA33 antigen previously described (38). The incidence of antibody to these antigens in our limited study of synovial fluids was 100%. whereas Hassfeld et al (38) reported a 36% incidence of antibodies against RA33. Those authors studied a far greater number of patients (n = 95); however. they did not study synovial fluid. Perhaps the incidence is higher in synovial fluid. Assuming these are, indeed, the same antigens, then it appears that this antigen (RA33) is expressed in diseased synovial tissue. In summary, antibodies in rheumatoid synovial fluid appear to react against several proteins in rheumatoid synovial tissues. Possibly these same proteins are responsible for eliciting the T cell response in RA. In this regard, although antibodies reactive against synovial tissue antigens may not have a direct pathologic role in RA (as has been generally suggested for the antibody response in RA, since an RA-like illness has been reported in patients with agammaglobulinemia), if RA is indeed antigen-driven, it nevertheless

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seems likely, given the important connections between B cell and T cell activation, including cognate T cell help, that activated T cells in RA will be directed against at least Some of the Same antigens as activated B cells. Given the much greater facility with which B cell antigens can be identified, further study of these antigens might allow identification of the elusive T cell antigen(s) responsible for RA.

ACKNOWLEDGMENTS We thank Dr. Baspeyre for help obtaining surgical specimens, Dr. Duthilleul for ANCA analysis, Dr. Fournier for help in anti-type I1 collagen antibody analysis, and Didier Monte and Dr. Lionel Prin for critical review of the manuscript.

REFERENCES I . Todd JA, Acha-Orbea H, Bell JI, Chao N, Fronek Z, Jacob CO, McDermott M, Sinha AA, Timmermann L, Steinman L, McDevitt HO: A molecular basis for MHC class I1 associated autoimmunity. Science 240: 10031009, 1988 2. Dougados M , Awada H. Amor B: Cyclosporin in rheumatoid arthritis: a double blind placebo controlled study in 52 patients. Ann Rheum Dis 47:127-133, 1988 3. Yocum DE, Klippel JH. Wilder RL. Gerber NL, Austin HA, Wahl SM, Lesko L. Minor JR. Preuss HG, Yarboro C, Berkebile C, Dougherty S: Cyclosporin A in severe treatment-refractory rheumatoid arthritis: a randomized study. Ann Intern Med 109:863-869, 1988 4. Steere AC, Brinckerhoff CE, Miller DJ, Drinker H, Harris ED Jr, Malawista SE: Elevated levels of collagenase and prostaglandin E2 from synovium associated with erosion of cartilage and bone in a patient with chronic Lyme arthritis. Arthritis Rheum 23:591-598, 1980 5. Collins DH, Goldie W: Observations on polyarthritis and on exDerimental ervsiDelothrix infection of swine. . Pathol Badteriol 50:323-353, 1940 Decker JL, Barden JA: Mycoplasma arthritis in the pig, Infection and Immunology in the Rheumatic Diseases. Edited by DC Dumond. Oxford, Blackwell Scientific Publications, 1976 Cromartie WJ, Craddock JG, Schwab JH, Anderle S K , Yang CH: Arthritis in rats after systemic injection of streptococcal cells or cell walls. J Exp Med 146:15851602, 1977 Pearson CM: Development of arthritis, periarthritis and periostitis in rats given adjuvant. Proc SOCExp Biol Med 91:95-101, 1956 Stuart JM, Townes AS, Kang AH: Collagen autoimmune arthritis. Annu Rev Immunol 2: 19%)-218, 1984

,.

