title: Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway.
abstract: Lipopolysaccharides (LPS) that lack acyloxyacyl groups can antagonize responses to LPS in human cells. Although the site and mechanism of inhibition are not known, it has been proposed that these inhibitory molecules compete with LPS for a common cellular target such as a cell-surface binding receptor. In the present study, we used an in vitro model system to test this hypothesis and to evaluate the role of CD14 in cellular responses to LPS. Cells of the THP-1 human monocyte-macrophage cell line were exposed to 1,25 dihydroxyvitamin D3 to induce adherence to plastic and expression of CD14, a binding receptor for LPS complexed with LPS-binding protein (LBP). The uptake of picograms of [3H]LPS (agonist) and enzymatically deacylated LPS [3H]dLPS (antagonist) was measured by exposing the cells to the radiolabeled ligands for short incubation periods. The amounts of cell-associated LPS and dLPS were then correlated with cellular responses by measuring the induction of nuclear NF-kappa B binding activity and the production of cell-associated interleukin (IL)-1 beta. We found that similar amounts of [3H]LPS or [3H]dLPS were taken up by the cells. The rate of cellular accumulation of the ligands was greatly enhanced by LBP and blocked by a monoclonal antibody to CD14 (mAb 60b), yet no cellular responses were induced by dLPS or dLPS-LBP complexes. In contrast, LPS stimulated marked increases of NF-kappa B binding activity and IL-1 beta. These responses were enhanced by LBP and inhibited by mAb 60b. dLPS and its synthetic lipid A counterpart, LA-14-PP (also known as lipid Ia, lipid IVa, or compound 406) strongly inhibited LPS-induced NF-kappa B and IL-1 beta, yet neither antagonist inhibited the uptake of LPS via CD14. dLPS did not inhibit NF-kappa B responses to tumor necrosis factor (TNF) alpha or phorbol ester. Our results indicate that (a) both stimulatory and nonstimulatory ligands can bind to CD14 in the presence of LBP; (b) the mechanism of inhibition by dLPS is LPS-specific, yet does not involve blockade of LPS binding to CD14; and (c) in keeping with previous results of others, large concentrations of LPS can stimulate the cells in the absence of detectable binding to CD14. The findings indicate that the site of dLPS inhibition is distal to CD14 binding in the LPS signal pathway in THP-1 cells, and suggest that molecules other than CD14 are important in LPS signaling.
authors.affiliation: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: R L
authors.initials: RL
authors.lastname: Kitchens
authors.forename: R J
authors.initials: RJ
authors.lastname: Ulevitch
authors.forename: R S
authors.initials: RS
authors.lastname: Munford
mesh_terms: D000209:Acute-Phase Proteins; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology / Q000502:physiology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology / Q000502:physiology*; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D002762:Cholecalciferol / Q000502:physiology; D004247:DNA; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D007700:Kinetics; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010969:Plastics; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D002352:Carrier Proteins; D002762:Cholecalciferol; D004247:DNA; D007375:Interleukin-1; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D010969:Plastics; D000099231:Lipopolysaccharide-Binding Protein