HECA-452 is a non-function blocking antibody for isolated sialyl Lewis x adhesion to endothelial expressed E-selectin under flow conditions
2012
E-selectin, expressed on inflamed endothelium, and sialyl Lewis x (sLeˣ), 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 sLeˣ 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 sLeˣ microspheres to investigate the hypothesis that HECA-452 is a non-function blocking mAb for isolated sLeˣ mediated adhesion to endothelial expressed E-selectin. Flow cytometric analysis revealed that HECA-452 recognizes and binds to the sLeˣ microspheres. Perfusion of the sLeˣ microspheres over human umbilical vein endothelial cells (HUVEC) at 1.5dyn/cm² revealed that the microspheres attach to 4h interleukin (IL)-1β activated HUVEC specifically via E-selectin. Pretreatment of the sLeˣ microspheres with HECA-452 did not influence sLeˣ microsphere initial tethering and accumulation on IL-1β activated HUVEC. Neuraminidase and fucosidase treatments of sLeˣ microspheres revealed that sialic acid and fucose are required for E-selectin binding, whereas HECA-452 recognition of sLeˣ 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 sLeˣ antigens for E-selectin and HECA-452. Combined, the data indicate that HECA-452 is a non-inhibitor of sLeˣ-mediated adhesion to endothelial expressed E-selectin.
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