title: Evidence for a CD14- and serum-independent pathway in the induction of endotoxin-tolerance in human monocytes and THP-1 monocytic cells.
abstract: Lipopolysaccharide (LPS) stimulation of macrophages or monocytes is believed to occur via a serum- and CD14-dependent signaling pathway via toll-like receptor 4 (TLR4). We sought to determine whether serum and/or CD14 are required for LPS to induce the endotoxin-tolerant state in human monocytes. LPS treatments were performed in the presence or absence of an anti-CD14 monoclonal antibody and with or without fetal bovine serum. Endotoxin tolerance was assessed after an 18-h exposure (pretreatment) to 10 ng/mL of LPS. Medium was discarded and cells were challenged with activating (1-1000 ng/mL) doses of LPS. LPS-stimulated tumor necrosis factor (TNF) secretion into culture supernatants was determined after 5 h by ELISA and p44/p42 ERK kinase activation was measured after 30 min by Western blot. Statistical analysis was by ANOVA. LPS induced endotoxin-tolerance with a significant inhibition of LPS-stimulated TNF secretion and less p44/p42 ERK kinase activation. When LPS-stimulation of naïve (nontolerant) monocytes was performed in medium with anti-CD14 antibody or without serum, there was marked blunting of TNF release. However, LPS pretreatment in medium without serum or in medium containing anti-CD14 antibody resulted in changes in monocyte activation and function characteristic of endotoxin tolerance. LPS-stimulated p44/p42 ERK kinase activation and TNF release were diminished whether or not anti-CD14 antibody was present during LPS pretreatment. LPS-stimulated TNF secretion and p44/p42 ERK kinase activation require the presence of serum and are inhibited by anti-CD14 antibody. Our findings suggest that LPS induces endotoxin tolerance in human monocytic cells via a pathway that does not require serum or cell surface CD14.
authors.affiliation: Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Wyrta
authors.initials: W
authors.lastname: Heagy
authors.forename: Christopher
authors.initials: C
authors.lastname: Hansen
authors.forename: Kimberly
authors.initials: K
authors.lastname: Nieman
authors.forename: Michael A
authors.initials: MA
authors.lastname: West
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002417:Cattle; D004361:Drug Tolerance; D004731:Endotoxins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005312:Fetal Blood / Q000502:physiology*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D004731:Endotoxins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; C044101:endotoxin, Escherichia coli; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases