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Osteoarthritis (OA) is characterized by progressive joint destruction, including synovial membrane alteration. EphB4 and its ligand ephrin-B2 were found in vitro to positively affect OA subchondral bone and cartilage. In vivo in an experimental mouse model overexpressing bone-specific Ephb4 (TgEphB4), a protective effect was found on both the subchondral bone and cartilage during OA. We investigated in the TgEphB4 mouse model the in vivo effect on synovial membrane during OA. Knee OA was surgically induced by destabilization of the medial meniscus (DMM). Synovial membrane was evaluated using histology, histomorphometry, IHC, and real-time PCR. Compared to DMM-wild-type (WT) mice, DMM-TgEphB4 mice had a significant decrease in synovial membrane thickness, vascular endothelial growth factor, and the profibrotic markers fibrin, type 1 procollagen, type 3 collagen, connective tissue growth factor, smooth muscle actin-α, cartilage oligomeric matrix protein, and procollagen-lysine, and 2-oxoglutarate 5-dioxygenase 2. Moreover, factors known to modulate transforming growth factor-β signaling, transforming growth factor receptor 1/ALK1, phosphorylated Smad-1, and heat shock protein 90β were significantly decreased in DMM-TgEphB4 compared with DMM-WT mice. Ephb4 overexpression also exhibited a protective effect on synovial membrane thickness of aged (24-month-old) mice. Overexpression of bone-specific Ephb4 clearly demonstrated prevention of the development and/or progression of fibrosis in OA synovial membrane, reinforcing the hypothesis that protecting the subchondral bone prophylactically and during OA reduces the pathologic changes in other articular tissues., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: To explore the disease-modifying effect, under therapeutic conditions, of strontium ranelate (SrRan) on the progression of joint structural changes and on the major pathophysiological pathways in an experimental osteoarthritis dog model., Methods: Dogs underwent sectioning of the anterior cruciate ligament, and 4 weeks after surgery received oral treatment of SrRan 25, 50 or 75 mg/kg per day, or placebo for 12 weeks. Methods included macroscopy, picrosirius red staining, histology, subchondral bone histomorphometry, quantitative PCR, and ELISA for CTX-II level in serum. Strontium plasma and synovial fluid levels were also measured., Results: At steady state, strontium blood exposures were within the clinical therapeutic range of osteoarthritis patients and correlated with strontium concentrations in synovial fluid. SrRan treatment significantly reduced the osteoarthritis cartilage lesions at all doses tested (p≤0.05). Significantly better preservation of the collagen network was also found in SrRan-treated dogs at 50 and 75 mg/kg per day (p=0.03). The osteoarthritis subchondral bone thickening observed in osteoarthritis-placebo dogs was significantly reduced by SrRan at 50 mg/kg per day (p=0.02). The increased gene expression levels of MMP-1, MMP-13 and cathepsin K in osteoarthritis cartilage were all significantly reduced by SrRan at 75 mg/kg per day (p≤0.03) as were, in osteoarthritis synovium, IL-1β at 50 and 75 mg/kg per day (p=0.05) and MMP-3 at all doses tested (p≤0.02). The serum level of CTX-II was reduced (p≤0.04) by SrRan at 16 weeks in dogs treated with 50 and 75 mg/kg per day., Conclusions: This study is the first to demonstrate in vivo in an animal model that SrRan reduced the progression of osteoarthritis structural changes. The inhibition of several key proteases as well as IL-1β may have contributed to the beneficial effect of SrRan.

Osteoarthritis (OA) is a progressive and disabling disease resulting from a combination of risk factors, including advancing age, genetics, trauma, knee malalignment, increased biomechanical loading of joints through obesity, augmented bone density and an imbalance in physiological processes resulting in catabolic cascades on a molecular level. This review will highlight the involvement early in the disease process of not only the cartilage but also the synovial membrane and subchondral bone and the pathophysiological mechanisms of each of these tissues that lead to joint degeneration. We will summarize the current pathological mechanisms that occur in the abovementioned articular tissues, and briefly discuss their interconnections during OA.

Introduction: High mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1β) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA)., Methods: HMGB1 was determined in human synovium using immunohistochemistry, comparing normal to OA. OA synoviocytes were incubated with HMGB1 at 15 or 25 ng/ml in the absence or presence of IL-1β (10 ng/ml). Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. Matrix metalloproteinase (MMP) activity was studied by fluorometric procedures and nuclear factor (NF)-κB activation by transient transfection with a NF-κB-luciferase plasmid., Results: In the normal synovium, HMGB1 was found in the synovial lining cells, sublining cells, and in the vascular wall cells. The distribution of HMGB1 in OA synovium was similar but the number of HMGB1 positive cells was higher and HMGB1 was also present in infiltrated cells. In normal synovial membrane cells, HMGB1 was found mostly in the nuclei, whereas in OA, HMGB1 was generally found mostly in the cytoplasm. In OA synoviocytes, HMGB1 alone at concentrations of 15 or 25 ng/ml did not affect the production of IL-6, IL-8, CCL2, CCL20, MMP-1 or MMP-3, but in the presence of IL-1β, a significant potentiation of protein and mRNA expression, as well as MMP activity was observed. HMGB1 also enhanced the phosphorylated ERK1/2 and p38 levels, with a lower effect on phosphorylated Akt. In contrast, JNK1/2 phosphorylation was not affected. In addition, HMGB1 at 25 ng/ml significantly potentiated NF-κB activation in the presence of IL-1β., Conclusions: Our results indicate that HMGB1 is overexpressed in OA synovium and mostly present in extracellular form. In OA synoviocytes, HMGB1 cooperates with IL-1β to amplify the inflammatory response leading to the production of a number of cytokines, chemokines and MMPs. Our data support a pro-inflammatory role for this protein contributing to synovitis and articular destruction in OA.

Objective: To investigate the distribution of the bone morphogenic protein (BMP) antagonist chordin in normal and osteoarthritic cartilage and synovial membranes, and its regulation in chondrocytes and synovial fibroblasts by inflammatory and growth factors., Methods: Localisation of chordin in tissues was undertaken by immunohistochemistry and gene regulation was determined by real time polymerase chain reaction., Results: In normal cartilage, chordin was found at low levels (mean (SD), 7.6 (1.3)%), mainly in the very superficial layers. In osteoarthritis, chordin was also found in the superficial layers (8.9 (1.1)%), though at a significantly higher level (24.7 (1.5)%) in the last two thirds of the cartilage. In contrast to normal cells, chordin mRNA and protein levels were significantly downregulated (p<0.01) in osteoarthritic chondrocytes by all the growth factors tested. Interferon gamma stimulated chordin expression in normal but not in osteoarthritic chondrocytes (p<0.0002), while interleukin 1 beta and tumour necrosis factor alpha did not affect the expression level. However, no difference was found in either the distribution or regulation of chordin in normal and osteoarthritic synovial membranes or synovial fibroblasts., Conclusions: The differential distribution and regulation of chordin in normal and osteoarthritic cartilage and chondrocytes suggests an involvement of this antagonist in the osteoarthritic process.

