title: Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein.
abstract: Recent work has established that bacterial endotoxin (LPS) binds to the plasma protein LPS-binding protein (LBP) forming high affinity complexes (LPS-LBP), that LBP is an opsonin for LPS-bearing particles, and that LPS-LBP complexes are potent agonists for monocytic cells (MO). mAb to the MO plasma membrane protein, CD14, inhibit LBP-dependent binding of LPS to MO, and LPS-LBP-dependent stimulation of cytokine release from MO. These data suggest that CD14 functions as a membrane receptor for LPS but do not demonstrate a direct association of LPS with CD14. Calcitriol was used to induce a high level of CD14 expression in the human monocyte-like cell line THP-1, resulting in enhanced responses of these cells to LPS-LBP complexes manifested by enhanced binding of LPS and a decrease in the amount of LPS needed to induce IL-8 release. An Re595 LPS derivative containing a radioiodinated, photoreactive, phenyl azide (125I-ASD-LPS) was used in cross-linking experiments to identify membrane proteins in calcitriol-treated THP-1 cells that interact with LPS. 125I-ASD-LPS was added to calcitriol-induced THP-1 cells in the presence or absence of LBP, the mixture photolyzed, and the resultant radioiodinated proteins analyzed by SDS-PAGE and autoradiography. We observed strong cross-linking of 125I-ASD-LPS to a 55-kDa membrane protein when LBP was present, but failed to observe radiolabeling of any other proteins with apparent molecular masses distinct from CD14. The cross-linked product was identified as CD14 by immunoprecipitation with anti-human CD14 mAb. Studies with human CD14 expressing transfectants of the murine B cell line 70Z/3 also revealed LBP-dependent cross-linking of 125I-ASD-LPS to CD14. These data document binding of LPS to a specific membrane protein that serves as an LPS receptor.
authors.affiliation: Scripps Research Institute, La Jolla, CA 92037.
authors.forename: P S
authors.initials: PS
authors.lastname: Tobias
authors.forename: K
authors.initials: K
authors.lastname: Soldau
authors.forename: L
authors.initials: L
authors.lastname: Kline
authors.forename: J D
authors.initials: JD
authors.lastname: Lee
authors.forename: K
authors.initials: K
authors.lastname: Kato
authors.forename: T P
authors.initials: TP
authors.lastname: Martin
authors.forename: R J
authors.initials: RJ
authors.lastname: Ulevitch
mesh_terms: D000209:Acute-Phase Proteins; D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism; D018821:CD18 Antigens; D002117:Calcitriol / Q000494:pharmacology; D002352:Carrier Proteins / Q000502:physiology*; D002460:Cell Line; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D006801:Humans; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D009000:Monocytes / Q000378:metabolism*; D014162:Transfection; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D018821:CD18 Antigens; D002117:Calcitriol; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004247:DNA; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D000099231:Lipopolysaccharide-Binding Protein