title: Superoxide potentiates NF-kappaB activation and modulates endotoxin-induced cytokine production in alveolar macrophages.
abstract: Gram-negative bacterial infection predisposes to the development of shock and acute lung injury with multiple organ dysfunction in the critically ill. Although overexpression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta, IL-6, IL-8, and other mediators is causally implicated in the pathogenesis of shock and lung injury, the underlying mechanisms following cellular exposure to gram-negative endotoxin remain unclear. De novo generation of reactive oxygen species (ROS) by monocytes/macrophages in particular has been proposed as a pivotal regulatory mechanism by which enhanced transactivation of redox-sensitive genes culminates in augmented cytokine expression within the lower respiratory tract. Here we sought to characterize the mechanism of action of a synthetic, nonpeptide, low-molecular-weight, Mn-containing superoxide dismutase mimetic (SODm), M40403, in modulating E. coli lipopolysaccharide serotype 0111:B4 (LPS)-induced cytokine production by cultured rat alveolar macrophages. Intracellular superoxide (O2) ion generation was measured using hydroethidine (HE) dye, and the dose-dependent effects of M40403 on TNF-alpha and IL-6 biosynthesis by ELISAs. Upstream redox-sensitive signaling events involving the pleiotropic transcription factor NF-kappaB were determined in nuclear extracts by electrophoretic mobility shift assays (EMSAs) and p65 subunit Western blot. The levels of the cytosolic inhibitory protein IkappaB-alpha were also assessed by Western analysis. We found that M40403 potently suppressed the production of superoxide, TNF-alpha, and IL-6 in LPS-stimulated alveolar macrophages, suggesting a key role for superoxide in endotoxin-induced cytokine production in the distal air spaces. In addition, M40403 decreased E. coli LPS-induced activation of NF-kappaB, and this effect was associated with modest suppression of cytoplasmic IkappaB-alpha degradation. Together, these results suggest that removal of superoxide by M40403 inhibits endotoxin-induced production of TNF-alpha and IL-6 in alveolar macrophages by a mechanism involving suppression of redox-sensitive NF-kappaB transactivation or signaling.
authors.affiliation: Metaphore Pharmaceutical, Inc., 1910 Inner Belt Business Center Drive, St. Louis, MO 63114, USA.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Ndengele
authors.forename: Carolina
authors.initials: C
authors.lastname: Muscoli
authors.forename: Zhi Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Doyle
authors.forename: George M
authors.initials: GM
authors.lastname: Matuschak
authors.forename: Daniela
authors.initials: D
authors.lastname: Salvemini
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004731:Endotoxins / Q000378:metabolism*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D015151:Immunoblotting; D007249:Inflammation; D007375:Interleukin-1 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009942:Organometallic Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010617:Phenanthridines / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D016207:Cytokines; D004731:Endotoxins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D009942:Organometallic Compounds; D010617:Phenanthridines; D017382:Reactive Oxygen Species; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D000072000:NF-KappaB Inhibitor alpha; C121876:imisopasem manganese; C058428:hydroethidine; D008345:Manganese; D013482:Superoxide Dismutase; D010100:Oxygen