Synovial tissues were obtained from five women (aged 45 to 66 years) with active RA or osteoarthritis (OA) whose disease had been diagnosed according to the criteria of the American College of Rheumatology 29303132 and who were treated by joint replacement surgery. All the enrolled patients with RA had more than six swollen joints, more than three tender joints, and an erythrocyte sedimentation rate (Westergren) of >28 mm/hour.

Samples were dissected under sterile conditions in PBS and were immediately prepared for culture of fibroblast-like synovial cells. Briefly, the tissue samples were minced into small pieces and digested with collagenase (Sigma Aldrich, Tokyo, Japan) in serum-free DMEM (Gibco BRL, Grand Island, NY, USA). The cells were filtered through a nylon mesh and then were washed extensively and suspended in DMEM supplemented with 10% FCS (Bio-Pro, Karlsruhe, Germany) and streptomycin/penicillin (10 units/ml; Sigma Aldrich). Finally, isolated cells were seeded in 25-cm² culture flasks (Falcon, Lincoln Park, NJ, USA) and cultured in a humidified 5% CO₂ atmosphere. After overnight culture, nonadherent cells were removed and incubation of adherent cells was continued in fresh medium. At confluence, the cells were trypsinized, passaged at a 1:3 split ratio, and recultured. The medium was changed twice each week, and the cells were used after 2 to 5 passages. We characterized cultured synovial cells derived from the synovium of RA patients. The cells were spindle-shaped and grew in a cobblestone pattern. Flow cytometric analysis of these cells indicated that they lacked macrophage markers such as major-histocompatibility-complex class II antigens CD14 and CD11b (data not shown). Thus, RA synovial cells are type B synovial-fibroblast-like cells.

Human thrombin was purchased from Sigma Aldrich. The following mAbs were used: fluorescein-isothiocyanate-conjugated control mAb anti-Thy 1.2 (Becton Dickinson, San Jose, CA, USA) and antithrombin receptor mAb ATAP2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). A Rho activation kit containing glutathione S-transferase (GST)-Rhotekin-Rho-binding domain (RBD) (GST-RBD) beads was purchased from Cytoskeleton (Denver, CO, USA).

Recombinant adenoviruses encoding green-fluorescent protein (GFP), the C-terminal regions of Gα12 (Gα12-ct), the C-terminal regions of Gα13 (Gα13-ct), and the regulator of the G-protein signaling domain of p115RhoGEF (p115RGS) were produced as described previously 33. RA SFs were plated onto a six-well culture dish and cultured in DMEM containing 10% FCS. After 24 hours, the cells were infected with recombinant adenoviruses at a multiplicity of infection of 30 for 1 hour at 37°C. Cells infected or not infected with adenovirus were then starved in DMEM with 1% FCS and cultured for an additional 48 hours before treatment. Under these conditions, infection with adenoviruses coding for GFP made almost 100% of cells GFP-positive. None of these vectors produced cytotoxic effects on RA SFs until 96 hours after infection, as confirmed by trypan blue staining (data not shown).

Staining and flow-cytometric analysis of RA SFs were conducted by standard procedures, as described previously 34, using a FACScan (Becton Dickinson, Mountain View, CA, USA). Briefly, cells (2 × 10⁵) were incubated with fluorescein-isothiocyanate-conjugated negative control mAb anti-Thy-1.2 or antithrombin receptor mAb at saturating concentrations in fluorescence-activated cell sorter (FACS) medium consisting of Hanks' balanced salt solution (Nissui, Tokyo, Japan), 0.5% human serum albumin (Mitsubishi Pharma, Osaka, Japan), and 0.2% NaN₃ (Sigma Aldrich) for 30 min at 4°C. After three washes in FACS medium, the cells were analyzed with the FACScan. Cell-surface antigens on single cells were quantified using standard beads, QIFKIT (Dako Japan, Kyoto, Japan), as described previously 3536. The data were used to construct the calibration curve of mean fluorescence intensity versus antibody-binding capacity. The cell specimen was analyzed on the FACScan and antibody-binding capacity calculated by interpolation on the calibration curve. When green-fluorescence laser detection was set at 450 nm in the FACScan used, antibody-binding capacity = 414.45 × exp (0.0092 × Mean fluorescence intensity) (R2 = 0.9999). Subsequently, specific antibody-binding capacity was obtained after corrections for background, apparent antibody-binding capacity of the negative control mAb anti-Thy-1.2. The specific antibody-binding capacity is the mean number of accessible antigenic sites per cell, referred to as antigen density and expressed in sites/cell.

RA SFs (1 × 10⁴) infected with or free of adenoviruses were seeded and incubated in 96-well flat-bottomed microfilter plates (Costar, Cambridge, MA, USA) in DMEM containing 1% FCS for 48 hours at 37°C and were then stimulated with the indicated amount of thrombin. At 24 hours after the thrombin stimulation, cells were stained with TetraColor One (Seikagaku, Tokyo, Japan) including tetrazolium and an electron-carrier mixture for detecting cell proliferation. After the cells had been stained in this way for 1 hour at 37°C, the optical density value of each well was measured using an ELISA plate reader at 450 nm.

RA SFs infected with or free of adenoviruses were cultured for 48 hours in DMEM containing 1% FCS and then stimulated with the indicated amount of thrombin. At 24 hours after thrombin stimulation, the cells were collected, washed with PBS, and fixed in 70% ethanol for 2 hours at 4°C. After treatment of cells with 10 μg/ml ribonuclease (Wako, Osaka, Japan) for 15 min at 37°C, the cells were stained with 50 μg/ml propidium iodide (Sigma Aldrich) for 2 minutes. The DNA content was subsequently measured by FACScan.

