A computer simulation approach to assessing therapeutic intervention points for the prevention of cytokine-induced cartilage breakdown.

OBJECTIVE: To use a novel computational approach to examine the molecular pathways involved in cartilage breakdown and to use computer simulation to test possible interventions for reducing collagen release. METHODS: We constructed a computational model of the relevant molecular pathways using the Systems Biology Markup Language, a computer-readable format of a biochemical network. The model was constructed using our experimental data showing that interleukin-1 (IL-1) and oncostatin M (OSM) act synergistically to up-regulate collagenase protein levels and activity and initiate cartilage collagen breakdown. Simulations were performed using the COPASI software package. RESULTS: The model predicted that simulated inhibition of JNK or p38 MAPK, and overexpression of tissue inhibitor of metalloproteinases 3 (TIMP-3) led to a reduction in collagen release. Overexpression of TIMP-1 was much less effective than that of TIMP-3 and led to a delay, rather than a reduction, in collagen release. Simulated interventions of receptor antagonists and inhibition of JAK-1, the first kinase in the OSM pathway, were ineffective. So, importantly, the model predicts that it is more effective to intervene at targets that are downstream, such as the JNK pathway, rather than those that are close to the cytokine signal. In vitro experiments confirmed the effectiveness of JNK inhibition. CONCLUSION: Our study shows the value of computer modeling as a tool for examining possible interventions by which to reduce cartilage collagen breakdown. The model predicts that interventions that either prevent transcription or inhibit the activity of collagenases are promising strategies and should be investigated further in an experimental setting.

Differential Toll-like receptor-dependent collagenase expression in chondrocytes.

OBJECTIVES: To characterise the catabolic response of osteoarthritic chondrocytes to Toll-like receptor (TLR) ligands. METHODS: Induction of the collagenases, matrix metalloproteinase (MMP)1 and MMP13, by TLR ligands was assessed in chondrocytes by real-time reverse transcriptase (RT)-PCR. TLR signalling pathway activation and their involvement in collagenase induction were confirmed by immunoblotting and use of pathway inhibitors and siRNA. TLR expression was compared in the femoral head cartilage of normal controls and patients with osteoarthritis (OA) by real-time RT-PCR. RESULTS: Ligands for TLR6/2 and TLR3 showed the greatest upregulation of MMP1 and MMP13 respectively, although all TLR ligands upregulated these MMPs. MMP1 and MMP13 induction by TLR3 and TLR1/2 or TLR6/2 ligands were dependent on Trif and MyD88, respectively. These inductions were dependent upon the nuclear factor (NF)kappaB pathway, but were differentially inhibited by various mitogen-activated protein kinase inhibitors, with MMP13 induction most reliant on the extracellular signal-regulated kinase pathway. In addition, ligands for TLR1/2 and TLR6/2, but not TLR3, induced significant collagenolysis in a cartilage resorption assay. Finally, TLR2 was significantly downregulated and TLR3 upregulated in OA, compared to normal, cartilage. CONCLUSIONS: Activation of chondrocyte TLRs leads to differential collagenase gene activation. Treatment of chondrocytes with TLR1/2 or TLR6/2 ligands resulted in collagen resorption. The modulated expression of chondrocyte TLR2 and TLR3 in OA cartilage, compared to normal, may reflect a response to repair cartilage or prevent further extracellular matrix destruction. These data suggest modulation of TLR-mediated signalling as a potential therapeutic strategy for the treatment of OA.

Gingival fibroblasts grown from cyclosporin-treated patients show a reduced production of matrix metalloproteinase-1 (MMP-1) compared with normal gingival fibroblasts, and cyclosporin down-regulates the production of MMP-1 stimulated by pro-inflammatory cytokines.

