Identification of a Biochemical Lesion, and Characteristic Response to Lipopolysaccharide (LPS) of a Cultured Macrophage-Like Cell Mutant with Defective LPS-Binding

Abstract

We have previously isolated a lipopolysaccharide (LPS)-resistant mutant (named LR-9) of a cultured macrophage-like cell line, J774.1. This mutant had defective LPS binding [Hara-Kuge, S., Amano, F., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 6606-6610]. In this study, we found that: (1) LPS-binding to parental J774.1 cells was dependent on a serum factor with a molecular weight of about 60 kDa, probably LPS binding protein (LBP); (2) LPS-binding to J774.1 cells was markedly reduced by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC); (3) mutant LR-9 cells were defective in LPS-binding even in the presence of serum; (4) LR-9 cells lacked CD14 protein on flow cytometric and immunoblot analyses, but retained normal CD14 mRNA levels on RNA blot analysis; (5) small amounts of LPS (1 to 10 ng/ml) activated J774.1, but not LR-9 cells, to secrete tumor necrosis factor-α and to release arachidonate metabolites, whereas both J774.1 and LR-9 were activated by large concentrations of LPS (100 to 1, 000 ng/ml). These results provide genetic evidence that CD14 molecules in J774.1 cells play a crucial role in LPS-binding and in LPS-triggered signal transduction, and indicate that large amounts of LPS can activate J774.1 cells without the participation of CD14 molecules.