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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.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has emerged as a major regulator of cellular functions and has been implicated in several pathologies involving remodeling of extracellular matrix (ECM). The end stage of inflammatory joint diseases is characterized by excessive ECM catabolism, and in this study we assess the role of PI3K signaling in the induction of collagenolytic matrix metalloproteinases (MMPs) in human chondrocytes. We used the most potent cytokine stimulus reported to promote cartilage ECM catabolism, namely interleukin-1 (IL-1) in combination with oncostatin M (OSM). Both OSM and IL-6 (in the presence of its soluble receptor), but not IL-1 nor leukemia inhibitory factor, induced Akt phosphorylation in human chondrocytes. Inhibition of PI3K signaling using LY294002 blocked IL-1+OSM-mediated Akt phosphorylation, induction of MMP-1 and MMP-13, and cartilage collagenolysis. To further explore the role of downstream substrates within the PI3K pathway, complementary use of small molecule inhibitors and specific small interfering RNAs demonstrated that the PI3K subunit p110alpha and Akt1 were required for MMP-1 mRNA induction. MMP-13 induction was also reduced by loss of function of these molecules and by a lack of p110delta, 3-phosphoinositide-dependent kinase-1 or Akt3. We therefore propose that the activities of specific elements of the PI3K signaling pathway, including Akt, are necessary for the synergistic induction of MMP-1 and MMP-13 and the cartilage breakdown stimulated by IL-1+OSM. Our data provide new insight into the mechanism of synergy between IL-1 and OSM and highlight new therapeutic targets for inflammatory joint diseases that aim to repress the expression of collagenases.

Assay of collagenase activity involves the use of radiolabeled collagen. Stimulation of cartilage with proinflammatory cytokines results in the upregulation of collagenases and the subsequent release of degraded collagen fragments. These enzymes can be localized in both osteoarthritic and rheumatoid arthritis cartilage and synovial tissues.

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.

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.

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.

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.

We have investigated proteinases that degrade cartilage collagen. We show that pro-inflammatory cytokines act synergistically with oncastatin M to promote cartilage collagen resorption by the up-regulation and activation of matrix metalloproteinases (MMPs). The precise mechanisms are not known, but involve the up-regulation of c-fos, which binds to MMP promoters at a proximal activator protein-1 (AP-1) site. This markedly up-regulates transcription and leads to higher levels of active MMP proteins.

Objective: Bovine and human cartilages in explant culture respond to proinflammatory cytokines with the up-regulation of procollagenases. In stimulated bovine nasal cartilage (BNC), >90% of collagen is released by day 14 of culture, but collagen release is rarely seen before day 7. The aim of this study was to investigate if activation of procollagenases is a rate-limiting step in cartilage collagen breakdown., Methods: BNC and human articular cartilage explants were cultured with interleukin-1alpha (IL-1alpha) and/or oncostatin M (OSM) with or without test reagents. Collagen levels were determined by assay of hydroxyproline. Collagenase activity was measured using the diffuse fibril assay., Results: The addition of procollagenase activators, matrix metalloproteinase 3 (MMP-3), and APMA to IL-1alpha/OSM-stimulated BNC resulted in early release of collagen. The release with APMA was completely blocked by the addition of tissue inhibitor of metalloproteinases 1. This shows that procollagenases are present early in the culture period, but cartilage collagen breakdown does not happen until activation occurs. The addition of plasminogen to IL-1alpha/OSM-stimulated cartilage produced early collagen release in bovine and a significant increase in human cartilage. Thus, plasminogen activators (PAs) are present and convert plasminogen to plasmin, a known activator of several MMPs, including collagenases. Addition of alpha1-proteinase inhibitor or a urokinase-type PA inhibitor, 7-amino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, partially blocked the breakdown of collagen from IL-1alpha/OSM-treated bovine cartilage. This suggests that serine proteinases are involved in the activation cascades of procollagenases that result in cartilage collagen breakdown., Conclusion: The activation of procollagenases is a key control point in cartilage collagen breakdown, and serine proteinase pathways activate MMPs.

