The major objectives of this proposal are: 1) to purify and characterize interstitial collagenase from human neutrophils; 2) to identify the factors modulating the selective release of the collagenolytic enzymes of the neutrophil; and 3) to examine the effects of neutrophils on fibroblast collagen production and collagenase secretion. The interstitial collagenase will be purified from culture supernatants of human neutrophils by a combination of ion-exchange, affinity and molecular sieve chromatography. The purified enzyme will be characterized in terms of its activity against various collagen substrates and its activity in the presence of proteinase inhibitors. The purification and characterization of this enzyme will permit the development of specific probes which can be used to assess the role of this enzyme in disease processes. The stimuli which modulate the selective release of the neutrophil collagenolytic enzymes, collagenase and elastase, will be studied. Human neutrophils will be incubated with various biologic and pharmacologic agents and the release of collagenase and elastase into the culture medium will be quantitated. Selective release of these collagen degrading enzymes may be important in the alteration of the type I:type III collagen ratio which has been observed in some fibrotic diseases. The effect of neutrophils on fibroblast collagen production and collagenase secretion will be examined. Fibroblasts will be cultured with neutrophils or neutrophil components and collagen production as well as collagenase secretion by the fibroblast will be quantitated. In addition, the relative amounts of type I and type III collagens produced by the fibroblast in the presence of such stimuli will be examined. Such neutrophil-induced qualitative and quantitative changes in fibroblast production of matrix components may play a role in the generation of the altered connective tissue matrix seen in inflammatory states.
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