Disruption of the delicate balance between proteases and proteinase inhibitors at tissue sites plays a role in the pathogenesis of emphysema, bronchiectasis, asthma, chronic bronchitis, sarcoidosis, respiratory distress syndrome, arthritis and some skin diseases. We have characterized, isolated and cloned a fast acting inhibitor of neutrophil elastase called """"""""Human Monocyte/Neutrophil Elastase Inhibitor"""""""" (HEI), a prevalent component of neutrophils, monocytes and macrophages, the cells associated with degradative lung diseases. We recently generated antibody reagents and also recombinant active HEI, which is biochemically indistinguishable from naturally occurring monocyte HEI. We propose to use these reagents in functional studies to define the spectrum of proteases inhibited by HEI by examining elastase, cathepsin-G, proteinase- 3, plasminogen activator and several granzymes. The cellular distribution of HEI will be defined by Northern analysis and ELISA of monocyte/macrophage and neutrophil lineage cells and also cytolytic T- lymphocytes and natural killer cells, all of which contain active serine proteases. Regulation of HEI expression will be examined in macrophages in comparison with regulation of expression of plasminogen activator inhibitor-2 (PAI-2), a closely related molecule. Extracellular fluids will also be examined for HEI content in the anticipation that HEI will be found in culture supernatant of activated monocytes. Substantial evidence places HEI as a member, with PAI-2, SCCA, maspin and ovalbumin, of the Ov- serpin family of proteins, characterized by internal noncleavable secretion signals and extracellular as well as intracellular location, depending on the activation status of the cell. The HEI gene will be characterized and its assignment to the Ov-serpin family verified by intron amplification and characterization. Chromosomal localization will be determined by the use of DNA from human-rodent hybrid cells. The promoter region of the HEI gene will be sequenced and characterized for regulatory elements after isolation of a genomic clone. Based on its structure, function and location, we propose that inherited defects of HEI are a causative or predisposing factor in unexplained lung dysfunction, including cases of progressive emphysema and progressive bronchiectasis of unknown cause. We will analyze the functional region of the HEI-encoding gene of patients with these diseases for sequence variations using single- strand conformation polymorphism and heteroduplex analysis.