Retinoic acid (RetA) and interleukin-1alpha (IL-1) together can induce a reproducible release of proteoglycan fragments from bovine nasal cartilage in culture. However, release of collagen fragments with either agent alone is often variable. In this study over 70% of the total collagen was released from bovine nasal cartilage in culture by day 14 when RetA and IL-1 were combined. This release was accompanied by the appearance of collagenolytic activity in the culture medium that cleaved collagen specifically at the (1/4)/(3/4) position. Tissue inhibitor of metalloproteinases (TIMP) activity was present at day 7 but low or absent in media from resorbing tissue at day 14. The breakdown of cartilage collagen could be prevented by the addition of BB-94, a specific metalloproteinase inhibitor. These results suggest that RetA promotes the early release of TIMP from the tissue and that IL-1 stimulates pro-collagenase secretion which, when activated, exceeds the local concentration of TIMP. Thus in the later stages of culture collagen destruction occurs. Both MMP-1 and MMP-13 were detected and appear to be involved in IL-1 + RetA induced bovine cartilage destruction. However, for the first time, we also present evidence to suggest that MMP-13 is the predominant collagenase in this system., (Copyright 2000 Wiley-Liss, Inc.)

We describe two alternative assays for measuring collagenolytic activity using (3)H-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble (3)H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity > or = 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added., (Copyright 1999 Academic Press.)

The primary agents responsible for cartilage and bone destruction in joint diseases are active proteinases that degrade collagen and proteoglycan. All four main classes of proteolytic enzymes are involved in either the normal turnover of connective tissue or its pathological destruction. These proteinases are made by different cells found within the joints. Both extracellular and intracellular pathways exist and individual enzymes can be inhibited by specific proteinaceous inhibitors that block their activity. Recent research has implicated the matrix metalloproteinases (MMPs) in many of the processes involved in joint diseases. The metalloproteinases are capable of degrading all components of the extracellular matrix. This family of proteinases contains a group of at least three collagenases that are capable of degrading native fibrillar collagen. Collagen degradation within joint disease is recognized as the irreversible step in the destruction of cartilage that leads to a failure in joint function. The collagenases are the enzymes necessary to initiate collagen turnover in normal connective tissue turnover and in disease.

This report shows for the first time that platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta (TGF-beta) can interact in a synergistic manner with retinoic acid to stimulate the production of tissue inhibitor of metalloproteinases (TIMP) from human skin and synovial fibroblasts. When cells are treated with 1, 10, and 100 ng/ml of either of these growth factors in combination with 10(-5) M retinoic acid, this results in a dose-dependent synergistic induction of TIMP protein secretion which is greater than the additive effect of the agents by up to fourfold. These responses can be inhibited by the presence of specific neutralising antibodies to the growth factors, demonstrating that they are not the result of an experimental artefact such as contamination with bacterial endotoxin. The mechanisms of these synergistic responses may involve the induction of receptors for retinoic acid, PDGF, or TGF-beta or may result from synergistic effects on TIMP gene transcription. We have also found that retinoic acid potently down-regulates PDGF-BB-stimulated collagenase in both types of fibroblast and that the effect of PDGF-BB alone on collagenase secretion from skin fibroblasts is biphasic. Finally, this study reports that retinoic acid and TGF-beta do not act in an additive fashion to inhibit the production of collagenase from skin fibroblasts.

During purification, human fibroblast collagenase breaks down into two major forms, an N-terminal 22000/25000-Mr fragment and a C-terminal 27000-Mr fragment; the most likely mechanism being autolysis. The cleavage site has been identified (Pro269- Ile270) and in an attempt to obtain full-length human collagenase (i.e., Mr 42570), this cleavage site and another potential cleavage site (Ala258- Ile259) have been mutated by PCR- directed mutagenesis: Ile270Ser and Ile259Leu. The mutated cDNA was then cloned into the expression vector, pGEX2T, and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). After cleavage with factor Xa, the mutated collagenase was purified on a peptide hydroxamic acid affinity column. The mutated recombinant collagenase is stable, remains full length and retains the ability to cleave collagen.

Objective: Collagen turnover in connective tissues is thought to be controlled by the balance between the levels of interstitial collagenase and tissue inhibitor of metalloproteinases (TIMP-1). The aim of this study was to measure the level of total collagenase (MMP-1), TIMP-1, collagenase approximately TIMP-1 complex and glycosaminoglycan (GAG) in sequential samples of osteoarthritic knee synovial fluid from well documented patients to determine if these parameters changed with time and correlated with clinical indices., Methods: Twenty-one patients were recruited and randomly allocated to receive tiaprofenic acid, indomethacin or naproxen. Total collagenase, TIMP-1, collagenase approximately TIMP-1 complex and GAG were measured in 80 osteoarthritic synovial fluids taken over a period of six months., Results: The majority of fluids contained a molar excess of TIMP-1 over collagenase, although in seven fluids collagenase was present in excess; six of these samples were from a single patient. GAG levels were relatively unchanged over the six months studied., Conclusion: The levels of collagenase and TIMP-1 varied between patients and over time in individual patients. No collagenase approximately TIMP-1 complex was found in any fluid. There was no significant difference in the median levels of collagenase, TIMP-1 or GAG in the different treatment groups. High levels of collagenase were found in one patient with a crystal related disease. These immunoassays give valuable information on the levels of collagenase and TIMP-1 in individual patients with time and may help to determine the mechanisms controlling the turnover of cartilage collagen in different arthritides.

