RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via toll‐like receptor 3

Objective

To assess the expression of Toll‐like receptor 3 (TLR‐3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR‐3 ligands.

Methods

TLR‐3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence‐activated cell sorting and real‐time polymerase chain reaction techniques. TLR‐3 signaling was assessed by incubating RASFs with poly(I‐C), lipopolysaccharide, palmitoyl‐3‐cysteine‐serine‐lysine‐4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon‐β (IFNβ), CXCL10, CCL5, and interleukin‐6 (IL‐6) protein production in the culture supernatants was performed by enzyme‐linked immunosorbent assays.

Results

TLR‐3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR‐3 expression was localized predominantly in the synovial lining, with a majority of the TLR‐3–expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR‐3 ligand poly(I‐C) resulted in the production of high levels of IFNβ, CXCL10, CCL5, and IL‐6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up‐regulation of these cytokines and chemokines in a TLR‐3–dependent manner.

Conclusion

Our findings demonstrate the expression of TLR‐3 in RA synovial tissue and the activation of RASFs in vitro by the TLR‐3 ligand poly(I‐C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR‐3 ligand for the stimulation of proinflammatory gene expression in RASFs.

     

Increased level of heme oxygenase-1 in rheumatoid arthritis synovial fluid

Abstract

We investigated the expression and localization of heme oxygenase-1 (HO-1) in synovial fluid and synovial tissue, and examined the stimulation of HO-1 production in rheumatoid synovial fibroblasts (RASFs). Synovial fluid samples were obtained from knee joints of 20 rheumatoid arthritis (RA) and 20 osteoarthritis (OA) patients, and concentration of HO-1 and matrix metalloproteinase-3 (MMP-3) were measured by enzyme-linked immunosorbent assay (ELISA). Synovial tissues obtained from RA or OA patients during total knee arthroplasty (TKA) were used for immunohistochemical analysis of HO-1. HO-1 production by RASFs in response to various cytokines was examined by ELISA. HO-1 levels in synovial fluid were higher in the RA group than in the OA group with significant difference (P < 0.001), and correlated with serum C-reactive protein (CRP) level (r = 0.80, P < 0.01) in the RA group. Higher levels of HO-1 were seen in the RA-L group (Larsen grade III–V) than in the RA-E (Larsen grade 0-II) group (P < 0.001). There was weak correlation between the levels of HO-1 protein and MMP-3 in synovial fluid in the RA group (r = 0.31, P < 0.01), while no positive correlation was observed in OA. Positive immunoreaction for HO-1 was observed in cells of synovial tissue including synovial fibroblasts and cells in synovial pannus. HO-1 protein levels in cultured media of RASFs were increased by stimulation by interleukin-1β at 6 h and tumor necrosis factor-alpha at 12 h, but suppressed by interferon-gamma at 12 and 24 h. These results indicated that HO-1 expression in synovial tissue might be stimulated by inflammatory cytokines. The correlation of HO-1 concentration in synovial fluid with serum CRP and MMP-3 in joint fluid indicated that HO-1 might be useful as a marker of joint inflammation in RA patients.     

Increased level of heme oxygenase-1 in rheumatoid arthritis synovial fluid

We investigated the expression and localization of heme oxygenase-1 (HO-1) in synovial fluid and synovial tissue, and examined the stimulation of HO-1 production in rheumatoid synovial fibroblasts (RASFs). Synovial fluid samples were obtained from knee joints of 20 rheumatoid arthritis (RA) and 20 osteoarthritis (OA) patients, and concentration of HO-1 and matrix metalloproteinase-3 (MMP-3) were measured by enzyme-linked immunosorbent assay (ELISA). Synovial tissues obtained from RA or OA patients during total knee arthroplasty (TKA) were used for immunohistochemical analysis of HO-1. HO-1 production by RASFs in response to various cytokines was examined by ELISA. HO-1 levels in synovial fluid were higher in the RA group than in the OA group with significant difference (P < 0.001), and correlated with serum C-reactive protein (CRP) level (r = 0.80, P < 0.01) in the RA group. Higher levels of HO-1 were seen in the RA-L group (Larsen grade III–V) than in the RA-E (Larsen grade 0-II) group (P < 0.001). There was weak correlation between the levels of HO-1 protein and MMP-3 in synovial fluid in the RA group (r = 0.31, P < 0.01), while no positive correlation was observed in OA. Positive immunoreaction for HO-1 was observed in cells of synovial tissue including synovial fibroblasts and cells in synovial pannus. HO-1 protein levels in cultured media of RASFs were increased by stimulation by interleukin-1β at 6 h and tumor necrosis factor-alpha at 12 h, but suppressed by interferon-gamma at 12 and 24 h. These results indicated that HO-1 expression in synovial tissue might be stimulated by inflammatory cytokines. The correlation of HO-1 concentration in synovial fluid with serum CRP and MMP-3 in joint fluid indicated that HO-1 might be useful as a marker of joint inflammation in RA patients.     

