Expression of interleukin-18 receptor in fibroblast-like synoviocytes

An excess of the proinflammatory substance IL-18 is present in joints of patients with rheumatoid arthritis (RA), and expression of IL-18 receptor (IL-18R) regulates IL-18 bioactivity in various cell types. We examined the expression of IL-18R alpha-chain and beta-chain and the biologic effects of IL-18 in fibroblast-like synoviocytes (FLS) after long-term culture. The presence of both IL-18R chains was a prerequisite for IL-18 signal transduction in FLS. However, all FLS cultures studied were either resistant or barely responsive to IL-18 stimulation as regards cell proliferation, expression of adhesion molecules ICAM-1 and vascular cell adhesion molecule (VCAM)-1, and the release of interstitial collagenase and stromelysin, IL-6 and IL-8, prostaglandin E2, or nitric oxide. We conclude that the presence of macrophages or IL-18R+ T cells that can respond directly to IL-18 is essential for the proinflammatory effects of IL-18 in synovitis in RA.     

Fibroblast-like synoviocytes from rheumatoid arthritis patients express functional IL-15 receptor complex: endogenous IL-15 in autocrine fashion enhances cell proliferation and expression of Bcl-x(L) and Bcl-2

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Ralpha at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rbeta and common gamma-chains. Interestingly, TNF-alpha or IL-1beta triggered significant elevation of IL-15Ralpha chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-x(L) expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-x(L). To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcgamma2a protein exerting properties of specific antagonist to the IL-15Ralpha chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-x(L), and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.     

Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis

Despite its potent ability to inhibit proinflammatory cytokine synthesis, interleukin (IL)-10 has a marginal clinical effect in rheumatoid arthritis (RA) patients. Recent evidence suggests that IL-10 induces monocyte/macrophage maturation in cooperation with macrophage-colony stimulating factor (M-CSF). In the present study, we found that the inducible subunit of the IL-10 receptor (IL-10R), type 1 IL-10R (IL-10R1), was expressed at higher levels on monocytes in RA than in healthy controls, in association with disease activity, while their expression of both type 1 and 2 tumour necrosis factor receptors (TNFR1/2) was not increased. The expression of IL-10R1 but not IL-10R2 was augmented on monocytes cultured in the presence of RA synovial tissue (ST) cell culture supernatants. Cell surface expression of TNFR1/2 expression on monocytes was induced by IL-10, and more efficiently in combination with M-CSF. Two-color immunofluorescence labeling of RA ST samples showed an intensive coexpression of IL-10R1, TNFR1/2, and M-CSF receptor in CD68⁺ lining macrophages. Adhered monocytes, after 3-day preincubation with IL-10 and M-CSF, could produce more IL-1β and IL-6 in response to TNF-α in the presence of dibutyryl cAMP, as compared with the cells preincubated with or without IL-10 or M-CSF alone. Microarray analysis of gene expression revealed that IL-10 activated various genes essential for macrophage functions, including other members of the TNFR superfamily, receptors for chemokines and growth factors, Toll-like receptors, and TNFR-associated signaling molecules. These results suggest that IL-10 may contribute to the inflammatory process by facilitating monocyte differentiation into TNF-α-responsive macrophages in the presence of M-CSF in RA.     

MiRNA-126 expression inhibits IL-23R mediated TNF-α or IFN-γ production in fibroblast-like synoviocytes in a mice model of collagen-induced rheumatoid arthritis

Both miR-126 and IL-23R affect rheumatoid arthritis (RA) procession. This study aimed to investigate the association of miR-126 and IL-23R and the possible modulation of miR-126 to RA pathogenesis. Serum, synovial tissue and synovial fluid were collected from patients with RA, and expression of miR-126, IL-23R, TNF-α and IFN-γ were detected. Fibroblast-like synoviocytes (FLS) was established using a collagen-induced arthritis mice model. The expression of miR-126 was manual intervened using pro-miR-126 and anti-miR-126 encoding lentivirus plasmids, or miR-126 agonists and corresponding negative controls. MiR-126 expression was inhibited in RA patients when compared with controls (P?<?0.05). TNF-α and IFN-γ production and IL-23R expression were significantly upregulated in RA patients when compared to controls (P?<?0.05). In pro-miR-126 treated FLS cells, the administration of pro-miR-126 plasmids upregulated miR-126, but inhibited IL-23R, TNF-α and IFN-γ expression or production. Moreover, the miR-126 agonist reversed the effects of the anti-miR-126 plasmid on FLS. These results revealed that miR-126 negative regulated the expression of IL-23R, TNF-α and IFN-γ. These results suggest the key impact of miR-126 on RA procession. Moreover, pro-miR-126 might be explored to be a potential therapy for RA.     

