Activation of fibroblast-like synoviocytes derived from rheumatoid arthritis via lysophosphatidic acid–lysophosphatidic acid receptor 1 cascade

Introduction Lysophosphatidic acid (LPA) is a bioactive lipid that binds to G protein–coupled receptors (LPA1–6). Recently, we reported that abrogation of LPA receptor 1 (LPA1) ameliorated murine collagen-induced arthritis, probably via inhibition of inflammatory cell migration, Th17 differentiation and osteoclastogenesis. In this study, we examined the importance of the LPA–LPA1 axis in cell proliferation, cytokine/chemokine production and lymphocyte transmigration in fibroblast-like synoviocytes (FLSs) obtained from the synovial tissues of rheumatoid arthritis (RA) patients. Methods FLSs were prepared from synovial tissues of RA patients. Expression of LPA1–6 was examined by quantitative real-time RT-PCR. Cell surface LPA1 expression was analyzed by flow cytometry. Cell proliferation was analyzed using a cell-counting kit. Production of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), chemokine (C-C motif) ligand 2 (CCL2), metalloproteinase 3 (MMP-3) and chemokine (C-X-C motif) ligand 12 (CXCL12) was measured by enzyme-linked immunosorbent assay. Pseudoemperipolesis was evaluated using a coculture of RA FLSs and T or B cells. Cell motility was examined by scrape motility assay. Expression of adhesion molecules was determined by flow cytometry. Results The expression of LPA1 mRNA and cell surface LPA1 was higher in RA FLSs than in FLSs from osteoarthritis tissue. Stimulation with LPA enhanced the proliferation of RA FLSs and the production of IL-6, VEGF, CCL2 and MMP-3 by FLSs, which were suppressed by an LPA1 inhibitor (LA-01). Ki16425, another LPA1 antagonist, also suppressed IL-6 production by LPA-stimulated RA FLSs. However, the production of CXCL12 was not altered by stimulation with LPA. LPA induced the pseudoemperipolesis of T and B cells cocultured with RA FLSs, which was suppressed by LPA1 inhibition. In addition, LPA enhanced the migration of RA FLSs and expression of vascular cell adhesion molecule and intercellular adhesion molecule on RA FLSs, which were also inhibited by an LPA1 antagonist. Conclusions Collectively, these results indicate that LPA–LPA1 signaling contributes to the activation of RA FLSs.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia with proliferation of fibroblast-like synoviocytes (FLSs), angiogenesis, infiltration of inflammatory cells such as lymphocytes and macrophages, and bone destruction of multiple joints [1]. FLSs are especially responsible for inflammation through cytokine and chemokine production and are also key cells of the invasive synovium, suggesting that they play a major role in the initiation and perpetuation of the destruction of inflamed joints [2].
It has previously been shown that expression of ATX by FLSs in the RA synovium and concentration of ATX in the RA synovial fluid are increased [12]. In addition, LPA 1-3 mRNA has been reported to be expressed in RA FLSs, and incubation with LPA induced cell motility and cytokine expression by the FLSs, indicating that LPA may contribute to the pathogenesis of RA by stimulation of FLSs [13,14]. We recently demonstrated that treatment with an LPA receptor 1 (LPA 1 ) antagonist, LA-01, ameliorated murine collagen-induced arthritis, probably via inhibition of inflammatory cell migration, Th17 differentiation and osteoclastogenesis [15].
In this study, we extensively analyzed the stimulatory effects of LPA for RA FLSs, as well as the effects of an LPA 1 antagonist, LA-01, against this stimulation.

Specimens
Synovial tissues were obtained from RA patients (n = 10) who fulfilled American College of Rheumatology criteria [16] and from patients with osteoarthritis (OA) (n = 5). RA patients were a median (range) of 67 years old (45 to 80), and had a disease duration of 14 years (2 to 30) and C-reactive protein level of 0.68 mg/dl (0.0 to 2.85). Seven patients (70%) were positive for rheumatoid factor, and seven (70%) were positive for anticitrullinated protein antibodies. All patients provided informed consent. The experimental protocol was approved by the ethics committee of the Tokyo Medical and Dental University.

Fibroblast-like synoviocytes
Synovial tissues from RA patients were minced and incubated with 0.5 mg/ml collagenase (Sigma-Aldrich, St Louis, MO, USA) for 1 hour at 37°C, then passed through a metal screen to obtain single-cell suspensions. Harvested cells were plated in cell culture plates and incubated with Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich). Adherent cells were maintained in the medium as FLSs and were used after five passages in the following experiments [17].

