David E. Woolley

Small molecular weight beta 1 serum protein which specifically inhibits human collagenases.

NEUTRAL collagenases are specific collagen-degrading enzymes which can be identified and isolated from the media of a wide variety of human tissues in culture. In vivo the enzymes function extracellularly at neutral pH and probably have a crucial role in the catabolism of collagen. Evidence for the involvement of the enzymes in diseases characterised by disordered collagen metabolism is substantial but our understanding of the mechanism of regulation of extracellular collagenase activity is poor. Since the specific inhibition of collagenase might offer a therapeutic approach to some connective tissue diseases we have attempted to identify the natural inhibitors of this enzyme in human serum. We report here our finding of a β1 serum protein which specifically inhibits human collagenase. Because of its small molecular size (molecular weight about 40,000) this inhibitor may be important in the regulation of tissue collagenase activity.

Inhibition of human collagenase activity by a small molecular weight serum protein

Abstract Fractionation of human serum proteins by gel filtration in Sephadex G-200 revealed two regions of collagenase inhibition which corresponded to α2-macroglobulin and a smaller serum component which eluted after α1-antitrypsin. The smaller collagenase inhibitor, having a molecular weight of 40,000 was separated from α1-antitrypsin by chromatography in Sephadex DEAE A.50. It was found to inhibit human collagenases derived from skin, rheumatoid synovium, gastric mucosa and granulocytes, but not the neutral proteases trypsin and papain. Purified preparations of α1-antitrypsin inhibiting trypsin and papain had no effect on the collagenase activities. The small collagenase inhibitor may have importance as a regulatory factor in the control of collagenase activity in vivo.

Mast cell products stimulate collagenase and prostaglandin E production by cultures of adherent rheumatoid synovial cells

Mast cells were purified from histologically-confirmed dog mastocytomas and extracted for whole mast cell products (MCP). When added to cultures of human adherent rheumatoid synovial cells MCP induced a 50-400 fold increase in prostaglandin E synthesis and a 10-50 fold stimulation of collagenase production. The mast cell stimulatory factor has not been identified and was not due to histamine, heparin or prostaglandin E. These results indicate a novel way in which mast cells might interact with synovial cells to promote the production of inflammatory mediators and proteolytic enzymes which might contribute to connective tissue degradation.

All three chains of 1α2α3α collagen from hyaline cartilage resist human collagenase

Abstract A previous report that the 3α collagen chain of hyaline cartilage was cleaved by human collagenase could not be confirmed when the 1α2α3α collagen fraction was freed of all contaminating type II collagen. All three minor collagen chains, 1α, 2α and 3α, were totally resistant to highly purified collagenases from both rheumatoid synovial and gastric mucosal tissues. This finding and CNBr-peptide patterns suggest that, despite the close homology with α1(II), the 3α chain is a unique collagen component, possibly combined with 1α and 2α in heterotrimeric molecules. In contrast, a 3α-like component from fibrocartilage was cleaved by collagenase and gave a CNBr-peptide pattern more typical of α1(II) than of the collagenase-resistant 3α of hyaline cartilage.

Cleavage of collagen type X by human synovial collagenase and neutrophil elastase.

Chick-derived native cartilage collagen type X and the pepsin-resistant 45 kDa fragment were susceptible to attack by human synovial collagenase and neutrophil elastase at 25 degrees C and 35 degrees C. Synovial collagenase cleaved type X collagen at two sites which were equally susceptible to the enzyme. In contrast, elastase produced three cleavages, but the sensitive loci showed different susceptibilities as judged by the sequential appearance of specific breakdown products. Both enzymes produced a major, enzyme-resistant fragment of approximately 32 kDa at 35 degrees C, and both of these end-products co-migrated in SDS polyacrylamide gels. Human chondrocyte-derived collagenase also degraded native, 59 kDa collagen type X in a similar manner to that shown by the synovial collagenase. From amino acid sequence data the enzyme cleavages probably occur at three regions of sequence imperfection. The specific cleavages brought about by synovial or chondrocyte collagenase, or neutrophil elastase, may have a functional catabolic role in vivo, and in vitro might provide useful tools with which to further analyse specific properties of the native collagen type X molecule.

Degradation of cartilage collagens type II, IX, X and XI by enzymes derived from human articular chondrocytes.

Conditioned culture medium derived from Interleukin-I alpha-activated human articular chondrocytes contained both collagen- and proteoglycan-degrading activities. Preparations of soluble type I collagen and the cartilage collagens type II, IX, X and XI were all degraded when incubated with the conditioned culture medium at 35 degrees C. Fractionation of the enzymic activities using column chromatography with Ultragel AcA 34 and Heparin-Sepharose allowed the separation and identification of neutral proteinase, collagenolytic and proteoglycan-degrading activities. Eluant fractions which contained type I collagenase activity effectively degraded collagen type II, but these fractions did not correspond precisely with those which degraded collagen types IX, X and XI. These observations indicate that chondrocytes have the potential to produce a conventional interstitial type II collagenase together with other enzymes having some specificity for the minor collagens. Thus IL-1-activated chondrocytes produce a range of collagenolytic and proteoglycan-degrading enzymes which can process most of the structural components of the cartilage matrix.

COLLAGENASE AND ITS NATURAL INHIBITORS IN RELATION TO THE RHEUMATOID JOINT

This report attempts to summarize our present knowledge of rheumatoid synovial collagenase and its natural serum inhibitors, beta1-anticollagenase and alpha2-macroglobulin, in relation to cartilage collagen resorption in the rheumatoid joint. Immunolocalization of collagenase across the cartilage/pannus junction is described, and in the light of the finding of the specific, small molecular weight beta1-anticollagenase we propose a model of cartilage erosion based on the interaction between collagenase and its natural inhibitors.

Plasminogen activator release from cultured murine mast cells.

Mast cells from the Furth murine mastocytoma tumour line were found to contain significant levels of plasminogen activator (PA). Cultured cells released PA activity into the culture medium in parallel with the release of histamine, and both were proportionately increased following exposure to degranulating agents. Pretreatment of the mast cells with cycloheximide did not alter their total PA content or their ability to release PA. These studies suggest that PA is a prestored granule constituent. The ability of PA to generate plasmin from plasminogen suggests an important role for mast cell PA in fibrinolysis and tissue degradation, observations that have been associated with mast cell degranulation and infiltration in vivo.