Inflammation is an essential process to eliminate pathogens, but it also has a fundamental role in causing disease (e.g. a relationship between inflammation and cardiovascular disease is well established, by which inflammation both contributes to its cause and results from it). Neutrophils are the most prominent leukocyte component of early inflammatory responses and their recruitment from the blood is amplified and prolonged by the leukocyte adhesion protein L-selectin (CD62L). Appropriately, L-selectin is highly regulated, which involves its very rapid ectodomain cleavage from the leukocyte surface (shedding). High levels of soluble L- selectin in normal serum of humans demonstrate this is a physiological process. Upon leukocyte activation, ADAM17 is the primary sheddase of L-selectin as well as of other leukocyte factors that modulate inflammation (e.g. TNF, TNFRI, and TNFRII). Currently, there are significant gaps in our understanding of how this proteolytic process is regulated and of ADAM17's role in L-selectin shedding during neutrophil extravasation and apoptosis. Therefore, our goal is to determine the underlying regulatory mechanisms of L- selectin shedding. Our overall hypothesis is that L-selectin shedding is modulated by a variety of molecular and cellular events. In turn, these events appear to control leukocyte recruitment in different ways, such as by influencing L-selectin adhesiveness upon neutrophil attachment to the vascular wall and by maintaining soluble L-selectin in the serum as an adhesion buffer. The goals of this study are to determine the regions of ADAM17 that are essential for L-selectin shedding (Aim 1), define the mechanisms that modulate L-selectin shedding by examining ADAM17 directly as well as determining their spatial distribution on the surface of neutrophils undergoing adhesion (Aim 2), and elucidate the role of ADAM17 in other means of inducing L- selectin shedding, such as neutrophil apoptosis (Aim 3). Our strategy will involve innovative concepts and approaches, including the use and manipulation of human neutrophils and mature leukocytes deficient in functional ADAM17. This research will help to answer fundamental questions in the cell biology of leukocyte adhesion and will provide new insights in to key aspects of inflammation regulation. This knowledge will be highly useful in the development of more specific therapies tailored to inflammatory processes and immunity. ? ? ? ?
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