Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance.
TLR4 and MD-2 are necessary for conferring cellular responsiveness to LPS. Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS re-stimulation, known as 'endotoxin tolerance'. While induction of LPS tolerance has been reported to correlate with down-regulation of cell surface expression of TLR4/MD-2, other mechanisms of LPS tolerance have been revealed that target intracellular intermediates downstream of the TLR4/MD-2 complex. In this study, we sought to examine whether endotoxin tolerance could be induced under conditions where expression of TLR4 and MD-2 proteins is not affected by LPS. Human HEK 293T cells are completely unresponsive to LPS, but acquire high LPS sensitivity following transient transfection with CD14, TLR4, and MD-2 (293T/CD14/TLR4/MD-2 cells), as judged by NF-kappaB activation, ERK 1/2 phosphorylation, and TNF-alpha gene expression. Prior exposure of 293T/CD14/TLR4/MD-2 cells to LPS resulted in a significant decrease of LPS-mediated responses, yet failed to affect expression levels of TLR4 and MD-2. Thus, altered expression and/or function of intracellular mediators downstream of the TLR4/MD-2 complex play an important role in mediating LPS tolerance.
Medvedev
Andrei E
AE
Department of Microbiology and Immunology, University of Maryland, Baltimore 21201, USA.
Vogel
Stefanie N
SN
D000954:Antigens, Surface / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000276:immunology; D004361:Drug Tolerance; D004926:Escherichia coli / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D007108:Immune Tolerance / Q000187:drug effects / Q000276:immunology; D007668:Kidney; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
D000954:Antigens, Surface; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases