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.     

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.     

Rheumatoid fibroblast-like synoviocytes overexpress the chemokine stromal cell-derived factor 1 (CXCL12), which supports distinct patterns and rates of CD4+ and CD8+ T cell migration within synovial tissue

OBJECTIVE: A characteristic feature of the inflammatory infiltrate in rheumatoid arthritis is the segregation of CD4 and CD8 T lymphocyte subsets into distinct microdomains within the inflamed synovium. The aim of this study was to test the hypothesis that chemokines in general and stromal cell-derived factor 1 (SDF-1; CXCL12) in particular are responsible for generating this distinctive microcompartmentalization. METHODS: We examined how synovial CD4/CD8 T cell subsets interacted in coculture assays with fibroblasts derived from chronic inflammatory synovial lesions and normal synovial tissue as well as from fetal lung and adult skin. We used the ability of T cells to migrate beneath fibroblasts (a process called pseudoemperipolesis) as an in vitro marker of T cell accumulation within synovial tissue. RESULTS: Rheumatoid fibroblast-like synoviocytes (FLS) displayed a unique ability to support high levels of CD4 and CD8 T cell pseudoemperipolesis. Nonrheumatoid FLS as well as fetal lung fibroblasts supported low levels of pseudoemperipolesis, while skin-derived fibroblasts were unable to do so. CD8 T cells migrated under fibroblasts more efficiently and at a higher velocity than CD4 T cells, a feature that was intrinsic to CD8 T cells. Rheumatoid fibroblasts constitutively produced high levels of SDF-1 (CXCL12), which was functionally important, since blocking studies showed reductions in T cell pseudoemperipolesis to levels seen in nonrheumatoid FLS. Rheumatoid fibroblasts also constitutively produced high levels of vascular cell adhesion molecule 1 (VCAM-1; CD106), but this did not contribute to T cell pseudoemperipolesis, unlike the case for B cells, which require SDF-1 (CXCL12)-CXCR4 and CD49d-VCAM-1 (CD106) interactions. Importantly, only combinations of rheumatoid FLS and rheumatoid-derived synovial fluid T cells supported pseudoemperipolesis when examined ex vivo, confirming the in vivo relevance of these findings. CONCLUSION: These studies demonstrate that features intrinsic to both fibroblasts (the production of SDF-1) and CD8/CD4 T cells (the expression of CXCR4) are responsible for the characteristic pattern of T lymphocyte accumulation seen in the rheumatoid synovium. These findings suggest that the SDF-1/CXCR4 ligand/receptor pair is likely to play an important functional role in T lymphocyte accumulation and positioning within the rheumatoid synovium.     

CD147分子和基质金属蛋白酶在类风湿关节炎滑膜中的表达

目的 探讨类风湿关节炎(RA)滑膜组织CD147的表达及其与基质金属蛋白酶(MMP)-01和MMP-2表达的相关性。方法 采用免疫组织化学SP(streptavidin／peroxidase)染色方法检测11例RA患者受损关节软骨-血管翳接合部（CPJ)滑膜组织中CD147和MMP-1及MMP-2的表达，并与3例骨关节炎（OA)患者滑膜组织CD147和MMP-1及MMP-2的检测相对照。结果 3例OA滑膜组织CD147和MMP-1及MMP-2的表达均为阴性，而11例RA滑膜组织中均有CD147和MMP-1及MMP-2的表达。其中，表达CD147的细胞为单核-巨噬细胞、淋巴细胞和滑膜成纤维样细胞，表达MMP-1及MMP-2的细胞为滑膜成纤维样细胞。统计学分析表明RA滑膜细胞CD147的表达和MMP-1及MMP-2的表达间存在显著相关性。结论 CD147在RA滑膜组织中表达增高，可能是导敏RA受损关节软骨、骨基质降解的重要因素之一。     

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.     