BACKGROUND AND OBJECTIVE: Cyclosporin-induced gingival overgrowth arises from an alteration in collagen homeostasis and is enhanced by inflammatory changes in the gingival tissues. The aim of this study was to investigate the interaction among interleukin-1, oncostatin M, cyclosporin and nifedipine in promoting the up-regulation of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase by gingival fibroblasts. MATERIAL AND METHODS: Fibroblast cultures (n = 5) were obtained from healthy controls and from patients with cyclosporin-induced gingival overgrowth, and cells were harvested between the fourth and ninth passages. Cells were stimulated with interleukin-1 and oncostatin M, alone or in combination, and with different concentrations of cyclosporin (0-2000 ng/mL) and nifedipine (0-200 ng/mL). MMP-1 and tissue inhibitor of metalloproteinase-1 production was determined using an enzyme-linked immunosorbent assay technique. A CyQuant cell proliferation assay was used to determine the DNA concentration in the sample. RESULTS: Fibroblasts obtained from patients with cyclosporin-induced gingival overgrowth produced significantly lower levels of MMP-1 than control fibroblasts (p < 0.001); tissue inhibitor of metalloproteinase-1 levels were significantly lower (p < 0.05), and the ratio of MMP-1 to tissue inhibitor of metalloproteinase-1 was reduced, in the conditioned medium of patients with cyclosporin-induced gingival overgrowth compared with controls. Interleukin-1 and oncostatin M produced a significant increase in the up-regulation of MMP-1, which was reversed when cyclosporin and nifedipine were added to the cell cultures (p < 0.05). CONCLUSION: Pro-inflammatory cytokines significantly up-regulate MMP-1 in cultured gingival fibroblasts. Up-regulation is attenuated by both cyclosporin and nifedipine. The interaction may account for the synergism between inflammation and cyclosporin-induced gingival overgrowth.

Matrix metalloproteinase 10 promotion of collagenolysis via procollagenase activation: implications for cartilage degradation in arthritis.

OBJECTIVE: We have previously reported the up-regulation of matrix metalloproteinase 10 (MMP-10) following treatment with the procatabolic stimulus of interleukin-1 (IL-1) and oncostatin M (OSM) in chondrocytes. Although MMP-10 is closely related to MMP-3, little is known about the role of MMP-10 in cartilage catabolism. The purpose of this study was to determine whether MMP-10 is expressed in connective tissue cells and to assess how it may contribute to cartilage collagenolysis. METHODS: MMP gene expression was assessed by real-time polymerase chain reaction using RNA from human articular chondrocytes and synovial fibroblasts stimulated with IL-1 plus OSM or tumor necrosis factor alpha (TNFalpha) plus OSM. Synovial fluid levels of MMP-10 were determined by specific immunoassay. Recombinant procollagenases were used in activation studies. Immunohistochemistry assessed MMP-10 expression in diseased joint tissues. RESULTS: MMP-10 expression was confirmed in both chondrocytes and synovial fibroblasts following stimulation with either IL-1 plus OSM or TNFalpha plus OSM, and MMP-10 was detected in synovial fluid samples from patients with various arthropathies. Exogenous MMP-10 significantly enhanced collagenolysis from IL-1 plus OSM-stimulated cartilage, and MMP-10 activated proMMP-1, proMMP-8, and proMMP-13. Immunohistochemistry revealed the presence of MMP-10 in the synovium and cartilage of an IL-1 plus OSM-induced model of arthritis as well as in samples of diseased human tissues. CONCLUSION: We confirm that both synovial fibroblasts and articular chondrocytes express MMP-10 following treatment with procatabolic stimuli. Furthermore, the detectable levels of synovial fluid MMP-10 and the histologic detection of this proteinase in diseased joint tissues strongly implicate MMP-10 in the cartilage degradome during arthritis. The ability of MMP-10 to superactivate procollagenases that are relevant to cartilage degradation suggests that this activation represents an important mechanism by which this MMP contributes to tissue destruction in arthritis.

Interleukin-6 signalling in juvenile idiopathic arthritis is limited by proteolytically cleaved soluble interleukin-6 receptor.

