Identification of a Biochemical Lesion, and Characteristic Response to Lipopolysaccharide (LPS) of a Cultured Macrophage-Like Cell Mutant with Defective LPS-Binding1

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-alpha 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.

Micrococcus luteusTeichuronic Acids Activate Human and Murine Monocytic Cells in a CD14- and Toll-Like Receptor 4-Dependent Manner

“…In the same experiment, the reference LPS induced TNF-␣ production in a concentration-dependent manner from 1 to 100 ng/ml and exhibited a plateau response at 100 ng/ml to 10 g/ml. In accordance with a previous report by Nishijima et al (27), LR-9 cells did not respond to the lower concentrations of LPS (1 to 100 ng/ml) and showed only a slight response to the higher concentrations (1 and 10 g/ml) (Fig. 3B).…”

“…LR-9 cells were originally isolated from J774.1 cells as an LPS-resistant mutant with a defect in LPS binding (13) and later were reported to lack mCD14 (27). Therefore, we first compared mCD14 expression by J774.1 cells with that by LR-9 cells by flow cytometry.…”

CD14 is a component of multiple recognition systems used by macrophages to phagocytose apoptotic lymphocytes

“…To test more specifically whether these effects can be entirely explained by the removal of CD14, advantage was taken of the LR-9 strain of J774 cells, which was selected for its resistance to LPS 16 and found to be deficient in the expression of CD14. 17 As shown in Figure 7, LR-9 cells are deficient in the surface expression of CD14 in comparison with the parental J774 cells. If the effects of PI-PLC treatment are a result of the removal of CD14, recognition of lipid-symmetric erythrocytes and apoptotic thymocytes by LR-9 cells should mimic that of PI-PLC-treated J774 cells.…”

Polyanion Induced Preferential Multinucleation in Macrophages at a Low Level of TNF-.ALPHA. Secretion.

“…It was reported that the complex of LPS and LPS binding protein interacted with the cell surface protein CD14 (22), and that this binding protein accelerated TNF-a production via high affinity binding sites (21). Thus, a certain serum component might be involved in the induction of TNF-a secretion by EP3 in vitro.…”

“…These phenomena are due to the effect of the lipopolysaccharide binding protein present in the serum on TNF-a induction (20,21). It was reported that the complex of LPS and LPS binding protein interacted with the cell surface protein CD14 (22), and that this binding protein accelerated TNF-a production via high affinity binding sites (21).…”