Objective: To study the mechanisms responsible for the cross-talk between lipoxygenase (LOX) and cyclooxygenase (COX) pathways in human osteoarthritic (OA) synovial explants, and to confirm the arachidonic acid (AA) shunting phenomenon and its influence on interleukin 1beta (IL-1beta) synthesis., Methods: Synovial membrane explants were cultured in the absence or presence of different drugs that inhibit COX and/or LOX activities. Concentrations of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), lipoxin A4 (LXA4), and IL-1beta were measured., Results: When membrane explants were incubated with naproxen (COX inhibitor) under unstimulated conditions, the production of LTB4 was dose-dependently enhanced, reaching a 5-fold increase over the control. This shunt could be partially reversed by the addition of exogenous PGE2. Under lipopolysaccharide (LPS) stimulation, both licofelone (COX/LOX inhibitor) at therapeutic concentrations and NDGA (LOX inhibitor) inhibited LTB4 production, whereas naproxen did not amplify the LPS-induced LTB4 production. Conversely, using NDGA, it was found that a shunt of AA from the LOX to the COX pathway did not occur. Under LPS conditions, both naproxen and licofelone inhibited LXA4, inducing an increase in the LTB4/LXA4 ratio with naproxen treatment but not with licofelone. Under these conditions, naproxen treatment induced a higher level of IL-1beta production., Conclusion: We demonstrated in OA synovium that a shunt from AA to the LOX pathway occurred and that treatment with a nonselective COX inhibitor could increase the production of LTB4 and secondarily the synthesis of IL-1beta. Therefore treatment with licofelone, which can act on both COX and LOX pathways, may have some interesting properties in the treatment of OA.

Objective: The cyclooxygenase (COX) metabolite, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), has been reported to inhibit the expression of a number of genes involved in the pathogenesis of arthritis. However, its effects on COX-2 remain controversial. We undertook this study to investigate the effects of 15d-PGJ(2) on interleukin-1beta (IL-1beta)-induced COX-2 expression in human synovial fibroblasts (HSFs)., Methods: HSFs were cultured with IL-1beta in the absence or presence of 15d-PGJ(2), and the levels of COX-2 protein and messenger RNA (mRNA) expression were evaluated using Western blotting and real-time reverse transcriptase-polymerase chain reaction, respectively. COX-2 promoter activity was analyzed in transient transfection experiments. Chromatin immunoprecipitation assays were performed to evaluate the level of histone acetylation and the recruitment of histone deacetylases (HDACs) 1, 2, and 3 and histone acetylase (HAT) p300 to the COX-2 promoter., Results: IL-1beta-induced COX-2 protein and mRNA expression, as well as COX-2 promoter activation, were inhibited by 15d-PGJ(2). Troglitazone, a selective peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, enhanced COX-2 expression, while GW9662, a specific PPARgamma antagonist, relieved the suppressive effect of 15d-PGJ(2). IL-1beta-induced histone H3 acetylation was selectively blocked by 15d-PGJ(2). The reduction of histone H3 acetylation did not correlate with the recruitment of HDACs to the COX-2 promoter. Also, treatment with the specific HDAC inhibitor, trichostatin A, did not relieve the suppressive effect of 15d-PGJ(2), indicating that HDACs are not involved in the inhibitory effect of 15d-PGJ(2). Furthermore, 15d-PGJ(2) blocked IL-1beta-induced recruitment of p300 to the COX-2 promoter, which may be the mechanism for decreased histone H3 acetylation and COX-2 expression. In accordance with this, overexpression of p300, but not of a mutant p300 lacking HAT activity, relieved the inhibitory effect of 15d-PGJ(2) on COX-2 promoter activation., Conclusion: These data suggest that 15d-PGJ(2) can inhibit IL-1beta-induced COX-2 expression by an HDAC-independent mechanism, probably by interfering with HAT p300.

Objective: To compare gene expression in normal and osteoarthritic (OA) human chondrocytes using microarray technology. Of the novel genes identified, we selected follistatin, a bone morphogenetic protein (BMP) antagonist, and investigated its expression/regulation as well as that of 3 other antagonists, gremlin, chordin, and noggin, in normal and OA chondrocytes and synovial fibroblasts., Methods: Basal and induced gene expression were determined using real-time polymerase chain reaction. Gene regulation was monitored following treatment with inflammatory, antiinflammatory, growth, and developmental factors. Follistatin protein production was measured using a specific enzyme-linked immunosorbent assay, and localization of follistatin and gremlin in cartilage was determined by immunohistochemical analysis., Results: All BMP antagonists except noggin were expressed in chondrocytes and synovial fibroblasts. Follistatin and gremlin were significantly up-regulated in OA chondrocytes but not in OA synovial fibroblasts. Chordin was weakly expressed in normal and OA cells. Production of follistatin protein paralleled the gene expression pattern. Follistatin and gremlin were expressed preferentially by the chondrocytes at the superficial layers of cartilage. Tumor necrosis factor alpha and interferon-gamma significantly stimulated follistatin expression but down-regulated expression of gremlin. Interleukin-1beta (IL-1beta) had no effect on follistatin but reduced gremlin expression. Conversely, BMP-2 and BMP-4 significantly stimulated expression of gremlin but down-regulated that of follistatin. IL-13, dexamethasone, transforming growth factor beta1, basic fibroblast growth factor, platelet-derived growth factor type BB, and endothelial cell growth factor down-regulated the expression of both antagonists., Conclusion: This study is the first to show the possible involvement of follistatin and gremlin in OA pathophysiology. The increased activin/BMP-binding activities of these antagonists could affect tissue remodeling. The data suggest that follistatin and gremlin might appear at different stages during the OA process, making them interesting targets for the treatment of this disease.

Membrane-associated prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the conversion of PGH(2) to PGE(2), which contributes to many biological processes. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. Here, we examined the effect of PPARgamma ligands on interleukin-1beta (IL-1beta)-induced mPGES-1 expression in human synovial fibroblasts. PPARgamma ligands 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinedione troglitazone (TRO), but not PPARalpha ligand Wy14643, dose-dependently suppressed IL-1beta-induced PGE(2) production, as well as mPGES-1 protein and mRNA expression. 15d-PGJ(2) and TRO suppressed IL-1beta-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPARgamma further enhanced, whereas overexpression of a dominant negative PPARgamma alleviated, the suppressive effect of both PPARgamma ligands. Furthermore, pretreatment with an antagonist of PPARgamma, GW9662, relieves the suppressive effect of PPARgamma ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPARgamma. We demonstrated that PPARgamma ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. The suppressive effect of PPARgamma ligands was enhanced in the presence of a PPARgamma expression plasmid. Electrophoretic mobility shift and supershift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both 15d-PGJ(2) and TRO suppressed IL-1beta-induced DNA-binding activity of Egr-1. These data define mPGES-1 and Egr-1 as novel targets of PPARgamma and suggest that inhibition of mPGES-1 gene transcription may be one of the mechanisms by which PPARgamma regulates inflammatory responses.

Objective: To investigate the regulation of osteoarthritis (OA) synovial fibroblast nitric oxide (NO) induced cell death., Methods: Cultured synovial fibroblasts from human OA synovium were incubated with NO donor sodium nitroprusside (SNP) in the absence or presence of specific inhibitors of different protein kinases, cyclooxygenase-2 (COX-2), caspase-3 and caspase-9, inducible NO synthase, and in the absence or presence of prostaglandin E2 (PGE2). Experiments were also performed using scavengers of NO (carboxy-PXTO), peroxynitrite (uric acid), and superoxide (taxifolin). The level of cell death was measured by MTT and DNA fragmentation., Results: Human OA synovial fibroblasts incubated with SNP decreased cell viability and increased DNA fragmentation in a dose dependent manner. This was associated with increased levels of both COX-2 and PGE2 production. Selective inhibition of COX-2 by NS-398 significantly inhibited SNP induced cell death, even in the presence of exogenously added PGE2. Experiments revealed that SNP treated cells expressed increased levels of active caspase-3 and caspase-9, while Bcl-2 was downregulated. Incubation of these treated cells with inhibitors of caspase-3 (Z-DEVD-FMK) or caspase-9 (Z-LEHD-FMK) protected viability of SNP treated OA synovial fibroblasts, indicating that NO mediated cell death was mainly related to apoptosis. This was also confirmed by measuring the DNA fragmentation (TUNEL method) and the level of active caspase-3 (immunocytochemistry) in these cells. Data also showed that SNP induces the activation of kinases MEK 1/2, p38, and tyrosine kinases. Specific inhibition of tyrosine kinases completely abrogated the SNP induced cell death. In turn, this cell death protection was associated with a marked inhibition of caspase-3 and caspase-9 activities, as well as COX-2/PGE2 production. Moreover, data showed that the NO donor SNP induced cell death was not solely related to the production of NO or peroxynitrite, but to the generation of reactive oxygen species (ROS) such as hydrogen peroxide and/or superoxide., Conclusion: Our results provided strong evidence of the role of tyrosine kinase and mitogen activated protein kinase activation, by upregulation of COX-2 expression, in NO induced OA synovial fibroblast death. The generation of ROS such as hydrogen peroxide and superoxide appeared to be a major factor in the death of these cells.

Objective: To determine the level of leukotriene B4 (LTB4) synthesized and released by synovium of patients with osteoarthritis (OA), and to study the role of lipoxygenase (LO)/cyclooxygenase (COX) products on proinflammatory cytokine and interstitial collagenase (MMP-1) synthesis., Methods: Human OA synovial explants were cultured in the presence of lipopolysaccharide (L) and the ionophores ionomycin (I) and thapsigargin (T) (LIT) for 72 h at 37 degrees C, and LTB4 released into the culture medium was measured in the absence or presence of a COX-2-specific inhibitor, NS-398, or the 5-LO activating protein inhibitor Bay-x-1005. Increasing concentrations of LTB4 (10(-9) to 10(-6) M) were incubated with explants for 24 h at 37 degrees C, and interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in the conditioned medium were quantitated by ELISA. The effect of endogenous eicosanoids on basal and induced levels of IL-1beta, TNF-alpha, and MMP-1 synthesis was examined by incubating explants in the presence of NS-398 and Bay-x-1005. The effect of antiinflammatory cytokines rhIL-4, IL-10, and IL-13 on basal and LTB4 dependent stimulation of IL-1beta/TNF-alpha synthesis was studied under titration conditions., Results: Physiologically relevant concentrations (10(-10) to 10(-9) mol/l) of LTB4 were produced in the presence of LIT. Bay-x-1005 abrogated LTB4 release, while NS-398 was without effect. LTB4 stimulated IL-1beta and TNF-alpha synthesis with an EC50 of 190 +/- 35 and 45 +/- 9 nmol/l, respectively. Significant concentrations of IL-1beta and TNF-alpha were released (100-200 and 500-600 pg/ml, respectively). Basal and LIT induced IL-1beta and TNF-alpha production were inhibited by Bay-x-1005 in a dose dependent manner, while the addition of NS-398 caused a potent stimulatory effect. The preferential COX-2 inhibitor also induced MMP-1 synthesis in a manner essentially identical to the proinflammatory cytokines. The antiinflammatory cytokine IL-4 blocked LTB4 dependent stimulation of IL-1beta and TNF-alpha synthesis. In contrast, IL-10 markedly stimulated both cytokines when incubated alone or in the presence of LTB4 where the effect was additive., Conclusion: Endogenous and locally produced eicosanoids regulate proinflammatory cytokine and MMP-1 synthesis under basal and stimulated conditions in vitro, with leukotrienes and prostaglandins having opposite effects in general. The clinical use of antiinflammatory drugs that inhibit eicosanoid synthesis requires an appreciation of their relative capacity to inhibit LO/COX in order to predict their effect on the synthesis of proinflammatory cytokines and matrix metalloproteases. IL-10 stimulated proinflammatory cytokine synthesis in our ex vivo culture system.

Objective: To characterize the inhibitory effects of therapeutic concentrations of the nonsteroidal antiinflammatory drug nimesulide (NIM) on peroxisome proliferator-activated receptor (PPAR)-induced cyclooxygenase 2 (COX-2) gene expression in human synovial fibroblasts (HSFs) from patients with osteoarthritis (OA) and to define the intracellular mechanisms mediating the response., Methods: PPARalpha and PPARgamma messenger RNA (mRNA) expression and protein synthesis in OA HSFs were measured by reverse transcription-polymerase chain reaction and electrophoretic mobility shift assay, respectively. Experiments investigating endogenous and overexpressed PPARalpha and PPARgamma activation of COX-2 mRNA and protein were conducted by incubating nontransfected and transfected cells with increasing concentrations of cognate ligands WY-14,643 (alpha agonist), ciglitasone (gamma agonist), and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) in the absence or presence of NIM and NS-398 (1 microM). COX-2 mRNA and protein were measured by Northern and Western blotting procedures, respectively. Receptor activation studies were evaluated by cotransfecting pSG5-Gal 4 DNA binding domain (DBD)-PPARalpha ligand binding domain (LBD) or pSG5-Gal 4 DBD-PPARgamma LBD chimeric constructs with a 5x Gal 4 enhancer site tk-tataa-luciferase reporter under ligand stimulation in the presence or absence of increasing concentrations of NIM. Gene transactivation analyses were conducted by treating cells overexpressing cytomegalovirus (CMV)-PPARalpha or CMV-PPARgamma expression constructs with either a PPAR response element (PPRE)-luciferase construct containing 3 DR1 acyl-coenzyme A (acyl-CoA) oxidase gene response elements or human COX-2 promoter constructs with WY-14,643, ciglitasone, and 15d-PGJ(2) in the presence or absence of increasing concentrations of NIM., Results: Human synovial cells expressed functional PPAR isoforms, PPARalpha and PPARgamma. Neither receptor agonists nor antagonists modulated the intracellular protein levels of PPAR. PPARalpha and, especially, PPARgamma mediated the induction of COX-2 gene expression by receptor agonists. Stimulation of COX-2 mRNA expression and protein synthesis by 15d-PGJ(2) appeared to occur through a receptor-independent process. NIM inhibited PPAR agonist stimulation of COX-2 expression and synthesis in a dose-dependent manner in both nontransfected cells and cells overexpressing both receptor isoforms. NIM potently abrogated basal and ligand-stimulated PPRE(3X) DR1 acyl-CoA oxidase-driven luciferase activity and also human PPRE-containing COX-2 promoter activity., Conclusion: PPAR-mediated induction of COX-2 expression and synthesis in human OA synovial fibroblasts is inhibited by therapeutic concentrations of NIM through the functional antagonism of ligand-dependent receptor activation, with the resultant suppression of PPAR-dependent transactivation of target genes (e.g., COX-2).

