Regulation of interleukin‐1β–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‐1β (IL‐1β)–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 α (GROα/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 GROα 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‐κB.

Results

EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 μM or 20 μM significantly inhibited IL‐1β–induced ENA‐78, RANTES, and GROα, but not MCP‐1 production in a concentration‐dependent manner. EGCG at 50 μM caused a complete block of IL‐1β–induced production of RANTES, ENA‐78, and GROα, and reduced production of MCP‐1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL‐1β–induced, and chemokine‐mediated MMP‐2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCδ and inhibited the activation and nuclear translocation of NF‐κB in IL‐1β–treated RA synovial fibroblasts.

Conclusion

These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.

     

Epigallocatechin-3-gallate suppresses TNF-α -induced production of MMP-1 and -3 in rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) synovial fibroblasts produce matrix metaloproteinases (MMPs), which destruct cartilage and bone in RA joint. Tumor necrosis factor-α (TNF-α) is one of the most important mediator leading to MMP production in RA synovial fibroblasts. Here we show that epigallocatechin-3-Gallate (EGCG) suppresses TNF-α-induced production of MMP-1 and MMP-3 in RA synovial fibroblasts, which was accompanied by inhibition of mitogen activated protein kinase (MAPK) and activator protein-1 (AP-1) pathways. EGCG treatment resulted in dose-dependent inhibition of TNF-α-induced production of MMP-1 and MMP-3 at the protein and mRNA levels in RA synovial fibroblast. EGCG treatment also inhibited TNF-α-induced phosphorylation of MAPKs, such as ERK1/2, p38, JNK. Electrophoretic mobility shift assay revealed that EGCG inhibits binding of AP-1 proteins to its response elements in synovial fibroblast treated. Thus, EGCG may play a role in regulating inflammation and bone destruction in RA patients.     

Oncostatin M stimulates monocyte chemoattractant protein-1- and interleukin-1-induced matrix metalloproteinase-1 production by human synovial fibroblasts in vitro

Objective. To measure levels of oncostatin M (OSM) in the synovial fluid of rheumatoid arthritis (RA) patients and to examine the activities of human OSM in the regulation of human synovial fibroblast (HSF) production of chemokines and matrix metalloproteinases (MMP-1 and MMP-3) in vitro. Methods. We examined the levels of OSM in the synovial fluids of patients with arthritis by an enzymelinked immunosorbent assay (ELISA). ELISA of cell culture supernatants and Northern blots were used to assess responses of HSF to interleukin-1α (IL-1α), OSM, and other members of the IL-6/leukemia inhibitory factor (IL-6/LIF) family of cytokines. Results. We detected variable levels of OSM antigen in 9 of 10 RA patient synovial fluids, but levels were not detectable in 9 of 10 osteoarthritis (OA) patient fluids. Upon examining the responses of HSF in culture, OSM stimulated monocyte chemoattractant protein 1 (MCP-1), whereas RANTES secretion (regulated upon activation, normal T expressed and presumably secreted) was not altered by OSM alone. In IL-1α induced cells, OSM costimulation further enhanced MCP-1 release, but inhibited the release of RANTES and IL-8. Other members of the IL-6/LIF family of cytokines did not show these effects. OSM induced a small elevation of MMP-1 production over 2 and 3 days of stimulation (2-fold), and acted significantly to enhance IL-1α-induced production of MMP-1 (to 8-fold and 9-fold at 48 and 72 hours, respectively). No effect of OSM was seen on MMP-3 secretion, either alone or in IL-1α-costimulated cells. Conclusion. These results suggest that OSM has potentially important functions in the modulation of chemokine and metalloproteinase production by synovial cells of the joint.     

Treatment with sulfasalazine or sulfapyridine, but not 5-aminosalicyclic acid, inhibits basic fibroblast growth factor-induced endothelial cell chemotaxis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression. METHODS: SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites. RESULTS: HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78. CONCLUSION: These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.     

Cordycepin inhibits IL-1{beta}-induced MMP-1 and MMP-3 expression in rheumatoid arthritis synovial fibroblasts

