Integrins are heterodimeric membrane molecules expressed on almost every cell in the body, which are involved in cell-cell and cell-extracellular matrix adhesion. In the immune system, integrins have become specialized to mediate cell-cell interactions as well. Leukocyte integrins are necessary for multiple immune response and effector functions, lymphocyte homing, and leukocyte migration to sites of inflammation. Unwanted integrin- mediated adhesion has been implicated in diseases such as sepsis syndrome, reperfusion injury, multiorgan failure after shock, thermal injury and myocardial infarction, in addition to a large number of idipathic inflammatory conditions. This application to continue the study of leukocyte integrins focuses on understanding two aspects of leukocyte integrin function which are central to their role in immunity. We propose to continue our studies first, of signal transduction by leukocyte integrins and second, of integrin interactions with cytoskeleton. For both aims, we have developed novel assays which uniquely enable us to answer questions about these basic integrin functions. This laboratory has generated data over several years that beta-3 integrin ligation can activate multiple functions of phagocytes and that this process is an important component of leukocyte responses in inflammation. Now we have developed a model system in which we can assay structure- function in beta-3 integrins specifically related to their role in signal transduction. Because it is the first transfectable system shown to be suitable for analysis of integrin signal transduction, this model is a major advance toward achieving a molecular understanding of the role of these integrins in inflammation and immunity. A large body of work on all integrins demonstrates that interaction with the actin cytoskeleton is critical for integrin functions. However, understanding integrin-cytoskeleton interaction has not progressed rapidly because of the lack of a kinetic assay of the dynamic interactions between integrins and cytoskeleton. To solve this problem, we have developed a new technique using single particle tracking (SPT) by video- enhanced interference contrast microscopy (DIC) to directly study this interaction. SPT can assay integrin-cytoskeleton interaction over a time course (seconds-to-minutes) during which integrin-mediated events such as adhesion and cell spreading occur. We propose to use this novel assay coupled with biochemical techniques to test the hypothesis that LFA-1 has two distinct mechanisms of association with the lymphocyte cytoskeleton. Completion of these two specific aims will greatly enhance understanding of the molecular mechanisms by which integrins participate in both normal and pathologic immunity and inflammation. These studies have implications for therapy in a wide range of inflammatory and infectious diseases.
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