Objective: To investigate the expression and activity of PPARgamma in human synovial fibroblasts and the effects of PPARgamma agonists on the expression of MMP-1. The molecular mechanisms by which PPARgamma agonists modulate MMP-1 expression were also examined., Methods: PPARgamma expression and activity were measured using reverse-transcription polymerase chain reaction (RT-PCR) and transient transfection assays. Human synovial fibroblasts were cultured with IL-1beta in the absence or presence of PPARgamma activators, and the expression and production of MMP-1 were evaluated by Northern blot and ELISA, respectively. The effect of 15d-PGJ(2) on MMP-1 promoter activation was analysed in transient transfection experiments, while electrophoretic mobility shift assays were performed to study the binding activity of the transcription factor AP-1., Results: PPARgamma was expressed and transcriptionally functional in human synovial fibroblasts. PPARgamma activators (15d-PGJ(2) and BRL 49653) inhibited IL-1beta-induced MMP-1 synthesis in a dose-dependent manner. Similarly, both activators inhibited IL-1-induced MMP-1 mRNA expression. Activation of the human MMP-1 promoter was also attenuated by 15d-PGJ(2), indicating that the inhibitory effect of 15d-PGJ(2) occurs at the transcriptional level. Interestingly, 15d-PGJ(2) reduced both basal and IL-1beta-induced AP-1 binding activity., Conclusions: These data indicate that PPARgamma agonists inhibit MMP-1 gene expression by transcriptional mechanisms, and suggest that they may be useful in reducing joint tissue destruction., (Copyright 2002 OsteoArthritis Research Society International.)

Osteoarthritic (OA) human synovial fibroblasts (HSF) in culture were treated with the preferential COX-2 inhibitors nimesulide or NS-398, the non-specific COX-1/COX-2 inhibitor naproxen, or dexamethasone, in the presence or absence of IL-1 beta or LPS. Nimesulide or NS-398 inhibited IL-1 beta-induced PGE2 production at all concentrations tested, and in addition they suppressed IL-1 beta-induced COX-2 mRNA expression and protein synthesis. These suppressive effects were most evident at therapeutic levels of the drugs. Mechanistic studies revealed that the drug-induced inhibition of COX-2 expression and synthesis was not promoter-based, but may be associated with the blockade of IL-1 beta-dependent calcium flux and increased cellular calcium levels.

Objective: We used the experimental dog model of osteoarthritis (OA) to examine the in vivo effects of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of the inducible nitric oxide synthase (iNOS), on the tissue level and distribution of interleukin-1beta (IL-1beta), collagenase-1, stromelysin-1, cyclooxygenase-2 (COX-2), iNOS and nitrotyrosine, through immunohistochemical and morphometric analysis., Methods: Cartilage and synovial membrane specimens were obtained from 3 experimental groups of dogs: Group I--unoperated dogs that received no treatment; Group II--dogs subjected to a sectioning of the anterior cruciate ligament of the right knee and given no treatment; and Group III--operated dogs that received oral treatment with L-NIL (10 mg/kg twice daily/po) for 10 weeks starting immediately after surgery. The operated dogs were killed 10 weeks post-surgery. The tissue distributions of IL-1beta, metalloproteases (MMP), COX-2, iNOS and nitrotyrosine were documented by immunohistochemistry using specific antibodies, and quantified by morphometric analysis., Results: In cartilage, the cell scores (percentage of chondrocytes staining positive for the antigen) for iNOS and 3-nitrotyrosine were dramatically enhanced in OA specimens compared to normal (p < 0.0001). However, the cartilage of dogs treated with L-NIL showed significantly lower cell scores for iNOS (p<0.0001, condyle; p<0.001, plateau), nitrotyrosine (p<0.0004; p<0.0001) and COX-2 (p<0.0001; p<0.001) compared to that of untreated OA dogs. Similar findings were observed for collagenase-1 and stromelysin-1, where the increased cell scores of these 2 MMP in OA cartilage were reduced after treatment with L-NIL (collagenase: p<0.002, condyle; p<0.0003, plateau; stromelysin: p<0.006; p<0.0001). The increased cell scores for the IL-1beta, COX-2, iNOS and nitrotyrosine found in the synovial lining and mononuclear cell infiltrate of operated animals were also found to be markedly reduced in dogs treated with L-NIL., Conclusion: Our study demonstrates for the first time in vivo in an experimental model of OA, that a selective inhibition of iNOS by L-NIL and the subsequent decreased production of NO also results in a marked decrease in production of major catabolic factors such as MMP, IL-1beta and peroxynitrite, as well as a reduction in COX-2 expression.

Fibroblasts from human synovial membranes were cultured with nimesulide, naproxen or dexamethasone. Nimesulide, but not naproxen, showed effects on the glucocorticoid system that may contribute importantly to its anti-inflammatory activity. Nimesulide at therapeutically relevant concentrations induced the intracellular phosphorylation and activation of glucocorticoid receptors, and activated their binding to the target genes. Naproxen or dexamethasone markedly reduced the number of glucocorticoid receptor binding sites, in contrast to nimesulide, which had no significant effect.

The pathophysiological and biological significance of tissue inhibitor of the metalloproteinases-3 (TIMP-3) gene compared to other TIMPs was investigated in osteoarthritic (OA) human and normal bovine joint tissues. Human OA synovial fibroblasts in culture constitutively expressed TIMP mRNAs. TIMP-3, TIMP-1 and gelatinase A mRNAs were elevated in most human OA synovia over controls, while TIMP-2 expression was similar. TIMP-3 and TIMP-1 mRNAs present in bovine cartilage were inducible by serum factors. Transforming growth factor beta (TGF-beta 1) induced TIMP-3 RNA and protein in human OA and normal bovine chondrocytes. TIMP mRNAs were low (TIMP-1) or undetectable in human fetal chondrocytes but were expressed at all other ages. Thus, the two main joint tissues, synovial membranes and cartilage, express TIMP genes. Due to their matrix protecting activities, the presence of multiple TIMPs may be beneficial for normal joints, while increased TIMP-3 and TIMP-1 expression in arthritic joints may be associated with pathological remodeling.

Objective: To evaluate the in vitro effects of diacerhein, a new drug for the treatment of osteoarthritis (OA), and its active metabolite, rhein, on interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) synthesis and expression in human OA synovial membrane, and on the IL-1beta and TNF-alpha receptors on human OA chondrocytes., Methods: Levels of IL-1beta and TNF-alpha were determined using specific ELISA in culture medium of human synovial membrane explants incubated in the presence of 1 microg/ml of lipopolysaccharide with or without therapeutic concentrations of diacerhein (1.4, 2.7, 5.4 x 10(-5) M) and rhein (1.7, 3.5, 7.0 x 10(-5) M). IL-1beta mRNA level was quantitated by Northern blotting. Using radioligand binding experiments, we determined the effects of these agents on the density and affinity of chondrocyte IL-1 and TNF receptors., Results: IL-1beta synthesis was significantly inhibited by diacerhein and rhein, with maximum inhibition at 5.4 x 10(-5) M for diacerhein (p < 0.02) and 3.5 x 10(-5) M for rhein (p < 0.05). The effect of both agents on IL-1beta was found to be translational and/or post-translational, judging by the absence of effect on gene expression level. Both agents produced dose and time dependent decreases in the number of IL-1 receptors (IL-1R) on OA chondrocytes. This effect was mediated through a reduction in the level of the type I IL-1R as shown by experiments using a blocking monoclonal antibody against this receptor type. Both agents also markedly reduced the IL-1 induced synthesis and expression of stromelysin 1. Neither diacerhein nor rhein significantly affected the level of synthesis of TNF-alpha or the level of TNF-R., Conclusion: Diacerhein and rhein can effectively inhibit the synthesis of IL-1beta on human OA synovium, as well as the action of this cytokine at the cartilage level, by reducing the number of chondrocyte IL-1R. The effects of these agents seemed "selective" to the IL-1 system.