Objective: To investigate the production of the matrix metalloproteinase (MMP), collagenase (MMP-1), and its natural inhibitor, the tissue inhibitor of metalloproteinases (TIMP) by diseased human tendon samples in organ culture., Methods: Portions of tendons were excised from the shoulders of patients undergoing shoulder surgery, classified as either proximal to the lesion (abnormal) or distal to the lesion (normal) according to their macroscopic appearance at surgery, and placed in organ culture for periods of up to 28 days. The release of collagenase and TIMP activity in the conditioned culture medium was measured., Results: Procollagenase and TIMP were both produced by all the tendon samples for an extended period of time. The levels of enzyme and inhibitor varied between patients, but in most of them TIMP levels were greater than collagenase levels. In one sample of calcified tendon, procollagenase levels were greater than those of TIMP. The mean level of collagenase produced by tendon proximal to the lesion and tendon distal to the lesion were not significantly different (95.2 (SD 106.8) U/g and 34.0 (45.3) U/g, respectively), while the corresponding figures for TIMP were 109.7 (62.3) U/g and 53.0 (27.9) U/g (p = < 0.05), although there was considerable variation in some samples. Western blotting and collagen fragment analysis confirmed that the collagenolytic activity detected was attributable to the metalloproteinase fibroblast collagenase (MMP-1)., Conclusions: Tendon tissue can actively secrete procollagenase, an enzyme that, once activated, is capable of remodelling collagen, the major connective tissue component of tendon. Collagenase is produced even in unstimulated cultures, although the concentrations of TIMP are usually greater than that of collagenase in most samples. Some activation of collagenase appeared to have occurred. These results indicate that tendon tissue cells are capable of producing a remodelling response, even in end stage tendon disease.

Background: The collagenases are members of the family of zinc-dependent enzymes known as the matrix metalloproteinases (MMPs). They are the only proteinases that specifically cleave the collagen triple helix, and are important in a large number of physiological and pathological processes. Structures are known for the N-terminal catalytic' domain of collagenases MMP-1 and MMP-8 and of stromelysin (MMP-3). This catalytic domain alone, which comprises about 150 amino acids, has no activity against collagen. A second domain, of 200 amino acids, is homologous to haemopexin, a haem-binding glycoprotein., Results: The crystal structure of full-length MMP-1 at 2.5 A resolution gives an R-factor of 21.7%. Two domains are connected by an exposed proline-rich linker of 17 amino acids, which is probably flexible and has no secondary structure. The catalytic domain resembles those previously observed, and contains three calcium-binding sites. The haemopexin-like domain contains four units of four-stranded antiparallel beta sheet stabilized on its fourfold axis by a cation, which is probably calcium. The domain constitutes a four-bladed beta-propeller structure in which the blades are scarcely twisted., Conclusions: The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.

Collagenase is a member of the matrix metalloproteinase family whose members are all capable of degrading extracellular matrix components. The mature form of porcine collagenase has been expressed in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N-terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to an active form of collagenase. Recombinant collagenase was biologically active in the form of a fusion protein; this was cleaved with factor Xa to yield collagenase with the authentic N terminus (phenylalanine) found in vivo and purified in a single step on a peptide hydroxamic acid affinity column. On purification the recombinant porcine collagenase undergoes autolysis at a number of different bonds in the region connecting the active site domain with the C-terminal hemopexin-like domain. This may represent a loop region of poor secondary structure, making it susceptible to relatively nonspecific cleavage. The N-terminal fragment retains a reduced level of collagenolytic activity, along with that against casein and gelatin.

