Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory and immunomodulatory cytokine implicated in inflammatory conditions such as rheumatoid arthritis, Crohn's disease, multiple sclerosis and the cachexia associated with cancer or human immunodeficiency virus infection. TNF-alpha is initially expressed as a 233-amino-acid membrane-anchored precursor which is proteolytically processed to yield the mature, 157-amino-acid cytokine. The processing enzyme(s) which cleave TNF-alpha are unknown. Here we show that the release of mature TNF-alpha from leukocytes cultured in vitro is specifically prevented by synthetic hydroxamic acid-based metalloproteinase inhibitors, which also prevent the release of TNF-alpha into the circulation of endotoxin challenged rats. A recombinant, truncated TNF-alpha precursor is cleaved to biologically active, mature TNF-alpha by several matrix metalloproteinase enzymes. These results indicate that processing of the TNF-alpha precursor is dependent on at least one matrix metalloproteinase-like enzyme, inhibition of which represents a novel therapeutic mechanism for interfering with TNF-alpha production.

The role of matrix metalloproteinases in tumor angiogenesis and growth is now well recognized for models of both human and animal cancer. Clinical studies currently under way with the prototype matrix metalloproteinase inhibitor, marimastat, will establish whether inhibitors of these enzymes are of benefit in the treatment of different types of human cancer. On chronic therapy in humans, marimastat induces a reversible tendinitis that can also be detected in certain animal species. This paper compares the ability of broad-spectrum and various types of selective matrix metalloproteinase inhibitors to induce tendinitis and to exhibit anticancer effects in an animal cancer model. Under conditions in which both systemic exposure and inhibitor potency are controlled, selective inhibitors are less pro-tendinitic, but are weaker anticancer agents than broad-spectrum agents such as marimastat. The clinical relevance of these findings is discussed.

1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.

A metalloproteinase, 'proteoglycanase', that degrades proteoglycan and insoluble type IV collagen as well as casein was purified to homogeneity from rabbit bone culture medium. The major form of this proteinase had a final specific activity of 2400 micrograms of casein degraded/min per mg of enzyme protein, and Mr 24 500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or 12 500 by gel-filtration chromatography. It was active over the pH range 5.0-9.0 against a number of substrates, and the rates of degradation were almost constant over the whole of this range. The products generated from proteoglycan-aggregate degradation by this enzyme indicated cleavage at multiple chondroitin sulphate-binding sites along the protein core. In a new assay to detect degradation of insoluble type IV collagen, the proteoglycanase generated large fragments, probably by cleavage in the non-helical regions. The enzyme degraded laminin, fibronectin and procollagen, removing the extension peptides of the last-mentioned. It also cleaved the 'weak region' of the type III collagen helix in a manner analogous to trypsin. The synthetic substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 was cleaved exclusively at the Gly-Ile bond. The proteoglycanase was inhibited by tissue inhibitors of metalloproteinases from rabbit bone culture medium, human amniotic fluid and bovine nasal-cartilage extracts, forming essentially irreversible inactive complexes. The importance of this tissue-derived enzyme, with such a wide-ranging degradative capacity, in normal and pathological connective-tissue matrix degradation is discussed.

1. Pure rabbit bone metalloproteinase inhibitor (TIMP) bound tightly to pure rabbit bone collagenase with an apparent Kd of 1.4 × 10(-10) M. 2. The molecular weight of the enzyme-inhibitor complex was found to be 54 000, but no enzyme activity could be recovered from the complex after treatment with either mercurials or proteinases. The complex thus differed from latent collagenase in terms of size, susceptibility to mercurials and behaviour on concanavalin A-Sepharose. 3. The interaction of the purified components was compared with that of crude collagenase and crude inhibitor in culture medium. Mercurial treatment partially reversed the inhibition in the crude system, but not when the purified components were used. 4. The significance of the results is discussed in relation to the extracellular control of the activity of collagenase.