Analysis of Tlr4-Mediated LPS Signal Transduction in Macrophages by Mutational Modification of the Receptor

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 × 104 copies of recombinant Tlr4 per cell enhances sensitivity to LPS, shifting the EC50 by 30-fold to the left. Expression of the Tlr4Lps−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 Tlr4Lps−d forms of protein lacking a cytoplasmic domain, 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.