Mast Cell.Derived Tryptase Inhibits Apoptosis of Human
Rheumatoid Synovial Fibroblasts via Rho-Mediated Signaling

Norifumi Sawamukai,1 Sonosuke Yukawa,2 Kazuyoshi Saito,2 Shingo Nakayamada,2 Taku Kambayashi,3 and Yoshiya Tanaka2

Objective. An abundance of mast cells are found in the synovium of patients with rheumatoid arthritis (RA). However, the role of mast cells in the pathogenesis of RA remains unclear. This study was undertaken to elucidate a role for mast cells in RA by investigating the antiapoptotic effects of tryptase, a major product of mast cells, on RA synovial fibroblasts (RASFs). Methods. RA synovial tissue was obtained from RA patients during joint replacement surgery, and histologic changes in the tissue were examined. The expression of cell surface molecules and apoptotic markers on RASFs were detected by flow cytometry. Rho activation was determined using a pull-down assay. Results. Mast cells, bearing both c-Kit and tryptase, accumulated in the sublining area of proliferating synovial tissue from RA patients. Proteaseactivated receptor 2 (PAR-2), a receptor for tryptase, was expressed on RASFs in the lining area, close to tryptase-positive mast cells in the RA synovium. Fasmediated apoptosis of RASFs was significantly inhibited, in a dose-dependent manner, by the addition of tryptase, and this effect correlated with increased activation of Rho kinase. Furthermore, Y27632, a Rho kinase inhibitor, reduced the antiapoptotic effect of tryptase on RASFs, suggesting that Rho was responsible for the antiapoptotic effects of tryptase. Conclusion. These results demonstrate that tryptase has a strong antiapoptotic effect on RASFs through the activation of Rho. Thus, we propose that the release of tryptase by mast cells leads to the binding of tryptase to PAR-2 on RASFs and inhibits the apoptosis of RASFs via the activation of Rho. Such mechanisms could play a pivotal role in the marked proliferation of RASFs and hyperplasia of synovial tissue seen in RA synovium. Rheumatoid arthritis (RA) is an inflammatory disease that is characterized by persistent joint inflammation, eventually leading to destruction of the joints, which results in significant impairment of daily activity. In addition to decreased mobility, joint destruction causes tenderness and pain, and the quality of life and life expectancy of RA patients is drastically reduced compared with that of healthy subjects (1). The recent emergence of biologic drugs that target inflammatory cytokines, including tumor necrosis factor (TNF), has greatly improved the treatment of RA. Despite such advances, the RA remission rate still remains low, at only 30–40%. Thus, further understanding of the pathogenesis of RA in order to yield new perspectives on RA treatment is necessary in those cases in which the current therapeutic strategy is insufficient. In the joints of RA patients, a proliferating mass in the synovium, known as pannus, covers the RA joint cartilage and contributes to joint erosion and fibrous ankylosis. Pannus consists of granulation tissue and proliferating synovial fibroblasts (SFs), accompanied by neoangiogenesis and inflammatory cell infiltrates (2). A main contributor to joint destruction is the RASF, which multiplies in the same manner as neoplastic cells and infiltrates into cartilage and bone, resulting in tissue destruction. However, it is puzzling that RASFs proliferate excessively in vivo despite a high level of expression of Fas and sensitivity to Fas-mediated killing in vitro (2,3). Thus, it is possible that RA joint cartilage contains a mechanism for the suppression of apoptosis of SFs, which leads to inappropriate SF hyperplasia. We have previously reported that mast cells may be an effective therapeutic target in the treatment of chronic inflammation in RA (4). Such notions stem from the observation that an abundance of mast cells is present in the synovial tissue of RA patients compared with those with other joint diseases (5). Mast cells appear to contribute to RA pathology in mouse studies, as was shown in a mouse model of autoantibody-induced arthritis in which mast cell–deficient mice exhibited attenuated joint inflammation (6). Moreover, mast cells produce cytokines that are of great relevance in RA, including TNF and interleukin-1 (7). Thus, although the specific details remain unclear (8), it is conceivable that mast cells also play an important role in the pathogenesis of human RA. In the present study, we hypothesized that mast cells may contribute to the pathogenesis of RA by inhibiting the apoptosis of RASFs. The results demonstrate that mast cells are found in close proximity to RASFs in the synovium of RA