title: Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4.
abstract: Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Medvedev
authors.forename: P
authors.initials: P
authors.lastname: Henneke
authors.forename: A
authors.initials: A
authors.lastname: Schromm
authors.forename: E
authors.initials: E
authors.lastname: Lien
authors.forename: R
authors.initials: R
authors.lastname: Ingalls
authors.forename: M J
authors.initials: MJ
authors.lastname: Fenton
authors.forename: D T
authors.initials: DT
authors.lastname: Golenbock
authors.forename: S N
authors.initials: SN
authors.lastname: Vogel
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics; D016466:CHO Cells / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D002999:Clone Cells; D006224:Cricetinae; D029721:Drosophila Proteins; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D006801:Humans; D007108:Immune Tolerance / Q000235:genetics; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis* / Q000235:genetics; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000276:immunology*; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; D029721:Drosophila Proteins; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; C050016:lipoarabinomannan; D020928:Mitogen-Activated Protein Kinases