Hypoxic suppression ofE. coli-induced NF-κB and AP-1 transactivation by oxyradical signaling

Abstract

/ajpregu. 00404.2003.-Transactivation of the DNA-binding proteins nuclear factor-B (NF-B) and activator protein (AP)-1 by de novo oxyradical generation is a stereotypic redox-sensitive process during hypoxic stress of the liver. Systemic trauma is associated with splanchnic hypoxia-reoxygenation (H/R) followed by intraportal gram-negative bacteremia, which collectively have been implicated in posttraumatic liver dysfunction and multiple organ damage. We hypothesized that hypoxic stress of the liver before stimulation by Escherichia coli serotype O55:B5 (EC) amplifies oxyradical-mediated transactivation of NF-B and AP-1 as well as cytokine production compared with noninfectious H/R or gram-negative sepsis without prior hypoxia. Livers from Sprague-Dawley rats underwent perfusion for 180 min with or without 0.5 h of hypoxia (perfusate PO 2, 40 Ϯ 5 mmHg) followed by reoxygenation and infection with 10 9 EC or 0.9% NaCl infusion. In H/R ϩ EC livers, nuclear translocation of NF-B and AP-1 was unexpectedly reduced in gel shift assays vs. normoxic EC controls, as were perfusate TNF-␣ and IL-1␤ levels. Preceding hypoxic stress paradoxically increased postbacteremic reduced-to-oxidized glutathione ratios plus nuclear localization of IB␣ and phospho-IB␣, but not JunB/FosB profiles. Notably, xanthine oxidase inhibition increased transactivation as well as cytokine production in H/R ϩ EC livers. Thus brief hypoxic stress of the liver before intraportal gram-negative bacteremia potently suppresses activation of canonical redox-sensitive transcription factors and production of inflammatory cytokines by mechanisms including xanthine oxidaseinduced oxyradicals functioning in an anti-inflammatory signaling role. These results suggest a novel multifunctionality of oxyradicals in decoupling hepatic transcriptional activity and cytokine biosynthesis early in the posttraumatic milieu.

Acute hypoxia decreases E. coli LPS-induced cytokine production and NF-κB activation in alveolar macrophages

“…We have extended that work here to confirm that the boundary conditions of superimposed oxygen limitation of LPS-stimulated alveolar cells here (atmospheric PO2 = 10 mm Hg and a liquid medium PO2 near that of mixed venous blood = 35 mm Hg) increased neither NF-κB or AP-1 activation (Figures 4 and 5), PGE 2 production, nor de novo generation of superoxide anion (Figure 6A). These results also agree with our finding in a different model of brief post-endotoxic oxygen limitation at the whole organ level using perfused rat livers, in which post-bacteremic H/R after intraportal infection with E. coli serotype O55:B5 led to downregulated TNF-α, IL-1α, and IL-1β expression without altering intrahepatic GSH redox balance (Loftis et al, 2000; Matuschak et al, 2004). Our finding here of suppressed LPS-induced TNF-α production in alveolar macrophages at the protein and mRNA levels by brief, well-tolerated hypoxic exposure is especially supported by a correlative in vivo rat model of acute brief hypoxia following co-stimulation by hematogenous infection with 5 × 10 9 live E. coli serotype O55:B5 (Matuschak et al, 2005).…”

“…Molecular biology-grade reagents (Sigma) were used for nuclear isolation, electrophoretic mobility shift assay (EMSA), and supershift assay procedures (Matuschak et al, 2004; Ndengele et al, 2005). The 32 P labeled radiolabeled nucleotides were from Promega (Madison, WI).…”

“…Cellular [GSH], a sensitive indicator of induced oxidative stress, was measured in cell lysates over the 6 h from onset of hypoxia through 4.5h of reoxygenation ( t = 6 h) using a modified Tietze assay as previously described (Ndengele et al, 2000; Matuschak et al, 2004; Loftis et al, 2000). Results are expressed as μmol GSH/mg cellular protein.…”

“…Samples of nuclear protein (10 µg) were incubated as previously described (Loftis et al, 2000; Matuschak et al, 2004) in binding buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 5 mM MgCl 2 , 25 mM NaCl, 5% glycerol, 5% sucrose, and 0.01% Nonidet P-40) for 10 min at 37°C. Poly(dIdC; 3 μg; Pharmacia, Piscataway, NJ) was added to samples before adding oligonucleotide probe.…”

Oxygenation Inhibits the Physiological Tissue-Protecting Mechanism and Thereby Exacerbates Acute Inflammatory Lung Injury

“…Hypoxia was also shown to strongly inhibit production of MCP-1 [32], IL-1β [33], granulocyte-macrophage colony-stimulating factor [34], and induced down-regulation of cosignaling molecules (CD80) [35]. Hypoxia was further shown to cause decreased expression of Toll-like receptor 4 receptors by inhibiting translocation of activator protein 1 [36] and caused suppression of Escherichia coli-induced nuclear factor κB and activator protein 1 transactivation [37]. Finally, hypoxia was shown to stimulate phosphatidylinositol 3-kinase activity and thereby protect human lung microvascular endothelial cells and epithelial type II-like A549 cells from subsequent oxygen toxicity [38].…”