Regulation of interleukin-1beta-induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts

OBJECTIVE: To evaluate the efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin-1beta (IL-1beta)-induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP-1/CCL2), epithelial neutrophil-activating peptide 78 (ENA-78/CXCL5), growth-regulated oncogene alpha (GROalpha/CXCL1), and matrix metalloproteinase 2 (MMP-2) activity in rheumatoid arthritis (RA) synovial fibroblasts. METHODS: Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP-1, ENA-78, and GROalpha produced in culture supernatants were measured by enzyme-linked immunosorbent assay. MMP-2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF-kappaB. RESULTS: EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 microM or 20 microM significantly inhibited IL-1beta-induced ENA-78, RANTES, and GROalpha, but not MCP-1 production in a concentration-dependent manner. EGCG at 50 microM caused a complete block of IL-1beta-induced production of RANTES, ENA-78, and GROalpha, and reduced production of MCP-1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL-1beta-induced, and chemokine-mediated MMP-2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCdelta and inhibited the activation and nuclear translocation of NF-kappaB in IL-1beta-treated RA synovial fibroblasts. CONCLUSION: These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.     

Dopamine induces platelet production from megakaryocytes via oxidative stress-mediated signaling pathways

Dopamine (DA), a catecholamine neurotransmitter, is known to for its diverse roles on hematopoiesis, yet its function in thrombopoiesis remains poorly understood. This study shows that DA stimulation can directly induce platelet production from megakaryocytes (MKs) in the final stages of thrombopoiesis via a reactive oxygen species (ROS)-dependent pathway. The mechanism was suggested by the results that DA treatment could significantly elevate the ROS levels in MKs, and time-dependently activate oxidative stress-mediated signaling, including p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and caspase-3 signaling pathways, while the antioxidants N-acetylcysteine and L-glutathione could effectively inhibit the activation of these signaling pathways, as well as the ROS increase and platelet production triggered by DA. Therefore, our data revealed that the direct role and mechanism of DA in thrombopoiesis, which provides new insights into the function recognition of DA in hematopoiesis.     

LIGHT induces cell proliferation and inflammatory responses of rheumatoid arthritis synovial fibroblasts via lymphotoxin beta receptor

OBJECTIVE: To investigate the effects of LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) on the proliferation and gene expression of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: We measured LIGHT levels in RA synovial fluids (SF) by ELISA, and compared them with those in osteoarthritis (OA) SF. Levels of LIGHT and its receptors in RA-FLS and synovium were assessed using real-time quantitative polymerase chain reaction (PCR). RA-FLS proliferation was examined by a bromodeoxyuridine assay. Expression of intercellular adhesion molecule-1 (ICAM-1) and several chemokines, such as interleukin 8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha), was examined by real-time quantitative PCR, ELISA, and flow cytometry. The effects of LIGHT on nuclear factor-kappaB (NF-kappaB) activation were investigated using immunofluorescence and Western blotting. RESULTS: LIGHT was upregulated in both SF and synovium of RA patients compared with OA patients. Herpes virus entry mediator (HVEM) and lymphotoxin beta receptor (LTbetaR), but not LIGHT, were detected in RA-FLS. LIGHT significantly promoted RA-FLS proliferation and induced expression of MCP-1, IL-8, MIP-1alpha, and ICAM-1 by RA-FLS. As well, LTbetaR small interfering RNA (siRNA), but not HVEM siRNA, inhibited these effects of LIGHT. LIGHT induced IkappaBa degradation and NF-kappaB translocation, and a NF-kappaB inhibitor suppressed the effects of LIGHT on RA-FLS. CONCLUSION: Our findings suggest that LIGHT signaling via LTbetaR plays an important role in the pathogenesis of RA by affecting key processes such as the proliferation and activation of RA-FLS. Regulation of LIGHT-LTbetaR signaling may represent a new therapeutic target for RA treatment.     

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-beta (IFNbeta), 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 IFNbeta, 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.     

Adiponectin stimulates prostaglandin E2 production in rheumatoid arthritis synovial fibroblasts

Objective

Adipokines may influence inflammatory and/or immune responses. This study was undertaken to examine whether adiponectin affects the production of prostaglandin E₂ (PGE₂) by rheumatoid arthritis synovial fibroblasts (RASFs).