OBJECTIVES: Interleukin-6 (IL-6) exerts multiple effects on chondrocytes and fibroblasts within the joint and is associated with disease activity in juvenile idiopathic arthritis (JIA). Although these cells express the ubiquitous signalling receptor for all IL-6-related cytokines, gp130, they do not express a cognate IL-6 receptor. Consequently, IL-6 responses within these cells occur via IL-6 trans-signalling relying on the presence of a soluble receptor (sIL-6R). Levels of sIL-6R in vivo are governed by either proteolytic cleavage (PC) of cognate receptor or by differential sIL-6R mRNA splicing (DS). The aim of this study was to evaluate the contribution of both isoforms to clinical parameters associated with IL-6 signalling in JIA. METHODS: IL-6, sIL-6R and DS-sIL-6R were measured by ELISA in serum and synovial fluid (SF) samples from 86 JIA patients. These data were related to indicators of inflammation-erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) and compared between patients stratified by subtype, age and disease duration. RESULTS: SF IL-6 significantly correlated with general indicators of activity (ESR and CRP) and SF PC-sIL-6R to a lesser degree with CRP. When the IL-6:sIL-6R ratio was calculated as an indicator of the potential for IL-6 signalling within the joint, 33% of SF samples showed a ratio >1 indicating saturation of sIL-6R by IL-6. Mean DS-sIL-6R levels were 0.71 ng/ml, whereas PC-sIL-6R levels constituted the majority of sIL-6R at 20.89 ng/ml. CONCLUSIONS: IL-6 trans-signalling within the joints of JIA patients is predominantly governed by the presence of PC-sIL-6R, and the data provided suggest that synovial levels of IL-6 and sIL-6R would be sufficient to drive IL-6 responses in chondrocytes and synovial fibroblasts.

Development of a novel 2D proteomics approach for the identification of proteins secreted by primary chondrocytes after stimulation by IL-1 and oncostatin M.

OBJECTIVES: To develop a proteomics approach to study changes in the secreted protein levels of primary human chondrocytes after stimulation by the pro-inflammatory cytokines interleukin-1 and oncostatin M. METHODS: Using both the primary human articular and bovine nasal chondrocyte-conditioned mediums, methods were investigated to enable the separation of proteins by two-dimensional (2D) gel electrophoresis. Differentially regulated proteins were identified using tandem electrospray mass spectrometery. RESULTS: We discovered that proteoglycans and glycosylaminoglycans (GAGs) secreted by chondrocytes significantly interfered with 2D gel focusing. Several different methods for GAG removal were attempted including enzymic digestion, cetyl pyridinium chloride precipitation and anion exchange in high salt. The anion exchange proved to be the most effective. Even from these initial gels, we were able to identify eight proteins produced by human chondrocytes: matrix metalloproteinase (MMP)-1, MMP-3, YKL40, cyclophilin A, beta2-microglobulin, transthyretin, S100A11, peroxidine 1 and cofilin. MMP-1, MMP-3, YKL40 and cyclophilin A were all identified as processed, smaller peptide fragments. CONCLUSIONS: We were able to develop a novel sample preparation protocol to allow the reproducible sample preparation of secreted proteins from human chondrocytes. From the initial data, we were able to show that at least some of the proteins produced were cleaved to smaller fragments as a result of proteolysis. Therefore, this technique provides valuable information about protein processing which gene-based arrays do not.

Interleukin-1 in combination with oncostatin M up-regulates multiple genes in chondrocytes: implications for cartilage destruction and repair.

