Suppressive effects of hyaluronan on MMP-1 and RANTES production from chondrocytes

The purpose of the study was to examine the effects of hyaluronan (HA) on human chondrocytes in terms of production of MMP-1 and RANTES. Chondrocytes were obtained from patients with osteoarthritis (OA) or femoral neck fracture (control). Chondrocytes in monolayer culture were treated with various molecular weights (1.2, 50, 800 and 1,900 kD) of HA and then stimulated with IL-1. Production and expression of MMP-1 and RANTES were quantified by ELISA and real-time polymerase chain reaction (PCR). The response was blocked by anti-CD44 antibody. Production of MMP-1 was significantly suppressed by both 800- and 1,900-kD HA, while production of RANTES was suppressed by 1,900-kD HA. Expression of MMP-1 and RANTES mRNA was inhibited by 1,900-kD HA. Suppressive effects of HA on production of MMP-1 were canceled by treatment of anti-CD44 antibody. Higher CD44 expression was found in OA chondrocytes than in those of control. High-molecular-weight HA suppressed MMP-1 and RANTES production, mediated partly by CD44–HA interaction.     

Effects of celecoxib on human chondrocytes--enhanced production of chemokines

OBJECTIVE: The purpose of this study was to examine the effects of a selective cyclooxigenase-2 (COX-2) inhibitor (celecoxib) comparing diclofenac. METHODS: Using chondrocytes derived from cartilage of non-arthritic (NA) subjects or patients with osteoarthritis (OA) or rheumatoid arthritis (RA), we examined the effects of celecoxib on incorporation of 3H-thymidine and 35S-sulfate, apoptosis, and production of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, and regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1a and nitric oxide (NO). RESULTS: Celecoxib and diclofenac tended to reduce 3H-tymidine incorporation of chondrocytes. Celecoxib induced apoptosis in a dose-dependent manner, but to a lesser degree than diclofenac. Celecoxib inhibited proteoglycan synthesis (indicated by 35S-sulfate incorporation) in NA chondrocytes, but not in OA and RA chondrocytes. Celecoxib increased interleukin-1 (IL-1)-induced production of RANTES and MIP-1alpha by chondrocytes and decreased IL-1-induced NO production by chondrocytes, whereas it did not affect MMP production. CONCLUSION: Celecoxib had both beneficial and adverse effects on chondrocytes. RA, OA and NA chondrocytes showed different responses. Interestingly, celecoxib enhanced the production of chemokines.     

Osteoarthritic articular chondrocytes stimulate autologous T cell responses in vitro

OBJECTIVE: To clarify the presence of specific T cell immune response to autologous chondrocytes in patients with osteoarthritis (OA). METHODS: Peripheral blood mononuclear cells obtained from OA or post-traumatic patients were co-cultured with irradiated autologous chondrocytes, and their proliferative response was assessed using 3H-thymidine incorporation. Expression of HLA-class II molecules was also assessed on chondrocytes by immunohistochemistry or flow cytometry. RESULTS: T cell responses to autologous chondrocytes in OA yielded a significantly greater mean stimulation index (6.35 +/- 1.63) compared to controls (1.21 +/- 0.09, p < 0.01). This response was partially blocked by antibodies against HLA class I, class II, CD4 or CD8. Increased expression of HLA-DP, -DQ, and -DR was observed. CONCLUSION: This study showed the autologous T cell-stimulating property of OA chondrocytes in vitro. The elucidation of the autoimmune responses may contribute to the understanding of immune-mediated mechanisms in OA.     

Transdifferentiation of polymorphonuclear neutrophils to dendritic-like cells at the site of inflammation in rheumatoid arthritis: evidence for activation by T cells

OBJECTIVES: To investigate infiltrated cells in the synovial fluid (SF) of inflamed joints of patients with rheumatoid arthritis (RA), with special reference to polymorphonuclear neutrophils (PMN) and their interaction with T cells. METHODS: Expression on PMN of activation associated receptors CD14, CD64, CD83, and major histocompatibility complex (MHC) class II was examined in the SF of 15 patients with RA, as were the infiltrated T cells. SF cytokines were determined by enzyme linked immunosorbent assay (ELISA). To mimic the in vivo situation, co-culture experiments were carried out using PMN and T cells of healthy donors. RESULTS: The SF contained activated T lymphocytes and abundant PMN. SF PMN expression of CD14 and CD64 was enhanced compared with peripheral blood. Of special interest was the observation that only the SF PMN expressed MHC class II antigens and CD83. Exposure to SF, which contained considerable amounts of cytokines (for example, interferon gamma (IFNgamma), tumour necrosis factor alpha, and interleukin 2), induced a similar receptor pattern on blood derived PMN of healthy donors. Furthermore, PMN acquired MHC class II and CD83 within 24 to 48 hours, when co-cultured with autologous T cells or T cell lines. This effect was also achieved by T cell supernatants, was dependent on protein synthesis, and could be inhibited by antibodies against IFNgamma. CONCLUSIONS: SF PMN from patients with RA undergo major alterations, including transdifferentiation to cells with dendritic-like characteristics, probably induced by T cell derived cytokines. Because MHC class II positive PMN are known to activate T cells, the mutual activation of PMN and T cells might contribute to the perpetuation of the local inflammatory process, and eventually to the destructive process in RA.     

Chemokines differentially induce matrix metalloproteinase-3 and prostaglandin E2 in human articular chondrocytes

OBJECTIVE: To explore prostaglandin (PG) E2 production by human articular chondrocytes induced by different chemokines. METHODS: Human chondrocytes were enzymatically isolated from the articular cartilage of patients with rheumatoid arthritis (RA), osteoarthritis (OA) or traumatic fracture (N) who underwent total joint replacement. They were cultured in vitro as monolayers and then exposed to MCP-1, RANTES or SDF-1 for 24 h. Levels of PGE2 and MMP-3 in the culture supernatant were then immunoassayed. RESULTS: PGE2 production was enhanced up to 2.7-fold in a subset of samples. Responses to different chemokines were heterogeneous even within the same disease groups. As previously reported, chemokines induced MMP-3 secretion by chondrocytes, but there was no significant correlation between levels of PGE2 and MMP-3. CONCLUSION: We here document the presence of "responders" among OA, RA and normal chondrocytes that produce enhanced levels of PGE2 upon stimulation by chemokines. The relationship between chemokines and prostaglandins could differentially influence the pathogenic network responsible for cartilage degradation in arthropathy.     

Synovial fluid and peripheral blood immune complexes of patients with rheumatoid arthritis induce apoptosis in cytokine-activated chondrocytes

The destruction of cartilage is an important characteristic of rheumatoid arthritis (RA). Immune complexes (IC) are usually found in high amounts in RA synovial fluids (SF) and in the superficial layers of RA cartilage. The objective of this study was to investigate if IC have a direct influence on proliferation, survival and production of nitric oxide (NO) of cytokine-activated chondrocytes. Primary bovine chondrocytes were incubated with cytokines (huIL-1α, bovIFN-γ, huTNF-α) and IC containing precipitates of peripheral blood (PB) and/or synovial fluid (SF) of 14 RA patients, 5 osteoarthritis (OA) patients and 10 healthy age and sex-matched controls. After 48 h, chondrocyte viability, proliferation, apoptosis, NO production and oxygen radical levels were measured. Staining with May-Grünwald-Giemsa after incubation with IC of RA PB and SF, showed apoptotic chondrocytes with condensation of the nuclei. The proliferation rates of cytokine-activated chondrocytes, incubated with sera and SF IC of RA patients were significantly decreased compared to chondrocytes, incubated with sera and SF IC of OA patients and compared to sera of controls. Quantitative evaluation of apoptotic cells by annexin-V/propidium iodide and TUNEL assays revealed a significant increase after incubation with sera and SF IC of RA patients, compared to control sera and OAs sera and SF. In all TUNEL positive samples, active-caspase-3-positive cells were found. There was a significant increase of chondrocyte NO production, after incubation with SF IC of RA patients, compared to OA SF. These results support the hypothesis that IC, present in serum and SF of RA patients, have a profound influence on chondrocyte growth, NO production and apoptosis, contributing to cartilage destruction in RA.     

Aberrant responsiveness to RANTES in synovial fluid T cells from patients with rheumatoid arthritis

OBJECTIVE: To study expression and function of the chemokine receptor CCR5 in synovial fluid (SF) T cells from patients with rheumatoid arthritis (RA). METHODS: Expression of CCR5 was studied by flow cytometry and immunoblotting. The chemotactic response of T cells to chemokines was studied in cell migration assay. Tyrosine phosphorylation of Crk-associated substrate lymphocyte-type (CasL) was evaluated in immunoprecipitation and immunoblotting. RESULTS: SF T cells showed an increase in the population of CCR5, CXCR4, and CD45RO positive cells and exhibited an increase in chemotactic activity, which was not augmented with RANTES but stromal cell-derived factor-1alpha. Tyrosine phosphorylation per CasL molecule was markedly enhanced in SF T cells. In H9 cells, tyrosine phosphorylation of not only focal adhesion kinase but also CasL was induced after treatment with RANTES. Downmodulation of CCR5 by RANTES was decreased and recycling of CCR5 was accelerated in SF T cells when compared with peripheral blood (PB) T cells. When CD45RO positive PB T cells were cultured with interleukin 2, blunted responsiveness to RANTES-induced chemotaxis was reproduced as well as spontaneous chemotaxis, increased expression of CCR5, and aberrant receptor dynamics, after RANTES stimulation as observed in SF T cells. CONCLUSION: Synovial fluid T cells highly positive for CCR5 show aberrant characteristics; resistant to RANTES in terms of migration, but responsive in terms of dynamics of CCR5.     

Cell-contact-dependent activation of CD4+ T cells by adhesion molecules on synovial fibroblasts

Objective: To determine how cell–cell contact with synovial fibroblasts (SF) influence on the proliferation and cytokine production of CD4^+?T cells.

Methods: Naïve CD4^+?T cells were cultured with SF from rheumatoid arthritis patients, stimulated by anti-CD3/28 antibody, and CD4^+?T cell proliferation and IFN-γ/IL-17 production were analyzed. To study the role of adhesion molecules, cell contact was blocked by transwell plate or anti-intracellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1(VCAM-1) antibody. To study the direct role of adhesion molecules for CD4^+?T cells, CD161^+?or CD161^- naïve CD4^+?T cells were stimulated on plastic plates coated by recombinant ICAM-1 or VCAM-1, and the source of IFN-γ/IL-17 were analyzed.

Results: SF enhanced naïve CD4^+?T cell proliferation and IFN-γ/IL-17 production in cell-contact and in part ICAM-1-/VCAM-1-dependent manner. Plate-coated ICAM-1 and VCAM-1 enhanced naïve CD4^+?T cell proliferation and IFN-γ production, while VCAM-1 efficiently promoting IL-17 production. CD161^+?naïve T cells upregulating LFA-1 and VLA-4 were the major source of IFN-γ/IL-17 upon interaction with ICAM-1/VCAM-1.

Conclusion: CD4^+?T cells rapidly expand and secrete IFN-γ/IL-17 upon cell-contact with SF via adhesion molecules. Interfering with ICAM-1-/VCAM-1 may be beneficial for inhibiting RA synovitis.     

Lectin-like oxidized low-density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low-density lipoprotein in rheumatoid arthritis cartilage

OBJECTIVE: To investigate for the presence of oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor 1 (LOX-1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox-LDL with LOX-1 can induce matrix metalloproteinase 3 (MMP-3) in articular cartilage explant culture. METHODS: Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX-1 and ox-LDL by confocal fluorescence microscopy. The association between ox-LDL and LOX-1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox-LDL, with or without preincubation with neutralizing anti-LOX-1 antibody. MMP-3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme-linked immunosorbent assay. RESULTS: The majority of the RA chondrocytes stained positively with both anti-LOX-1 and anti-ox-LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti-LOX-1 antibody suppressed the binding of DiI-labeled ox-LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX-1. In contrast to native LDL, ox-LDL induced MMP-3 synthesis by articular chondrocytes in association with the induction of LOX-1, which resulted in enhanced secretion of MMP-3 into the culture medium. Anti-LOX-1 antibody reversed ox-LDL-stimulated MMP-3 synthesis to control levels. CONCLUSION: Ox-LDL, principally mediated by LOX-1, enhanced MMP-3 production in articular chondrocytes. Increased accumulation of ox-LDL with elevated expression of LOX-1 in RA cartilage indicates a specific role of the receptor-ligand interaction in cartilage pathology in RA.     

类风湿关节炎患者血清和关节液趋化因子RANTES的测定及其意义

目的：探讨血清、关节液中激活正常T细胞表达和分泌因子（RANTES）水平与类风湿关节炎（RA）病变活动的相关性。方法：有和酶联免疫吸附法（ELISA）测定28例类风湿关节炎患者血清及关节液中RANTES水平,并与骨关节炎（OA）患者对照,结果：RA患者血清RANTES水平明显高于OA,且RA滑液中RANTES水平高于血清,血清RANTES水平与关节肿胀指数,血沉,C反应蛋白呈正相关,结论：RA病变关节组织是RANTES产生的主要部位;血清RANTES水平与RA的活动性病变相关。     

CTLA‐4IG suppresses reactive oxygen species by preventing synovial adherent cell–induced inactivation of rap1, a ras family GTPASE mediator of oxidative stress in rheumatoid arthritis T cells

Objective

Oxidative stress contributes to the inflammatory properties of rheumatoid arthritis (RA) synovial T lymphocytes. This study was undertaken to investigate the mechanisms leading to production of reactive oxygen species (ROS) and oxidative stress in RA synovial T lymphocytes.

Methods

ROS production in T lymphocytes from the peripheral blood (PB) of healthy donors and from the PB and synovial fluid (SF) of RA patients was measured by ROS‐dependent fluorescence of 6‐carboxy‐2′,7′‐dichlorofluorescein. Rap1 GTPase activation was assessed by activation‐specific probe precipitation. Proliferation of RA PB and SF T lymphocytes was assayed by ³H‐thymidine incorporation. In some experiments, RA PB T cells were preincubated with autologous SF or with PB or SF adherent cells. Experiments were performed in the absence or presence of transwell membranes or CTLA‐4Ig fusion proteins. Short‐ and long‐term stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti‐CD28 antibodies.

Results

T lymphocyte ROS production and Rap1 inactivation were mediated by cell–cell contact with RA synovial adherent cells, and this correlated with T cell mitogenic hyporesponsiveness. CTLA4‐Ig blockade of synovial adherent cell signaling to CD28 T cells reversed the inhibition of Rap1 activity and prevented induction of ROS. Introduction of active RapV12 into T cells also prevented induction of ROS production. Coincubation of T cells with stimulating anti‐CD28 antibodies and inflammatory cytokines synergistically increased T cell ROS production.

Conclusion

Cell–cell contact between T cells and RA synovial adherent cells mediates Rap1 inactivation and subsequent ROS production in T lymphocytes following exposure to inflammatory cytokines. This process can be blocked by CTLA4‐Ig fusion protein.

     

Inhibition of interleukin-1beta-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

OBJECTIVE: To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin-1beta (IL-1beta)-stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage. METHODS: IL-1beta was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800-kd HA to assess its effect on IL-1beta-induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti-CD44 antibody were performed to investigate the mechanism of action of HA. RESULTS: Treatment and pretreatment with 800-kd HA at 1 mg/ml resulted in significant suppression of IL-1beta-stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA-binding blocking experiments using anti-CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti-CD44 antibody, which suppressed IL-1beta-stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL-1beta in normal and OA cartilage. CONCLUSION: This study demonstrates that HA effectively inhibits IL-1beta-stimulated production of MMP-1, MMP-3, and MMP-13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL-1beta may involve direct interaction between HA and CD44 on chondrocytes.     

