Analysis of Tlr4-mediated LPS signal transduction in macrophages by mutational modification of the receptor

Blood Cells Mol Dis. Oct-Dec 1999;25(5-6):328-38. doi: 10.1006/bcmd.1999.0262.

Abstract

In mouse macrophages (RAW 264.7 cells), toll-like receptor 4 (Tlr4) is a limiting factor in lipopolysaccharide (LPS) signal transduction. The expression of only 1-2 x 10(4) copies of recombinant Tlr4 per cell enhances sensitivity to LPS, shifting the EC50 by 30-fold to the left. Expression of the Tlr4(Lps-d) isoform of Tlr4 (found in C3H/HeJ mice) shifts the EC50 2600-fold to the right, essentially abolishing LPS responses. A truncated form of Tlr4, lacking a cytoplasmic domain, exerts only a weak inhibitory effect on signal transduction. Similarly, the normal or Tlr4(Lps-d) forms of protein lacking an ectodomain [corrected], cause modest inhibition of LPS signaling. Manipulations of Tlr4 structure and expression cause changes in LPS sensitivity that range over 3 to 4 orders of magnitude. These findings support the view that Tlr4 is an integral component of a solitary pathway for LPS signal transduction in macrophages and permit inferences related to the mechanism of signaling and its blockade.

MeSH terms

  • Animals
  • Cell Line
  • Dose-Response Relationship, Drug
  • Drosophila Proteins*
  • L Cells
  • Lipopolysaccharides / metabolism*
  • Macrophages / chemistry*
  • Macrophages / physiology
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / pharmacology*
  • Membrane Glycoproteins / physiology*
  • Mice
  • Mutation
  • Protein Structure, Quaternary
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / physiology*
  • Signal Transduction / drug effects*
  • Toll-Like Receptor 4
  • Toll-Like Receptors
  • Transfection
  • Tumor Necrosis Factor-alpha / biosynthesis
  • Tumor Necrosis Factor-alpha / drug effects

Substances

  • Drosophila Proteins
  • Lipopolysaccharides
  • Membrane Glycoproteins
  • Receptors, Cell Surface
  • Toll-Like Receptor 4
  • Toll-Like Receptors
  • Tumor Necrosis Factor-alpha