OBJECTIVE: To identify the genes up-regulated by interleukin-1 (IL-1) in combination with oncostatin M (OSM) in chondrocytes that may be involved in mechanisms of cartilage repair and degradation. METHODS: Gene microarray and real-time polymerase chain reaction (PCR) experiments were performed using RNA from SW1353 chondrocytes and primary human articular chondrocytes. Sections prepared from murine joints, injected with adenovirus vectors overexpressing IL-1 and/or OSM, were analyzed by immunohistochemistry for selected proteins. RESULTS: The combination of IL-1 and OSM markedly up-regulated the expression of various genes, including matrix metalloproteinases (MMPs), cytokines, chemokines, extracellular matrix components, and genes involved in signal transduction. Real-time PCR confirmed a synergistic induction of several MMPs, activin A, pentraxin 3 (PTX-3), and IL-8. The in vivo findings further indicated that stimulation with IL-1 plus OSM induced protein expression of activin A, PTX-3, and KC (the murine homolog of IL-8), as compared with the changes induced by individual cytokine treatment and unstimulated controls. CONCLUSION: The results confirm that the potent proinflammatory cytokine combination of IL-1 plus OSM synergistically and coordinately up-regulates many genes and several MMPs. Moreover, chondrocytes exhibit a potential repair response following this procatabolic stimulus such that the repair mechanisms are ultimately overwhelmed by degradative processes in the cartilage. This gene-profiling study provides insight into the complex processes that mediate joint disease in the inflammatory arthritides through the coordinated expression of multiple genes.

Retinoic acid and oncostatin M combine to promote cartilage degradation via matrix metalloproteinase-13 expression in bovine but not human chondrocytes.

OBJECTIVES: Retinoic acid (RetA) and oncostatin M (OSM) have both been shown to mediate potent effects with respect to extracellular matrix integrity. This study assesses the effects of a RetA + OSM combination on cartilage catabolism. METHODS: Animal and human cartilage samples were used to assess the ability of RetA + OSM to promote the release of collagen and proteoglycan fragments, which was determined by measuring glycosaminoglycan and hydroxyproline, respectively. Total collagenolytic and tissue inhibitor of metalloproteinases (TIMP) inhibitory activities were determined by bioassay, whilst gene expression of matrix metalloproteinases (MMPs) and TIMP-1 were determined by northern blotting. Immunohistochemistry was used to assess the presence of MMP-1 and -13 in resorbing cartilage explants. RESULTS: Both agents alone induced proteoglycan release from bovine cartilage, whilst RetA-induced collagen release was variable. Reproducible and synergistic collagenolysis was observed with RetA + OSM, which appeared to be due to MMP-13. Similar collagen release was observed from porcine cartilage. Conversely, no collagen release was seen with human articular cartilage. In primary human chondrocytes, RetA + OSM failed to induce MMP-1 or -13 but caused a significant increase in TIMP-1 expression. CONCLUSIONS: These novel observations show that the combination of RetA + OSM has profound effects on cartilage matrix turnover, but these effects are species-specific. A better understanding of the mechanism by which this combination differentially regulates MMP and TIMP expression in human chondrocytes could provide valuable insight into new therapeutic strategies aimed at the prevention of cartilage destruction.

Human nasal cartilage responds to oncostatin M in combination with interleukin 1 or tumour necrosis factor alpha by the release of collagen fragments via collagenases.

BACKGROUND: The synergistic degradation of cartilage by oncostatin M (OSM) in combination with either interleukin 1 (IL1) or tumour necrosis factor alpha (TNFalpha) has been previously demonstrated using bovine nasal cartilage (BNC). OBJECTIVES: (a) To investigate if human nasal cartilage (HNC) responds in the same way as BNC to these cytokine combinations, particularly in collagen degradation. (b) To compare the response of human nasal and articular cartilages. METHODS: Collagen release was assessed by measuring the hydroxyproline content of culture supernatants and proteoglycan release by the dimethylmethylene blue assay. Matrix metalloproteinase (MMP)-1, MMP-13, and tissue inhibitor of metalloproteinase 1 release were measured by specific enzyme linked immunosorbent assays (ELISAs), and collagenolytic activity was measured by a bioassay using radiolabelled collagen. RESULTS: OSM in combination with either IL1 or TNFalpha acted synergistically to induce collagenolysis from HNC, with a maximum of 79% collagen release. This degradation strongly correlated with MMP-1 and MMP-13 levels and collagenolytic activity. CONCLUSION: Collagen release from human cartilage is marked and implicates both MMP-1 and MMP-13 in the synergistic degradation of human cartilage by OSM in combination with either IL1 or TNFalpha. HNC responds in the same way as BNC, thus validating the bovine cartilage degradation assay as a model relevant to human disease.

