HECA-452 is a non-function blocking antibody for isolated sialyl Lewis x adhesion to endothelial expressed E-selectin under flow conditions.

Abstract E-selectin, expressed on inflamed endothelium, and sialyl Lewis x (sLe x ), present on the surface of leukocytes, play a key role in leukocyte–endothelial interactions during leukocyte recruitment to sites of inflammation. HECA-452 is a monoclonal antibody (mAb) that recognizes sLe x and is routinely used by investigators from diverse fields who seek to unravel the mechanisms of leukocyte adhesion. The data regarding the ability of HECA-452 to inhibit carbohydrate-mediated leukocyte adhesion to E-selectin remains conflicted, in part due to the presence of a variety of potential E-selectin reactive moieties on leukocytes. Recognizing this, we utilized a complementary approach to gain insight into HECA-452 adhesion assays. Specifically, we used sLe x microspheres to investigate the hypothesis that HECA-452 is a non-function blocking mAb for isolated sLe x mediated adhesion to endothelial expressed E-selectin. Flow cytometric analysis revealed that HECA-452 recognizes and binds to the sLe x microspheres. Perfusion of the sLe x microspheres over human umbilical vein endothelial cells (HUVEC) at 1.5 dyn/cm 2 revealed that the microspheres attach to 4 h interleukin (IL)-1β activated HUVEC specifically via E-selectin. Pretreatment of the sLe x microspheres with HECA-452 did not influence sLe x microsphere initial tethering and accumulation on IL-1β activated HUVEC. Neuraminidase and fucosidase treatments of sLe x microspheres revealed that sialic acid and fucose are required for E-selectin binding, whereas HECA-452 recognition of sLe x does not depend on the fucose moiety to the extent required for E-selectin recognition. This latter finding suggests there are potential subtle differences between the sLe x antigens for E-selectin and HECA-452. Combined, the data indicate that HECA-452 is a non-inhibitor of sLe x -mediated adhesion to endothelial expressed E-selectin.