The aim of the study was to immunolocalise interstitial collagenase (MMP-1) and the tissue inhibitor of metalloproteinases (TIMP-1) in ulcerating corneas from patients with rheumatoid arthritis, to determine whether changes in expression are associated with destructive corneal disease. Collagenase was expressed by stromal cells in 8 of 8 ulcerating corneas but was not seen in normal tissue (n = 3). TIMP-1 was abundant throughout the normal stroma, but was much reduced or absent from diseased corneas. Collagenase staining was frequently more intense near the epithelial surface and associated with a cellular infiltrate consisting of activated antigen-presenting cells (HLA-DR+), many of which were macrophages (CD68+) and derived from the epithelium or limbus (S100+). Interstitial collagenase produced by infiltrating macrophages and/or stimulated corneal fibrocytes is probably a major mediator of collagen degradation in rheumatoid corneal ulceration. In addition, reduced levels of TIMP-1 expression are consistent with collagenase activity and tissue destruction. Epithelial-stromal cell interactions and the production of local inflammatory mediators are of major importance in the pathogenesis of corneal destruction, although the precise nature of the antigenic stimulation and/or cellular interactions remains to be elucidated.

On purification, active human fibroblast collagenase breaks down by an autolytic mechanism into two major forms (M(r) 22,000 and M(r) 27,000) and one minor form (M(r) 25,000). The ability of human collagenase to bind to the tissue inhibitor of metalloproteinases (TIMP) and to TIMP-2 resides mainly in the active site area of the 22,000 M(r) N-terminal domain of the molecule, but the 27,000 M(r) C-terminal domain also has a role in stabilizing these interactions. The 22,000 M(r) fragment is able to form a complex with TIMP and TIMP-2 which is stable to gel filtration in a similar manner to the whole molecule, but no such complexes are formed by the 27,000 M(r) fragment. Complex formation with the whole molecule is prevented by EDTA and by 1,10-phenanthroline demonstrating the importance of the active site; additionally TIMP and TIMP-2 will compete with a reversibly bound peptide hydroxamic acid inhibitor for the active site. The inhibition of enzyme activity by TIMP and TIMP-2 is less pronounced in the 22,000 M(r) fragment when compared to the whole molecule and a similar effect is seen with the peptide hydroxamic acid inhibitor and also with alpha 2-macroglobulin, suggesting a role for the C-terminal domain in interacting with these inhibitors. Whole molecule collagenase and the 27,000 M(r) fragment bind to type 1 collagen-Sepharose while the 22,000 M(r) fragment exhibits no such binding, suggesting that the C-terminal domain has an important role in the binding of enzyme to substrate.

This study aimed to investigate possible feedback mechanisms in the control of collagenase (matrix metalloproteinase-1) and tissue inhibitor of metalloproteinases (TIMP-1). Procollagenase, active collagenase, TIMP-1 and collagenase-TIMP complex were applied to human skin and synovial fibroblasts for 48 hours, the cells were washed and the resulting collagenase and TIMP-1 secretion was measured over the following 72 hours using ELISAs. Of the additions, only TIMP-1 showed any measurable effect, stimulating collagenase secretion over a range of 1-8 micrograms/ml. Endotoxin contamination was ruled out as an explanation and the active conformation of the TIMP-1 molecule was shown to be necessary.

Serum levels of tissue collagenase, matrix metalloproteinase-1, were measured in both longitudinal and cross-sectional studies, in 332 pregnant women and 27 non-pregnant volunteers. The enzyme-linked immunosorbent assay (ELISA) used is the first described to measure collagenase in serum directly, is specific, and is rapid and reproducible. Levels were determined throughout pregnancy, during term and preterm labour, and in the post-partum period. Serum tissue collagenase levels were elevated in pregnancy (P < 0.001). There was no difference between levels of serum collagenase prior to labour at term and those observed during labour. Similarly, there was no significant difference in levels obtained during preterm labour and those at a similar gestation in women who subsequently delivered at term. No significant decrease in levels had occurred by the 4th post-partum day. In view of these findings of unaltered matrix metalloproteinase-1 levels in association with labour, previous reports of raised serum collagenase activity in association with the onset of spontaneous labour, at term and preterm gestation periods, may be due to increased neutrophil collagenase activity.

Objective: To compare the levels of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). This study aims to clarify existing data from previously used enzyme or inhibitor activity assays performed following separation by gel filtration, by using a 1-step enzyme-linked immunosorbent assay (ELISA) for each component., Methods: Total collagenase, free TIMP, and collagenase-TIMP complex were measured using a newly developed, specific double-antibody sandwich ELISA:, Results: Levels of both collagenase and TIMP were significantly higher in RA patients (collagenase 1,560 +/- 150 ng/ml [mean +/- SEM], TIMP 1,610 +/- 130 ng/ml; n = 80) than OA patients (collagenase 420 +/- 90 ng/ml, TIMP 1,050 +/- 60 ng/ml; n = 80), with the difference being especially striking for collagenase. Sixteen RA fluids had detectable levels of collagenase-TIMP complex, compared with only 3 OA fluids., Conclusion: The level of total collagenase in SF is greater in RA than OA, while levels of free TIMP show more overlap between the 2 diseases; this may simply reflect the increased inflammation seen in RA, or it may reflect a different disease mechanism.