Assessment of the clinical significance of gelatinase activity in patients with juvenile idiopathic arthritis using quantitative protein substrate zymography.

OBJECTIVE: To measure gelatinase activities in paired synovial fluid (SF) and serum of patients with juvenile idiopathic arthritis (JIA), and to assess how these activities relate to clinical and laboratory measures of disease activity. METHODS: A quantitative protein substrate zymography method was adapted and validated for use with serum and SF. Bands of activity were measured by densitometry and correlated with standard laboratory indicators of inflammation: erythrocyte sedimentation rate and platelet count. RESULTS: Gelatinase activity was found consistently in patients with JIA, with reproducible, quantified bands of activity corresponding to pro-matrix metalloproteinase-9 (pro-MMP-9), including the neutrophil associated lipocalin complex, and pro- and active forms of MMP-2. Both active MMP-2 and pro-MMP-9 were higher in JIA serum than in controls, though no differences were seen between patients grouped according to age, disease duration, or JIA subtype. However, SF MMP-9 correlated significantly with the laboratory indicators of inflammation, as did the relative level of active MMP-2. CONCLUSIONS: Both MMP-2 and MMP-9 gelatinolytic activities are raised during active JIA and associated with inflammatory activity regardless of age and disease duration, supporting a role for MMPs in the breakdown of joint components from early in disease. These MMPs may be specific markers of active joint destruction linked to inflammatory JIA, MMP-9 as a product of infiltrating cells, and the activation of MMP-2 produced within the joint.

Primary airway epithelial cell culture from lung transplant recipients.

Long-term survival in lung transplantation is limited by the development of obliterative bronchiolitis, a condition characterised by inflammation, epithelial injury, fibroproliferation and obliteration of bronchioles leading to airflow obstruction. To investigate the role of the bronchial epithelium in the pathogenesis of obliterative bronchiolitis the current study aimed to establish primary bronchial epithelial cell cultures (PBEC) from lung allografts. Four to six bronchial brushings were obtained from sub-segmental bronchi of lung allografts. Cells were seeded onto collagen-coated plates and grown to confluence in bronchial epithelial growth medium. Bronchial brushings (n=33) were obtained from 27 patients. PBECs were grown to confluence from 12 out of 33 (39%) brushings. Failure to reach confluence was due to early innate infection. Bacteria were usually isolated from both bronchoalveolar lavage and culture media, but a separate population was identified in culture media only. Primary culture of bronchial epithelial cells from lung transplant recipients is feasible, despite a high rate of early, patient-derived infection. Latent infection of the allograft, identified only by bronchial brushings, may itself be a persistent stimulus for epithelial injury. This technique facilitates future mechanistic studies of airway epithelial responses in the pathogenesis of obliterative bronchiolitis.

Levels of matrix metalloproteinase (MMP)-1 in paired sera and synovial fluids of juvenile idiopathic arthritis patients: relationship to inflammatory activity, MMP-3 and tissue inhibitor of metalloproteinases-1 in a longitudinal study.

OBJECTIVES: To measure levels of the collagenases matrix metalloproteinase (MMP)-1 and -13 in the synovial fluid (SF) and serum of patients with juvenile idiopathic arthritis (JIA), and to correlate these measurements with inflammatory activity, levels of the collagenase activator MMP-3 and the tissue inhibitor of metalloproteinases-1 (TIMP-1). METHODS: Levels of MMP-1, -3, -13 and TIMP-1 were measured in paired SF and serum from 82 JIA patients using enzyme-linked immunsorbent assay and compared between subtypes and patients of different ages and disease durations. These levels were also correlated to the active joint count (AJC) and standard measures of inflammatory activity and therapeutic response, including erythrocyte sedimentation rate (ESR) and platelet count (PLT). RESULTS: MMP-1 was detected in JIA SF and correlated with PLT. MMP-3 levels were high in SF and detectable in serum where they correlated with PLT, ESR and AJC. MMP-13, however, was not detected in SF or serum. No differences were observed between patients grouped by subtype, age or disease duration. MMP-3 contributed the majority of total MMP in SF samples resulting in excess MMP levels over TIMP-1. CONCLUSIONS: MMP-1 is up-regulated in SF concordant with inflammatory activity in JIA. This was true for patients in all JIA subtypes and age groups, suggesting that the capability for degradation of type II collagen is present in early disease, and throughout the disease course. MMP-3 may be important in the activation of collagenases and the saturation of exogenous inhibitors. Serum MMP-3 may therefore be a useful, measurable and specific marker of active disease in JIA.

Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients.

BACKGROUND: Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling. METHODS: Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters. RESULTS: A median 15% (0-48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-beta1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-beta1 stimulation. CONCLUSIONS: This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.

Oncostatin M in combination with tumour necrosis factor {alpha} induces a chondrocyte membrane associated aggrecanase that is distinct from ADAMTS aggrecanase-1 or -2.

OBJECTIVE: To determine whether oncostatin M (OSM) + tumour necrosis factor alpha (TNFalpha) induces aggrecanase activity in chondrocyte membranes, to determine the effects of transforming growth factor beta1 (TGFbeta1), interleukin 4 (IL4), and tissue inhibitor of metalloproteinases (TIMPs) on this activity, and to determine whether this activity is due to a known ADAMTS aggrecanase. METHODS: Aggrecanase activity and ability of agents to prevent membrane associated aggrecanase activity were assessed by Western blotting. Expression of known aggrecanases was measured by real time polymerase chain reaction in bovine nasal and human articular chondrocytes. RESULTS: Chondrocyte membrane associated aggrecanase activity and increased mRNA expression of ADAMTS-1, -4, -5, and -9, but not ADAMTS-4 or -15, were enhanced after stimulation by OSM+TNFalpha in bovine chondrocytes. This activity was inhibited by TIMP-3. In human chondrocytes, OSM+TNFalpha also enhanced ADAMTS-1 and -4 expression, but not that of other ADAMTSs. TNFalpha alone induced ADAMTS-9 expression, whereas OSM addition caused suppression. Both TGFbeta1 and IL4 blocked membrane associated aggrecanase activity and decreased OSM+TNFalpha-induced expression of ADAMTS-9 in bovine and human chondrocytes. IL4 down regulated ADAMTS-4 mRNA, whereas TGFbeta1 increased this expression in both bovine and human chondrocytes. CONCLUSIONS: OSM+TNFalpha up regulates membrane associated aggrecanase activity and several ADAMTS aggrecanase mRNAs in chondrocytes. The chondroprotective effects of IL4 and TIMP-3 suggest that they may have therapeutic benefit for aggrecanolysis, whereas the differential inhibitory effects of TGFbeta1 may limit its therapeutic potential. Induced membrane associated aggrecanase activity is distinct from known soluble ADAMTS aggrecanases and merits further investigation.

Serum MMP-1 and TIMP-1 levels are increased in patients with psoriatic arthritis and their siblings.