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.     

Sinomenine reduces invasion and migration ability in fibroblast-like synoviocytes cells co-cultured with activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, CD147

CD147 expressed by monocytes, macrophages, and synoviocytes cells can stimulate the production of matrix metalloproteinases (MMPs) associated with the development of rheumatoid arthritis (RA). We investigated the effects of Sinomenine (SIN) on invasion and migration ability and gene expression of CD147, MMP-2, MMP-9 of fibroblast-like synoviocytes cells (FLS) co-cultured with activated human monocytic THP-1 cells (A-THP-1) in vitro. SIN is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum. FLS cells were co-cultured with THP-1 cells which were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Cells were treated with different concentrations of SIN. Invasion and migration ability of cells was tested by transwell assays. Western blot analysis and zymographic analysis were adopted to detect the expression of CD147 and MMPs, respectively. RT–PCR was used to determine the expression of mRNA of CD147, MMP-2, and MMP-9. The invasion and migration ability of the co-cultured cells was significantly inhibited by SIN in a concentration-dependent fashion, and at the same time, the levels of CD147, MMP-2, MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 and 1.00 mM (P < 0.01). Our results point to a possible mechanism of SIN on treatment of RA is the inhibitory effect of SIN on cell invasion and migration ability, which strongly correlates with repressing the expression of CD147, MMP-2, and MMP-9.     

CTLA-4IG suppresses reactive oxygen species by preventing synovial adherent cell-induced inactivation of Rap1, a Ras family GTPASE mediator of oxidative stress in rheumatoid arthritis T cells

OBJECTIVE: Oxidative stress contributes to the inflammatory properties of rheumatoid arthritis (RA) synovial T lymphocytes. This study was undertaken to investigate the mechanisms leading to production of reactive oxygen species (ROS) and oxidative stress in RA synovial T lymphocytes. METHODS: ROS production in T lymphocytes from the peripheral blood (PB) of healthy donors and from the PB and synovial fluid (SF) of RA patients was measured by ROS-dependent fluorescence of 6-carboxy-2',7'-dichlorofluorescein. Rap1 GTPase activation was assessed by activation-specific probe precipitation. Proliferation of RA PB and SF T lymphocytes was assayed by 3H-thymidine incorporation. In some experiments, RA PB T cells were preincubated with autologous SF or with PB or SF adherent cells. Experiments were performed in the absence or presence of transwell membranes or CTLA-4Ig fusion proteins. Short- and long-term stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti-CD28 antibodies. RESULTS: T lymphocyte ROS production and Rap1 inactivation were mediated by cell-cell contact with RA synovial adherent cells, and this correlated with T cell mitogenic hyporesponsiveness. CTLA4-Ig blockade of synovial adherent cell signaling to CD28 T cells reversed the inhibition of Rap1 activity and prevented induction of ROS. Introduction of active RapV12 into T cells also prevented induction of ROS production. Coincubation of T cells with stimulating anti-CD28 antibodies and inflammatory cytokines synergistically increased T cell ROS production. CONCLUSION: Cell-cell contact between T cells and RA synovial adherent cells mediates Rap1 inactivation and subsequent ROS production in T lymphocytes following exposure to inflammatory cytokines. This process can be blocked by CTLA4-Ig fusion protein.     

CD4+ PD-1+ T cells accumulate as unique anergic cells in rheumatoid arthritis synovial fluid

OBJECTIVE: The PD-1 receptor, whose deficiency in mice causes autoimmune diseases such as arthritis, is considered to be a negative regulator of activated T cells and to play a crucial role in peripheral tolerance. To clarify the involvement of the PD-1 system in rheumatoid arthritis (RA), we investigated PD-1 expression on synovial fluid (SF) T cells from patients with RA. METHODS: FACS analysis for PD-1 was performed on SF T cells from 44 patients with RA and 6 with osteoarthritis (OA), and also on peripheral blood (PB) T cells from 12 RA patients and 7 healthy controls. Two-color analysis of cell surface PD-1 expression and the intracellular concentration of cytokine production was used to investigate CD4+ T cells from SF of patients with RA and PB from controls. RESULTS: Scarcely any PD-1 expression was detected on control PB T or OA SF T cells. In contrast, PD-1+ cells made up 20.9 +/- 8.6% (mean +/- SD) of RA SF T cells. In RA SF, PD-1 was expressed more predominantly on CD4+ T cells than on CD8+ T cells. As well as expressing CD45RO and CXCR3, CD4+ PD-1+ T cells were mostly CTLA-4 positive and CD26 negative, and were enriched in CD45RB(low) cells. Intracellular cytokine staining revealed that CD4+ PD-1+, but not CD4+ PD-1-, T cells produced interleukin 10 (IL-10), and that CD4+ PD-1+ T cells produced less IL-2 than CD4+ PD-1- T cells. CONCLUSION: PD-1+ T cells in RA SF are enriched, and phenotypic analysis suggests that these cells constitute a unique anergic T cell subset in RA SF.     

Stromelysin-1 (MMP-3) in synovial fluid of patients with rheumatoid arthritis has potential to cleave membrane bound Fas ligand

OBJECTIVE: To investigate the relationship between matrix metalloproteinases (MMP) and the soluble form of Fas ligand (sFasL) in the synovial fluid (SF) of patients with rheumatoid arthritis (RA), and to determine which MMP have a major role in cleaving FasL. METHODS: The concentrations of sFas and sFasL in SF from 48 patients with RA and 43 patients with osteoarthritis (OA) were measured using specific ELISA. The levels of different MMP (MMP-1, 2, 3, 7, 9) in SF were also measured by ELISA. The active forms of gelatinases were detected by gelatin zymogram. Human FasL-expressing transfected cells (hFasL/L5178Y) were used to investigate whether FasL is cleaved from membrane bound FasL. RESULTS: Significantly higher levels of MMP-1, 3, and 9 were found in SF from RA patients compared to OA patients, but MMP-7 was not detectable in either group. The concentrations of sFas and sFasL in SF were also higher in RA than in OA patients. However, there was no relationship between the concentration of sFas and sFasL. Among MMP, MMP-3 concentrations in SF were closely correlated with the level of sFasL and with disease activity of RA. Enzymatic cleavage assay indicated that MMP-3 has potential to cleave the FasL expressed on hFasL/L5178Y cells and to produce sFasL. CONCLUSION: There was significant correlation between the concentration of sFasL and MMP-3 in SF of patients with RA. In addition, our data indicate that the shedding of FasL may be regulated by MMP-3 in the joint of patients with RA.     

Effects of glucosamine hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteases by chondrocytes and synoviocytes in osteoarthritis

OBJECTIVES: To determine the response of glucosamine hydrochloride on chondrocytes and synoviocytes in terms of prostaglandin E2 (PGE2), nitric oxide (NO) and matrix metalloproteases (MMPs). METHODS: Chondrocytes and synoviocytes were prepared from joint specimens of patients who underwent total knee arthroplasty for osteoarthritis (OA). Chondrocytes from patients with femoral neck fracture were served as a normal control. Culture cells were stimulated by 5 ng/ml of IL-1beta and treated with various concentration of glucosamine hydrochloride (from 1 microg/ml to 500 microg/ml). PGE2, NO, MMP-1, MMP-3, and MMP-13 levels were evaluated in the culture supernatant. Further, the expression of COX-2 mRNA was studied by semiquantitative PCR. RESULTS: With IL-1beta stimulation, the levels of these mediators increased dramatically, except for NO from synoviocytes. After stimulation, levels of these mediators in OA chondrocytes were higher than synoviocytes and normal chondrocytes, and the level of MMP-3 was higher than those of MMP-1 and MMP-13. Glucosamine hydrochloride at a concentration of 100 microg/ml suppressed PGE2 production, and partly suppressed NO production. It also suppressed the production of MMPs from normal chondrocytes and synoviocytes but not from OA chondrocytes. CONCLUSION: Glucosamine modulates the metabolism of chondrocytes and synoviocytes and its mode of action differs between cells and conditions.