patients. Furthermore, RASFs express the receptor for mast cell tryptase, known as protease-activated receptor 2 (PAR-2), and are protected from Fas-mediated apoptosis by tryptase in a Rho GTPase–dependent manner. We propose that such mechanisms could play a pivotal role in the marked proliferation of RASFs and hyperplasia seen in RA synovium. PATIENTS AND METHODS Human studies. The study protocol was approved by the Human Ethics Review Committee of the University of Occupational and Environmental Health in Japan. Signed informed consent was obtained from each subject involved in this study. Synovial tissue and culture of SFs. Synovial tissue was obtained from 5 women (ages 47–60 years) with active RA, whose disease had been diagnosed according to the criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (9) and who had undergone joint replacement surgery. All enrolled patients had 6 swollen joints, 3 tender joints, and an erythrocyte sedimentation rate (Westergren) of 28 mm/hour. Synovial tissue samples were dissected under sterile conditions in phosphate buffered saline, and fibroblast-like synovial cells were isolated and cultured. Briefly, the tissue samples were minced into small pieces and digested with collagenase (Sigma-Aldrich, Tokyo, Japan) in serum-free Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY). The cells were filtered through a nylon mesh, washed extensively, and suspended in DMEM supplemented with 10% fetal calf serum (FCS; Bio-Pro, Karlsruhe, Germany) and streptomycin/penicillin (10 units/ml; Sigma- Aldrich). Finally, isolated cells were seeded in tissue culture flasks (Falcon, Lincoln Park, NJ), and nonadherent cells were removed. The medium was changed biweekly, and the cells were used after 5 passages. The resulting synovial cells were spindle-shaped and grew in a cobblestone pattern. Flow cytometric analysis of these cells indicated that they lacked macrophage markers, such as class II major histocompatibility complex, CD14, and CD11b (results not shown). Thus, the RA synovial cells obtained appeared to represent type B synovial fibroblast-like cells. Reagents. Human -tryptase was purchased from Promega (Madison, WI). The following monoclonal antibodies (mAb) were used: fluorescein isothiocyanate–conjugated control mAb anti-Thy1.2 (Becton Dickinson, San Jose, CA) and anti-human PAR-2 mAb (R&D Systems, Minneapolis, MN). A Rho activation kit containing glutathione S-transferase– Rhotekin-Rho binding domain (GST-RBD) beads was purchased from Cytoskeleton (Denver, CO). Immunohistochemistry. Synovial tissue was stained as previously described (10). Briefly, sections (6 m) were fixed in ice-cold acetone, and endogenous peroxidase was quenched with 3% H2O2/methanol. Sections were incubated with blocking buffer and then with the SAM-11 mAb (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 g/ml for 12 hours at 4°C. SAM-11 is a previously characterized (11) highly specific mAb to human PAR-2 that binds the membrane-bound part of the receptor, in both cleaved and uncleaved states. Endogenous biotin was blocked using an avidin–biotin kit (Vector, Peterborough, UK), and biotinylated secondary antibody (Autogen Bioclear, Wiltshire, UK) was then applied, followed by the addition of peroxidase-conjugated streptavidin. Antigen–antibody complexes were visualized utilizing 3,3-diaminobenzidine. Sections were also probed with an antibody specific for mast cell tryptase (Dako, Ely, UK). Flow cytometry. Staining and flow cytometric analyses of RASFs were performed using a FACScan (BD PharMingen, San Diego, CA) and standard procedures as described elsewhere (12). The RASFs (2 105 cells) were incubated with a negative control antibody (mAb anti-Thy1.2; Becton Dickinson) or phycoerythrin-conjugated anti–PAR-2 mAb (Mouse- Mono 344222; R&D Systems) in fluorescence-activated cell sorting (FACS) medium consisting of Hanks’ balanced salt solution (Nissui, Tokyo, Japan), 0.5% human serum albumin (Yoshitomi, Osaka, Japan), and 0.2% NaN3 (Sigma, St. Louis, MO) for 30 minutes at 4°C. After washing the cells 3 times with FACS medium, the fluorescence intensity was detected using a FACScan. Apoptosis assay. Apoptosis was evaluated by flow cytometry utilizing annexin V binding (Annexin V–Fluorescein Isothiocyanate Apoptosis Detection Kit I; Becton Dickinson). Briefly, RASFs were cultured under starved conditions for 24 hours with 1% DMEM, and were then incubated with or without CH11 (1 g/ml), tryptase (1–4 g/ml), and Y27632 (0.1–10 M; Calbiochem, La Jolla, CA) for 12 hours in DMEM containing 1% FCS. In some experiments, E11 fibroblasts (immortalized RASF cell line [13]) were treated with CH11 (1 g/ml) with or without tryptase (2 