Generation and Characterization of a Novel Adhesion Function Blocking Monoclonal Antibody Recognizing Both Rat and Mouse E-Selectin

Abstract

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 IgG1 kappa 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 condition. This novel antibody thus recognizes a function-related epitope on rodent E-selectin.

Heterogeneity of Endothelial Cells from Different Organ Sites in T-Cell Subset Recruitment

“…Purified rat antimouse ICAM-1 (YN1.1) and anti-mouse VCAM-1 (MK 2) were acquired from the American Type Culture Collection (Rockville, MD). Purified mouse anti-mouse E-selectin (RME-1) 16 and mouse anti-mouse P-selectin (RMP-1) 17 were kind gifts from Dr. Andrew Issekutz, Dalhousie University (Halifax, Canada). Unless otherwise stated, all other reagents were obtained from M.A.…”

A down-regulatable E-selectin ligand is functionally important for PSGL-1–independent leukocyte–endothelial cell interactions

“…19,20 The recordings for the intravital microscopy experiments were made between 3 and 4 hours after TNF-␣ injection. The following monoclonal antibodies were used intravenously: RB40.34 against mouse P-selectin 21 (20 g/mouse), 2PH1 against mouse PSGL-1 10 (50 g/mouse), 4RA10 against mouse PSGL-1 22 (50 g/ mouse), and RME-1 against mouse E-selectin 23 (100 g/mouse; kindly supplied by Dr A. C. Issekutz). These concentrations of antibodies have been shown to be optimal in our preliminary work and in published results.…”

The α4β1 (Very Late Antigen (VLA)-4, CD49d/CD29) and α5β1 (VLA-5, CD49e/CD29) Integrins Mediate β2 (CD11/CD18) Integrin-Independent Neutrophil Recruitment to Endotoxin-Induced Lung Inflammation

“…The hamster IgG mAb HM␣ 5 reacts with and blocks the rat ␣ 5 of VLA-5 (27,28) and HRL-3 (a gift from D. Anderson, Pharmacia Upjohn, Kalamazoo, MI, and M. Miyasaka, Osaka University) reacts with and blocks rat L-selectin (29). The mouse IgG2a mAb RMP-1 recognizes and blocks rat P-selectin, and the mouse IgG1 mAb RME-1 blocks rat E-selectin (30,31). These were generated in our laboratory.…”