Norepinephrine from synovial tyrosine hydroxylase positive cells is a strong indicator of synovial inflammation in rheumatoid arthritis

OBJECTIVE: Density of sympathetic nerve fibers in synovial tissue was lower in patients with rheumatoid arthritis (RA) compared to those with osteoarthritis (OA). This was accompanied by norepinephrine (NE) release from synovial tyrosine hydroxylase positive cells (TH+ cells). We investigated the role of TH+ cells and NE in synovial inflammation. METHODS: Synovial tissue of 34 patients with RA and 36 with OA who underwent knee joint replacement surgery was characterized using immunohistochemistry and a synovial tissue superfusion technique, respectively. In culture experiments with mixed synoviocytes, the effect of NE on secretion of interleukin 6 (IL-6), IL-8, tumor necrosis factor (TNF), and matrix metalloproteinase-3 (MMP-3) was investigated. RESULTS: Tissue density of TH+ cells was higher in RA compared to OA (63.9 vs 34.2 cells/mm2; p = 0.017). Basal NE release from synovial tissue correlated highly significantly with density of TH+ cells in RA (Rrank = 0.573, p = 0.001) but not in OA (Rrank = 0.102, NS). Basal NE release correlated with the degree of inflammation in RA (Rrank = 0.420, p = 0.021) but not in OA (Rrank = 0.174, NS), and with spontaneous IL-8 secretion in RA (Rrank = 0.581, p = 0.001) but not in OA (Rrank = 0.160, NS). Only in RA, density of TH+ cells correlated positively with spontaneous secretion of IL-6, IL-8, and MMP-3. We confirmed the extensive loss of sympathetic nerve fibers in RA compared to OA (0.32 vs 3.1 nerve fiber/mm2; p < 0.001). The ratio of sympathetic to sensory nerve fibers was 1 to 5 in RA and 2 to 1 in OA. A ratio of 1.0 separates almost all patients into 2 diseases groups (RA vs OA). Prior prednisolone treatment of RA patients was related to decreased spontaneous cytokine secretion, a lower density of T cells, CD163+ macrophages and TH+ cells, a lower degree of inflammation, and reduced synovial NE secretion. NE was able to inhibit secretion of IL-6 (in OA), IL-8 (in RA), and TNF (in RA and OA) in culture experiments. CONCLUSION: TH+ cells and release of NE are strongly linked to a higher degree of synovial inflammation. Culture experiments indicate that NE has antiinflammatory properties at higher concentrations (10(-5) M). NE secretion of TH+ cells may be an antiinflammatory mechanism to counteract local inflammation. Thus, TH+ cell derived NE can be an important local factor of immunomodulation in synovial inflammation.     

Turnover of type II collagen and aggrecan in cartilage matrix at the onset of inflammatory arthritis in humans: relationship to mediators of systemic and local inflammation

OBJECTIVE: To determine in vivo the extent of damage to, and changes in turnover of, articular cartilage type II collagen (CII) and the proteoglycan aggrecan following the onset of inflammatory arthritis in humans, and to examine the hypothesis that there are direct relationships between cartilage biomarkers of damage/turnover and clinical, histologic, and molecular markers of inflammation. METHODS: Synovial fluid (SF) and synovial membrane (SM) were obtained by arthroscopy, and a synovitis score was determined, in 32 patients with rheumatoid arthritis (RA) (13 with early untreated disease, 19 with established disease), 18 with psoriatic arthritis (PsA), and 10 with osteoarthritis (OA). Systemic disease activity markers were recorded, and SM CD3+ T cells, CD4+ T cells, CD68+ macrophages, and lining layer hyperplasia were quantified. SF levels of tumor necrosis factor alpha (TNFalpha), interleukin-10 (IL-10), matrix metalloproteinase 1 (MMP-1), MMP-3, Col2-3/4C(Long mono) neoepitope (C2C) (reflecting collagenase cleavage of cartilage CII), C-propeptide of type II procollagen (PIICP) (a biosynthesis marker), keratan sulfate (KS), and the 846 epitope of aggrecan (turnover) were measured by enzyme-linked immunosorbent assay or radioimmunoassay. RESULTS: Levels of cartilage degradation products in early RA or early PsA were not elevated above levels in OA, although in early inflammatory arthritis, TNFalpha and MMP-1 levels were similar to those observed in late inflammatory disease and higher than those in OA. PIICP was reduced in early RA. Correlations were observed between the SF C2C neoepitope level and the Health Assessment Questionnaire score, C-reactive protein level, plasma viscosity, synovitis score, and SF TNFalpha and MMP-1 levels. KS epitope content was reduced in direct relation to SM macrophage infiltration in the sublining and lining layers and in the presence of elevated SF MMP-3. Both SF MMP-1 and SF MMP-3 levels correlated with CD4+ T cell infiltration and lining layer hyperplasia in the SM, and MMP-1 levels correlated with lining layer CD68 levels, but TNFalpha and IL-10 levels did not. CONCLUSION: Except for CII synthesis, there were no significant changes in extracellular matrix turnover of aggrecan or CII in the early stages of human inflammatory arthritis. However, the direct correlation between the increases in TNFalpha and MMP-1 production and collagen degradation suggests that collagenase cleavage of cartilage collagen is related to the activities of TNFalpha and MMP-1. The reduction in CII synthesis in early RA may contribute to the developing pathology, since a lack of synthesis of this molecule would inhibit maintenance of cartilage matrix.     