Rho activation was determined by a pull-down assay using GST-RBD beads 3738. Forty-eight hours after adenovirus infection, RA SFs were stimulated with 10 units/ml thrombin, quickly washed with ice-cold Tris-buffered saline, and lysed in 500 μl of lysis buffer (50 mM Tris, pH 7.5, 10 mM MgCl₂, 0.5 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 μg/ml tosyl arginine methyl ester, and 10 μg/ml each of leupeptin and aprotinin). Cell lysates were immediately centrifuged at 8,000 rpm at 4°C for 5 min and equal volumes of lysates were incubated with 30 μg GST-RBD beads for 1 hour at 4°C. The beads were washed twice with wash buffer (in mmoles: 25 Tris, pH 7.5; 30 MgCl₂; 40 NaCl), and bound Rho was eluted by boiling each sample in Laemmli sample buffer. Eluted samples from the beads and total cell lysate were then electrophoresed on 12% SDS–PAGE gels, transferred to nitrocellulose, blocked with 5% nonfat milk, and analyzed by western blotting using a polyclonal anti-Rho antibody.

Data are expressed as mean ± standard deviation (SD) of the number of indicated patients. Differences from the control were examined for statistical significance by the Mann–Whitney U test. A P value less than 0.05 denoted the presence of a statistically significant difference.

First, we assessed the expression of thrombin receptor on synovial fibroblasts using FACScan. Fig. 1a shows the histogram of thrombin receptor expressed on RA and OA synovial fibroblasts. Although thrombin receptor was expressed on both types of synovial fibroblasts, its level was significantly higher in RA than OA fibroblasts (Fig. 1b). These results were identified in synovial fibroblasts from patients with RA and OA (n = 5 each).

To assess the effect of thrombin on the proliferation of RA SFs, we performed proliferation assay and cell-cycle analysis. After cells were starved for 48 hours in DMEM containing 1% FCS, cells were stimulated with the indicated amount of thrombin for 24 hours. Thrombin significantly induced cell proliferation in a dose-dependent manner (Fig. 2a). As shown in Fig. 2b, the vast majority of the starved cells existed at the propidium-iodide-low G0/G1 phase and showed little progression to S phase. However, thrombin significantly increased the S/G₂/M phase of the cell in a dose-dependent manner (Fig. 2b,c). The maximum effects of thrombin on cell proliferation and progression to S phase were noted at 10 units/ml. These results indicate that thrombin acts as an important stimulator of RA synovial proliferation.

We next compared cell growth and the cell cycle of RA SFs with those of OA SFs. As shown in Fig. 3a, thrombin induced cell growth and cell-cycle progress in both RA and OA SFs, whereas unstimulated cells did not proliferate well. However, thrombin-induced proliferation of RA SFs was significantly higher than that of OA SFs after incubation for 24 or 48 hours. As shown in Fig. 3b, thrombin induced cell-cycle progress to S phase in both RA and OA SFs, but the responses of RA SFs were significantly higher than those of OA SFs after 24 hours of incubation.

Thrombin is known to bind to protease-activated receptor-1 such as thrombin receptor, and binding of thrombin to receptors leads to activation of the G protein Gα13 and induces activation of p115RhoGEF, a Rho-specific GEF, and thereby activates Rho 9101112. We measured Rho activation and its inhibition in RA SFs using the GST-Rhotekin fusion protein. We used the C-terminal regions of Gα12 and Gα13, which inhibit Gα12 and Gα13 from coupling with each receptor, or the regulator of the G-protein signaling domain of p115RhoGEF, which inhibits endogenous p115RhoGEF function by blocking the interaction of p115RhoGEF with Gα12/13 and by its GTPase-activating-protein activity on Gα12/13 333940. After adenoviral infection, cells were stimulated with 10 units/ml thrombin, and the lysates from cells were incubated with Rhotekin bound to GST beads to determine Rho activation. As shown in Fig. 4, thrombin stimulation increased the amount of activated Rho in RA SFs infected or not infected with adenoviruses encoding control vector, suggesting that thrombin activates Rho in RA SFs. However, the expression of Gα13-ct or p115RGS completely prevented thrombin-induced Rho activation (Fig. 4). These results suggest that Rho is involved in signaling via thrombin-stimulation, which leads to synovial proliferation.

Thrombin (10 unit/ml) significantly induced synovial proliferation and S-phase progression in RA SFs infected or not infected with adenoviruses encoding control vector and Gα12-ct (Fig. 5a,b). In contrast, thrombin failed to induce both cell proliferation and S-phase progression in RA SFs expressing Gα13-ct and p115RGS (Fig. 5a,b). These data suggest that Gα13 (but not Gα12) and RhoGEF are involved in signaling via thrombin-stimulation, which leads to synovial proliferation.

Finally, we assessed IL-6 secretion by RA SFs, using ELISA. Fig. 6 shows the concentrations of IL-6 in supernatants of thrombin-stimulated RA SFs. Stimulation with 10 units/ml thrombin significantly increased IL-6 secretion at 6 hours (Fig. 6b), and this effect was dose-dependent (Fig. 6a). The same dose of thrombin produced a significant induction of IL-6 secretion by RA SFs infected or not infected with adenoviruses encoding control vector (Fig. 6c). However, the thrombin-induced IL-6 secretion in RA SFs transfected with Gα12-ct and Gα13-ct was partially reduced and that in RA SFs expressing p115RGS was markedly inhibited (Fig. 6c). These results suggest that thrombin-induced IL-6 secretion by RA SFs is mainly mediated through RhoGEF.