(645c) Elucidating the Sequential Binding of Lfa-1 and Mac-1 to Icam-1 under Shear Conditions: Possible Role of E-Selectin Signaling

LFA-1 and Mac-1 are the integrins involved in the firm arrest of leukocytes to the endothelium during inflammation response. Previous work using a cone-plate viscometer showed that the adhesion kinetics of LFA-1 and Mac-1 varies with shear rate and that LFA-1 binding was transient, lasting ~2 min, while Mac-1 sustained longer adhesion. This work suggests that LFA-1 binding is necessary for Mac-1 adhesion and supports the notion that LFA-1 and Mac-1 binding to ICAM-1 is a cooperative and sequential process. While this work highlights important shear dependent kinetics of LFA-1 and Mac-1 with their ligands, it does not accurately mimic the in vivo neutrophil encounter with the endothelium. Furthermore, not much has been done to fully characterize the suggested sequential binding of LFA-1 and Mac-1 to ICAM-1. Here, we explored the adhesion of FMLP stimulated human neutrophils to mouse L-cells co-transfected with human E-selectin and ICAM-1 in a parallel plate flow chamber in the presence or absence of function blocking antibodies to LFA-1 and/or Mac-1. Our results show that LFA-1 dependent firm adhesion of neutrophils in laminar flow partially degrades over a period of ~1 min while Mac-1 mediated adhesion is stable over a period greater than 10-min. Furthermore, when E-selectin interaction is removed physically (L-cells expressing ICAM-1 alone) or via blockage of MAPK signaling, a complete degradation of LFA-1 mediated adhesion is observed in the same 1-min window. This result suggests a signaling role of E-selectin in the dynamics of LFA-1 and Mac-1 interaction with ICAM-1.