Objective: In this study we investigated the effect of interleukin-13 (IL-13), an anti-inflammatory cytokine, for potential therapeutic use in osteoarthritis (OA)., Design: We examined the effect of IL-13 on the synthesis and expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-1 receptor antagonist (IL-1Ra) and stromelysin-1 on human OA synovial membrane in ex vivo cultures. In addition, we explored the effect of IL-13 on both the IL-1 receptor (IL-1R) and TNF-receptor (TNF-R) systems on OA synovial fibroblasts. This included determination of the levels of IL-1 beta and TNF-alpha receptor binding, IL-1Ra and TNF-soluble receptors 55 and 75 (TNF-sR55 and TNF-sR75)., Results: In OA synovial membrane treated with LPS, IL-13 inhibited the synthesis of IL-1 beta, TNF-alpha and stromelysin-1, but increased IL-1Ra production. In addition, IL-13 reduced the level of IL-1 beta mRNA and stimulated the level of IL-1Ra mRNA. In synovial fibroblasts, IL-13 decreased the level of IL-1 binding, an effect related to the increased production of IL-1Ra. Although IL-13 had no effect on the TNF-R level, this cytokine markedly decreased the shedding of TNF-R75., Conclusion: These experiments suggest that IL-13 is potentially useful in the therapeutic treatment of OA, as it could regulate the major pathological process of this disease by reducing the production of proinflammatory cytokines and metalloproteases, and favoring the production of IL-1Ra.

Objective: To investigate the presence, number, and level of expression of tumor necrosis factor receptors (TNF-R) in normal and osteoarthritic (OA) human synovial fibroblasts; to examine which receptor isotype mediates the biological response of these cells to TNF-alpha; and to study homologous regulatory mechanisms of TNF-R by TNF-alpha., Methods: We used radioligand binding assay with [125I]TNF-alpha and flow cytometric analysis with specific antireceptor antibodies to characterize receptor populations, densities, and ligand induced internalization of TNF-R. Inducible cyclooxygenase (COX-2) synthesis, prostaglandin E2 (PGE2) release, and TNF-R shedding (soluble receptors, TNF-sR) were measured after incubation with TNF-alpha the presence or absence of receptor specific blocking antibodies., Results: Although radioligand binding assays showed no difference in the density or affinity of TNF-R in OA synovial fibroblasts compared with normal cells, flow cytometric analysis revealed that OA cells express a significantly higher level of TNF-R55 (p < 0.04) than normal cells. The TNF-R55 was found to be the major receptor species responsible for ligand binding activity, such as COX-2 induction and PGE2 synthesis, since a specific antireceptor TNF-R55 blocking antibody inhibited about 76% of TNF-alpha binding and TNF-alpha stimulated COX-2 induction and PGE2 production. Further experiments revealed that TNF-R55 was the only receptor type internalized after binding TFN-alpha, whereas TNF-R75 was concomitantly shed. Moreover, reducing the shedding of TNF-sR, particularly the TNF-sR75, with a synthetic inhibitor decreased TNF-alpha induced PGE2 production., Conclusion: TNF-R55 is the major receptor isoform transducing PGE2 and COX-2 responses to TNF-alpha in OA synovial fibroblasts; soluble receptors could be involved in facilitating the binding of TNF-alpha to its receptor.

Recently, a new human collagenase, collagenase-3 has been identified. Since collagen changes are of particular importance in cartilage degeneration, we investigated if collagenase-3 plays a role in osteoarthritis (OA). Reverse transcriptase-PCR analysis revealed that in articular tissues collagenase-3 was expressed by the chondrocytes but not by the synoviocytes. Northern blot analysis of the chondrocyte mRNA revealed the presence of two major gene transcripts of 3.0 and 2.5 kb, and a third one of 2.2 kb was occasionally present. Compared to normal, OA showed a significantly higher (3.0 kb, P < or = 0.05; 2.5 kb, P < or = 0.03) level of collagenase-3 mRNA expression. Collagenase-3 had a higher catalytic velocity tate (about fivefold) than collagenase-1 on type II collagen. With the use of two specific antibodies, we showed that human chondrocytes had the ability to produce collagenase-3 as a proenzyme and as a glycosylated doublet. The chondrocyte collagenase-3 protein is produced in a significantly higher (P < or = 0.04) level in OA (approximately 9.5-fold) than in normal. The synthesis and expression of this new collagenase could also be modulated by two proinflammatory cytokines, IL-1 beta and TNF-alpha, in a time- and dose-dependent manner. This study provides novel and interesting data on collagenase-3 expression and synthesis in human cartilage cells and suggest its involvement in human OA cartilage patho-physiology.

Objective: To investigate the effects of tenidap, a new antirheumatic drug, sodium diclofenac, a non-steroidal antiinflammatory drug, and a disease modifying antirheumatic drug, hydroxychloroquine, on the level and expression of interleukin 1 receptors (IL-1R) on synovial fibroblasts from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, the effect of tenidap on IL-1 stimulated collagenase gene expression was studied., Methods: Binding assays were performed using [125I]-IL-1 beta as radioligand. Flow cytometry was done using a specific antibody against type I IL-1R. Protein synthesis was determined by [3H]-leucine incorporation. Levels of expression were determined by Northern Blot for collagenase and by reverse transcription polymerase chain reaction (RT-PCR) for type I IL-1R., Results: Tenidap produced for both OA and RA synovial fibroblasts a dose dependent decrease in the number of IL-1 binding sites/cell. A reduction of 41% (2.5 micrograms/ml) to 81% (at therapeutic concentration, 20 micrograms/ml) was noted for OA, while 29% (2.5 micrograms/ml) to 89% (20 micrograms/ml) was found for RA cells. Diclofenac produced no effect on OA cells, and minimal inhibition of RA synovial fibroblasts was observed only at pharmacological concentration (12%, 300 mg/ml). Hydroxychloroquine had effects similar to diclofenac. The decreased number of IL-1 binding sites/cell by tenidap was time dependent and reached 93% inhibition after 48 h. The effect of tenidap appears to be posttranscriptional, judged by the marked reduction of the type I IL-1R protein/cell and the absence of effect on its mRNA level. Tenidap also markedly reduced the IL-1 induced collagenase expression in synovial fibroblasts., Conclusion: At therapeutic concentrations tenidap is a potent inhibitor of type I IL-1R in OA and RA synovial fibroblasts. The effect of tenidap was considerably more marked than diclofenac or hydroxychloroquine.

Background: Cytokines, in particular IL-1, are believed to be responsible for mediating cartilage degradation in osteoarthritis (OA). To investigate the role of the IL-1 system in this disease, we studied in normal and OA human synovial fibroblasts the nature, the number, and the level of expression of the IL-1 receptor (IL-1R) and through which receptor the biologic stimulation of these cells by IL-1 is mediated., Experimental Design: We determined the IL-1R level by radioligand assay, the type of IL-1R with the use of specific antibodies and by the reverse transcriptase-PCR (RT-PCR), and the mRNA level of the type I IL-1R by slot blot analysis. Biologic activity was measured on the synovial fibroblasts via IL-1 binding and prostaglandin E2 production., Results: Binding data revealed the presence of a single class of high affinity IL-1R in both normal (kD, 21 +/- 4.5 pM) and OA (kD, 23 +/- 5.0 pM) human synovial fibroblasts. The number of receptors was significantly higher (p < 0.004) in OA synovial fibroblasts (2534 +/- 187 sites/cell) than in normal cells (1310 +/- 96 sites/cell). This increase was transient; OA synovial fibroblasts in second and third passages had a normal level of IL-1R. Analysis of the mRNA species by RT-PCR revealed that both type I and type II IL-1R are coexpressed in normal and OA synovial fibroblasts; the type I mRNA was the most predominant in all samples. No difference in the relative amount of type I IL-1R mRNA level was found between normal and OA cells. A blocking Ab against the type I IL-1R completely inhibited, in both normal and OA cells, the receptor binding and IL-1 beta stimulated PGE2 production, whereas the treatment with anti-type II IL-1R was ineffective., Conclusions: These results indicate that the type I IL-1R is up-regulated in OA synovial fibroblasts and is responsible for mediating the biologic activation of these cells by IL-1. This phenomenon is probably secondary to an abnormality in the post-transcriptional regulation of the type I IL-1R. Although type II IL-1R is also expressed, its translation seems to be inoperative, or this receptor is already shed.