Induction of RANKL expression and osteoclast maturation by the binding of fibroblast growth factor 2 to heparan sulfate proteoglycan on rheumatoid synovial fibroblasts

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by progressive joint destruction. The aim of this study was to clarify the relevance of RA synovial fibroblasts (RASFs) and fibroblast growth factor 2 (FGF-2), which is produced abundantly by RASFs, to the osteoclastogenesis and bone resorption in RA. METHODS: Synovial fibroblasts were prepared from the synovial tissues of 10 patients with active RA and 7 patients with osteoarthritis (OA). The expression of RANKL, intercellular adhesion molecule 1 (ICAM-1), FGF receptor 1 (FGFR-1), and heparan sulfate proteoglycan (HSPG) on synovial fibroblasts was measured by FACScan. Osteoclast formation in cocultures of RASFs and peripheral blood mononuclear cells (PBMCs) was evaluated by tartrate-resistant acid phosphatase staining and a pit-formation assay using dentin slices. RESULTS: FGF-2 induced the expression of both RANKL and ICAM-1 on RASFs more so than on OA synovial fibroblasts (OASFs). FGF-2-induced up-regulation of RANKL and ICAM-1 was inhibited by anti-FGF-2 antibody. Although FGFR-1 was equally expressed on RASFs and OASFs, HSPG was highly expressed on RASFs. Up-regulation of RANKL by FGF-2 on RASFs was diminished by the removal of heparan sulfate with heparitinase. Osteoclast formation from PBMCs induced by RASFs was inhibited by the addition of either heparitinase, anti-ICAM-1 antibody, anti-FGF-2 antibody, or osteoprotegerin. FGF-2-induced RANKL on RASFs and osteoclast formation were suppressed by an inhibitor of ERK. CONCLUSION: FGF-2 was transferred to FGFR-1 through binding to HSPG, which is characteristically expressed on RASFs, resulting in RANKL- and ICAM-1-mediated maturation of osteoclasts via ERK activation. Thus, we propose that FGF-2 not only augments the proliferation of RASFs, but also is involved in osteoclast maturation, which leads to bone destruction in RA.     

CC motif chemokine ligand 13 is associated with rheumatoid arthritis pathogenesis

Objectives

CC motif chemokines are considered to be implicated in the pathogenesis of rheumatoid arthritis (RA) via recruitment of monocytes and lymphocytes. CC motif chemokine ligand 13 (CCL13)/monocyte chemoattractant protein-4 (MCP-4) is postulated to be a potent RA inducer. We conducted a study to more precisely clarify the role of CCL13 in RA pathogenesis.

Methods

CCL13 expression was evaluated by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining in serum samples and synovial tissues from RA patients. The effects of CCL13 against apoptosis were monitored on cultured synovial fibroblasts. The chemoattractant activity of CCL13 was evaluated by the Boyden chamber assay in monocytes (THP-1 cells) and human umbilical vein endothelial cells (HUVECs).

Results

We found that CCL13 serum level and synovial tissue expression were increased in RA patients. CCL13 had chemoattractant activity for both THP-1 cells and HUVECs. Interestingly, CCL13 expression was positively regulated by tumor necrosis factor-alpha (TNF-α). Furthermore, apoptosis induced by hydrogen peroxide (H₂O₂) and serum deprivation was inhibited by CCL13 on the cultured synovial fibroblasts.

Conclusions

CCL13 may be associated with disease progression as a result of its antiapoptotic effects, increased macrophage infiltration, and synovial tissue angiogenesis in RA patients.     

Expression of interleukin-21 receptor, but not interleukin-21, in synovial fibroblasts and synovial macrophages of patients with rheumatoid arthritis

OBJECTIVE: To determine the role and expression of the cytokine/receptor pair interleukin-21 (IL-21)/IL-21 receptor (IL-21R) in rheumatoid arthritis (RA). METHODS: The expression of IL-21R and IL-21 was analyzed by TaqMan real-time polymerase chain reaction (PCR) and in situ hybridization of synovial biopsy samples from patients with RA and osteoarthritis (OA). Double labeling by immunohistochemistry after in situ hybridization was performed with anti-CD68 antibodies. The expression of IL-21R at the protein level was confirmed by Western blotting. Stimulation experiments were performed with recombinant IL-1beta, tumor necrosis factor alpha (TNFalpha), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGFbeta). The role of IL-21R in cartilage destruction was analyzed in the SCID mouse coimplantation model of RA. RESULTS: IL-21R was found in total RNA extracts and in synovial biopsy samples from RA patients, whereas no expression or only minimal expression was seen in samples from OA patients. Double labeling indicated that both synovial macrophages and synovial fibroblasts expressed IL-21R. Western blotting with anti-IL-21R antibodies confirmed the expression of IL-21R protein in RA synovial fibroblasts (RASFs). Of note, IL-21 was not detectable by real-time PCR and in situ hybridization in the same samples in vivo as in vitro. The level of expression of IL-21R messenger RNA (mRNA) was not altered by stimulation with IL-1beta, TNFalpha, PDGF, or TGFbeta. Interestingly, in the SCID mouse coimplantation model, RASFs did not maintain their expression of IL-21R at sites of invasion into the cartilage. Similarly, IL-21R mRNA was not expressed at sites of invasion into cartilage and bone in RA synovium. CONCLUSION: Our data demonstrate that IL-21R is expressed in RA synovium by RASFs and synovial macrophages. IL-21R is associated with the activated phenotype of RASFs independently of the major proinflammatory cytokines IL-1beta and TNFalpha, but correlates negatively with the destruction of articular cartilage and bone.     

微小RNA-146a对类风湿关节炎患者滑膜成纤维细胞抑制作用的研究

目的 研究微小RNA-146a(miR-146a)对类风湿关节炎(RA)患者滑膜成纤维细胞增殖及细胞因子分泌的影响.方法 体外分离培养RA患者滑膜成纤维细胞,脂质体转染化学合成的miR-146a,~3H掺入法检测滑膜成纤维细胞增殖.酶联免疫吸附试验(ELISA)法检测细胞上清白细胞介素(IL)-8及IL-6水平.实时定量聚合酶链反应(PCR)检测滑膜成纤维细胞中miR-146a靶分子肿瘤坏死因子受体相关因子6(TRAF6)、IL-受体相关激酶(IRAKI)的mRNA水平.应用独立样本t检验进行统计学分析.结果 与阴性对照小RNA相比,miR-146a转染后的滑膜成纤维细胞增殖能力明显下降(2015±545与8799±1922,P<0.01),IL-8及IL-6分泌受到明显抑制[(153±49)pg/ml与(311±123)pg/ml,P<0.01和(295±95)pg/ml与(459±126)pg/ml,P<0.05].定量PCR检测IRAK1的mRNA水平显示,miR-146a转染可明显下调滑膜成纤维细胞IRAK1的表达(0.28±0.07与1,P<0.01).结论 miR-146a可能通过下调IRAKI表达,进而抑制滑膜成纤维细胞增殖及IL-8、IL-6等炎性细胞因子分泌,从而发挥抑制RA滑膜炎症的作用.     

Simvastatin inhibits cysteine‐rich protein 61 expression in rheumatoid arthritis synovial fibroblasts through the regulation of sirtuin‐1/FoxO3a signaling

Objective

To examine the role of sirtuin‐1 (SIRT‐1)/FoxO3a in the expression of cysteine‐rich protein 61 (CYR‐61) in rheumatoid arthritis synovial fibroblasts (RASFs) and the influence of simvastatin on this pathway, and to determine the relationship between disease progression and FoxO3a/CYR‐61 signaling in synovial fibroblasts in vivo using a rat model of collagen‐induced arthritis (CIA).

Methods

In RASFs, the expression of CYR‐61 and SIRT‐1, the localization of FoxO3a in the nucleus/cytoplasm, and the phosphorylation/acetylation of FoxO3a were examined by Western blotting. Secretion of CCL20 was assessed by enzyme‐linked immunosorbent assay. Promoter activity of the Cyr61 gene was evaluated by luciferase assay, with or without forced expression of FoxO3a and SIRT‐1 by lentiviral transduction. FoxO3a–Cyr61 promoter interaction was examined by chromatin immunoprecipitation. In rats with CIA, the expression of CYR‐61 and phosphorylated FoxO3a in synovial fibroblasts was examined by immunohistochemistry.

Results

In RASFs, simvastatin suppressed the tumor necrosis factor α (TNFα)–induced production of CYR‐61 and CCL20. Nuclear levels of FoxO3a were decreased after TNFα stimulation of RASFs, and forced expression of FoxO3a reversed the inductive effects of TNFα on CYR‐61. Simvastatin inhibited the nuclear export, phosphorylation, and acetylation of FoxO3a and maintained its binding to the Cyr61 promoter. Forced expression of SIRT‐1 in RASFs led to decreased levels of CYR‐61 and deacetylation of FoxO3a. Following treatment with simvastatin, the expression of SIRT‐1 was up‐regulated and SIRT‐1/FoxO3a binding was enhanced in RASFs. In rats with CIA, intraarticular injection of simvastatin alleviated arthritis and suppressed CYR‐61 expression and FoxO3a phosphorylation in synovial fibroblasts.

Conclusion

CYR‐61 is important in the pathogenesis of RA, and SIRT‐1/FoxO3a signaling is crucial to induction of CYR‐61 in RASFs. Simvastatin plays a beneficial role in inflammatory arthritis through its up‐regulation of SIRT‐1/FoxO3a signaling in synovial fibroblasts. Continued study of the pathways linking sirtuins, FoxO proteins, and the inflammatory responses of RASFs may provide new insights into the pathophysiology of RA.

     

Evaluation of SV40-transformed synovial fibroblasts in the study of rheumatoid arthritis pathogenesis

The SV40 T antigen has been used to generate immortalized cells from rheumatoid arthritis (RA) synovial fibroblasts (RASFs) that are commonly used in lieu of primary RASFs. In the current study, we investigated the effect of stimulation by tissue necrosis factor alpha (TNF-α) and interleukin 17 (IL-17) on primary and immortalized RASFs in order to gauge the appropriateness of the use of immortalized RASFs, the MH7A cell line, in the study of RA pathogenesis. Changes in the levels of secretion and expression of 8 proteins associated with RA upon stimulation were assessed by multiplex immunoassay. IL-17 stimulation had a minimal impact on protein secretion and expression for primary and immortalized cells. Basic fibroblast growth factor (FGF-2) was not detectable for the primary cells but was detectable for the immortalized cells. In contrast, monocyte chemoattractant protein 1 (MCP-1) was detectable for primary cells but was undetectable for immortalized cells. In general, protein expression and secretion by cells stimulated with TNF-α were significantly increased. For primary cells, several proteins were below the limit of detection for unstimulated cells and cells stimulated with IL-17, while levels for TNF-α-stimulated cells were within the detectable range. For the same proteins, expression was observed for immortalized cells, regardless of stimulation, suggestive of constitutive activation of the NF-κB signaling pathway. The current study therefore provides strong evidence that immortalized and primary RASFs differ in regard to protein expression and secretion and therefore may not be appropriate for use in the study of RA pathogenesis.     