Cyr61 induces IL-6 production by fibroblast-like synoviocytes promoting Th17 differentiation in rheumatoid arthritis

Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17-dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvβ5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4(+) T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.     

A novel role for interleukin-18 in adhesion molecule induction through NF kappa B and phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathways

Interleukin-18 (IL-18) is a novel proinflammatory cytokine found in serum and joints of patients with rheumatoid arthritis (RA). We studied a novel role for IL-18 in mediating cell adhesion, a vital component of the inflammation found in RA and other inflammatory diseases. We examined the expression of cellular cell adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and RA synovial fibroblasts using flow cytometry. Adhesion of the monocyte-like cell line HL-60 to endothelial cells was determined by immunofluorescence. IL-18 significantly enhanced ICAM-1 and VCAM-1 expression on endothelial cells and RA synovial fibroblasts. In addition, IL-18 induced E-selectin expression on endothelial cells and promoted the adhesion of HL-60 cells to IL-18-stimulated endothelial cells. Neutralizing anti-VCAM-1 and anti-E-selectin could completely inhibit HL-60 adherence to endothelial cells. IL-18-induced adhesion molecule expression appears to be mediated through nuclear factor kappa B (NF kappa B) and phosphatidyl-inositol 3 kinase (PI 3-kinase) since addition of inhibitors to either NF kappa B (pyrrolidine dithiocarbamate and N-acetyl-l-cysteine) or PI 3-kinase (LY294002) inhibited RA synovial fibroblast VCAM-1 expression by 50 to 60%. Addition of both inhibitors resulted in inhibition of VCAM-1 expression by 85%. In conclusion, the ability of IL-18 to induce adhesion molecule expression on endothelial cells and RA synovial fibroblasts indicates that IL-18 may contribute to RA joint inflammation by enhancing the recruitment of leukocytes into the joint. IL-18 requires NF kappa B as well as PI 3-kinase to induce VCAM-1 on RA synovial fibroblasts, suggesting that there may be two distinct pathways in IL-18-induced adhesion molecule expression.     

Local treatment with the selective IκB kinase β inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-κB is a key regulator of synovial inflammation. We investigated the effect of local NF-κB inhibition in rat adjuvant arthritis (AA), using the specific IκB kinase (IKK)-β blocking NF-κB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-β-induced IκBα phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-α and IL-1-β in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-β-induced TNF-α production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-α-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-κB blockade using a small peptide inhibitor of IKK-β has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-β-targeted NF-κB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.     

Alpha 4/beta 1 integrin (VLA-4) ligands in arthritis. Vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes.

Expression of vascular cell adhesion molecule-1 (VCAM-1) in synovial tissue was determined using the immunoperoxidase technique. Normal, rheumatoid arthritis (RA), and osteoarthritis (OA) synovia bound VCAM-1 antibodies in the intimal lining as well as blood vessels. The amount of VCAM-1 was significantly greater in the synovial lining of RA and OA tissues compared with normal synovium (p less than 0.002). There was also a trend toward greater levels of VCAM-1 staining in blood vessels of arthritic tissue (RA greater than OA greater than normal). Because VCAM-1 staining was especially intense in the synovial lining, VCAM-1 expression and regulation was studied on cultured fibroblast-like synoviocytes (FLS) derived from this region. Both VCAM-1 and intercellular adhesion molecule 1 were constitutively expressed on FLS. VCAM-1 expression was further increased by exposure to IL-1 beta, TNF-alpha, IL-4, and IFN-gamma. These cytokines (except for IL-4) also induced intercellular adhesion molecule 1 expression on FLS. ELAM was not detected on resting or cytokine-stimulated FLS. The specificity of VCAM-1 for FLS was demonstrated by the fact that only trace amounts were detected on normal and RA dermal fibroblasts. Cytokines induced intercellular adhesion molecule 1 display on dermal fibroblasts but had minimal effect on VCAM-1 expression. Finally, in adherence assays, Jurkat cell binding to resting FLS monolayers was inhibited by antibody to alpha 4/beta 1 integrin (VLA-4), CS-1 peptide from alternatively spliced fibronectin (which is another VLA-4 ligand), and, to a lesser extent, anti-VCAM-1 antibody. After cytokine stimulation of FLS, Jurkat-binding significantly increased, and this increase was blocked by anti-VCAM-1 antibody. Therefore, both CS-1 and VCAM-1 participate in VLA-4-mediated adherence to resting FLS in vitro, and VCAM-1 is responsible for the increase in Jurkat binding mediated by cytokines.     

IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn''s disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin⁺ fibroblast-like synoviocytes and CD68⁺ macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t⁺ Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R⁻ or CCR6⁻ CD161⁻) CD4⁺ memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.     

A cell-cycle independent role for p21 in regulating synovial fibroblast migration in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and destruction of cartilage and bone. The fibroblast-like synoviocyte (FLS) population is central to the development of pannus by migrating into cartilage and bone. We demonstrated previously that expression of the cell cycle inhibitor p21 is significantly reduced in RA synovial lining, particularly in the FLS. The aim of this study was to determine whether reduced expression of p21 in FLS could alter the migratory behavior of these cells. FLS were isolated from mice deficient in p21 (p21^(-/-)) and were examined with respect to growth and migration. p21^(-/-) and wild-type (WT) FLS were compared with respect to migration towards chemoattractants found in RA synovial fluid in the presence and absence of cell cycle inhibitors. Restoration of p21 expression was accomplished using adenoviral infection. As anticipated from the loss of a cell cycle inhibitor, p21^(-/-) FLS grow more rapidly than WT FLS. In examining migration towards biologically relevant RA synovial fluid, p21^(-/-) FLS display a marked increase (3.1-fold; p < 0.05) in migration compared to WT cells. Moreover, this effect is independent of the cell cycle since chemical inhibitors that block the cell cycle have no effect on migration. In contrast, p21 is required to repress migration as restoration of p21 expression in p21^(-/-) FLS reverses this effect. Taken together, these data suggest that p21 plays a novel role in normal FLS, namely to repress migration. Loss of p21 expression that occurs in RA FLS may contribute to excessive invasion and subsequent joint destruction.     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

Resistance of rheumatoid synovial dendritic cells to the immunosuppressive effects of IL-10.

IL-10 down-regulates the APC function of many dendritic cells (DC), including human peripheral blood (PB) DC. In rheumatoid arthritis (RA), synovial fluid (SF) DC express markers of differentiation and are effective APC despite abundant synovial IL-10. The regulation of DC responsiveness to IL-10 was therefore examined by comparing the effect of IL-10 on normal PB and RA SF DC. Whereas IL-10 down-modulated APC function and MHC class II and B7 expression of PB DC, IL-10 had no such effect on SF DC. Since SF DC have differentiated in vivo in the presence of proinflammatory cytokines, PB DC were cocultured in the presence of IL-10 and either GM-CSF, IL-1beta, TNF-alpha, IL-6, or TGF-beta. GM-CSF, IL-1beta, and TNF-alpha were all able to restore APC function. Whereas the effects of IL-10 on PB DC were shown to be mediated by IL-10R1, neither PB nor RA SF DC constitutively expressed IL-10R1 mRNA or detectable surface protein. In contrast, IL-10R1 protein was demonstrated in PB and SF DC whole cell lysates, suggestive of predominant intracellular localization of the receptor. Thus, DC responsiveness to IL-10 may be regulated through modulation of cell surface IL-10R1 expression or signaling.     