Objective. MMP is a key enzyme in the degradation of extracellularmatrices, and its expression plays important roles in inflammatorydiseases. Cordycepin (3'-deoxyadenosine), a bioactive compoundof Cordyceps militaris, has been shown to exhibit many pharmacologicalactivities, such as anti-cancer, anti-inflammatory and anti-infectionactivities. In this study, we aimed at the inhibitory effectof cordycepin on IL-1β-induced MMP-1 and MMP-3 expressionas well as the molecular basis using RA synovial fibroblasts(RASFs). Methods. RASFs were isolated from synovial tissue obtained from12 patients with RA and cultured in monolayer. Expression ofMMP-1 and MMP-3 was evaluated using western blotting and real-timePCR. Chemokines were analysed by ELISA. The phosphorylationof mitogen-activated protein kinase was measured by westernblotting. Electrophoretic mobility shift assay was performedto evaluate binding activities of DNA to nuclear factor-B (NF-B)and activator protein-1 (AP-1). Results. Cordycepin inhibited IL-1β-induced MMP-1 and MMP-3expressions in RASFs in a dose-dependent manner. Among variouschemokines [such as monocyte chemoattractant protein-1 (MCP-1),GRO-, regulated upon activation, normal T-cell expressed andpresumably secreted (RANTES) and epithelial neutrophil activatingpeptide 78 (ENA-78)], cordycepin specifically blocked IL-1β-inducedENA-78 production in RASF. Moreover, cordycepin significantlyinhibited IL-1β-induced p38/JNK and AP-1 activation, butnot extracellular signal-regulated kinase (ERK) and NF-B activation. Conclusions. Cordycepin is a potent inhibitor of IL-1β-inducedchemokine production and MMP expression and strongly blocksthe p38/JNK/AP-1 signalling pathway in RASFs. KEY WORDS: Cordycepin, Interleukin-1β, Matrix metalloproteinase, p38 mitogen-activated protein kinase, Activator protein-1     

Intercellular adhesion molecule-1 mediates the inhibitory effects of hyaluronan on interleukin-1beta-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts via down-regulation of NF-kappaB and p38

OBJECTIVE: In rheumatoid arthritis (RA), it is well known that rheumatoid synovial fibroblasts (RSF) produce matrix metalloproteinases (MMPs) when stimulated with proinflammatory cytokines such as interleukin-1beta (IL-1beta), which causes joint destruction. We have previously shown that hyaluronan (HA) inhibits IL-1beta actions in RSF via CD44, the principal HA receptor. However, CD44 mediates HA effects only partially, and intracellular events after the HA binding to its receptors remain unclear. We investigated the role of intercellular adhesion molecule-1 (ICAM-1), another cell surface receptor for HA, and the intracellular signalling pathways in the actions of HA. METHODS: RSF were isolated from rheumatoid synovial tissues by enzymatic digestion and cultured in monolayers. The confluent cells were incubated for 48 h with IL-1beta, IL-1beta in the presence of HA, or IL-1beta in the presence of HA with pretreatment with anti-ICAM-1 antibody. Secretion of MMP-1 and MMP-3 was analysed by immunoblotting and immunofluorescence cytochemistry. Immunofluorescence cytochemistry was also performed to evaluate binding of HA to ICAM-1. The phosphorylation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases (MAPKs) was analysed by immunoblotting. RESULTS: Production of MMP-1 and MMP-3 by RSF was stimulated by IL-1beta. HA at > or =2 mg/ml significantly inhibited MMP production induced by IL-1beta in a dose-dependent manner. Moreover, pretreatment with anti-ICAM-1 antibody at 50 mug/ml significantly blocked the effects of HA on the actions of IL-1beta on RSF, as shown by immunoblotting and immunofluorescence cytochemistry. Another immunofluorescence cytochemistry study demonstrated that HA bound RSF via ICAM-1. Inhibition studies revealed the requirement of NF-kappaB, p38 and c-jun NH2-terminal kinase (JNK) for IL-1beta-induced MMP production. IL-1beta activated all three pathways, whereas HA down-regulated their phosphorylation. Pretreatment with anti-ICAM-1 antibody reversed the inhibitory effects of HA on the activation of NF-kappaB and p38 without affecting JNK. CONCLUSION: HA suppresses IL-1beta-enhanced MMP-1 and MMP-3 synthesis in RSF via ICAM-1 through down-regulation of NF-kappaB and p38. Intra-articular injection of HA of high molecular weight may work through such a mechanism in RA joints.     

Platelet chemokines and chemokine receptors: linking hemostasis,inflammation, and host defense

Blood platelets play critical roles in hemostasis, providing rapid essential protection against bleeding and catalyzing the important slower formation of stable blood clots via the coagulation cascade. They are also involved in protection from infection by phagocytosis of pathogens and by secreting chemokines that attract leukocytes. Platelet function usually is activated by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas secondary agonists like adrenalin do not induce aggregation on their own but become highly effective in the presence of low levels of primary agonists. Current research has revealed that chemokines represent an important additional class of agonists capable of causing significant activation of platelet function. Early work on platelet alpha-granule proteins suggested that platelet factor 4, now known as CXCL4, modulated aggregation and secretion induced by low agonist levels. Subsequent reports revealed the presence in platelets of messenger RNA for several additional chemokines and chemokine receptors. Three chemokines in particular, CXCL12 (SDF-1), CCL17 (TARC), and CCL22 (MDC), recently have been shown to be strong and rapid activators of platelet aggregation and adhesion after their binding to platelet CXCR4 or CCR4, when acting in combination with low levels of primary agonists. CXCL12 can be secreted by endothelial cells and is present in atherosclerotic plaques, whereas CCL17 and CCL22 are secreted by monocytes and macrophages. Platelet activation leads to the release of alpha-granule chemokines, including CCL3 (MIP-1alpha), CCL5 (RANTES), CCL7 (MCP-3), CCL17, CXCL1 (growth-regulated oncogene-alpha), CXCL5 (ENA-78), and CXCL8 (IL-8), which attract leukocytes and further activate other platelets. These findings help to provide a direct linkage between hemostasis, infection, and inflammation and the development of atherosclerosis.     

Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy

OBJECTIVE: To investigate the potential role of IkappaB kinase 1 (IKK-1) and IKK-2 in the regulation of nuclear factor kappaB (NF-kappaB) activation and the expression of tumor necrosis factor alpha (TNFalpha), as well as interleukin-1beta (IL-1beta), IL-6, IL-8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA). METHODS: Recombinant adenoviruses expressing beta-galactosidase, dominant-negative IKK-1 and IKK-2, or IkappaBalpha were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte-derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus-induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined. RESULTS: IKK-2 was not required for lipopolysaccharide (LPS)-induced NF-kappaB activation or TNFalpha, IL-6, or IL-8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFalpha, and IL-1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK-2 was also required for LPS-induced NF-kappaB activation and IL-6 or IL-8 production. In RA synovial membrane cells, IKK-2 inhibition had no effect on spontaneous TNFalpha production but significantly reduced IL-1beta, IL-6, IL-8, VEGF, and MMPs 1, 2, 3, and 13. CONCLUSION: Our study demonstrates that IKK-2 is not essential for TNFalpha production in RA. However, because IKK-2 regulates the expression of other inflammatory cytokines (IL-1beta, IL-6, and IL-8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.     

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.     

Differential gene expression during capillary morphogenesis in a microcarrier-based three-dimensional in vitro model of angiogenesis with focus on chemokines and chemokine receptors

AIM: To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogeness system with affymetrix oligonucleotide array. METHODS: A microcarrier-based in vitro angiogenesis system was developed, in which ECs migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed networks. The total RNA samples from the HMVECs at the selected time points (0.5, 24, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the softwares provided by the manufacturers. The expression patterns of some genes were validated and confirmed by semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines and chemokine receptors was specially examined and their functional implications were analyzed. RESULTS: A total of 1 961 genes were up- or downreg-ulated two-folds or above, and among them, 468 genes were up- or down-regulated three-folds or above. The regulated genes could be grouped into categories based on their molecular functions, and were also clustered into six groups based on their patterns of expression. As for chemokines and chemokine receptors, CXCL1/GRO-α, CXCL2/GRO-β, CXCL5/ENA-78, CXCL6/GCP2, IL-8/CXCL8, CXCL12/SDF-1, CXCL9/Mig, CXC11/ITAC, OOCL1/fractalkine, CCL2/MCP-1, CCL3, CCL5/RANTES, CCL7, CCL15, CCL21, CCL23, CCL28, and CCR1, CCR9, CXCR4 were identified. Moreover, these genes demonstrated different changing patterns during the capillary morphogenesis, which implied that they might have different roles in the sequential process. Among the chemokines identified, CCL2/MCP-1, CCL5/RANTES and CX3CL1 were specially up-regulated at the 24-h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis. CONCLUSION: The present study demonstrates a global profile of gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be conducted.     

Rosiglitazone induces interleukin-1 receptor antagonist in interleukin-1beta-stimulated rat synovial fibroblasts via a peroxisome proliferator-activated receptor beta/delta-dependent mechanism

