Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance

J Endotoxin Res. 2003;9(1):60-4. doi: 10.1179/096805103125001360.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Surface / metabolism*
  • Blotting, Western
  • Cell Line / drug effects
  • Cell Line / immunology
  • Cell Line / metabolism
  • Dose-Response Relationship, Drug
  • Down-Regulation / immunology
  • Drug Tolerance
  • Escherichia coli / immunology
  • Gene Expression
  • Humans
  • Immune Tolerance* / drug effects
  • Immune Tolerance* / immunology
  • Kidney
  • Lipopolysaccharide Receptors / metabolism*
  • Lipopolysaccharides / pharmacology
  • Lymphocyte Antigen 96
  • Membrane Glycoproteins / metabolism*
  • Mitogen-Activated Protein Kinase 1 / genetics
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases / genetics
  • Mitogen-Activated Protein Kinases / metabolism
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Phosphorylation
  • RNA, Messenger / metabolism
  • Receptors, Cell Surface / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Toll-Like Receptor 4
  • Toll-Like Receptors
  • Transfection
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Antigens, Surface
  • LY96 protein, human
  • Lipopolysaccharide Receptors
  • Lipopolysaccharides
  • Lymphocyte Antigen 96
  • Membrane Glycoproteins
  • NF-kappa B
  • RNA, Messenger
  • Receptors, Cell Surface
  • TLR4 protein, human
  • Toll-Like Receptor 4
  • Toll-Like Receptors
  • Tumor Necrosis Factor-alpha
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases