Abstract
Although oxidative stress has been thought to play a general role in the activation of NF-kappaB, the involvement of reactive oxygen species (ROS) in facilitating nuclear translocation of NF-kappaB in neutrophils has not been described. In addition, the mechanisms by which ROS modulate the transcriptional activity of NF-kappaB in response to Toll-like receptor 4 (TLR4)-dependent signaling are not well characterized. To examine these issues, oxidant-dependent signaling events downstream of TLR4 were investigated in neutrophils stimulated with LPS. Pretreatment of neutrophils with the antioxidants N-acetylcysteine or alpha-tocopherol prevented LPS-induced nuclear translocation of NF-kappaB. Antioxidant treatment of LPS-stimulated neutrophils also inhibited the production of proinflammatory cytokines (TNF-alpha, macrophage inflammatory protein-2, and IL-1beta), as well as activation of the kinases IkappaB kinase alpha, IkappaB kinase beta, p38, Akt, and extracellular receptor-activated kinases 1 and 2. The decrease in cytoplasmic levels of IkappaBalpha produced by exposure of neutrophils to LPS was prevented by N-acetylcysteine or alpha-tocopherol. Activation of IL-1R-associated kinase-1 (IRAK-1) and IRAK-4 in response to LPS stimulation was inhibited by antioxidants. These results demonstrate that proximal events in TLR4 signaling, at or antecedent to IRAK-1 and IRAK-4 activation, are oxidant dependent and indicate that ROS can modulate NF-kappaB-dependent transcription through their involvement in early TLR4-mediated cellular responses.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Acetylcysteine / pharmacology
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Active Transport, Cell Nucleus / immunology
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Animals
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Antioxidants / pharmacology
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Cell Nucleus / immunology
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Cell Nucleus / metabolism
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Cells, Cultured
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Cytokines / metabolism
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Enzyme Activation / drug effects
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Enzyme Activation / immunology
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Enzyme Inhibitors / pharmacology
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Enzyme Stability / drug effects
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Enzyme Stability / immunology
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I-kappa B Kinase
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I-kappa B Proteins / metabolism
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Inflammation Mediators / metabolism
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Interleukin-1 Receptor-Associated Kinases
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Lipopolysaccharides / pharmacology
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Male
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Membrane Glycoproteins / physiology*
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Mice
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Mice, Inbred BALB C
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Mitogen-Activated Protein Kinases / metabolism
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NF-KappaB Inhibitor alpha
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NF-kappa B / metabolism*
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Neutrophils / enzymology
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Neutrophils / immunology
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Neutrophils / metabolism
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Oxidants / physiology
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Phosphotransferases (Alcohol Group Acceptor) / metabolism
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Protein Kinases / metabolism
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-akt
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Reactive Oxygen Species / metabolism*
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Receptors, Cell Surface / physiology*
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Toll-Like Receptor 4
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Toll-Like Receptors
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Up-Regulation / immunology
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alpha-Tocopherol / pharmacology
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p38 Mitogen-Activated Protein Kinases
Substances
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Antioxidants
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Cytokines
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Enzyme Inhibitors
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I-kappa B Proteins
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Inflammation Mediators
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Lipopolysaccharides
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Membrane Glycoproteins
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NF-kappa B
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Nfkbia protein, mouse
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Oxidants
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Proto-Oncogene Proteins
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Reactive Oxygen Species
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Receptors, Cell Surface
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Toll-Like Receptor 4
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Toll-Like Receptors
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NF-KappaB Inhibitor alpha
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Protein Kinases
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Phosphotransferases (Alcohol Group Acceptor)
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Interleukin-1 Receptor-Associated Kinases
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Irak4 protein, mouse
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Chuk protein, mouse
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I-kappa B Kinase
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Ikbkb protein, mouse
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Ikbke protein, mouse
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases
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alpha-Tocopherol
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Acetylcysteine