Activation of interleukin-1 receptor-associated kinase by gram-negative flagellin.
Flagellin from various species of gram-negative bacteria activates monocytes to produce proinflammatory cytokines. We have analyzed the pathway by which Salmonella enteritidis flagellin (FliC) activates murine and human monocyte/macrophage-like cell lines. Since lipopolysaccharide (LPS), the principal immune stimulatory component of gram-negative bacteria, is known to signal through Toll-like receptor 4 (TLR4), we tested the possibility that FliC also signals via TLR4. When murine HeNC2 cells were stimulated with LPS in the presence of a neutralizing anti-TLR4 monoclonal antibody, tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production were markedly reduced. In contrast, FliC-mediated TNF-alpha and NO production were minimally affected by the anti-TLR4 antibody. Furthermore, FliC, unlike LPS, stimulated TNF-alpha production in the TLR4 mutant cell line, GG2EE, indicating that TLR4 is not essential for FliC-mediated signaling. To test the possibility that FliC signals via another TLR, we measured FliC-mediated activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK), a central component in IL-1R/TLR signaling. FliC induced IRAK activation in HeNC2 and GG2EE cells as well as in the human promonocytic cell line THP-1. IRAK activation was rapid in HeNC2 cells, with maximal activity observed after 5 min of treatment with FliC. In addition, FliC-mediated IRAK activation exhibited the same concentration dependence as was demonstrated for the induction of TNF-alpha. These results represent the first demonstration of IRAK activation by a purified bacterial protein and strongly suggest that a TLR distinct from TLR4 is involved in the macrophage inflammatory response to FliC.
Moors
M A
MA
Department of Microbiology and Immunology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
Li
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Mizel
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SB
D000818:Animals; D002460:Cell Line; D029721:Drosophila Proteins; D004789:Enzyme Activation; D005408:Flagellin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases; D007700:Kinetics; D008262:Macrophage Activation; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000378:metabolism*; D012477:Salmonella enteritidis / Q000378:metabolism; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D029721:Drosophila Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D017472:Receptors, Interleukin-1; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D005408:Flagellin; D011494:Protein Kinases; D053592:Interleukin-1 Receptor-Associated Kinases