LEUKOCYTE ADHESION TO VASCULAR ENDOTHELIUM INDUCES E-SELECTIN LINKAGETO THE ACTIN CYTOSKELETON

We have examined functions of the cytoplasmic domain of E-selectin, an inducible endothelial transmembrane protein, especially its ability t o associate with the cytoskeleton during leukocyte adhesion. Confocal microscopy of interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells (HUVEC) visualized clustering of E-selectin mo lecules in the vicinity of leukocyte-endothelial cell attachment sites , A detergent based extraction and Western blotting procedure demonstr ated an association of E-selectin with the insoluble (cytoskeletal) fr action of endothelial monolayers that correlated with adhesion of leuk ocytes via an E-selectin-dependent mechanism. A mutant form of E-selec tin lacking the cytoplasmic domain (tailless E-selectin) was expressed in COS-7 cells and supported leukocyte attachment (in a nonstatic adh esion assay) in a fashion similar to the native E-selectin molecule, b ut failed to become associated with the cytoskeletal fraction. To iden tify the cytoskeletal components that associate with the cytoplasmic d omain of E-selectin, paramagnetic beads coated with the adhesion-block ing anti-E-selectin monoclonal antibody H18/7 were incubated with IL-1 beta-activated HUVEC, and then subjected to detergent extraction and magnetic separation. Certain actin-associated proteins, including alph a-actinin, vinculin, filamin, paxillin, as well as focal adhesion kina se (FAK), were copurified by this procedure, however talin was not, Wh en a mechanical stress was applied to H18/7-coated ferromagnetic beads bound to the surface of IL-1 beta-activated HUVEC, using a magnetical twisting cytometer, the observed resistance to the applied stress was inhibited by cytochalasin D, thus demonstrating transmembrane cytoske letal mechanical linkage. COS-7 cells transfected with the tailless E- selectin failed to show resistance to the twisting stress, Taken toget her, these data indicate that leukocyte adhesion to cytokine-activated HUVEC induces transmembrane cytoskeletal linkage of E-selectin throug h its cytoplasmic domain, a process which may have important implicati ons for cell-cell signaling as well as mechanical anchoring during leu kocyte-endothelial adhesive interactions.