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. Immunostaini
ng with 4Cl was inhibited by treatment of the cells with phosphatidylinosit
ol specific phospholipase C, suggesting that the antigen is GPI-anchored. I
mmunoprecipitates from biotin-labeled RAW264.7 cell lysate with 4Cl were ar
ound 55 kDa and were visualized with rmC5-3, the only commercially availabl
e anti-murine CD14 mAb. 4Cl positively stained COS7 cells transfected with
an expression vector containing cDNA of murine CD14. Pretreatment of macrop
hages with 4Cl reduced LPS-mediated production of TNF alpha, IL-6, and nitr
ite. 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 b
ut 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.