g/ml) and/or nafamostat mesylate (1 nM; Tocris Biosciences, Ellsville, MO) for 12 hours in DMEM containing 10% FCS. Cells were then stained with annexin V and propidium iodide (PI), according to the manufacturer’s instructions, and analyzed using a FACScan flow cytometer (Becton Dickinson). All PI-positive cells were considered dead. PI-negative and annexin V–positive cells were considered early apoptotic cells, and the remaining doublenegative cells were considered viable. Rho activation assay. Rho activation was determined with the use of a pull-down assay with GST-RBD beads (14,15). RASFs were stimulated with 0.1–2 g/ml tryptase, quickly washed with ice-cold Tris buffered saline, and lysed in 500 l of lysis buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 0.5M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 500 g/ml tosyl arginine methyl ester, and 10 g/ml each of leupeptin and aprotinin). Cell lysates were immediately centrifuged at 8,000 revolutions per minute at 4°C for 5 minutes, and equal volumes of lysates were incubated with 30 g GST-RBD beads for 1 hour at 4°C. The beads were washed twice with wash buffer (25 mM Tris, pH 7.5, 30 mM MgCl2, 40 mM 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–polyacrylamide electrophoresis gels, transferred to nitrocellulose, blocked with 5% nonfat milk, and analyzed by Western blotting using a polyclonal anti-Rho antibody. Statistical analysis. Results are expressed as the mean SD. Differences in comparison with the control group were examined for statistical significance by the Mann- Whitney U test. P values less than 0.01 were considered statistically significant. RESULTS Detection of mast cells in close proximity to PAR-2–expressing SFs in RA synovial tissue. Synovial tissue specimens were surgically removed from the joints of patients with RA, and the samples were used to investigate the localization of mast cells and SFs in the synovial tissue. Histologic examination of the tissue by hematoxylin and eosin staining revealed the presence of pannus, represented by detection of SFs, inflammatory cell infiltrates, and finer vessels (Figure 1A). Moreover, immunohistochemical staining of the tissue samples demonstrated numerous c-Kit–positive cells (Figure 1B) and tryptase-positive cells (Figure 1C), which are characteristic of mast cells, in the pannus and sublining area. This suggested that large numbers of mast cells are present in hyperplastic synovial tissue. Since we hypothesized that tryptase, a mast cell– specific protease, may be involved in the pathogenesis of RA, we next determined which cells could respond to tryptase, by examining the expression of PAR-2, the receptor for tryptase. PAR-2 was expressed in spindleshaped cells, most likely representing RASFs, which were present in the lining area (Figure 1D). Notably, in serial sections, PAR-2–expressing cells were found in close proximity to the area in which tryptase was expressed (Figure 1E). Similar results were obtained in synovial tissue samples from 5 other patients with RA (results not shown). To verify that PAR-2 is expressed on RA fibroblasts, SFs were isolated from the RA synovial tissue samples and PAR-2 expression was detected with the use of flow cytometry. Consistent with the findings on immunohistochemical analysis, flow cytometry revealed the expression of PAR-2 on isolated RASFs obtained from 5 separate RA patients (Figure 2), thus confirming that PAR-2 is expressed on RASFs. Inhibition of anti-Fas antibody–induced cell death by tryptase in RASFs. We previously reported that RASFs express Fas and are susceptible to Fasinduced cell death (3). Nevertheless, in the synovial tissue of RA patients, RASFs proliferate, rather than undergo apoptosis, suggesting that there might be a mechanism that prevents RASFs from undergoing apoptosis in situ. Given that mast cells lie in close proximity to RASFs and that RASFs express PAR-2, we questioned whether a mast cell–specific PAR-2 activator such as tryptase would suppress apoptosis induction in RASFs. To test this notion, RASFs were treated with or without anti-Fas antibody (CH11) in the presence or absence of tryptase, under starved conditions. As expected, cell death was morphologically apparent and increased 12 hours after incubation with CH11, as compared with that in cultures with untreated cells (Figure 3). In contrast, the addition of tryptase significantly inhibited such morphologic changes in the RASFs (Figure 3). To enumerate the proportion of live cells remaining in each well, the cells were removed from the wells with the use of trypsin, and live cells were counted using trypan blue exclusion. Compared with untreated cells, a significant