Identification of Cystatin C, a Cysteine Proteinase Inhibitor, as a Major Secretory Product of Human Alveolar MacrophagesIn Vitro

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Abstract
The major inhibitor of the cysteine class of proteinases found in human body fluids, such as spinal fluid, milk, and seminal plasma, is cystatin C. In this study we show that human bronchoalveolar fluid also contains cystatin C and examine cystatin C expression by alveolar macrophages in vitro. Immunoprecipitation of extracts of metabolically labeled cells and immunoblotting of cellular extracts and culture media show that cystatin C is synthesized as a 14 (.+-. 0.5) kilodalton (kD) protein and that greater than 90% of the protein is released as the 14 kD product into the culture supernatant (26.5 .+-. 6.8 ng per 106 cells per 24 h). Cystatin C is one of the most abundant proteins secreted during the first 24 h in vitro, representing .apprx. 10 to 12% of the total protein released by normal nonsmoker macrophages. Alveolar macrophages obtained from cigarette smokers or nonsmoker macrophages exposed to zymosan in vitro released 10 to 55% less cystatin C than nonsmoker macrophages. We also assayed culture supernatants from macrophages of smokers and nonsmokers for functional cystatin C. Supernatants of nonsmoker macrophages inhibited cathespin B-like amidolytic activity in a fluorometric assay at pH 5.5. The inhibition was blocked by adsorption with Sepharose-coupled cystatin C antibodies and the inhibitor subsequently recovered from the Sepharose beads. In contrast, supernatants from smoker macrophages had obvious cathespin B-like activity. Following adsorption of smoker cell supernatants with the solid-phase cystatin c antibodies, cathepsin B-like activity increased over 100%, confirming the immunoblot data indicating that smoker macrophages also release cystatin C. These data indicate that cystatin C is a major constitutive secretory product of alveolar macrophages and normally masks the activity of cysteine proteinases also secreted by these cells. During inflammatory processes, such as that produced by cigarette smoking, cystatin C release is downregulated, contributing to increased acidic proteinase activity in the macrophage microenvironment. This altered balance of enzyme and inhibitor release may contribute to the macrophage-mediated connective tissue remodeling known to be a part of inflammatory processes.

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