Methods

Synovial tissue was obtained from patients with RA who were undergoing joint replacement surgery. Fibroblast‐like cells from the third or fourth passage were used as RASFs. Expression of adiponectin receptor messenger RNA (mRNA) and protein was detected. PGE₂ (converted from arachidonic acid) was measured by enzyme‐linked immunosorbent assay (ELISA). Expression of mRNA and protein for cyclooxygenase 2 (COX‐2) and membrane‐associated PGE synthase 1 (mPGES‐1), key enzymes involved in PGE₂ synthesis, was detected in RASFs. The effects of RNA interference (RNAi) targeting the adiponectin receptor genes and the receptor signal inhibitors were examined. The influence of adiponectin on NF‐κB activation in RASFs was measured with an ELISA kit.

Results

Adiponectin receptors were detected in RASFs. Adiponectin increased both COX‐2 and mPGES‐1 mRNA and protein expression by RASFs in a time‐ and concentration‐dependent manner. PGE₂ production by RASFs was also increased by the addition of adiponectin, and this increase was inhibited by RNAi for the adiponectin receptor gene, or coincubation with the receptor signal inhibitors. Enhancement of NF‐κB activation by adiponectin as well as by interleukin‐1β was observed in RASFs.

Conclusion

Our findings indicate that adiponectin induces COX‐2 and mPGES‐1 expression, resulting in the enhancement of PGE₂ production by RASFs. Thus, adiponectin may play a role in the pathogenesis of synovitis in RA patients.

     

Interleukin-1β induces cytosolic phospholipase a2 and prostaglandin h synthase in rheumatoid synovial fibroblasts

Objective. In order to investigate potential regulatory mechanisms for the increased production of prostaglandin E₂ (PGE₂) in interleukin-1β (IL-1β)–stimulated rheumatoid synovial fibroblasts (RSF), this study examined the induction of phospholipase A₂ (PLA₂) and prostaglandin H synthase (PGHS) enzymes and the correlation of these events with PGE₂ production in IL-1β–stimulated RSF. Methods. Protein and messenger RNA (mRNA) levels of cytosolic PLA₂ (cPLA₂) and PGHS-2 enzymes in IL-1β–stimulated RSF were measured by Western and Northern blotting, respectively, using specific antisera and complementary DNA probes. Enzymatic activity of cPLA₂ was determined in cell-free reaction mixtures utilizing mixed micelles of ¹⁴C-phosphatidylcholine and Triton X-100 as the substrate. PGE₂ levels were quantitated using a commercial enzyme immunoassay kit. Results. Incubation of RSF with IL-1β increased the mRNA and protein levels for the high molecular weight cPLA₂ as well as for the mitogen/growth factor–responsive PGHS (PGHS-2). The IL-1 receptor antagonist completely abolished the induction of these two enzymes and the stimulation of PGE₂ production by IL-1β in RSF. In contrast, levels of the other known forms of these enzymes, i.e., the 14-kd secretory group II PLA₂ (sPLA₂) and the constitutive form of PGHS (PGHS-1), were unaffected by IL-1β treatment. Conclusion. These are the first data to demonstrate the coordinate induction by IL-1 of cPLA₂ and PGHS-2 in RSF. The time-course for the induction of these enzymes suggests that their increase contributes to the increased production of PGE₂ in IL-1–treated RSF, and may help explain the capacity of RSF to produce large amounts of PGE₂.     

Interferon-gamma enhances interleukin 12 production in rheumatoid synovial cells via CD40-CD154 dependent and independent pathways

OBJECTIVE: To determine the role of interferon-gamma (IFN-gamma) in CD40-CD154 dependent production of interleukin 12 (IL-12) by synovial cells of patients with rheumatoid arthritis (RA). METHODS: We examined the effects of IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) on CD40 expression on CD68+ synovial macrophage-lineage cells (SMC). The effects of IFN-gamma and soluble CD154 (sCD154) on IL-12 production by RA synovial cells were determined by ELISA. RESULTS: CD68+ SMC expressed substantial levels of CD40. IFN-gamma, but not TNF-alpha or GM-CSF, markedly upregulated CD40 expression on CD68+ SMC. IFN-gamma also dose dependently increased IL-12 production by synovial cells. The effects of IFN-gamma on CD40 expression (EC50 = 127.4 U/ml) were observed at a concentration 19 times lower than the effects on IL-12 production (EC50 = 6.8 U/ml). Treatment with IFN-gamma at a concentration low enough to augment CD40 expression but not IL-12 production enhanced spontaneous IL-12 production synergy with sCD 154. The synergistic enhancement of spontaneous IL-12 production was abrogated by CD40-Fc. In contrast, IL-12 production induced by high concentration of IFN-gamma was not neutralized by CD40-Fc. CONCLUSION: IFN-gamma enhanced IL-12 production via both CD40-CD154 dependent and independent pathways in RA synovium. IFN-gamma may play a crucial role in the development of RA synovitis through regulation of IL-12 production.     