decrease in the proportion of live cells was found in cells treated with CH11, which was reversed by the addition of tryptase (Figure 3). Of note, cell death was also observed in 50% of the untreated cells, which was attributable to the starved culture conditions necessary to make the cells more sensitive to CH11-induced apoptosis. To confirm the results obtained by trypan blue exclusion, we next used a flow cytometric approach involving annexin V and PI staining to detect apoptotic cells. The fraction of PIlowannexin Vhigh cells (early apoptotic) and PIhighannexin Vhigh cells (late apoptotic) increased after treatment of RASFs with CH11 (Figure 4A). Both early and late apoptosis were inhibited by tryptase in a concentration-dependent manner (Figure 4B). Of note, apoptosis of primary dermal fibroblasts could not be induced under the same conditions (results not shown), suggesting that this phenomenon might be specific to RASFs. Taken together, these results suggest that tryptase inhibits Fas-induced apoptosis in RASFs. We next tested whether tryptase inhibits Fasmediated apoptosis of fibroblasts via the activation of PAR-2. Since PAR-2 is activated by proteolytic cleavage of the receptor, we tested whether the addition of the protease inhibitor nafamostat mesylate would reverse the antiapoptotic effects of tryptase. As expected, treatment of fibroblasts with anti-Fas antibody resulted in cell death, and this was attenuated by the addition of tryptase (Figure 4C). However, the protective effect of tryptase was lost when the cells were cotreated with tryptase and nafamostat mesylate (Figure 4C), suggesting that the proteolytic function of tryptase and subsequent cleavage of PAR-2 is responsible for the antiapoptotic effects of tryptase against Fas-mediated apoptosis of fibroblasts. Involvement of Rho activation in the antiapoptotic effect of tryptase on RASFs. Activation of Rho, which is a low molecular weight G protein, is related to cell survival (16,17). We have previously reported that activation of Rho through the ligation of PAR-1 by thrombin promotes proliferation of RASFs (18). Since Rho also mediates downstream signaling of PAR-2 (19), the activation of Rho in tryptase-stimulated RASFs was evaluated. The activation of Rho was examined with the use of a pull-down assay for the detection of GTP-bound Rho (active form of GTPases) followed by Western blot analysis of the Rho protein. An increase in GTP-bound Rho was observed in RASFs after treatment with tryptase (Figure 5), suggesting that PAR-2 stimulation induces the activation of Rho. We then tested whether the activation of Rho is involved in the protection of RASFs against CH11- induced apoptosis. After the addition of CH11 to the RASF cultures, an increase in annexin Vhigh cells was observed, and this was again inhibited by tryptase (Figure 6A). The protective effect of tryptase was abrogated by the addition of a Rho kinase–specific inhibitor, Y27632, in a dose-dependent manner (Figures 6A and B). Similar results were obtained in RASFs from 5 separate RA patients (results not shown). Taken together, these data suggest that tryptase inhibits Fasinduced apoptosis of RASFs through a mechanism that involves Rho kinase. DISCUSSION Although numerous mast cells are present in RA synovial tissue, their involvement in the pathogenesis of RA remains unclear. In this report, we shed light on one potential mechanism by which mast cells may contribute to RA. Since tryptase is a protease that is specifically produced by mast cells and is considered one means by which mast cells can convey information to surrounding cells, we hypothesized that tryptase and its receptor, PAR-2, may play a role in RA pathogenesis. Through our studies, we have demonstrated that tryptaseexpressing mast cells lie in close proximity to RASFs in the synovial tissue of RA patients. Furthermore, RASFs express the receptor for mast cell tryptase (PAR-2) and are protected from Fas-mediated apoptosis by tryptase in a Rho kinase–dependent manner. Such a mechanism could play an important role in the marked proliferation of RASFs and hyperplasia seen in RA synovium, leading to disease progression. One reason that we focused on the effect of mast cell mediators on SF apoptosis was to yield insight into the apparently paradoxical finding that RASFs proliferate vigorously in vivo despite the high expression of Fas (2,3). Moreover, RASFs are readily susceptible to anti- Fas–mediated apoptosis in vitro (20). These findings suggest that a mechanism that prevents Fas-mediated cell death exists in RASFs, and that this excessive proliferation may contribute to disease pathogenesis. Indeed, we were able to