In order for inflammatory cells to leave the circulation and enter extravascular sites in the lungs and other tissues, they must cross endothelium and subendothelial basement membranes composed of extracellular matrix (BCM). Major advances have occurred in understanding the interactions between neutrophils (PMN) and endothelium, but relatively little is known about interactions between PMN and the subendothelial basement membrane. This project concerns interactions between inflammatory cells and basement membranes. The focus is primarily upon PMN and entactin, an integral basement membrane component which plays a role in basement membrane assembly through its ability to bind avidly to both laminin and type IV collagen. Recently, we reported that both wild type and recombinant forms of entactin are chemotactic and adhesive for PMN and are degraded by matrix metalloproteinases released by PMN and other inflammatory cells. In this project we will define further the responses of PMN to entactin by looking at the effects of entactin upon PMN phagocytic activity and the release of primary granule contents from PMN. We will identify the entactin domains and PMN receptors responsible for entactin-PMN interactions, quantify entactin binding to PMN, and study signal transduction involved in PMN responses entactin. Using domain- specific anti-entactin antibodies that we will raise to recombinant entactin domains, we will determine the circumstances under which domains of entactin that interact with inflammatory cells are exposed in basement membranes. To extend our observations about degradation of pure entactin by proteinases, we will determine the susceptibility of entactin that is assembled in basement membranes to degradation by matrilysin and other inflammatory cell proteinases. To define the cellular expression and distribution of entactin in normal lung and in lung injured by inflammation, we will use molecular and immunologic techniques in developing lung and animal models that mimic human cigarette smoke-induced emphysema, diffuse alveolar injury, and fibrosing alveolitis.
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