Identification of a biochemical lesion, and characteristic response to lipopolysaccharide (LPS) of a cultured macrophage-like cell mutant with defective LPS-binding.
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.
Nishijima
M
M
Department of Biochemistry and Cell Biology, National Institute of Health, Tokyo.
Hara-Kuge
S
S
Takasuka
N
N
Akagawa
K
K
Setouchi
M
M
Matsuura
K
K
Yamamoto
S
S
Akamatsu
Y
Y
D000209:Acute-Phase Proteins; D015703:Antigens, CD / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D016227:Benzoquinones; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D047029:Lactams, Macrocyclic; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins; D009154:Mutation; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D016718:Arachidonic Acid; D016227:Benzoquinones; D002352:Carrier Proteins; D047029:Lactams, Macrocyclic; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D011809:Quinones; D017828:Rifabutin; D014409:Tumor Necrosis Factor-alpha; D000099231:Lipopolysaccharide-Binding Protein; C020754:herbimycin