Pseudoemperipolesis
FLSs were seeded onto 96-well plates (2 × 10 4 cells/well) and cultured for 48 hours. CD4-and CD8-positive (CD4 + and CD8 + , respectively) T cells and CD19 + B cells were purified from human peripheral blood of healthy volunteers by using MACS microbeads (>95% purity; Miltenyi Biotec, Auburn, CA, USA) and added to the FLS-cultured wells (1 × 10 5 cells/well). The cells were treated with LA-01 (0, 1 or 10 nM) for 30 minutes, followed by stimulation with LPA (10 μM) in FCS-free DMEM. After 12 hours, the wells were washed three times with medium. Pseudoemperipolesis was assessed by counting the number of cells beneath FLSs in three independent fields under a microscope.
Scrape motility assay RA FLSs were plated at a density of 1 × 10 5 cells/ml in 12-well plates in DMEM with 10% FCS. After overnight incubation, FLSs was washed twice with FCS-free medium. The tip of a plastic pipette was drawn across the center of the well to produce a scraped area. Culture wells were washed twice with PBS, and free cells were removed. After pretreatment with LA-01 (0, 1 or 10 nM) for 30 minutes, cells were incubated with LPA (10 μM) in FCS-free DMEM. A cell-free area was measured by using ImageJ software (National Institutes of Health, Bethesda, MD, USA) at 0 and 48 hours, and the ratio was then calculated (cell-free area at 48 hours per cell-free area at 0 hours).

Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM). The comparison of the data from the two groups was conducted by using Student's t-test. P-values less than 0.05 were considered significant.

Expression of lysophosphatidic acid receptors in RA fibroblast-like synoviocytes
The expression of LPA 1-6 mRNA in FLSs from RA and OA patients was analyzed by quantitative real-time RT-PCR. The expression of LPA 1 mRNA in RA FLSs was significantly higher than that in OA FLSs ( Figure 1A). The expression of LPA 3 and LPA 4 was also significantly higher in RA FLSs than that in OA FLSs, although the ratios of LPA 3   the expression level was substantially higher than that of OA FLSs ( Figure 1B).

Lysophosphatidic acid receptor 1 inhibitor suppressed lysophosphatidic acid-induced proliferation and cytokine production in RA fibroblast-like synoviocytes
We analyzed the effects of LPA on the proliferation and production of inflammatory mediators by RA FLSs. Stimulation with LPA dose-dependently induced the proliferation of FLSs ( Figure 1C). LPA stimulation also induced the production of IL-6 and CCL2 from FLSs in a dose-dependent manner ( Figures 1E and 1G), which supports a previous report that LPA upregulated IL-6 mRNA expression by RA FLSs [18]. Stimulation with LPA also induced the production of VEGF and MMP-3 by RA FLSs in vitro (Figures 1I and 1J).
Next, we analyzed the effect of an LPA 1 inhibitor on LPA stimulation for RA FLSs. Enhanced cell proliferation by 10 μM LPA was significantly suppressed by LA-01, the LPA 1 -selective antagonist ( Figure 1D). The treatment with LA-01 significantly reduced the production of IL-6, CCL2, VEGF and MMP-3 by LPA-stimulated RA FLSs ( Figures 1F and 1H through 1J). In contrast, the production of CXCL12 by RA FLSs was not altered by stimulation with LPA ( Figure 1K). We used Ki16425, another LPA 1 antagonist, to confirm the effects of LPA 1 inhibition on IL-6 production from LPA-stimulated RA FLSs. Incubation with Ki16425 suppressed IL-6 production from LPA-stimulated RA FLSs as well as LA-01 (IL-6 concentrations: vehicle = 299.413 ± 28.084 pg/ml; Ki16425 = 116.785 ± 11.162 pg/ml (P < 0.05 vs vehicle); LA-01 = 145.715 ± 15.921 pg/ml (P < 0.05 vs vehicle)). These results suggest that LPA-LPA 1 signaling plays important roles in proliferation and cytokine production of RA FLSs in vitro.
LPA-LPA 1 signaling promoted pseudoemperipolesis RA FLSs have been shown to promote the spontaneous migration of leukocytes beneath them, a process termed pseudoemperipolesis [21]. We examined the effect of LPA on pseudoemperipolesis. Stimulation with 10 μM LPA significantly increased the number of CD4 + and CD8 + T cells, as well as CD19 + B cells, beneath RA FLSs (Figures 2A to 2F). Moreover, incubation with LA-01 suppressed the LPA-enhanced pseudoemperipolesis of CD4 + and CD8 + T and CD19 + B cells (Figures 2A  through 2F), suggesting that interaction of LPA and LPA 1 promotes pseudoemperipolesis of leukocytes.

