Toll-like receptor 4-mediated nuclear factor kappa B activation is essential for sensing exogenous oxidants to propagate and maintain oxidative/nitrosative cellular stress.
The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments.
Division of Pharmacology and Toxicology, University of Missouri-Kansas City, Missouri, United States of America.
Rajendra
R
Karki
Orisa J
OJ
Igwe
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D060846:Cell Engineering; D002470:Cell Survival / Q000187:drug effects; D002840:Chromates / Q000494:pharmacology*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D009942:Organometallic Compounds / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D010146:Pain / Q000503:physiopathology / Q000517:prevention & control; D017288:Pain Threshold; D010545:Peroxides / Q000494:pharmacology*; D026361:Reactive Nitrogen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D012459:Salicylates / Q000494:pharmacology; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000378:metabolism
D000975:Antioxidants; D002840:Chromates; C433928:EUK-134; D053583:Interleukin-1beta; D016328:NF-kappa B; D009942:Organometallic Compounds; D016877:Oxidants; D010545:Peroxides; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D012459:Salicylates; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; C019537:potassium tetraperoxochromate; D000469:Alkaline Phosphatase