Regulation of annexin I in rheumatoid synovial cells by glucocorticoids and interleukin-1

The glucocorticoid (GC)-induced antiinflammatory molecule annexin I is expressed in leukocytes and has antiinflammatory effects in animal models of arthritis, but the expression of annexin I in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is unknown. We report the constitutive and dexamethasone (DEX)-inducible expression of annexin I in RA FLS. DEX increased FLS annexin I protein translocation and mRNA expression. Interleukin (IL)-1beta also induced annexin I translocation and mRNA but also increased intracellular protein. DEX and IL-1 had additive effects on annexin I mRNA, but DEX inhibited the inducing effect of IL-1beta on cell surface annexin I. These results indicate that glucocorticoids and IL-1beta upregulate the synthesis and translocation of annexin I in RA FLS, but interdependent signalling pathways are involved.     

IL-17 receptor and its functional significance in psoriatic arthritis

To delineate the functional significance of IL-17 Receptor (IL-17RA) and characterize the IL-17 producing T cell (Th17) subpopulation in psoriatic arthritis (PsA). Mononuclear cells from blood and synovial fluid (SF) were obtained from PsA (n=20), rheumatoid arthritis (RA, n=20) and osteoarthritis (OA, n=20) patients. Synoviocytes (FLS) were isolated from the synovium of RA (n=5), PsA (n=5) and OA (n=5) patients. IL-17RA expression in FLS was identified by western blotting (WB) and flowcytometry. T lymphocytes derived from the SF of these patients were studied to identify and phenotype the Th17 cells. The functional significance of IL-17RA was determined by evaluating its regulatory role on the production of proinflammatory cytokines and endopeptidase. IL-17RA expression was found to be significantly higher in FLS of RA (15.7%±4.9) and PsA (4.5%±0.9) in comparison to OA (1.14%±0.9). Western blot analyses showed that the relative intensity (RI) of IL-17RA protein was higher in RA and PsA compared to OA (Fisher exact, P<0.01). A significant enrichment of IL-17-producing CD4+ T cells (7.9%±2.8) was observed in the SF of PsA patients compared to that of OA patients (P<.001). Compared to OA-FLS, recombinant IL-17 induced higher levels of IL-6, IL-8, and MMP-3 production in PsA-FLS. Blockage of IL-17RA with an anti-IL-17RA antibody inhibited the production of IL-6, IL-8, and MMP-3. This is the first report to demonstrate the functional significance of IL-17RA in PsA. Results of this study support the hypothesis that IL-17RA blocking antibodies have the potential to be a therapeutic option for psoriatic arthritis.     

Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium

Introduction

Acid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).

Methods

Experiments were performed in cultured FLS from wild-type (WT) and ASIC3-/- mice, ASIC1-/- mice, and people with rheumatoid arthritis. We assessed the effects of acidic pH with and without interleukin-1β on FLS and the role of ASICs in modulating intracellular calcium [Ca²⁺]_i, mitogen activated kinase (MAP kinase) expression, and cell death. [Ca²⁺]_i was assessed by fluorescent calcium imaging, MAP kinases were measured by Western Blots; ASIC, cytokine and protease mRNA expression were measured by quantitative PCR and cell death was measured with a LIVE/DEAD assay.

Results

Acidic pH increased [Ca²⁺]_i and decreased p-ERK expression in WT FLS; these effects were significantly smaller in ASIC3-/- FLS and were prevented by blockade of [Ca²⁺]_i. Blockade of protein phosphatase 2A (PP2A) prevented the pH-induced decreases in p-ERK. In WT FLS, IL-1β increases ASIC3 mRNA, and when combined with acidic pH enhances [Ca²⁺]_i, p-ERK, IL-6 and metalloprotienase mRNA, and cell death. Inhibitors of [Ca²⁺]_i and ERK prevented cell death induced by pH 6.0 in combination with IL-1β in WT FLS.

Conclusions

Decreased pH activates ASIC3 resulting in increased [Ca²⁺]_i, and decreased p-ERK. Under inflammatory conditions, acidic pH results in enhanced [Ca²⁺]_i and phosphorylation of extracellular signal-regulated kinase that leads to cell death. Thus, activation of ASIC3 on FLS by acidic pH from an inflamed joint could limit synovial proliferation resulting in reduced accumulation of inflammatory mediators and subsequent joint damage.