Elevated levels of tissue inhibitor of metalloproteases-1 (TIMP-1) have been demonstrated in inflamed synovial membranes, and it is believed that the inhibitor may play a critical role in the regulation of connective tissue degradation. The present study was undertaken to define the cellular mechanism of action of the inflammatory mediators, interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE2), in the control of TIMP-1 synthesis and expression in human synovial fibroblasts. Recombinant human IL-1 beta induced a time- and dose-dependent saturable response in terms of TIMP-1 mRNA expression (effective concentration for 50% maximal response, EC50 = 31.5 +/- 3.3 pg/ml) and protein synthesis (EC50 = 30 +/- 3.3 pg/ml). The protein kinase C (PKC) inhibitors, H-7, staurosporine, and calphostin C, reversed the rhIL-1 beta induction of TIMP-1 mRNA. PGE2 also inhibited rhIL-1 beta-stimulated TIMP-1 mRNA expression and protein secretion in a dose-dependent fashion. The concentration of PGE2 necessary to block 50% of rhIL-1 beta-stimulated TIMP-1 secretion, IC50, was 1.93 ng/ml (4.89 nM). Forskolin, and other stable derivatives of cAMP, mimicked, to a large extent, the effects of PGE2. The phorbol ester, PMA, up-regulated considerably the mRNA expression of TIMP-1 but had no effect on protein production. Calphostin C substantially reduced PMA-activated TIMP-1 expression. Staurosporine, calphostin C, H-7, and substances that elevate cellular levels of cAMP, like PGE2, also reduced basal expression and synthesis of TIMP-1. Taken together, the data suggest that PKA and C may mediate opposing effects in terms of TIMP-1 expression and secretion in human synovial fibroblasts.

Tumor necrosis factor alpha (TNF alpha) is suggested to be of importance in the pathogenesis of inflammatory diseases. One mechanism that modulates the action of TNF is binding to specific soluble receptors. Using human synovial fibroblasts, we investigated the effect of cytokines and growth factors, known to be present in increased amounts in arthritic disorders, on the release of the TNF soluble receptors TNFsR75 and TNFsR55. Levels of TNFsR75 and TNFsR55 were determined using conditioned medium from human synovial fibroblasts incubated in increasing concentrations of cytokines, IL-1 beta, TNF alpha, IL-6, and IL-2, and platelet derived growth factor BB (PDGF-BB), transforming growth factor beta (TGF beta), and insulin like growth factor I (IGF-I), alone or in combination with IL-1 beta. The levels of both TNFsR were measured by specific immunoassays. Both TNFsR demonstrated similar levels under basal conditions. IL-1 and TNF alpha induced a significant enhancement of TNFsR75 compared to TNFsR55. When cells were treated with both IL-1 beta and TNF alpha, a marked inhibition in the release of TNFsR55 was observed, while TNFsR75 did not show any changes. IL-6 and IL-2 produced no effect on the release of TNFsR75 and a minimal increase of TNFsR55. PDGF-BB and IGF-I demonstrated a dose dependent increased level of TNFsR55, and both soluble receptors released were inhibited by TGF beta. TGF beta and IL-1 beta together produced a greater inhibition of the release of the TNFsR. These data support the notion that both TNFR in synovial fibroblasts are differently regulated.

We examined the common signal transduction mechanisms governing collagenase (MMP-1), stromelysin-1 (MMP-3), and tissue inhibitor of metalloproteases (TIMP-1) gene expression in human synovial fibroblasts for insight into the pathophysiology of arthritis. MMP-1, MMP-3, and TIMP-1 expression and synthesis were induced in cultured human synoviocytes with recombinant human interleukin 1 beta in the absence or presence of either chemical inhibitors of protein kinase A and C (PKA, PKC), or prostaglandin E2, or cyclic AMP (cAMP) mimetics. We used enzyme immunoassays (EIA) to determine MMP-1, MMP-3, and TIMP-1 antigen levels in spent culture medium and Northern hybridization to measure steady state mRNA expression levels. Extracellular signals (e.g., IL-1, phorbol myristic acetate) that result in the activation of cytoplasmic PKC augment in tandem the expression and synthesis of MMP-1, MMP-3, and TIMP-1 in human synovial fibroblasts. In addition, such signals induce nuclear transcription factors (e.g., activator protein 1) that bind to common gene regulatory elements and augment promoter activity of MMP-1, MMP-3, and TIMP-1 gene promoter constructs. In contrast, signals that activate PKA oppose PKC mediated signals, in that the expression of MMP-1, MMP-3, and TIMP-1 are suppressed. Experimental data suggest that the expression of MMP-1, MMP-3, and TIMP-1 are coordinated through a series of common cytoplasmic signal transducing pathways, cis regulatory elements, and nuclear trans acting factors.

We investigated the nature of cytokines synthesized by human osteoarthritic (OA) synovium, particularly interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha). We examined the capacity of recombinant human interleukin 1 receptor antagonist (rhIL-1ra) to block the synthesis of metalloproteases (collagenase and stromelysin), IL-1 beta, and IL-6 in osteoarthritis (OA) synovium. Human OA synovium were incubated in the presence or absence of lipopolysaccharide (LPS) or increasing concentrations of rhIL-1ra. The determinations of IL-1 alpha, IL-1 beta, TNF alpha, IL-6, and IL-1ra in culture medium were carried out using specific ELISA. Although both IL-1 isoforms and TNF alpha could be produced by OA synovium, IL-1 beta was the predominant cytokine synthesized either in the presence or absence of LPS. Treatment of the OA synovium with an increasing concentration of rhIL-1ra (0-10 micrograms/ml) showed a dose dependent reduction of both metalloproteases and IL-6. Maximal inhibition was 70% for collagenase, 80% for stromelysin, and 76% for IL-6. LPS treated synovium also showed a consistent suppression of metalloproteases and IL-6, although a higher IL-1ra concentration was required. Conversely, IL-1 beta production was not inhibited by IL-1ra, irrespective of the concentration used and whether the membranes were LPS stimulated. These data showed that IL-1 appears to be the major autocrine cytokine involved in the stimulation of metalloproteases and IL-6 synthesis in OA synovium.

Objective: We examined the effect of 3 commonly used nonsteroidal antiinflammatory drugs (NSAID), indomethacin, naproxen and tiaprofenic acid, and a prostaglandin E1 analog, misoprostol, on the glucocorticoid receptor level in synovial fibroblasts., Methods: Synovial fibroblasts were isolated by enzymatic digestion from human normal synovial membranes. These cells were incubated with therapeutic and pharmacological concentrations of NSAID in the presence or absence of misoprostol (0.1-100 ng/ml). The glucocorticoid receptor (GR) level was measured by binding assay using 3H-dexamethasone., Results: Naproxen and indomethacin but not tiaprofenic acid, at therapeutic concentrations, significantly reduced the level of GR in synovial cells. This effect was reversed with the addition of misoprostol., Conclusion: Our findings bring insight into the differential effects of NSAID on the GR system and may provide an explanation for the reduced level of GR found in OA chondrocytes. The possible interference with the action of therapeutically administered corticosteroids is discussed.