EMMPRIN/CD147对类风湿关节炎患者滑膜成纤维细胞合成基质金属蛋白酶的刺激作用

目的 观察细胞外基质金属蛋白酶诱导剂（EMMPRIN／CD147）对类风湿关节炎（RA）患者滑膜成纤维细胞（FLS）合成基质金属蛋白酶（MMPs）的作用。方法 手术切除的RA患者的滑膜组织，分离成FLS，传代培养。明胶酶谱法测FLS的MMPs含量将FLS与高表达EMMPRIN／CD147的单核细胞株（THP）-1共培养，观察THP-1细胞对FLS MMPs表达量的影响，同时观察EMMPRIN／CD147拮抗肽激活蛋白（AP）-9对FLS MMPs表达的影响。结果 THP-1细胞表达高水平的CD147，而MMP-2、MMP-9的表达水平很低；分离的FLS表达低水平的CD147和一定水平的MMP-2、MMP-9。二者混合培养后，FLS表达MMP-2、MMP-9水平显著升高，随着THP-1细胞数的增加，MMP-2、MMP-9的合成量也增加THP-1对FLS合成MMP-2、MMP-9的刺激作用可被CD147拮抗肽AP-9所抑制。结论 CD147对RA患者FLS合成MMP-2、MMP-9有刺激作用，而这种刺激作用呵被CD147拮抗肽AP-9所抑制。     

The TNF superfamily member LIGHT contributes to survival and activation of synovial fibroblasts in rheumatoid arthritis

OBJECTIVES: The TNF superfamily member LIGHT has a T-cell co-stimulatory role and has previously been associated with inflammation and autoimmunity. To investigate its role in rheumatoid arthritis (RA), a disease where activated T cells contribute in a prominent way, we have analysed the expression of LIGHT and its receptors in RA and analysed its effects on synovial fibroblasts in vitro. METHODS: The expression of LIGHT was measured in synovial tissues and fluids and the receptors of LIGHT were detected on synovial fibroblasts derived from patients with RA and osteoarthritis (OA). The effects of recombinant LIGHT on the production of proinflammatory cytokines and proteases and on the apoptosis of synovial fibroblasts was assessed. RESULTS: LIGHT mRNA was present in synovial tissues of patients with RA but not with OA. Correspondingly, soluble LIGHT protein could be detected in RA synovial fluid samples at much higher levels than in synovial fluid from patients with OA. Immunohistochemical detection of LIGHT and analysis of synovial fluid cells by flow cytometry revealed CD4 T cells as the major source of LIGHT in the rheumatoid joint. Synovial fibroblasts from RA patients were found to express the LIGHT receptors HVEM and LTbetaR. Recombinant LIGHT induced RA synovial fibroblasts to upregulate MMP-9 mRNA, CD54 and IL-6 in an NF-kappaB-dependent fashion. In vitro, exposure of cultured synovial fibroblasts to LIGHT reduced FAS-mediated apoptosis significantly, without affecting the rate of spontaneous apoptosis. CONCLUSIONS: The results provide evidence for a novel T-cell-dependent activation of synovial fibroblasts by LIGHT in joints of patients with RA, contributing to an inflammatory and destructive phenotype.     

