Plasmin, a broad spectrum protease, may be important to the pathogenesis of inflammatory tissue injury and the subsequent repair process. Plasmin activity is regulated partly by plasminogen activators (PA) and plasminogen activator inhibitors (PAI). This proposal is based on the hypothesis that abnormal expression of plasmin activity may influence the course of acute an chronic lung inflammation. further, pulmonary macrophages may regulate plasmin activity in the lung by producing both PA and PAI proteins. The proposed experiments are designed to determine whether PA and PAI activities are expressed abnormally in human lung diseases, including the adult respiratory distress syndrome, pulmonary sarcoidosis, usual interstitial pneumonitis, and emphysema. Secondly, human alveolar macrophages and macrophage-like U937 cells will be studied in vitro to characterize regulatory steps in the biosynthesis and secretion of PA and PAI proteins. Cells will be cultured in vitro and subjected to a range of stimuli designed to alter PA and PAI expression. The proteins will be characterized by functional (esterolytic) assays, fibrin-agar enzymography, ELISA, and Western blotting. To characterize the biosynthesis of these proteins, steady-state mRNA will be characterized by Northern blot analysis and dot-blotting. The rate of gene transcription will be determined by measuring pulse-labeled transcription by isolated nuclei. Alveolar macrophages and fluid will be obtained by bronchoalveolar lavage of the human subjects, and the PA and PAI activities characterized by the immunochemical and biochemical methods used in the in vitro studies. Specific mRNA for PA and PAI proteins will also be measured in alveolar macrophages. If abnormalities in PA or PAI expression can be identified in any of these lung diseases, this information will contribute greatly to understanding the role of plasmin in lung inflammation. These studies should also expand on present knowledge of the mechanisms by which macrophages regulate expression of inflammatory proteases.
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