Monoclonal antibodies have been raised against purified human fibroblast collagenase and characterised. One of these antibodies has been used in combination with a polyclonal anticollagenase antibody in a double antibody sandwich ELISA to measure collagenase. The assay range was 2-50 ng/ml collagenase. The assay measures total collagenase, i.e. pro- and active enzyme as well as collagenase in complex with TIMP. The level of collagenase has been measured in sera samples from patients with rheumatoid arthritis and compared with age- and sex-matched controls. The levels measured were: rheumatoid arthritis, 69 +/- 29 ng/ml; normal, 49 +/- 14 ng/ml.

The matrix metalloproteinases are a family of enzymes involved in the turnover of the connective tissues. The regulation of these enzymes is complex, involving the control of synthesis, the activation of proenzyme forms and the presence of specific inhibitors. Retinoids have been reported to inhibit the production of metalloproteinases by human and rabbit synovial fibroblasts and by human skin fibroblasts. The production of the highly specific tissue inhibitor of metalloproteinases (TIMP) by connective tissue cells may be crucial in the regulation of connective tissue breakdown and this present study was undertaken to determine if retinoic acid (RA) could modulate TIMP and collagenase production by synovial fibroblasts. The results show that RA at concentrations from 10(-7) to 10(-5) M significantly stimulated the secretion of TIMP by two of three human synovial cell lines. The effect of mononuclear cell factor (MCF) on TIMP and collagenase levels was also investigated. The apparent reduction of collagenase levels in the presence of RA, could result from a failure to accurately measure this enzyme in the presence of increased levels of TIMP.

IL-1 stimulates the secretion of metalloproteinases by a variety of connective tissue cells and is thought to be the primary inducing agent of connective tissue breakdown in rheumatoid arthritis. Transforming growth factor-beta (TGF-beta) is known to be capable of inhibiting the synthesis of metalloproteinases and to be able to partially inhibit interleukin-1 (IL-1) induced cartilage degradation. The present paper examines the ability of TGF-beta to modulate the action of IL-1 on fibroblasts of synovial and skin origin and investigates the secretion of the tissue inhibitor of metalloproteinases (TIMP) by these cells after exposure to TGF-beta and IL-1. The principal findings are that when four out of five fibroblast lines were exposed to TGF-beta and IL-1 in combination they displayed a significant increase in TIMP secretion; furthermore, in two of these cell lines a significant stimulation of TIMP secretion was induced by TGF-beta alone.

The production of collagenase by human articular chondrocytes in response to interleukin-1 beta is inhibited in a dose-dependent manner by interferon-gamma (1-1,000 units/ml). The analysis of culture medium samples by Western blotting and the measurement of levels of tissue inhibitor of metalloproteinases suggest that the decrease in measurable collagenase activity is primarily due to the inhibition of procollagenase production. These results provide evidence of a role for interferon-gamma in limiting connective tissue degradation.

Pig synovial and human skin fibroblast procollagenases were treated with highly purified tryptase, the major proteinase of human mast cells, to determine whether this trypsin-like proteinase could activate the latent form of collagenase and so be involved in connective tissue breakdown. No significant activation of either human or pig procollagenase was found, but the highest concentration of tryptase partially destroyed procollagenase. Tryptase did not degrade type I collagen or proteoglycan. These data indicate that human mast cell tryptase does not contribute to connective tissue breakdown via procollagenase activation or via proteoglycan degradation.

The ability of the anabolic steroid, stanozolol, to stimulate procollagenase production by human synovial and skin fibroblasts was examined in an in vitro assay system. Stanozolol is used therapeutically to treat a variety of connective tissue and vascular disorders and its clinical effects suggest that it can modulate connective tissue breakdown. The results showed that stanozolol was capable, in a dose dependent manner, of significantly stimulating procollagenase production by skin fibroblasts. However, in three synovial fibroblast lines no evidence was found of increased collagenase production following treatment with stanozolol; although the synovial fibroblasts secreted significantly increased amounts of procollagenase in response to IL-1. These results may shed some light on the mechanism of action in vivo of stanozolol in the treatment of connective tissue disorders.