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

Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan, adachiyo{at}ps.toyaku.ac.jp

C. Satokawa

Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan

M. Saeki

Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan

N. Ohno

Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan

H. Tamura

Seikagaku Corporation, Tokyo Institute, Tokyo, Japan

S. Tanaka

Seikagaku Corporation, Tokyo Institute, Tokyo, Japan

T. Yadomae

Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan

We have established an anti-CD14 mAb named 4C1 against murine macrophages. 4C1 can bind to thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages and casein-induced peritoneal neutrophils. Immunostaining with 4C1 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 4C1 were around 55 kDa and were visualized with rmC5-3, the only commercially available anti-murine CD14 mAb. 4C1 positively stained COS7 cells transfected with an expression vector containing cDNA of murine CD14. Pretreatment of macrophages with 4C1 reduced LPS-mediated production of TNF, IL-6, and nitrite. The binding of FITC-LPS to RAW264.7 cells was blocked by pretreatment with 4C1 but not with rmC5. Pretreatment of cells with unlabeled 4C1 mAb but not unlabeled rmC5-3 reduced binding of FITC-4C1. These results suggest that the 4C1 epitope on murine CD14 plays an important role in LPS binding and is distinct from the rmC5-3 epitope.

Journal of Endotoxin Research, Vol. 5, No. 3, 139-146 (1999)
DOI: 10.1177/09680519990050030701


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