Induction of macrophage nitric oxide production by Gram-negative flagellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes

J Immunol. 2003 Jun 15;170(12):6217-23. doi: 10.4049/jimmunol.170.12.6217.


The induction of cytokine synthesis by flagellin is mediated by a Toll-like receptor 5 (TLR5) signaling pathway. Although flagellin activation of the IL-1R-associated kinase and induction of TNF-alpha synthesis are dependent on TLR5 and not TLR4, we have found that flagellin stimulates NO in macrophages via a pathway that requires TLR5 and TLR4. Flagellin induced NO synthesis in HeNC2 cells, a murine macrophage cell line that expresses wild-type TLR4, but not in TLR4-mutant or -deficient GG2EE and 10ScNCr/23 cells. Flagellin stimulated an increase in inducible NO synthase (iNOS) mRNA and activation of the iNOS promoter. TLR5 forms heteromeric complexes with TLR4 as well as homomeric complexes. IFN-gamma permitted GG2EE and 10ScNCr/23 cells to produce NO in response to flagellin. Flagellin stimulated IFN-beta synthesis and Stat1 activation. The effect of flagellin on iNOS gene expression was inhibited by a Stat1 mutant protein. Taken together, these results support the conclusions that flagellin induces distinct patterns of inflammatory mediators depending on the nature of the TLR5 signaling complex and that the induction of NO by flagellin involves signaling via TLR5/TLR4 complexes.

Publication types

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

MeSH terms

  • Animals
  • COS Cells
  • Cell Line
  • DNA-Binding Proteins / metabolism
  • Flagellin / pharmacology*
  • Interferon-beta / biosynthesis
  • Interferon-gamma / physiology
  • Macromolecular Substances
  • Macrophages / enzymology
  • Macrophages / metabolism*
  • Macrophages / microbiology*
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / deficiency
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / physiology*
  • Mice
  • Mice, Inbred C3H
  • Mice, Inbred C57BL
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide Synthase / genetics
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase Type II
  • Promoter Regions, Genetic
  • RNA, Messenger / metabolism
  • Receptors, Cell Surface / chemistry*
  • Receptors, Cell Surface / deficiency
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / physiology*
  • STAT1 Transcription Factor
  • Salmonella enteritidis / physiology
  • Signal Transduction / physiology*
  • Toll-Like Receptor 4
  • Toll-Like Receptor 5
  • Toll-Like Receptors
  • Trans-Activators / metabolism
  • Transcriptional Activation
  • Transfection


  • DNA-Binding Proteins
  • Macromolecular Substances
  • Membrane Glycoproteins
  • RNA, Messenger
  • Receptors, Cell Surface
  • STAT1 Transcription Factor
  • Stat1 protein, mouse
  • Toll-Like Receptor 4
  • Toll-Like Receptor 5
  • Toll-Like Receptors
  • Trans-Activators
  • Flagellin
  • Nitric Oxide
  • Interferon-beta
  • Interferon-gamma
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse