Rheumatoid Synovial Cells Research Articles

Mizoribine, an inhibitor of inosine monophosphate dehydrogenase, inhibits interleukin-6 production by freshly prepared rheumatoid synovial cells

Mizoribine, an immunosuppressive drug, has been used for treatment in organ transplantation, lupus nephritis, and rheumatoid arthritis (RA). On the basis of in vitro experiments, mizoribine has been postulated to be an inhibitor of inosine monophosphate (IMP) dehydrogenase, a pivotal enzyme in the formation of guanine ribonucleotides from IMP. To further characterize the mechanism of the antirheumatic action of this drug, we examined the effect of mizoribine on the production of interleukin (IL)-6, a major inflammatory cytokine in rheumatoid synovia, by freshly prepared rheumatoid synovial cells (RSC). Mizoribine (1.25–5 μg/ml) was able to inhibit the spontaneous production of IL-6 by fresh RSC in a dose–response fashion. The addition of guanosine monophosphate (GMP) reversed its inhibitory effects. In addition, mizoribine inhibited the enhanced production of IL-6 by the IL-1α and/or tumor necrosis factor α-stimulated RSC. Inhibition was also observed at the mRNA level, determined by Northern blot analysis. In contrast, mizoribine did not affect IL-8 production by these cells. These data suggest that mizoribine inhibits IL-6 production by fresh RSC, possibly owing to the depletion of intracellular GMP, and that this inhibitory effect of the drug on rheumatoid synovial cells may be related to its efficacy in RA.

Mizoribine, an inhibitor of inosine monophosphate dehydrogenase, inhibits interleukin-6 production by freshly prepared rheumatoid synovial cells

Mizoribine, an immunosuppressive drug, has been used for treatment in organ transplantation, lupus nephritis, and rheumatoid arthritis (RA). On the basis of in vitro experiments, mizoribine has been postulated to be an inhibitor of inosine monophosphate (IMP) dehydrogenase, a pivotal enzyme in the formation of guanine ribonucleotides from IMP. To further characterize the mechanism of the antirheumatic action of this drug, we examined the effect of mizoribine on the production of interleukin (IL)-6, a major inflammatory cytokine in rheumatoid synovia, by freshly prepared rheumatoid synovial cells (RSC). Mizoribine (1.25–5 μg/ml) was able to inhibit the spontaneous production of IL-6 by fresh RSC in a dose–response fashion. The addition of guanosine monophosphate (GMP) reversed its inhibitory effects. In addition, mizoribine inhibited the enhanced production of IL-6 by the IL-1α and/or tumor necrosis factor α-stimulated RSC. Inhibition was also observed at the mRNA level, determined by Northern blot analysis. In contrast, mizoribine did not affect IL-8 production by these cells. These data suggest that mizoribine inhibits IL-6 production by fresh RSC, possibly owing to the depletion of intracellular GMP, and that this inhibitory effect of the drug on rheumatoid synovial cells may be related to its efficacy in RA.

Involvement of ErbB‐2 in rheumatoid synovial cell growth

Involvement of ErbB‐2 in rheumatoid synovial cell growth

Intracellular expression of CXCR3 on rheumatoid arthritis synovial tissue cells

Inflammatory cell infiltration and synovial activation are important processes in rheumatoid arthritis. Chemotactic gradients of various chemokines are responsible for cell attraction and possibly for their activation. We have previously detected strong expression of chemokine receptor CXCR3 in the rheumatoid joint by immunostaining.

Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.

Involvement of ErbB-2 in rheumatoid synovial cell growth.

The synovial tissue affected by rheumatoid arthritis (RA) is characterized by hyperproliferation of synovial cells. High amounts of epidermal growth factor (EGF) in the synovial fluid of RA patients contribute to the growth of rheumatoid synovial cells. To characterize the receptor for EGF in rheumatoid synovial cells, the expression and function of ErbB family members were examined. Synovial tissues were obtained from surgical excisions. The expression of ErbB products was examined by immunohistochemistry and immunoblotting by using specific antibodies. Primary cultures were established from the surgical materials. Cell growth was measured using MTT. The levels and phosphorylation state of the ErbB-2 protein were analyzed by immunoprecipitation and immunoblotting. The expression of ErbB-2, but not other ErbB-related products, was detected in synovium with RA as compared with that with osteoarthritis (OA) and ligament injury. Growth of primary synovial cells with RA was inhibited by genistein, a tyrosine kinase inhibitor, and herceptin, a specific monoclonal antibody against ErbB-2. Herceptin showed a small effect on growth of primary synovial cells with OA. EGF stimulated the phosphorylation of ErbB-2 in primary synovial cells with RA. This EGF-stimulated phosphorylation was completely abrogated by genistein and herceptin. ErbB-2 is expressed in rheumatoid synovial cells and may function as the receptor for EGF. Our data suggest that mitotic signals from EGF family members are transduced by ErbB-2 in these cells. Inhibition of ErbB-2 may provide a new approach to the effective treatment for RA.

Synovial stromal cells from rheumatoid arthritis patients attract monocytes by producing MCP-1 and IL-8.

Macrophages that accumulate in the synovium of rheumatoid arthritis patients play an important role in the pathogenesis of this inflammatory disease. However, the mechanism by which macrophages are attracted into the inflamed synovium and accumulate there has not been completely delineated. The results of this study show that rheumatoid arthritis synovial stromal cells produce the chemokines monocyte chemotactic protein-1 and IL-8, and these have the capacity to attract peripheral monocytes. These results suggest that one of the mechanisms by which macrophages accumulate in the inflamed synovium is by responding to the chemokines produced locally.

Induction of Matrix Metalloproteinase-8 in Human Fibroblasts by Basic Calcium Phosphate and Calcium Pyrophosphate Dihydrate Crystals: Effect of Phosphocitrate

Although matrix metalloproteinase-8 (MMP-8) was regarded as the exclusive product of the neutrophils, recent studies have shown that it is also expressed in articular chondrocytes, rheumatoid synovial fibroblasts and endothelial cells. Our aim was to determine the expression of MMP-8 in human fibroblasts (HF) by reverse transcription/polymerase chain reaction (RTIPCR), Northern and Western blotting methods and MMP-8 activity assay. We have shown the expression of MMP-8 in HF and its dose-dependent upregulation by basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD) crystals which arc markers of severe joint degeneration in ostcoarthritis. These effects require new protein synthesis and are reversed by phosphocitrate (PC). The results also show that this fibroblast MMP-8 is distinct from the neutrophil MMP-8 and from the fibroblast MMP-1. These results indicate that MMP-8 may play a significant role in the pathogenic effects of the crystals in ostcoarthritis.

Interleukin-4 inhibits interleukin-11 production by rheumatoid synovial cells.

To examine the effect of interleukin-4 (IL-4) on IL-11 production by rheumatoid synovial cells. Freshly isolated rheumatoid synovial cells (FRS) were obtained by collagenase digestion of rheumatoid arthritis (RA) synovial tissue specimens taken at the time of operation. Rheumatoid synovial cells at four to eight passages were used as cultured rheumatoid synovial fibroblasts (RSF). IL-11 concentration was measured by ELISA. IL-4 inhibited the production of IL-11 by FRS in a dose-dependent manner. This inhibition was observed in FRS obtained from six patients, and the mean inhibition was 46.5%. The inhibitory effect of IL-4 on IL-11 production was cancelled by the addition of anti-IL-4 antibody. IL-4 also inhibited IL-11 production by IL-1alpha-stimulated cultured RSF. IL-4 inhibited IL-11 production by rheumatoid synovial cells. IL-4 has a protective effect on bone resorption. On the contrary, IL-11 participates in bone resorption via osteoclastogenesis. Therefore, IL-4 may exert its protective effect on bone resorption, at least in part, via inhibition of IL-11 production in rheumatoid joints.

Enhanced expression and DNA binding activity of two CCAAT/enhancer-binding protein isoforms, C/EBPbeta and C/EBPdelta, in rheumatoid synovium.

To investigate the activation and expression of CCAAT/enhancer-binding proteins (C/EBP), especially C/EBPbeta and -delta, in rheumatoid synovium, and their pathogenic implications in rheumatoid arthritis (RA). The activation of C/EBPbeta and -delta was assessed in synovial tissues from patients with RA by electrophoretic mobility shift assay (EMSA); DNA binding activity of C/EBPs was evaluated by measuring EMSA band density. The expression and distribution of C/EBPbeta and -delta in synovial tissues were examined by immunohistochemistry analysis. As a control, synovial tissues from patients with osteoarthritis (OA) were studied. Enhanced DNA binding activity of C/EBPbeta and -delta, 2 major members of the C/EBP family, was detected in synovial tissues from RA patients, while synovial tissues from the patients with OA showed only faint or marginal activity (mean +/- SEM arbitrary units [AU] RA 23.3 +/- 11.7 in RA versus 4.5 +/- 1.3 in OA; P < 0.05). Moreover, the binding activities of the C/EBP proteins were correlated with both serum C-reactive protein levels (r = 0.62, P < 0.05) and synovial interleukin-6 messenger RNA levels (r = 0.60, P < 0.05). In immunohistochemistry studies, C/EBPbeta and -delta were detected predominantly in the rheumatoid synovial lining cells (both CD14+ and CD14- cells). C/EBPbeta and -delta may contribute to the pathology of rheumatoid synovitis.

Enhanced expression and DNA binding activity of two CCAAT/enhancer-binding protein isoforms, C/EBPβ and C/EBPδ, in rheumatoid synovium

Objective To investigate the activation and expression of CCAAT/enhancer-binding proteins (C/EBP), especially C/EBPβ and -δ, in rheumatoid synovium, and their pathogenic implications in rheumatoid arthritis (RA). Methods The activation of C/EBPβ and -δ was assessed in synovial tissues from patients with RA by electrophoretic mobility shift assay (EMSA); DNA binding activity of C/EBPs was evaluated by measuring EMSA band density. The expression and distribution of C/EBPβ and -δ in synovial tissues were examined by immunohistochemistry analysis. As a control, synovial tissues from patients with osteoarthritis (OA) were studied. Results Enhanced DNA binding activity of C/EBPβ and -δ, 2 major members of the C/EBP family, was detected in synovial tissues from RA patients, while synovial tissues from the patients with OA showed only faint or marginal activity (mean ± SEM arbitrary units [AU] RA 23.3 ± 11.7 in RA versus 4.5 ± 1.3 in OA; P < 0.05). Moreover, the binding activities of the C/EBP proteins were correlated with both serum C-reactive protein levels (r = 0.62, P < 0.05) and synovial interleukin-6 messenger RNA levels (r = 0.60, P < 0.05). In immunohistochemistry studies, C/EBPβ and -δ were detected predominantly in the rheumatoid synovial lining cells (both CD14+ and CD14− cells). Conclusion C/EBPβ and -δ may contribute to the pathology of rheumatoid synovitis.

Expression and Tissue Localization of Membrane-Types 1, 2, and 3 Matrix Metalloproteinases in Rheumatoid Synovium

In vitro, membrane-type matrix metalloproteinases (MT-MMP) are known to activate the zymogen of MMP-2 (proMMP-2, progelatinase A), which is one of the key MMP in joint destruction in rheumatoid arthritis. In the present study, we examined the production and activation of proMMP-2, and the expression of MT1-MMP, MT2-MMP, and MT3-MMP, their correlation with proMMP-2 activation, and their localization in rheumatoid synovial tissue. Using sandwich enzyme immunoassay and gelatin zymography techniques, proMMP-2 production levels and activation ratios were found to be significantly higher in rheumatoid synovium compared with normal synovium (p < 0.01). Quantitative RT-PCR analyses demonstrated that MT1-MMP and MT3-MMP were expressed in all rheumatoid synovial tissue (30 of 30 cases), but that the mean expression level of MT1-MMP was approximately 11-fold higher than MT3-MMP. Significant correlation was found between the mRNA expression level of MT1-MMP and the activation ratio of proMMP-2 (p < 0.01). In situ hybridization indicated that the hyperplastic lining cells of rheumatoid synovium expressed MT1-MMP. Immunohistochemistry demonstrated that MT1-MMP was co-localized with MMP-2 and with a tissue inhibitor of metalloproteinase-2, and was mainly located in the rheumatoid synovial lining cells. In situ zymography of rheumatoid synovium showed gelatinolytic activity, predominantly in the lining cell layer. This activity was blocked when incubated with BB94, a specific MMP inhibitor. These results demonstrate that MT1-MMP plays an important role in the activation of proMMP-2 in the rheumatoid synovial lining cell layer, and suggest that its activity may be involved in the cartilage destruction of rheumatoid arthritis.

Preferential inhibition of cyclooxygenase-2 by meloxicam in human rheumatoid synoviocytes

The aim of this study was to evaluate the anti-inflammatory effect of 4-hydroxy-2-methyl- N-[5-methyl-2-thiazolyl]-2 H-1,2-benzothiazine-3-carboxamide-1,1-dioxide (meloxicam) using cultured rheumatoid synovial fibroblast-like cells (synoviocytes). Synoviocytes were treated with meloxicam in the presence or absence of interleukin-1β. Meloxicam had no effect on both cyclooxygenase-1 and -2 expression as determined by Western blot analysis, immunohistochemical staining, and reverse transcription polymerase chain reaction (RT-PCR). Even the lower doses of meloxicam inhibited cyclooxygenase-2 activity, but only the higher doses of meloxicam inhibited cyclooxygenase-1 activity as determined by prostaglandin E 2 synthesis assay. So meloxicam had a preferential inhibitory effect of cyclooxygenase-2 relative to cyclooxygenase-1 on cultured rheumatoid synoviocytes without affecting cyclooxygenase expression. On the other hand, indomethacin had no selectivity and dexamethasone inhibited the expression of cyclooxygenase-2. Our data indicate that clinical efficacy and safety of meloxicam for rheumatoid arthritis may result from its preferential inhibition of cyclooxygenase-2 activity relative to cyclooxygenase-1 on rheumatoid synoviocytes.

Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates.

Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappaB/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic mobility shift-Western immunoblotting assays. Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB. However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.

TUMOUR NECROSIS FACTOR α (TNF-α) INTERFERES WITH Fas-MEDIATED APOPTOTIC CELL DEATH ON RHEUMATOID ARTHRITIS (RA) SYNOVIAL CELLS: A POSSIBLE MECHANISM OF RHEUMATOID SYNOVIAL HYPERPLASIA AND A CLINICAL BENEFIT OF ANTI-TNF-α THERAPY FOR RA

To investigate the mechanism of rheumatoid synovial hyperplasia (RASH), the influence of tumour necrosis factor α (TNF-α) on Fas-mediated apoptotic cell death (Fas-ACD) was examined on cultured rheumatoid synovial cells (RASCs). RASCs were obtained from the synovial tissues of eight patients with rheumatoid arthritis (RA) and SCs from eight patients with osteoarthritis (OA) were used as a control. To examine the influence of TNF-α on Fas-ACD, SCs were cultured with anti-Fas antibody (CH11) for 16h in the absence or presence of different doses of recombinant TNF-α. ACD was determined by electron microscopic analysis and the percentage of apoptotic cells was calculated by trypan blue staining. In addition, the expression of Fas and Bcl-2 on RASCs was examined by flow cytometry. As a result, RASCs were more susceptible to Fas-ACD in vitro than OASCs. TNF-α interfered with Fas-ACD on RASCs in a dose-dependent manner. Moreover, removal of TNF-α activity by a neutralizing anti-TNF-α antibody (cA2) restored Fas-ACD. Flow cytometric analysis showed no significant changes in either Fas or Bcl-2 expression on RASCs after the culture with TNFα.These results suggest the following: (1) Fas-ACD might be diminished in vivo by local excessive TNF-α and this might contribute in part to RASH. (2) The inhibition of Fas-ACD on RASCs by TNF-α might not be associated with changes in the expression of Fas or Bcl-2. (3) In addition, considering a magnetic resonance imaging (MRI) finding of marked reduction in the RASH after cA2 treatment, blockade of TNF-α activity could restore Fas-ACD in RA synovium, implicating a clinical benefit of anti-TNF-α therapy for RA.

Regulation of Rheumatoid Synovial Cell Growth by Ceramide

Overgrowth of rheumatoid synoviocytes, which results in joint destruction, is due to impaired balance between cell proliferation and cell death (apoptosis). Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. We investigated the effects of ceramide on growth-promoting anti-apoptotic signals in rheumatoid synovial cells. Human synovial cells isolated from patients with rheumatoid arthritis (RA) were stimulated with platelet-derived growth factor (PDGF) in the presence or absence of C2-ceramide. The kinase activity of Akt, MEK, and ERK1/2 was analyzed in PDGF-stimulated synovial cells by Western blot analysis. Pretreatment with C2-ceramide completely inhibited PDGF-induced cell cycle progression of rheumatoid synovial cells. PDGF stimulation induced phosphorylation and activation of Akt, MEK, and ERK1/2 in rheumatoid synovial cells. C2-ceramide inhibited the activation of Akt, MEK and ERK1/2 in PDGF-stimulated synovial cells. Our data demonstrated that inhibition of anti-apoptotic kinases, such as Akt and ERK1/2, may play an important role in ceramide-mediated apoptosis of rheumatoid synovial cells.

