Degradation of extracellular proteins by inflammatory cells is a fundamental element of effective inflammatory responses. However, when inflammatory responses are excessive, inappropriate, or prolonged, proteolytic tissue injury becomes a feature of a variety of diseases that together affect essentially every organ of the body. For example, in pulmonary emphysema, it is clear that proteolytic injury to extracellular matrix is a pivotal pathogenic event. Inhibitors confine the proteolytic activity of inflammatory cells to the immediate pericellular microenvironment, but cannot inhibit degradation of proteins that are in direct contact with the cells. Our understanding of the cell biology of this confined proteolytic activity has lagged far behind our knowledge of the structure and biochemistry of the enzymes and inhibitors themselves. Studies begun during the present funding period have produced important new concepts and new insights into mechanisms by which catalytic activity of enzymes can be confined to (or near) the cell surface when an inflammatory cell is bathed in a medium containing high-affinity inhibitors of the enzymes. For example, our success in imaging quantum bursts of proteolytic activity from single azurophil granules of neutrophils has led to new insights and a diffusion-based construct for understanding localized catalytic activity. Further we have identified catalytically active serine proteinases on the cell surface of neutrophils that are resistant to inhibition by naturally-occurring proteinase inhibitors. In the work proposed, we will pursue these observations in logical directions. Specifically, we will address the following aims: 1) Study proteolysis in zones of close contact between cells and subjacent substrates, through: a) Examining evanescent bursts of proteolytic activity from release of single azurophil granules; b) Simulating dispersion of enzymes, and non-isotropic inhibition, in space and time following degranulation; and c) Exploring mechanisms of tissue injury in alpha1-antitrypsin deficiency through experiments and simulations. 2) Study serine proteinases bound to the cell surface of neutrophils and monocytes, with regard to: a) Mechanism(s) of binding to the cell surface; and b) Modulation and focal targeting. The proposed work will provide important new information about mechanisms by which inflammatory cells normally degrade extracellular proteins with little damage to surrounding tissues, and also ways in which their proteolytic mechanisms may suffer from dysregulation.
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