Laminin-dependent inflammatory response in synovial fibroblasts of rheumatoid arthritis patients

Elevated expression of matrix-metalloproteinases (MMP) contributes to cartilage destruction in rheumatoid arthritis. We report on a novel pathway of inflammatory activation of synovial fibroblasts that is induced by TGF-beta and laminin (extracellular matrix) and leads to increased expression of the proteases MMP-3 and MMP-10. Neither costimulation by the central inflammatory cytokines TNF-alpha and IL-1beta nor NFkB signalling is needed for this pathway.     

The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles

Rheumatoid arthritis is a chronic inflammatory disease characterized by destruction of cartilage and bone that is mediated by synovial fibroblasts. To determine the mechanisms by which these cells are activated to produce matrix metalloproteinases (MMPs), the effects of microparticles were investigated. Microparticles are small membrane-bound vesicles whose release from immune cells is increased during activation and apoptosis. Because microparticles occur abundantly in the synovial fluid in rheumatoid arthritis, they could represent novel stimulatory agents. Microparticles derived from T cells and monocytes strongly induced the synthesis of MMP-1, MMP-3, MMP-9, and MMP-13 in fibroblasts. The induction was time-dependent, with effects primarily observed after 36 h; under these conditions, MMP-2, MMP-14, and tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and TIMP-3 were not induced. Microparticles also increased the synthesis of inflammatory mediators including IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), and MCP-2. In Ikappa-B-transfected synovial fibroblasts, MMPs were less inducible by microparticles compared with wild-type fibroblasts. Blocking of TNFalpha and IL-1beta with antibodies against TNFalpha and with IL-1 receptor antagonist did not abrogate stimulation by microparticles. These data provide evidence for a novel mechanism by which vesicles derived from activated or apoptotic immune cells can promote the destructive activity of synovial fibroblasts in rheumatoid arthritis.     

Platelet-released growth factors enhance the secretion of hyaluronic acid and induce hepatocyte growth factor production by synovial fibroblasts from arthritic patients

OBJECTIVES: Autologous platelet-secreted growth factors (GFs) may have therapeutic effects in osteoarthritis (OA) capsular joints via multiple mechanisms. Our aim was to examine the effect of a platelet-derived preparation rich in growth factors (PRGFs) in OA synovial cell biology. METHODS: Synovial cells were isolated from 10 osteoarthritic patients and cultured in serum-free media (basal conditions) and exposed to either a platelet-poor preparation or PRGF for 72 h. Cells activated with interleukin-1beta (IL-1beta) for 48 h were also exposed to PRGF. Changes in several events relevant to joint homeostasis including (i) hyaluronic acid (HA) secretion, (ii) the balance between metalloproteinase-1, -3 and -13 (MMP-1, MMP-3 and MMP-13) and tissue inhibitor-1 (TIMP-1) and (iii) the secretion of transforming growth factor-beta1(TGF-beta1), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), were all assessed. RESULTS: PRGF significantly enhanced HA secretion compared with platelet-poor preparations, P < 0.05; at the same time release of TIMP-1, MMP-1, MMP-3 and MMP-13 were not affected. An increased HGF production was observed (P < 0.05) but VEGF and TGF-beta1 levels remained unchanged. PRGF significantly enhanced the secretion of HA induced by IL-1beta activation, P < 0.05, but it did not modify the IL-1beta-induced rise in MMP-1, MMP-3 and VEGF. In contrast, PRGF-induced HGF production was abolished by the presence of IL-1beta during PRGF treatment, P < 0.05. CONCLUSIONS: Intra-articular administration of PRGF might be beneficial in restoring HA concentration and switching angiogenesis to a more balanced status but does not halt the effects of IL-1beta on synovial cells.