Background: Cytokine modulated matrix metalloprotease (MMP, i.e., collagenase and stromelysin) synthesis may be associated etiologically with osteoarthritic diseases. The aim of this study was to investigate the mode of action by which interleukin-1 beta (IL-1 beta) induced collagenase and stromelysin mRNA expression and synthesis in normal human synoviocytes and to explore mechanisms of suppression of these proteolytic enzymes by prostaglandins (PG)., Experimental Design: Collagenase and stromelysin expression and synthesis were induced in cultured human synoviocytes with rhIL-1 beta in the absence or presence of either chemical inhibitors of protein kinase (PK) A and C, PGE2 or PGE1, or cAMP mimetics. We used enzyme immunoassays to determine MMP antigen levels in spent culture medium and Northern hybridization to measure steady-state MMP mRNA expression., Results: Dose-response experiments revealed that 10 pg/ml of interleukin-1 beta was an effective sub-saturating concentration for the induction of collagenase and stromelysin expression in normal human synovial fibroblasts. The rate of collagenase synthesis and expression peaked at 18 to 24 hours after rhIL-1 beta stimulation, whereas stromelysin output increased steadily within the experimental time frame (72 hours). Protein kinase C inhibitors, H-7 and staurosporine, prevented the rhIL-1 beta induction of MMP mRNA expression and protein synthesis. Pretreatment of synoviocytes with phorbol myristate acetate for 18 hours abrogated the ability of rhIL-1 beta to induce MMP synthesis. Prostaglandins E2 and E1 potently inhibited in a dose-dependent fashion rhIL-1 beta induced MMP synthesis: PGE2, IC50, collagenase, 2.3 ng/ml; stromelysin, 21.2 ng/ml; PGE1, IC50, collagenase, 2.5 ng/ml; stromelysin, 13.4 ng/ml. MMP mRNA steady-state levels were suppressed in a fashion similar to that of the protein synthesis. Forskolin, dibutyryl cAMP, and 3-isobutyl-1-methyl xanthine mimicked the effects of the prostaglandins (PGs). [N-(2-methyl-amino)-5-isoquinoline-sulfonamide dihydrochloride] (H-8), an inhibitor of PKA activity, could reverse to a large extent, the suppressive effects of the PGs as did cycloheximide when preincubated with PGE2 before rhIL-1 beta activation of synoviocytes., Conclusions: We conclude that IL-1 beta stimulates MMP synthesis by activating PKC but not PKA, and that the synthesis of collagenase and stromelysin is discoordinate on a temporal and quantitative basis. PGs inhibit rhIL-1 beta-induced MMP expression and synthesis by virtue of their ability to increase cAMP intracellular levels and subsequent activation of signal transduction mechanisms involving PKA. Homeostasis may be maintained in acute episodes of joint inflammation through feedback processes involving locally produced eicosanoids.

Differential expression of PAI-1 in connective tissues has been associated etiologically with some forms of arthritis. Our objective was to delineate the mechanisms by which PGE2 and IL-1 beta, inflammatory mediators commonly found at sites of inflammation, regulate the expression and synthesis of PAI-1 in human synoviocytes. PGE2 (and PGE1) inhibited PAI-1 mRNA expression and secretion in a dose-dependent manner with an IC50 (for antigen secretion) of 4.6 x 10(-10) M and 8.7 x 10(-10) M, respectively. Cyclic AMP agonists forskolin, Sp-cAMP, and IBMX mimic the effects of the PGEs. rhIL-1 beta stimulated the secretion of PAI-1 in a dose-dependent fashion under basal culture conditions; the effect was reversed by actinomycin D and the protein kinase inhibitors H7 and staurosporine but not KT-5720. PMA, an activator of protein kinase C, transiently increased (maximum 3 h) the expression of PAI-1 mRNA by approximately 10-fold, especially the 3.2 kb species. However, there was no significant increase in PAI-1 antigen secreted into the culture medium after PMA (100-300 nM) treatment. The half-life (t1/2) of PAI-1 mRNA, both the 3.2 and 2.2 transcripts was about 9.6 h (mean n = 3) and PGE2 has no affect on the stability of both messages. PGE2 reduced the rate of PAI-1 gene transcription as judged by run-off assays. The NSAID naproxen (30 micrograms/ml) induced the expression of PAI-1 mRNA over basal levels and super-induced the inhibitor's expression above rhIL-1 beta stimulated levels. Our results suggest that PGE2 suppresses PAI-1 expression and synthesis by activation of the cAMP/PKA system and inhibition of the rate of gene transcription. Data concerning the activation of PKC suggest that the expression, synthesis and release of the PAI-1 may be differentially regulated in normal human synoviocytes.

A sandwich enzyme immunoassay using a chemiluminescent detection has been developed for the quantification of total human (high and low molecular weight) kininogens in tissue extracts. This assay uses monospecific polyclonal IgG labelled with alkaline phosphatase and the commercially available dioxetane derivatives as substrates, for the detection of immune complexes. This method exhibits a sensitivity level of 1 fmol/ml and allows a precise quantification of total kininogens in synovium and cartilage extracts. When characterized by Western blot, the immunoreactive material reveals the presence of both high and low molecular weight kininogens.

The significance of activating proteins (AP-1), c-fos, c-jun and jun B relative to the AP-1 responsive metallothionein, collagenase and stromelysin gene expression in the pathophysiology of osteoarthritis (OA) was investigated. The 'early' c-fos, c-jun and jun-B mRNAs were ubiquitously expressed in normal and OA human knee synovial membranes. There was no strict correlation between expression of these and the AP-1 responsive, collagenase and stromelysin gene expression. Interestingly, the total metallothionein (MT) and the AP-1 responsive, MT-IIA gene-specific mRNAs were greatly diminished in OA compared with normal synovial membranes. The possible role of reduced expression of MT and trace metals in OA pathophysiology is discussed. Collectively, these data demonstrate a discoordinate expression of AP-1 encoding and their target genes in synovium.

The levels of metalloproteases and inhibitor expression in synovial membranes were measured by analyzing mRNA of collagenase, stromelysin, and tissue inhibitor of metalloproteinase (TIMP-1) in this tissue from 20 healthy persons and 20 patients with osteoarthritis (OA). Our results indicated that while most of the normal synovia expressed TIMP-1 mRNA at low levels, very few expressed metalloproteinase mRNA. In contrast 40% of patients with OA expressed a moderate level of collagenase compared to 55 and 80% cases with considerably elevated stromelysin and TIMP-1 mRNA, respectively. The mRNA expression of collagenase (p < 0.032), stromelysin (p < 0.0001) and TIMP-1 (p < 0.008) was significantly higher in OA than in normals. The greater abundance of stromelysin mRNA relative to collagenase indicates differential regulation of the 2 genes. We also demonstrated an association of IL-1 with metalloproteinase gene expression.