10. Holoshitz J, Naparstek Y, Ben-Nun A, Cohen IR: Lines of T lymphocytes induce or vaccinate against autoimmune arthritis. Science 219:56-58, 1983 1 1 . Victor KD, Randen Thompson K* Forre 0- Natvk JB. Fu SM, Capra JD: Rheumatoid factors isolated from patients with autoimmune disorders are derived from germline genes distinct from those encoding the Wa, Po, and Bla cross-reactive idiotypes. J Clin Invest 87: 16031613. 1991 12. Carson DA, Chen PP, Kipps TJ: New roles for rheumatoid factor. J Clin Invest 87:379-383, 1991 13. Roosnek E. Lanzavecchia A: Efficient and selective presentation of antigen-antibody complexes by rheumatoid factor B cells. J Exp Med 173:487489, 1991 14. Smolen JS, Menzel EJ, Scherak 0, Kojer M, Kolarz G , Steffen C, Mayr WR: Lymphocyte transformation to denatured type 1 collagen and B lymphocyte alloantigens in rheumatoid arthritis. Arthritis Rheum 23:424-432. 1980 15. Rowley M , Tait B, Mackay IR, Cunningham T, Phillips B: Collagen antibodies in rheumatoid arthritis: significance of antibodies to denatured collagen and their association with HLA-DR4. Arthritis Rheum 29: 174184. 1986 16. Trentham DE. Dynesius MS, Rocklin RE, David JR: Cellular sensitivity to collagen in rheumatoid arthritis. N Engl J Med 299:327-332, 1980 17. Londei M, Savill CM, Verhoef A, Brennan F, Leech ZA, Duance V. Maini RN, Feldmann M: Persistence of collagen type 11-specific clones in the synovial membrane of patients with rheumatoid arthritis. Proc Natl Acad Sci USA 86:636-640, 1989 18. Mottonen T. Hannonen P, Oka M, Rautiainen J, Jokinen I , Arvilommi H, Palosuo T , Aho K: Antibodies against native type I1 collagen do not precede the clinical onset of rheumatoid arthritis. Arthritis Rheum 31:776-779. I988 19. Jasin HE: Autoantibody specificities of immune complexes sequestered in articular cartilage of patients with rheumatoid arthritis and osteoarthritis. Arthritis Rheum 28:241-248, 1985 20. Alspaugh MA, Tan EM: Serum antibody in rheumatoid arthritis reactive with a cell-associated antigen: demonstration by precipitation and immunofluorescence. Arthritis Rheum 19:711-719, 1976 21. Alspaugh MA, Jensen FC, Rabin H, Tan EM: Lymphocytes transformed by Epstein-Barr virus: induction of nuclear antigen reactive with antibody in rheumatoid arthritis. J Exp Med 147:1018-1027, 1978 22. Alspaugh MA, Henle G , Lennette ET, Henle W: Elevated levels of antibodies to Epstein-Barr virus antigens in sera and synovial fluids of patients with rheumatoid arthritis. J Clin Invest 67: 1134-1 140, 1981 23. Catalan0 MA, Carson DA, Neiderman JC. Feorino P. 1 9

ANTIGENS IN RA

24.

25.

26.

27.

28.

29.

30.

31.

32.

33

I

34.

35

I

Vaughan JH: Antibody to the rheumatoid arthritis nuclear antigen. J Clin Invest 65:1238-1242, 1980 Catalan0 MA, Carson DA, Slovin SF, Richman DD, Vaughan JH: Antibodies to the Epstein-Barr virusdetermined antigens in normal subjects and in patients with rheumatoid arthritis. Proc Natl Acad Sci USA 1 1 :5825-5828, 1979 Tosato G, Steinberg AD, Yarchoan R, Heilman CA, Pike SE, DeSeau V, Blaese RM: Abnormally elevated frequency of Epstein-Barr virus-infected B cells in the blood of patients with rheumatoid arthritis. J Clin Invest 73:1789-1795, 1984 Silverman SL, Schumacher HR: Antibodies to EpsteinBarr viral antigens in early rheumatoid arthritis. Arthritis Rheum 24:1465-1468, 1981 Roudier J, Petersen J, Rhodes GH, Luka J, Carson DA: Susceptibility to rheumatoid arthritis maps to a T-cell epitope shared by the HLA-Dw4 DR beta-I chain and the Epstein-Barr virus glycoprotein gpl10. Proc Natl Acad Sci USA 86:51045108. 1989 Fox R, Sportsman R, Rhodes G, Luka J , Pearson G, Vaughan J: Rheumatoid arthritis synovial membrane contains a 62,000-molecular-weight protein that shares an antigenic epitope with the Epstein-Barr-virusencoded associated nuclear antigen. J Clin Invest 77: 1539-1547, 1986 Van den Broek MF, Hogervorst EJM, van Bruggen MCJ, van Eden W. van der Zee R, van den Berg WB: Protection against streptococcal cell wall-induced arthritis by pretreatment with the 65-kD mycobacterial heat 1989 shock protein. J Exp Med 170:44-66, Van Eden W, Thole JER, van der Zee R, Noordzij A, van Embden JDA, Hensen EJ, Cohen IR: Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature 331:171-173, 1988 Holoshitz J , Klajman A, Drucker I , Lapidot Z, Yaretzky A, Frenkel A, van Eden W, Cohen 1R: T lymphocytes of rheumatoid arthritis patients show augmented reactivity to a fraction of mycobacteria cross-reactive with cartilage. Lancet II:305-309, 1986 Holoshitz J , Koning F, Coligan JE, DeBruyn J, Strober S: Isolation of CD4-CD8-mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 339:22&229, 1989 Gaston JSH, Life PF, Jenner PJ, Colston MJ, Bacon PA: Recognition of a mycobacteria-specific epitope in the 65-kD heat shock protein by synovial fluid derived T cell clones. J Exp Med 171:831-841, 1990 Res PCM, Orsini DLM. van Laar JM, Janson AAM, Abou-Zeid C, Vries RRP: Diversity in antigen recognition by mycobacterium tuberculosis-reactive T-cell clones from synovial fluid of rheumatoid arthritis patients. Eur J Immunol 21:1297-1302. 1991 Carson DA: Rheumatoid factor, Textbook of Rheuma-