demonstrate that RASFs isolated from 5 independent patients with RA exhibited apoptosis when incubated with anti-Fas antibody. However, Fas-mediated apoptosis was inhibited by a mast cell–specific protease, tryptase. Thus, we propose that the accumulation of mast cells in RA synovium creates an environment that is rich in tryptase and allows RASFs to counteract Fas-mediated killing. In fact, the interplay between RASFs and mast cells may be important for the maintenance of chronic inflammation in the RA synovium. The PARs represent a unique family of receptors that are activated by proteolytic cleavage (21). The ligand for these receptors is encoded in the N-terminal region of the receptor itself but is unable to bind until the N-terminus is cleaved at specific sites by serine proteases, such as thrombin and tryptase (22). The proteolytic cleavage of PARs creates a new N-terminus that can now bind to this G protein–coupled receptor, and subsequently activates the small G protein Rho. In our present study, several lines of evidence suggest that tryptase protects RASFs against Fas-mediated apoptosis through PAR-2. First, PAR-2 was found to be expressed both in vivo and ex vivo on RASFs. Second, the inhibition of the protease function of tryptase by the protease inhibitor nafamostat mesylate reversed the protective effect conferred by tryptase. Third, Rho was activated upon tryptase treatment of RASFs. Finally, the antiapoptotic effect of tryptase was abrogated by the addition of the Rho kinase inhibitor Y27632. We have previously reported that thrombin-mediated PAR-1 activation also allows RASF survival and proliferation through a similar mechanism (18), by inhibiting apoptosis through the activation of Rho (16,17). Taken together, these results suggest the possibility that a series of protease-mediated signals is important in the pathogenesis of RA. Obviously, RASFs are not the only cells that contribute to joint destruction and inflammation, since the pannus is a complex inflammatory granulation tissue (23) consisting of RASFs, vascular tissue, and an inflammatory cell infiltrate. However, it is noteworthy that PAR-2 is also expressed on other cells in the pannus, including vascular endothelial cells and inflammatory cells (24,25). Thus, it is tempting to speculate that mast cells also affect other cell types in the synovium of RA patients, through a tryptase/PAR-2– dependent mechanism. Our present findings are consistent with those from previous studies in animal models, in which it has been demonstrated that tryptase is involved in the pathogenesis of murine joint inflammation. For example, mice lacking monocyte chemotactic protein 6 (analogous to human tryptase) show resistance to antibodymediated arthritis (26), suggesting that mouse mast cell proteases play an important role in joint inflammation. Furthermore, the injection of tryptase directly into the joints of mice results in inflammation and swelling. However, this inflammation is not observed when tryptase is injected into PAR-2–deficient mice, suggesting that tryptase can cause joint inflammation through the activation of PAR-2 (27). Our study demonstrates a potential mechanism by which mast cells contribute to RA pathogenesis, through their communication with RASFs. Numerous mast cells reside in close proximity to PAR-2–expressing RASFs in the synovium of RA patients. We believe that the interaction of mast cell–associated tryptase and PAR-2 on RASFs inhibits the apoptosis of RASFs, causing hyperplasia of RA synovial tissue. This notion is consistent with the observation that, similar to neoplastic cells, RASFs multiply, and this occurs even though RASFs express high levels of Fas (2,3). Although further studies are required to test whether such interactions indeed occur in vivo, we propose that therapy aimed at inhibiting the mast cell/tryptase/PAR-2/Rho pathway may be a new treatment target for patients with RA. ACKNOWLEDGMENT The authors thank Ms T. Adachi for providing excellent technical assistance. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Tanaka had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Sawamukai, Tanaka. Acquisition of data. Sawamukai, Yukawa. Analysis and interpretation of data. Sawamukai, Saito, Nakayamada, Kambayashi. REFERENCES 1. Gabriel SE. Why do people with rheumatoid arthritis still die prematurely? Ann Rheum Dis 2008;67 Suppl 3:iii30–4. 2. Nakayamada S, Saito K, Fujii K, Yasuda M, Tamura M, Tanaka Y. 1 integrin–mediated signaling induces intercellular adhesion molecule 1 and Fas on rheumatoid synovial cells and Fas-mediated apoptosis. Arthritis Rheum 2003;48:1239–48. 3. 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