LPA-LPA 1 signaling promoted cell motility of RA fibroblast-like synoviocytes
We also analyzed the effect of LPA 1 on RA FLS migration by scrape motility assay. Incubation with 10 μM LPA significantly decreased the cell-free area, indicating that LPA induced cell migration in vitro ( Figures 3A and 3B), as reported previously [22]. In addition, LA-01 significantly increased the cell-free area of RA FLSs ( Figures 3A and 3B), suggesting that LPA-LPA 1 signaling also contributes to the promotion of RA FLS motility.

LPA-LPA 1 signaling induced adhesion molecule expression on RA fibroblast-like synoviocytes
It has been reported that signaling from VCAM and ICAM in RA FLSs supports pseudoemperipolesis [21]. Therefore, we next analyzed the expression of VCAM and ICAM on RA FLSs by flow cytometry. We found that stimulation with 10 μM LPA induced the expression of VCAM and ICAM on RA FLSs (Figure 4). Moreover, LA-01 decreased the expression of VCAM and ICAM induced by LPA on RA FLSs ( Figure 4). However, the expression of E-selectin on RA FLSs was not altered by LPA simulation (data not shown).

Discussion
In this study, we found that LPA 1 was highly expressed in RA FLSs. LPA stimulated RA FLSs to enhance proliferation, production of inflammatory mediators, pseudoemperipolesis, migration and the expression of adhesion molecules, which are attributable to signaling through LPA 1 .
RA FLSs express inflammatory cytokines, chemokines and matrix-degrading enzymes, which contribute to the pathogenesis of RA. LPA has been reported to induce IL-6 mRNA expression on RA FLSs, as well as cell motility [13]. However, the corresponding LPA receptor on RA FLSs has not been identified. We show that LPA augmented IL-6, CCL2, VEGF and MMP-3 production by RA FLSs. Moreover, the LPA-induced production of the inflammatory mediators was inhibited by a LPA 1selective inhibitor. Therefore, the LPA-LPA 1 cascade plays an important role in cytokine, chemokine and matrixdegrading enzyme production by RA FLSs. Although IC 50 of LA-01 was 86 nM, which was determined by using LPA 1 -transfected CHO cells, 10 nM LA-01 significantly inhibited stimulation of LPA in RA FLSs. The IC 50 may be dependent on cell type or on the expression level of LPA 1 .
Pseudoemperipolesis contributes to the chronic inflammation induced by lymphocyte recruitment in the inflamed joints and protects lymphocytes from apoptosis [21,23,24]. We show that LPA enhanced the pseudoemperipolesis of T and B cells, which is also attributable to LPA 1 . It has been reported that stimulation with CXCL12 and signaling from VCAM and ICAM in RA FLSs support pseudoemperipolesis [21]. Our results indicate that LPA upregulated the expression of VCAM and ICAM on RA FLSs, which was blocked by the LPA 1 antagonist. Thus, LPA may enhance pseudoemperipolesis via the upregulation of VCAM and ICAM expression on RA FLSs through LPA 1 . Interestingly, CXCL12 production by RA FLSs was not altered by LPA simulation. Stimulation of lymphocytes by LPA via LPA 1 may also contribute to the enhanced pseudoemperipolesis. In this regard, it has been reported that LPA induced chemokinesis in T cells [25] and lymphocyte transmigration through high endothelial venules [26,27]. Further studies are needed to clarify the effects of LPA-LPA 1 signaling for the lymphocytes on pseudoemperipolesis.
The hyperplastic rheumatoid pannus is characterized by an overabundance of FLSs [2]. This cellular excess stems largely from an imbalance between the proliferation and apoptosis of FLSs [2]. The migration of RA FLSs may also contribute to pannus formation [2]. Our results show that LPA induced the proliferation and migration of FLSs, which was inhibited by the LPA 1 antagonist. Moreover, in a recent study, researchers reported that LPA suppressed tumor necrosis factor-induced apoptosis on RA FLSs via LPA 1 [28]. Therefore, it is suggested that the LPA-LPA 1 signaling also contributed to the cellular excess and migration of FLSs in the RA synovium.
It was shown that conditional genetic ablation of ATX, which generates LPA via hydrolysis of lysophosphatidylcholine, in mesenchymal cells resulted in disease attenuation in animal models of arthritis [12]. We have also found that LPA 1 is essential for the development of arthritis in collagen-induced arthritis [15]. The ATX-LPA-LPA 1 axis may play an important role in the development of arthritis.

Conclusion
Our study suggests that LPA-LPA 1 signaling in FLSs may contribute to the pathogenesis of RA by inducing proliferation, production of inflammatory mediators, pseudoemperipolesis and migration on RA FLSs. Thus, LPA 1 could be a promising therapeutic target for RA.