1027

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

tology. Edited by WN Kelley. ED Harris Jr, S Ruddy, CB Sledge. Philadelphia, WB Saunders, 1985 Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, Medsger TA Jr, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder RL, Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315-324, 1988 Antibodies: A Laboratory Manual. Edited by E Harlow, D Lane. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1988 Hassfeld W, Steiner G, Hartmuth K, Kolarz G, Scherak 0, Graninger W, Thumb N, Smolen JS: Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis. Arthritis Rheum 32: 1515-1520, 1989 Gripenberg M, Wafin F, Isomaki H, Linder E: A simple immunoassay for the demonstration of rheumatoid factor. J Immunol Methods 31:109-118, 1979 Wilk A: Delineation of a standard procedure for indirect immunofluorescence of ANCA. Acta Pathol Microbiol lmmunol Scand 97:12-15, 1988 Boissier M , Feng X-Z, Carlioz A, Roudier R, Fournier C: Experimental autoimmune arthritis in mice. I. Homologous type 11 collagen is responsible for selfperpetuating chronic polyarthritis. Ann Rheum Dis 46: 691-700, 1987 Riches PG, Poke B, Hong R: Contaminant bands on SDS-polyacrylamide gel electrophoresis are recognised by antibodies in normal serum and saliva. J Immunol Methods 110:117-121, 1988 Harris ED Jr: Antinuclear antibodies, Textbook of Rheumatology. Edited by WN Kelley, ED Harris Jr, S Ruddy, CB Sledge. Philadelphia, WB Saunders, 1985 Savige JA, Gallicchio MC, Stockman A, Cunningham TJ, Rowley MJ, Georgiou T, Davies D: Anti-neutrophil cytoplasmic antibodies in rheumatoid arthritis. Clin Exp lmmunol 86:92-98, 1991 De Rooij DJ, van de Putte LB, Habets WJ, Verbeek AL, van Venrooij WJ: The use of immunoblotting to detect antibodies to nuclear and cytoplasmic antigens. Scand J Rheumatol 17:353-364, 1988 Youinou P, LeGoff P, Colaco CB, Thivolet J, Tater D, Viac J , Shipley M: Antikeratin antibodies in serum and synovial fluid show specificity for rheumatoid arthritis in a study of connective diseases. Ann Rheum Dis 44:45& 454, 1985 Hoet RMA, Boerbooms AMT, Arends M, Ruiter DJ, van Venrooij WJ: Antiperinuclear factor, a marker autoantibody for rheumatoid arthritis: colocalisation of the perinuclear factor and profilaggrin. Ann Rheum Dis 50:611-618, 1991

Antibodies in rheumatoid synovial fluids bind to a restricted series of protein antigens in rheumatoid synovial tissue.

By searching the synovial fluid of patients with rheumatoid arthritis (RA) for antibodies that react to protein antigens in synovial tissue, we sought...
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