β2‐integrin affinity and valence in binding ICAM‐1 regulates contact mediated emigration of PMN in shear flow.

Polymorphonuclear leukocytes (PMN) circulate to inflamed vascular endothelium by homing to a gradient of chemokine activators secreted from injured tissue. During activation, the β 2 ‐integrin Leukocyte Functional Antigen‐1 (LFA‐1) shifts conformation into a high affinity state primed for binding its primary ligand on the endothelium, Intercellular Adhesion Molecule‐1 (ICAM‐1). After neutrophil capture by the endothelium, LFA‐1 is redistributed on the cellular membrane into high density clusters, a harbinger to cellular polarization where LFA‐1 is found at the leading and trailing regions of membrane contact. We have previously reported that dimeric clusters of LFA‐1 bind 10‐fold longer to ICAM‐1 dimers than monomers. Further, the affinity state of LFA‐1 regulates the lifetime and stability of PMN adhesion. We show here that formation of dimeric LFA‐1/ICAM‐1 clusters can trigger outside‐in signaling through LFA‐1. Capture of dimeric ICAM‐1 beads by PMN induced active CD18, F‐actin, and Rap‐1 clustering at the bead/cell interface much more efficiently than monomer. Outside‐in signaling via LFA‐1 was supported by an intermediate or high affinity state, but not a low affinity conformation. Activated Src‐family kinases were also involved in transmembrane signaling and colocalized with LFA‐1/ICAM‐1 clusters. We conclude that assembly of an inflammatory synapse involves dimerization of high affinity LFA‐1 with ICAM‐1, thereby providing a substrate for direction signaling of cytoskeletal mediated cell polarization and transmigration.