The regulation of inhibitors which block the action of IL-1 has significant implications in the control of inflammation. Different modulators of cellular metabolism were studied for their effect on the synthesis of IL-1 receptor antagonist (IL-1ra). Among the different growth factors and cytokines studied, only the inflammatory mediators IL-1 and TNF-alpha and one growth factor, PDGF-BB, were found to significantly increase, in an additive manner, the synthesis and secretion of IL-1ra.

The mechanism(s) of action of NSAIDs and corticosteroids in arthritic diseases has been the subject of intensive investigation in recent years. Although NSAIDs and corticosteroids have many effects, their possible ability to modify the disease course in patients has not been fully documented. In an attempt to characterize the mechanism(s) involved in the effect of some NSAIDs in joint diseases, we investigated the effect of three concentrations within the pharmacological (260 micrograms/ml) and therapeutic (26 and 2.6 micrograms/ml) ranges of tiaprofenic acid in the synthesis and release of interleukin (IL-1) alpha and beta in human OA synovial membranes. The effect of tiaprofenic acid was compared to the effect of two other NSAIDs, sodium salicylate (160 micrograms/ml) and indomethacin (1.5 micrograms/ml), and to a corticosteroid, hydrocortisone (0.725 and 7.25 micrograms/ml). This study was carried out using human OA synovium explants incubated in the presence or absence of LPS. In the absence of LPS and at therapeutic concentration, tiaprofenic acid decreased both the synthesis and release of IL-1 beta. A less marked effect of the drug was noted under LPS treatment, and inhibition of the production/secretion of IL-1 beta was found only at pharmacological concentration. Sodium salicylate and indomethacin did not share this action, and demonstrated either no effect or enhancement of IL-1 beta synthesis, respectively, in the presence of LPS. As expected, hydrocortisone demonstrated a marked decrease on IL-1 alpha and IL-1 beta, both in the presence and absence of LPS. These results bring forth new information on the action of these drugs and their effects on the OA pathophysiological process.

The effect of therapeutic and pharmacological concentrations of tiaprofenic acid, a non-steroidal anti-inflammatory drug (NSAID), on the synthesis of the plasminogen activators, urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA), and the plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), by human synovial membranes isolated from osteoarthritis (OA) and rheumatoid arthritis (RA) sufferers was evaluated. Both forms of plasminogen activator (PA) and PA inhibitor (PAI) were synthesized by the arthritic synovium. PAI-1 and PAI-2 were both synthesized in greater amounts than the plasminogen activators. Tiaprofenic acid induced a dose-dependent decrease in uPA synthesis in both OA and RA, particularly in OA synovium, but had no true effect on tPA. Tiaprofenic acid also exerted a suppressive effect on the synthesis of PAI-1 in both OA and RA synovial membranes, and on the release of PAI-2 in RA synovium. The results of this study indicate that a decrease in uPA synthesis may be one of the mechanisms by which tiaprofenic acid could exert its effects on the arthritic process. The suppressive action of tiaprofenic acid on PAI is not likely to have a significant impact on the balance of plasminogen activators and plasminogen activator inhibitors, as plasminogen activator inhibitors are synthesized in greater amounts than plasminogen activators.

We examined the histologic and immunopathologic features of the synovial membrane of 18 patients with rheumatoid arthritis (RA) and 12 patients with osteoarthritis (OA) who had undergone total knee arthroplasty. Patients were classified into 5 groups according to therapeutic regimen and disease: RA treated with nonsteroidal antiinflammatory drugs (NSAIDs), RA treated with NSAIDs and prednisone, RA treated with NSAIDs and methotrexate (MTX), OA treated with analgesics, and OA treated with NSAIDs. There were no significant between-group differences in the percentages or the distribution pattern of the infiltrating T cell subsets (CD4, CD8), HLA-DR, or interleukin-2 receptor-bearing cells. However, inflammatory indices, which included the thickness of the lining cell layer and the density of the mononuclear cell infiltrate, were significantly higher in the RA patients treated with prednisone and those treated with MTX (P less than 0.05). Similarly, fibrosis was markedly reduced in these 2 groups. The RA patients treated with NSAIDs alone and the 2 groups of patients with OA demonstrated similar profiles. These data suggest that prednisone and MTX may inhibit the development of fibrosis without altering the subsets of the inflammatory cell population. This observation raises the possibility that the action of these 2 drugs may be partly mediated by the suppression of inflammatory mediators that are responsible for fibroblast activation.

The destruction of joints in rheumatoid arthritis (RA) is thought to be related in part to an increased synthesis of proteolytic enzymes. We have determined neutral collagenolytic protease activity levels in human RA synovia and articular cartilage and examined the in vivo effects of various therapeutic regimens on enzyme levels. Neutral metallocollagenolytic enzyme (NMCE) was measured in 29 RA cartilages and synovial membranes. In addition, synovial serine protease levels were determined. Specimens were divided into 4 groups according to prescribed medications: (1) nonsteroidal antiinflammatory drugs alone; (2) steroids alone; (3) steroids and gold; and (4) steroids and methotrexate (MTX). Ten normal specimens were used as controls. Total and active NMCE measured in both RA cartilage and synovial membrane specimens showed a significantly higher level of activity than in controls (p less than 0.0001, p less than 0.005; p less than 0.004, p less than 0.02). MTX was found to markedly decrease NMCE activity; cartilage NMCE level in patients with RA receiving MTX was reduced, compared to the other subgroups. This was particularly noted for the active form. Synovial NMCE levels from the MTX subgroup for both enzyme forms were much lower than in any other RA subgroup, significantly lower than in the RA group as a whole (p less than 0.05), and similar to controls. RA synovial membrane serine protease activity showed an increase compared to controls. Again, MTX markedly decreased the activity of this class of enzyme. Our data strongly support the role of neutral proteases in the destruction of RA joints. MTX was the only drug to consistently decrease these enzyme levels in joint tissues.

The activities of neutral collagenolytic enzymes (CE) and neutral proteoglycan-degrading enzymes (PE) in the synovial membranes of osteoarthritis (OA) patients were determined. The total neutral metallo-CE activity showed a significantly higher level of activity when the membranes of OA patients were compared with those of controls. The severely and moderately inflamed synovia had significantly more enzyme activity than did either mildly inflamed or control synovia. Steroids reduced the total metallo-CE activity. In specimens with severe inflammation, the active form of the neutral metallo-CE was significantly elevated over that found in controls. The serine-CE activity was also significantly elevated in OA synovia with severe inflammation and synovial hypertrophy. The total and active neutral metallo-PE was significantly elevated in synovial membranes of OA patients with severe inflammation. Moreover, the serine-PE showed much more activity in OA patients than in controls. The enzyme activity remained at a significantly high level in the OA synovium, regardless of the presence or absence of macroscopic synovial hypertrophy or the histologic grading of the synovium (mild, moderate, severe). Our data indicate that, in OA, an increased level of neutral proteases in the synovia could be involved in the local tissue destruction of the periarticular structures. Because of the very high level of serine proteases, their diffusion may render plausible a degradative action on the cartilage surface.

In this study, we measured the levels of neutral metallo- and serine proteases in human osteoarthritic synovium. These enzymes degrade both collagen and proteoglycan macromolecules. They were analyzed by tissue culture methodology and direct extraction. We have demonstrated that in human osteoarthritic synovium, there is a correlation between neutral enzyme activity and the severity of synovial inflammation. Tissue culture studies have shown that the human osteoarthritic synovial membrane produces metalloproteases, such as collagenase, proteoglycanase and gelatinase. These enzymes were further characterized by their molecular weight. Extracts of osteoarthritic synovial tissues showed the presence of serine proteases, with apparent Mr of 25,000.