Integrin activation during neutrophil adhesion and vascular inflammation

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Neutrophils, which are essential for host defense against bacterial and fungal infections, induce inflammation following tissue necrosis or ischemic injury. If not properly resolved, neutrophilic inflammation is the driving force behind a plethora of human inflammatory diseases. Neutrophils arrive at the site of injury from the bloodstream by first rolling and then arresting. Arrest is triggered by chemokines that induce the high-affinity conformation of ..2 integrins. Two FERM domain proteins, kindlin-3 and talin-1, are required for neutrophil arrest. It is well known that talin-1, an adaptor protein that binds the actin cytoskeleton, activates integrins by binding and altering the topology of the .. transmembrane domain. We recently reported that kindlin-3 is recruited to the plasma membrane through its pleckstrin homology domain prior to neutrophil arrest. However, the mechanism underlying neutrophil spreading and the role of kindlin-3 in this process are poorly understood. Moreover, how kindlin-3 cooperates with talin-1 to activate integrins and whether kindlin-3 also functions as an adaptor protein by directly binding to actin are completely unknown. Our overarching hypothesis is that, during neutrophil spreading, both kindlin-3 and talin-1 are simultaneously recruited to the plasma membrane, where kindlin-3 organizes high-affinity integrin activation. To test this hypothesis, we generated reporter mouse lines for simultaneous detection of ..2 integrin activation and imaging of kindlin-3 and talin-1 in mouse neutrophils. In Aim 1, we will test the hypothesis that kindlin-3 organizes a ring of clustered high-affinity ..2 integrins during neutrophil spreading under flow conditions; in Aim 2, we will test the hypothesis that kindlin-3 regulates integrin activation by directly binding to the actin cytoskeleton; and in Aim 3, we will determine the role of ..2 integrin activation in ischemia-reperfusion injury (IRI). The proposed research is conceptually innovative and highly significant because it will resolve the enigma of how kindlin-3 organizes high-affinity integrin activation and define the role it plays in IRI-induced inflammation. Successful completion of this proposal will establish molecular mechanisms of integrin activation and provide mechanistic insight into neutrophil spreading and vascular inflammation.