Duffy antigen receptor for chemokines and CXCL5 are essential for the recruitment of neutrophils in a multicellular model of rheumatoid arthritis synovium

OBJECTIVE: The role of chemokines and their transporters in rheumatoid arthritis (RA) is poorly described. Evidence suggests that CXCL5 plays an important role, because it is abundant in RA tissue, and its neutralization moderates joint damage in animal models of arthritis. Expression of the chemokine transporter Duffy antigen receptor for chemokines (DARC) is also up-regulated in early RA. The aim of this study was to investigate the role of CXCL5 and DARC in regulating neutrophil recruitment, using an in vitro model of RA synovium. METHODS: To model RA synovium, RA synovial fibroblasts (RASFs) were cocultured with endothelial cells (ECs) for 24 hours. Gene expression in cocultured cells was investigated using TaqMan gene arrays. The roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, in which their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents. RESULTS: EC-RASF coculture induced chemokine expression in both cell types. Although the expression of CXC chemokines was modestly up-regulated in ECs, the expression of CXCL1, CXCL5, and CXCL8 was greatly increased in RASFs. RASFs also promoted the recruitment of flowing neutrophils to ECs. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on ECs or when used to immunodeplete coculture-conditioned medium. DARC was also induced on ECs by coculture, and anti-Fy6 antibody or small interfering RNA targeting of DARC expression effectively abolished neutrophil recruitment. CONCLUSION: This study is the first to demonstrate, in a model of human disease, that the function of DARC is essential for editing the chemokine signals presented by ECs and for promoting unwanted leukocyte recruitment.     

Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll-like receptor signaling

OBJECTIVE: To test the hypothesis that bacterial products acting as adjuvants, such as CpG oligodeoxynucleotides (ODNs) and peptidoglycans (PGs), are able to activate synoviocytes, and to determine the involvement of Toll-like receptors (TLRs) in this activation process. METHODS: Cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were stimulated with CpG ODNs or PGs. The expression of various integrins was determined by fluorescence-activated cell sorting. TLR and matrix metalloproteinase (MMP) messenger RNA (mRNA) was measured by real-time polymerase chain reaction. Additionally, levels of interleukin-6 (IL-6) and IL-8 in the culture supernatants were assessed by enzyme-linked immunosorbent assay. Blocking experiments were performed by adding anti-TLR-2 and anti-TLR-4 monoclonal antibodies to cultures stimulated with bacterial PGs. RESULTS: Incubation of synovial fibroblasts with CpG ODNs resulted in neither up-regulation of the expression of integrins on the cell surface, up-regulation of MMP mRNA expression, nor IL-6 and IL-8 production. However, incubation of RA synovial fibroblasts as well as OA synovial fibroblasts with staphylococcal PGs led to an up-regulation of CD54 (ICAM-1) surface expression and to increased expression of MMP-1, MMP-3, and MMP-13 mRNA. Furthermore, production of the proinflammatory cytokines IL-6 and IL-8 was increased by treatment with PGs. We demonstrated that cultured synovial fibroblasts express low levels of TLR-2 and TLR-9 mRNA. TLR-2 was up-regulated after stimulation with PGs, whereas TLR-9 mRNA remained at baseline levels after stimulation with CpG ODNs. Anti-TLR-2 monoclonal antibodies significantly inhibited production of IL-6 and IL-8 induced by stimulation with PGs. CONCLUSION: We demonstrate that bacterial PGs activate synovial fibroblasts, at least partially via TLR-2, to express integrins, MMPs, and proinflammatory cytokines. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction.     

Inhibition of fibroblast activation protein and dipeptidylpeptidase 4 increases cartilage invasion by rheumatoid arthritis synovial fibroblasts

Objective

Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP‐4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage.

Methods

Expression of FAP and DPP‐4/CD26 by RASFs was analyzed using fluorescence‐activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L‐glutamyl L‐boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real‐time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell–derived factor 1 (SDF‐1 [CXCL12]), MMP‐1, and MMP‐3 protein levels were measured using enzyme‐linked immunosorbent assay. The impact of FAP and DPP‐4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry.

Results

Inhibition of serine protease activity of FAP and DPP‐4/CD26 in vitro led to increased levels of SDF‐1 in concert with MMP‐1 and MMP‐3, which are downstream effectors of SDF‐1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP‐expressing RASFs and marked by a significantly higher accumulation of MMP‐1 and MMP‐3, when compared with controls.

Conclusion

Our results indicate a central role for the serine protease activity of FAP and DPP‐4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.

     

Mast cell–derived tryptase inhibits apoptosis of human rheumatoid synovial fibroblasts via rho‐mediated signaling

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. Protease‐activated 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. Fas‐mediated 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.