To elucidate the mechanism of synovial damage in rheumatoid arthritis, we studied the activation of latent collagenases released from adherent rheumatoid synovial cells in culture. Latent enzyme was not complexed with alpha2 macroglobulin, the prinicpal proteinase inhibitor in serum, and could be activated by trypsin in the presence of alpha2 macroglobulin if sufficient proteinase was added to saturate inhibitor. Latent collagenase bound half as effectively to collagen fibrils as active enzyme. Plasmin was a threefold better activator of latent enzyme than trypsin and could be generated by addition of plasminogen to synovial-cell cultures. Production of both collagenase and plasminogen activator was inhibited by dexamethasone (10(-9) M). These studies emphasize in importance of control of activation in regulation collagenase activity, It is likely that rheumatoid synovium produces both latent collagenase and plasminogen activator; plasmin is activated from its zymogen, plasminogen, present in inflamed tissues, and in turn activates collagenase.

Objective. To assess the presence of fibroblast collagenase (MMP-l), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) in osteoarthritic (OA) cartilage, with particular emphasis on areas of macroscopic cartilage erosion.Methods. Messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blot analysis.Results. MMP-1 and MMP-13 were expressed at higher levels by OA chondrocytes than by normal chondrocytes. In addition, mRNA for MMP-8 was present in OA cartilage but not normal cartilage by PCR and Northern blot analyses. Chondrocytes from areas surrounding the OA lesion expressed greater quantities of MMP-1 and MMP-13 compared with normal chondrocytes, suggesting local modulation by mechanical and inflammatory factors. Tumor necrosis factor a stimulated the expression of all 3 collagenases. Retinoic acid, an agent which induces autodigestion of cartilage in vitro, stimulated only the expression of MMP-13.Conclusion. These findings suggest a key role of MMP-13 and MMP-8, as well as MMP-1 in osteoarthritis.The matrix metalloproteinase (MMP) family of enzymes consists of at least 15 distinct entities, including

The family of matrix metalloproteinases is a family of closely related enzymes that play an inportant role in physiological and pathological processes of matrix degradation. The most distinctive characteristic of interstitial collagenases (fibroblast and neutrophil collagenases) is their ability to cleave interstitial coilagens at a single peptide bond; however, the precise region of the enzyme responsible for this substrate specificity remains to be defined. To address this question, we generated truncated mutants of neutrophil collagenase with various deletions in the COOH-terminal domain and chimeric molecules between neutrophil collagenase and stromelysin and assayed the expressed enzymes against type I collagen and the general substrate, casein. Our data suggest that substrate specificity for interstitial collagen is determined by a 16-aa sequence in the COOH-terminal domain of neutrophil collagenase and is influenced by the integrity of a disulfide-defined loop at the COOH terminus for maximal activity. It was found that a relatively large region of 62-aa residues influenced the relative efficiency of collagenolytic activity. In addition to the region that conferred this specificity, a site at the COOH side of the presumptive zinc-binding locus was found to be necessary for general catalytic activity. Mutation of a critical aspartic residue at position 253 within this area resulted in complete loss of proteolytic activity, suggesting that Asp-253 might function as one of the ligands for divalent cations, which are essential for enzymatic activity.The family of matrix metalloproteinases (MMPs) is a family of closely related enzymes that play an important role in a variety of physiological and pathological processes, including embryonic development (1), tumor invasion (2), and arthritis (3, 4). The human MMP gene family contains at least two distinct interstitial collagenases (5, 6), three types of stromelysins (7-9), putative metalloproteinase 1 (10), and two gelatinases, 72-kDa type IV collagenase (11, 12) and 92-kDa type V collagenase (13,14). When the primary structures of MMPs are compared, it is apparent that they are structurally homologous molecules consisting of defined functional domains (13,15 MATERIALS AND METHODSPlasmid Construction of Truncated Mutants of NC (TrNCs). The NC 7.2 cDNA containing a full-length coding region for NC was used to create TrNCs with various deletions in the COOH-terminal sequence (6). The size of the TrNC is identified by amino acid residue numbers starting from the initiating Met (Fig. 1). A premature stop codon was introduced by PCR. The primer (5'-GCTCGAATTCGGGC-TCGCCAGGGAAGGGCCCTACCC-3') complementary to the 5' end of NC 7.2 incorporated a unique EcoRI restriction site and was used for construction of all the mutants. Primers at the 3' end contained sequences for a stop codon at various intervals and a unique Not I restriction site. The isolated fragments were digested with EcoRI and Not I and then ligated into these sites in the expression vector pcDNA I (...

Interleukin 1 (IL-1) or lymphocyte-activating factor, a monokine released in vitro by cultured monocytes or macrophages, acts on a variety of somatic and immunerelated target cells in a genetically unrestricted manner (1-5).It has long been recognized that fibroblasts appear at sites of inflammatory reactions after macrophages and play a pivotal role in the repair of damaged connective tissue by synthesizing and remodeling components of the matrix (e.g., collagens, fibronectin, and proteoglycans) (6). In this report, we have observed that IL-1 is a potent stimulator of fibroblast collagenase production in vitro. This finding suggests that ILl may be a major effector molecule by which macrophages modulate not only fibroblast growth but collagenase production as well (4, 5). Materials and Methods IL-1 Purification.In preliminary studies, we found that IL-1 isolated from normal human peripheral blood monocytes was contaminated by lymphokines (personal observation by A. E. Postlethwaite and L. Lachman). However, IL-I from monoblasts from patients with acute monocytic or myelomonocytic leukemia was free of contaminating lyrnphokines and its physicochemical properties were identical to those of IL-1 obtained from normal monocytes (7). IL-1 was purified from supernatants from cultures of monoblasts by diafiltration, uhrafihration, and isoelectric focusing (IEF) as previously described (7,8). The protein content of IL-1 prepared in this manner was below the limit detectable by the Lowry assay (<50 ng/ml).High Performance Liquid Chromatography. Analytical gel filtration high performance liquid chromatography (HPLC) was performed on two 1-125 protein analysis columns (Waters Associates, Inc., Milford, MA) connected in tandem. The columns were equilibrated and run with 0.0075 M glycylglycine:0.14 M NaCI (GGBS) at pH 7.2.Analytical anion exchange-HPLC was also performed on a 250-mm × 4. l-ram SynChropak AX300 column (SynChrom, Inc., Linden, IN). The starting buffer was 0.02 M Tris acetate, pH 8, and the limiting buffer was 0.02 M Tris acetate:0.5 M sodium acetate, pH 8.Thymocyte Proliferation Assay. IL-1 activity was quantitated by measuring the uptake of tritiated thymidine ([3H]TdR) by thymoeytes from 6-to 10-wk-old Swiss-Webster mice (Charles River Breeding Laboratories, Inc., Boston, MA) (7). Standard errors were < 10% of the mean of triplicate determinations,

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