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Conditions were established to stimulate human gingival fibroblast explant cultures to synthesize milligram quantities of the metalloproteinase proenzymes, prostromelysin and procollagenase. To stimulate enzyme production, cells were treated with 1 nM recombinant human IL-1 beta for approximately 7 days under serum free conditions. Using a combination of rapid column chromatography steps, approximately 10 milligrams of prostromelysin and 5 milligrams of procollagenase were purified from 1 liter of conditioned media. Prostromelysin electrophoresed as a doublet with molecular weights of 55,57 kD, whereas, procollagenase migrated with slightly lower molecular weights of 52, 54 kD. Both proenzymes were treated with trypsin or aminophenylmercuric acetate to generate active species. The molecular weights of the active enzymes were approximately 10 kD smaller than the proenzymes. Active enzymes were inhibited by metal chelators and the natural metalloproteinase inhibitor, tissue inhibitor of metalloproteinase (TIMP), but not by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). Activated stromelysin degraded a number of substrates including transferrin, proteoglycan monomer, proteoglycan aggregated with hyaluronic acid, and substance P. By contrast, collagenase degraded interstitial type I collagen and the peptide thioester, Ac-Pro-Leu-Gly-SCH(iBu)Co-Leu-GlyOEt. Identity of both enzymes were confirmed by amino-terminal protein sequence analysis as well as by immunoblot analysis using monoclonal antibodies.

Several known chemical compounds were shown to selectively inhibit the interaction between immune aggregates and C1q, the activation of C1r-C1s complex by immune aggregate-bound C1q, and the esterolytic activity of the activated C1s, C1s. These reactions are relevant to the functions of the first complement component, C1, and its activation induced by immune complexes. The effects of these inhibitors on tissue injury mediated by immune complex-induced complement activation, such as immune hemolysis, passive cutaneous anaphylaxis, and experimental glomerulonephritis were examined. The results suggest an approximate correlation between the activity shown on the molecular level and that obtained in vivo. One such compound, suramin, was shown to be an effective inhibitor of PCA and the proteinuria manifestation of EGN while not affecting antibody fixation to tissue or histamine-mediated skin reaction. These results suggest that effective suppression of the initial steps of complement activation may be of value of controlling immune complex-mediated tissue injuries in disease.

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