Generation and characterization of a novel adhesion function blocking monoclonal antibody recognizing both rat and mouse E-selectin

The prerequisite for the recruitment of circulating leukocytes to sites of inflammation is adhesion to vascular endothelial cells. Selectins play a significant role in the initiation of this multistep process by mediating 'rolling' of the leukocytes on the endothelium. Investigation of selectin- dependent cell interactions using function blocking monoclonal antibodies (MAb) provides insights into the mechanisms involved in leukocyte migration into inflammation. Until now most studies in inflammation models in rats have relied on cross-reactive or polyclonal antibodies against rat E-selectin. In an E-selectin knockout mouse, we aimed to generate an adhesion function blocking MAb to rat E-selectin by immunization with rat E-selectin transfected Chinese hamster ovary cells (RESEC). An IgG1κ MAb was identified that reacts with RESEC but not with untransfected Chinese hamster ovary cells, as well as with recombinant mouse E-selectin protein as assessed by ELISA. This MAb is designated RME-1. It does not cross-react with rat L- selectin or rat P-selectin or E-selectin expressed on human umbilical vein endothelium. Adhesion of the HL-60 myeloid cells to immobilized mouse E- selectin was completely inhibited by MAb RME-1 under static conditions and adhesion of rat polymorphonuclear leukocytes to recombinant mouse E-selectin was blocked under rotation conditions. This novel antibody thus recognizes a function-related epitope on rodent E-selectin.