Inhibition by a CD14 monoclonal antibody of lipopolysaccharide binding to murine macrophages

We have established an anti-CD14 mAb named 4Cl against murine macrophages. 4Cl can bind to thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages and casein-induced peritoneal neutrophils. Immunostaining with 4Cl was inhibited by treatment of the cells with phosphatidylinositol specific phospholipase C, suggesting that the antigen is GPI-anchored. Immunoprecipitates from biotin-labeled RAW264.7 cell lysate with 4Cl were around 55 kDa and were visualized with rmC5-3, the only commercially available anti-murine CD14 mAb. 4Cl positively stained COS7 cells transfected with an expression vector containing cDNA of murine CD14. Pretreatment of macrophages with 4Cl reduced LPS-mediated production of TNF alpha, IL-6, and nitrite. The binding of FITC-LPS to RAW264.7 cells was blocked by pretreatment with 4C l but not with rmC5. Pretreatment of cells with unlabeled 4Cl mAb but not unlabeled rmC5-3 reduced binding of FITC-4Cl. These results suggest that the 4Cl epitope on murine CD14 plays an important role in LPS binding and is distinct from the rmC5-3 epitope.