OBJECTIVE: To determine matrix metalloproteinase-1 (MMP-1) and tissue-inhibitor metalloproteinase-1 (TIMP-1) serum levels in patients with psoriatic arthritis (PsA) and to compare this with their siblings and local blood donor controls. PsA is an interesting condition in which to study metalloproteinases because there are variations in the level of destructiveness, including a significant proportion of cases without destructive change. This is unlike rheumatoid arthritis (RA) which is more uniformly destructive and where MMP-1/TIMP-1 levels are known to be elevated. METHODS: MMP-1 and TIMP-1 serum levels were determined by enzyme-linked immunosorbent assay (ELISA) in (a) index cases with PsA (subtype: RA n = 43, distal interphalangeal disease n = 2, oligoarticular n = 15, spondyloarthropathy n = 9, enthesitis n = 1), (b) siblings with PsA, (c) siblings with psoriasis (Ps), (d) unaffected siblings and (e) local controls. Patients with Ps were divided according to the onset of disease: type I disease, onset before age 40 yr and type II, onset after age 40 yr. RESULTS: MMP-1 and TIMP-1 levels were significantly increased in both the index cases and the group including all siblings compared with the controls (P < 0.0001). There was no statistical difference in MMP-1 or TIMP-1 levels between index cases and their siblings. There was no difference in serum MMP-1 level between the different subtypes (Moll and Wright) of PsA, but there was an increased level of serum TIMP-1 in patients with rheumatoid pattern (P = 0.05). In the index cases there were increased levels of TIMP-1 in type II onset psoriasis (P = 0.03) but no difference in MMP-1 levels. CONCLUSION: MMP-1 and TIMP-1 serum levels are elevated in PsA. This is greatest in RA pattern PsA. These levels were also elevated in unaffected siblings suggesting that genetic factors may be important. TIMP-1 levels were elevated in psoriasis alone, more so in late onset psoriasis, suggesting that the pathological processes of early and late onset psoriasis may be different.

Oncostatin M in combination with tumor necrosis factor alpha induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo.

OBJECTIVE: To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha) on cartilage destruction in both in vitro and in vivo model systems. METHODS: The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFalpha. RESULTS: The combination of OSM + TNFalpha induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 mRNA. The in vivo data clearly indicated that OSM + TNFalpha overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP-1). Specifically, OSM + TNFalpha induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP-1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone. CONCLUSION: This study demonstrates that OSM + TNFalpha represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.

Inhibition of furin-like enzymes blocks interleukin-1alpha/oncostatin M-stimulated cartilage degradation.

OBJECTIVE: To investigate the role of furin-like enzymes in the proteolytic cascades leading to cartilage breakdown and to examine which collagenase(s) contribute to collagen degradation. METHODS: Bovine nasal cartilage was stimulated to resorb with the addition of interleukin-1alpha (IL-1alpha)/oncostatin M (OSM) in the presence or absence of a furin inhibitor, Dec-RVKR-CH(2)Cl, or selective matrix metalloproteinase 1 (MMP-1) inhibitors. Collagen and proteoglycan levels were determined by assay of hydroxyproline and sulfated glycosaminoglycan, respectively. Collagenase and gelatinase activity were measured using (3)H-acetylated collagen and gelatin zymography, respectively. RESULTS: The addition of Dec-RVKR-CH(2)Cl to stimulated cartilage reduced the release of collagen fragments and the levels of active collagenase and MMP-2, suggesting that furin-like enzymes are involved in the cascades leading to activation of procollagenases. At MMP inhibitor concentrations that selectively inhibit MMP-1, no inhibition of collagen release was observed, but increasing the concentration to the 50% inhibition concentration for MMP-13 resulted in a 50% blockage of collagen release. The addition of Dec-RVKR-CH(2)Cl to resorbing cartilage also partially blocked proteoglycan release, thus demonstrating a role for furin-activated enzymes in the pathways leading to proteoglycan degradation. CONCLUSION: Furin-like enzymes are involved in cascades leading to activation of procollagenases and degradation of collagen. MMP-13, which can be activated by furin-processed membrane-type 1 MMP-1, appears to be a major collagenase involved in collagen degradation induced by IL-1alpha/OSM. Furin-like enzymes also appear to play a role in the pathways leading to proteoglycan degradation. These findings are of importance when considering proteinase inhibition as a target for therapeutic intervention in arthritic diseases.

Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines.

OBJECTIVE: To investigate whether interleukin 17 (IL17), derived specifically from T cells, can promote type II collagen release from cartilage. The ability of IL17 to synergise with other proinflammatory mediators to induce collagen release from cartilage, and what effect anti-inflammatory agents had on this process, was also assessed. METHODS: IL17 alone, or in combination with IL1, IL6, oncostatin M (OSM), or tumour necrosis factor alpha (TNFalpha), was added to bovine nasal cartilage explant cultures. Proteoglycan and collagen release were determined. Collagenolytic activity was determined by bioassay. Chondroprotective effects of IL4, IL13, transforming growth factor beta1 (TGFbeta1) and insulin-like growth factor-1 (IGF1) were assessed by inclusion in the explant cultures. RESULTS: IL17 alone stimulated a dose dependent release of proteoglycan and type II collagen from bovine nasal cartilage explants. Suboptimal doses of IL17 synergised potently with TNFalpha, IL1, OSM, and IL6 to promote collagen degradation. This collagen release was completely inhibited by tissue inhibitor of metalloproteinase-1 and BB-94 (a synthetic metalloproteinase inhibitor), and was significantly reduced by IL4, IL13, TGFbeta1, and IGF1. In IL17 treated chondrocytes, mRNA expression for matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 was detected. Moreover, a synergistic induction of these MMPs was seen when IL17 was combined with other proinflammatory cytokines. CONCLUSIONS: IL17 can, alone and synergistically in combination with other proinflammatory cytokines, promote chondrocyte mediated MMP dependent type II collagen release from cartilage. Because levels of all these proinflammatory cytokines are raised in rheumatoid synovial fluids, this study suggests that IL17 may act as a potent upstream mediator of cartilage collagen breakdown in inflammatory joint diseases.

The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin-1 and oncostatin M: a time-course study using real-time quantitative reverse transcription-polymerase chain reaction.

OBJECTIVE: Previous studies have reported elevated levels of interleukin-1 (IL-1) and oncostatin M (OSM) in rheumatoid joints, as well as the synergistic degradation of human articular cartilage by this cytokine combination. The present study was undertaken to investigate the ability of IL-1 and OSM to modulate gene expression of matrix metalloproteinase (MMP), ADAM, and ADAM-TS (ADAM with thrombospondin motifs) family members in human chondrocytes. METHODS: T/C28a4 human chondrocytes were stimulated for 2-48 hours with IL-1 and/or OSM. Total RNA was harvested, reverse transcribed, and assessed by real-time polymerase chain reaction for the expression of various MMP, ADAM, and ADAM-TS messenger RNAs (mRNA). Results were normalized to 18S ribosomal RNA. RESULTS: IL-1 and OSM synergized to markedly induce the expression of the collagenases MMP-1, MMP-8, and MMP-13 as well as MMP-3, an activator of proMMPs. Expression of mRNA for MMPs 1, 3, and 13 was induced early, whereas that of MMP-8 mRNA occurred late. Gene expression of MMP-14, an MMP that degrades collagen and activates proMMP-13, was elevated by this combination. IL-1 and OSM also synergized to induce gene expression of the aggrecanase ADAM-TS4, but not ADAM-TS5. CONCLUSION: These data indicate that the potent cartilage-degrading properties of the combination of IL-1 and OSM are potentially mediated by a synergistic induction of the aggrecan-degrading enzyme ADAM-TS4 and the collagen-degrading enzymes MMP-1, MMP-8, MMP-13, and MMP-14, although differences in the magnitude of response and in the time course of induction were observed. A role for MMPs 3 and 14 in the activation of proMMPs may also be implicated.