OBJECTIVE: To study the potency of 2 peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-deoxy-PGJ(2)) and rosiglitazone, to modulate the expression of interleukin-1 receptor antagonist (IL-1Ra) in rat synovial fibroblasts. METHODS: Levels of messenger RNA for IL-1Ra and PPAR isotypes (alpha, beta/delta, gamma) were assessed by real-time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL-1beta. PPAR levels were assessed by Western blotting and secreted IL-1Ra levels by immunoassay. The potency of PPARgamma agonists and the PPARbeta/delta agonist GW-501516 on IL-1Ra levels was tested in the range of 1-10 microM and at 100 pM, respectively. The contribution of PPARgamma to the effects of rosiglitazone on IL-1Ra secretion was examined either by its overexpression or by inhibition using wild-type or dominant-negative constructs and the antagonist GW-9662 (10 microM), respectively. The dominant-negative strategy was also performed to investigate the possible contribution of PPARbeta/delta and NF-kappaB activation. RESULTS: IL-1beta-induced IL-1Ra production was increased by 10 microM rosiglitazone but was reduced dose-dependently by 15-deoxy-PGJ(2). Both agonists lowered IL-1beta secretion, but rosiglitazone alone reduced the imbalance of IL-1beta/IL-1Ra toward basal levels. Enhancement of IL-1beta-induced IL-1Ra production by rosiglitazone was not affected by PPARgamma overexpression or by its inhibition with dominant-negative PPARgamma or GW-9662. Inhibition of NF-kappaB was also ineffective against rosiglitazone but abolished the stimulating effect of IL-1beta on IL-1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARgamma decreased dramatically upon IL-1beta exposure, whereas PPARbeta/delta remained stable. Dominant-negative PPARbeta/delta abolished the enhancement of IL-1Ra by rosiglitazone, whereas GW-501516 reproduced the effect of rosiglitazone on IL-1Ra secretion. CONCLUSION: Rosiglitazone stimulates IL-1Ra production by a PPARbeta/delta mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.     

类风湿关节炎炎性关节中高水平脂联素促进白细胞介素-6单核细胞趋化因子-1和核因子-κB受体活化因子配体表达

目的 研究类风湿关节炎(RA)炎性关节中高水平脂联素(AD)表达的意义.方法 酶联免疫吸附试验(ELISA)测定关节液中AD及白细胞介素(IL)-1β、IL-6、IL-8、肿瘤坏死因子(TNF)-α、单核细胞趋化因子(MCP)-1和基质金属蛋白酶(MMP)-9等促炎因子/趋化因子的含量,并分析AD与其相关性.双重免疫标记分析RA滑膜中AD在滑膜成纤维细胞(SFLs)、T淋巴细胞、单核细胞、中性粒细胞和肥大细胞的表达.ELISA测定重组AD因子刺激SFLs后培养上清液中细胞因子含量变化.实时荧光定量反转录-聚合酶链反应(real-time PCR)分析AD和IL-6共刺激对人滑膜细胞系MH7A中核因子-κB受体活化因子配体(RANKL)mRNA表达影响.3组间采用多变量方差分析,2组比较采用Student's t检验.Mann-Whitney U检验,相关性分析采用Spearman检验.结果 RA关节液中AD与IL-6含量呈正相关(r=0.27,P=0.03).AD与成纤维细胞双重免疫标记呈阳性反应.以重组AD因子30g/ml刺激SFLs 24 h后,培养上清液中IL-6和MCP-1分泌量与刺激前相比分别增加了11.4倍和8倍(P<0.05).AD和IL-6共刺激后,MH7A中RANKL mRNA表达显著增加(82±26).结论 炎性关节中高水平AD可促进IL-6、MCP-1和RANKL表达,其可能在RA慢性炎症和骨侵蚀过程中发挥重要作用.     

Inflammatory status and cartilage regenerative potential of synovial fibroblasts from patients with osteoarthritis and chondropathy

OBJECTIVES: To evaluate the inflammatory status and the cartilage regenerative potential of pathological synovial fibroblasts from patients with osteoarthritis (OA) compared with non-inflamed synovium (NS)-derived cells from patients with chondropathy. METHODS: The inflammatory cell phenotype was investigated based on the constitutive and inducible surface expression and secretion of various effector molecules using flow cytometry or ELISA assays. The capacity of cells to produce cartilage-like extracellular matrix was assessed using acid Alcian blue staining and type II collagen immunostaining after treatment with transforming growth factor beta1 (TGF-beta1). RESULTS: OA and NS fibroblasts consistently expressed CD29, CD44, CD49e, CD54, CD90 and CD106. Expression of high-affinity receptors for IL-4, IL-15, CXCL8 and CXCL12 was also detected but only intracellularly. All types of fibroblasts spontaneously released abundant amounts of CXCL12, CCL2, IL-6 and tissue inhibitor of metalloproteinase 1, while the production of IL-11, TGF-beta1, matrix metalloproteinase 1 (MMP-1) and MMP-9 was detected at moderate levels. Several other secreted factors remained undetectable. No statistically significant differences were noted between the two groups of fibroblasts. Treatment with the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) up-regulated the same set of surface and secreted molecules, including CD54, CD106, membrane IL-15, CCL2 and CCL5. Under TGF-beta1 treatment and adipogenic culture conditions, both OA and NS fibroblasts displayed chondrogenic and adipocytic activities that were reduced in OA compared with NS cells. CONCLUSIONS: OA synovial fibroblasts did not display a distinct activated inflammatory phenotype compared with NS cells. However, they did differ in their reduced ability to produce cartilage-like matrix. This difference may be an additional important factor contributing to OA pathogenesis.