The production of CXCR3-agonistic chemokines by synovial fibroblasts from patients with rheumatoid arthritis

The inflamed synovial tissue of rheumatoid arthritis (RA) is characterized by an infiltration with Th1 cells that predominantly express the chemokine receptors CXCR3 and CCR5. In this study, we investigated the production of the CXCR3-agonistic chemokines CXCL9, CXCL10, and CXCL11 by synovial tissue cells and synovial fibroblast-cell lines (fourth or fifth passage) from RA patients. Concentrations of all CXCR3 ligands in synovial fluids were markedly higher in RA patients than in osteoarthritis (OA) patients. Synovial tissue cells from RA patients more strongly expressed mRNAs for CXCR3 ligands and spontaneously secreted larger amounts of these chemokine proteins than the cells from OA patients. The mRNA expression of all CXCR3 ligands was induced in synovial fibroblasts from RA patients after stimulation with interferon gamma (IFN-), tumor necrosis factor alpha (TNF-), or interleukin-1 beta (IL-1). However, synovial fibroblasts significantly secreted CXCL9 and CXCL10 proteins, but not CXCL11 protein, after IFN- stimulation and secreted only CXCL10 protein after TNF- or IL-1 stimulation. When stimulated with a combination of IFN- and TNF-, these cells were able to secrete large amounts of all three chemokines. These results indicate that synovial fibroblasts may be involved in perpetuating the Th1 immune response by producing the Th1-associated CXCR3 ligands, and the synergistic effect of IFN- and TNF- may be important for their chemokine production in RA joints.     

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.

     

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.

     

Galectin 3 and its binding protein in rheumatoid arthritis

OBJECTIVE: To characterize the expression pattern and role of galectin 3 and galectin 3 binding protein (G3BP) in rheumatoid arthritis (RA), in comparison with galectin 1, and to explore whether soluble galectin 3 and G3BP, investigated in serum, synovial fluid, or cell culture supernatant, are associated with disease. METHODS: Synovial tissues from patients with RA or osteoarthritis (OA), as well as from healthy controls, were analyzed for galectins 1 and 3 and G3BP by in situ hybridization and immunohistochemistry. Levels of galectin 3 and G3BP in serum and synovial fluid from patients with RA and OA and controls, as well as in cell culture supernatants, were determined by enzyme-linked immunosorbent assay (ELISA). In vitro, the intracellular expression of galectin 3 in RA and OA synovial fibroblasts after modulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and anti-CD40 monoclonal antibodies was measured by flow cytometry. RESULTS: In RA, galectin 3 messenger RNA and protein stained throughout the synovial membrane, whereas G3BP was particularly expressed at sites of bone destruction. In contrast, the expression of galectin 1 was not uniform in different RA specimens, and was never found at sites of invasion. In OA and normal synovial tissues, only a small number of cells were positive for galectins and/or G3BP. Galectin 3 was elevated in RA sera and synovial fluids, whereas G3BP was increased in RA synovial fluids only. In RA, serum galectin 3 correlated with C-reactive protein levels, whereas G3BP was associated with joint destruction and/or synovial cell activation as measured by the levels of cartilage oligomeric matrix protein. In vitro, RA synovial fibroblasts showed an increased release of galectin 3 into culture medium, as measured by ELISA, but decreased secretion of G3BP. In RA synovial fibroblasts with low basal expression of galectin 3, TNFalpha increased its intracellular level in a dose-dependent manner. In contrast, IL-1beta or anti-CD40 monoclonal antibodies showed no effect. CONCLUSION: Our data indicate that galectin 3 and G3BP are not only involved in inflammation, but also contribute to the activation of synovial fibroblasts. The intracellular accumulation of galectin 3 can be enhanced by TNFalpha. Thus, galectin 3 and G3BP represent novel markers of disease activity in RA.     

Physiology of cytokine pathways in rheumatoid arthritis

This review has summarized the physiology of some cytokine pathways in RA, emphasizing the redundant and synergistic nature of this network. However, it is important to understand that this system is self-regulating through the action of anti-inflammatory cytokines, opposing cytokines, cytokine receptor antagonists, and possibly naturally occurring antibodies to cytokines (Figure 1). Disease results when an imbalance in the cytokine network develops, either from excess production of pro-inflammatory cytokines or from inadequate presence of natural anti-inflammatory mechanisms. The current therapeutic approaches to RA that are aimed at restoring this balance include the use of monoclonal antibodies to TNFalpha, soluble TNFalpha receptors, and IL-1Ra. Other therapeutic agents that interfere with the cytokine network are in various stages of preclinical and clinical evaluation.