Polymorphonuclear cells (PMN, neutrophilis ) are primary generators and effectors of inflammation. Inhibition of their migration, activation, or effector mechanisms can be used to control inflammatory diseases. We have defined a PMN activation pathway that is involved in PMN migration into inflamed tissue and their activation once there. The experiments proposed here aim at understanding the mechanism of action of this activation pathway, with regulating inflammation through its inhibition as an ultimate goal. We present background information and preliminary data showing that the extracellular domain of Integrin- Associated Protein (IAP/CD47) is the primary integrator of this activation pathway, constituting an extracellular and specific target for potential intervention. We propose biochemical and genetical experiments that will define how the IAP extracellular domain interacts with its ligands: the alphavbeta3 integrin and thrombospondin. Our cell biological experiments will correlate this information with PMN migration, transendothelial migration, and activation, resulting in an understanding of the mechanism of IAP action, concrete information on how to inhibit it, and information on the result of such inhibition. Specifically, we will in specific aim 1 use surface plasmon resonance to analyze binding of the mutant of the IAP extracellular domain to thrombospondin and the alphavbeta3 integrin. We will then determine the effect of IAP and thrombospondin binding on integrin function.
In specific aim 2 we will use IAP-deficient cells, control cells, and mutant transfectants to investigate the function of IAP in systems of vitronectin particle binding, PMN migration, and transendothelial migration.
