Brief hypoxic stress suppresses postbacteremic NF-κB activation and TNF-α bioactivity in perfused liver

Abstract

Reductions in hepatic O(2) delivery are common early after gram-negative bacteremic sepsis owing to cardiopulmonary dysfunction and derangements in sinusoidal perfusion. Although gram-negative endotoxin and cellular hypoxia independently enhance activation of nuclear factor-kappaB (NF-kappaB) via generation of reactive O(2) species (ROS), the combination of these stimuli downregulates hepatic TNF-alpha gene expression. Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF-alpha gene expression is transcriptionally mediated by reduced activation of NF-kappaB. Buffer-perfused rat livers (n = 52) were studied over 180 min after intraportal infection at t = 0 with 10(9) live Escherichia coli (EC), serotype O55:B5, or 0.9% NaCl controls under normoxic conditions, compared with 0.5 h of constant-flow hypoxia (PO(2) approximately 41 +/- 7 Torr) beginning at t = 30 min, followed by 120 min of reoxygenation. In parallel studies, tissue was obtained at peak hypoxia (t = 60 min). To determine the role of xanthine oxidase (XO)-induced ROS in modulating NF-kappaB activity after hypoxia/reoxygenation (H/R), livers were pretreated with the XO inhibitor allopurinol, with results confirmed in organs of tungstate-fed animals. Electrophoretic mobility shift assays were performed on nuclear extracts of whole liver lysates using (32)P-labeled oligonucleotides specific for NF-kappaB. Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-kappaB nuclear translocation and TNF-alpha bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O(2) consumption. XO inhibition reversed the hypoxic suppression of NF-kappaB nuclear translocation and ameliorated decreases in cell-associated TNF-alpha. Thus decreases in hepatic O(2) delivery reduce postbacteremic nuclear translocation of NF-kappaB and hepatic TNF-alpha biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.

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

“…Molecular biology-grade reagents (Sigma Chemical, St. Louis, MO) were used for nuclear isolation, electrophoretic mobility shift assay (EMSA), and supershift assay procedures (19). The transcription protein/DNA array for NF-B and AP-1 was obtained from Panomics (Redwood City, CA).…”

“…coli cultures. Strain 12014 of E. coli (EC) serotype O55:B5 (American Type Culture Collection, Bethesda, MD) was maintained in trypticase soy broth and grown up over 18 -24 h in fresh cultures (10,19,22). Organisms sedimented at 1,000 g for 10 min at 4°C were washed twice in sterile 0.9% NaCl (NS) and resuspended in NS to 1 ϫ 10 9 colony-forming units (cfu) in 1 ml.…”

“…Ex situ liver perfusion. Perfusates were freshly prepared in pyrogen-free glassware as previously described (10,19,22) by addition of 10 mM glucose, 5% (vol/vol) sterile rat serum, sodium taurocholate (50 M), 10 5 U of penicillin, and 100 mg of streptomycin to 1 liter of Krebs-Henseleit Ringer bicarbonate (297 Ϯ 2 mosM final osmolarity, pH 7.4). After equilibration with 95% O 2-5% CO2 gas, perfusate arterial PO 2 values were 550 -620 mmHg (IL-1306 blood gas analyzer, Instrumentation Laboratories, Lexington, MA).…”

“…After anesthesia was induced in rats with pentobarbital sodium (50 mg/kg ip), aseptic organ harvesting was performed (10,19,22). Livers were equilibrated for 30 min before the end of each experiment at time 0 to ensure steady-state conditions of hepatic O 2 consumption (V O2) and portal venous pressure.…”

“…We therefore performed these studies to test the hypothesis that brief and otherwise well-tolerated hypoxic stress of the liver, followed by reoxygenation and intraportal Escherichia coli bacteremia, amplifies transactivation of NF-B and AP-1 by a xanthine oxidase-derived oxyradical signaling mechanism (14,19). As a corollary, we postulated that hypoxia would modulate subsequent bacteremic transcriptional events and proportionately increase downstream cytokine production, as reflected by increases in TNF-␣ and IL-1␤ in hepatic venous effluent.…”