A major metabolite of aceclofenac, 4′-hydroxy aceclofenac, suppresses the production of interstitial pro-collagenase/proMMP-1 and pro-stromelysin-1/proMMP-3 by human rheumatoid synovial cells

We examined the effects of aceclofenac and its metabolites on the production of pro-collagenase-1/pro-matrix metalloproteinase-1 (proMMP-1), pro-gelatinase A/proMMP-2, pro-stromelysin-1/proMMP-3 and tissue inhibitor of metalloproteinases-1 (TIMP-1) by rheumatoid synovial cells. Synovial cells were obtained from patients with rheumatoid arthritis. Cultures of confluent cells were treated with interleukin-1beta (IL-1beta)(1 ng/ml) and/or test drugs (0.3-30 microM) for 48 h. Production of proMMPs and TIMP-1 was monitored by Western blotting or gelatin zymography. Prostaglandin E2 (PGE2) was measured by an enzyme immunoassay. 4'-Hydroxy aceclofenac, a major metabolite of aceclofenac, down-regulated both basal and IL-1beta-induced production of proMMP-1 and proMMP-3 at a concentration sufficient to suppress PGE2 production without modulating proMMP-2 or TIMP-1, whereas aceclofenac itself had no marked effect on the production of proMMPs. Down-regulation of proMMP-1 and proMMP-3 production by 4'-hydroxy aceclofenac may contribute to the therapeutic effect of aceclofenac on rheumatoid arthritis and osteoarthritis.

Novel gene therapy for rheumatoid arthritis by FADD gene transfer: induction of apoptosis of rheumatoid synoviocytes but not chondrocytes.

Synovial cells in the rheumatoid synovium show abnormal proliferation, leading to joint destruction. Rheumatoid synovial cells express functional Fas antigen and are susceptible to Fas-mediated apoptosis. We have proposed the induction of apoptosis by Fas/Fas ligand system of proliferative rheumatoid synovium as a novel therapy for rheumatoid arthritis (RA). We have recently reported that Fas-associated death domain protein (FADD) plays a key role in Fas-mediated apoptosis of synovial cells in patients with RA. In this study, we determined whether FADD gene transfer could induce apoptosis of RA synoviocytes in vitro and in vivo. Transfection of FADD gene by adenoviral vector into cultured RA synoviocytes induced up-regulation of FADD expression and apoptosis. In addition, local injection of FADD adenovirus (Ad-FADD) eliminated synoviocytes in vivo by induction of apoptosis of proliferating human rheumatoid synovium engrafted in severe combined immunodeficiency mouse, which is the most suitable animal model of RA for the evaluation of treatment strategy in vivo. In addition, Ad-FADD-induced apoptosis was limited to cells of the synovium tissue and did not affect chondrocytes. Our results strongly suggest that FADD gene transfer can induce apoptosis of RA synoviocytes both in vitro and in vivo, suggesting that FADD gene transfer might be effective in the treatment of RA.

Induction of COX-2 Expression by Nitric Oxide in Rheumatoid Synovial Cells

Prostaglandins formed by cyclooxygenase (COX) enzymes are important mediators of inflammation. The contribution of inducible COX-2 in the rheumatoid synovium is well documented. In this study, we evaluated the contribution of nitric oxide (NO) to COX-2 expression in rheumatoid synovial cells. Exposure of rheumatoid synovial cells to a NO donor, SNAP, induced COX-2 protein expression in a dose-dependent manner. RT-PCR analysis also demonstrated that COX-2 mRNA was induced in SNAP-treated synovial cells. Dexamethasone at therapeutic concentrations markedly inhibited this NO-mediated COX-2 expression in synovial cells. In contrast to its effect on COX-2 expression, SNAP did not affect the constitutive expression of COX-1 in rheumatoid synovial cells. Our findings suggest that NO is an important modulator of COX-2 expression and that glucocorticoids exert their anti-inflammatory action in rheumatoid synovium, at least in part, by suppression of COX-2 induction.

Increased expression of CD23 in rheumatoid synovitis

Objective. Soluble (s)CD23 is a potent macrophage stimulator. High levels of this molecule have been reported in rheumatoid arthritis (RA) serum. We investigated the expression of CD23 and its ligands in rheumatoid synovial fluid and cells. Methods. Levels of sCD23, and cellular expression of CD23 and its ligands CD21, CD11b, and CD11c were measured in synovial fluid (SF) of RA patients and in blood of RA patients and controls. Results. SF contained higher levels of sCD23 than either rheumatoid or normal sera (median 4.8, 3.16,and 1.13 ng/ml respectively, p<0.01). Synovial CD23 was found to be expressed principally on macrophages. While little CD21 expression was detected, CD11b and CD11c were both expressed at high levels, particularly on macrophages. Conclusions. Soluble CD23 is present in high levels in RA synovial fluid. Macrophages appear to be the principal source. Macrophages also express ligands for sCD23, and may therefore also be the targets of this potent pro-inflammatory molecule.