Brief hypoxic stress downregulates E. coli-induced IL-1 alpha and IL-1 beta gene expression in perfused liver

Abstract

Hepatic cytokine gene expression is independently stimulated by circulating microbial products and reductions in the cellular O2 supply. Although these stimuli occur sequentially after gram-negative bacteremia, it is unknown whether their interplay augments production of interleukin (IL)-1 by the liver. We studied the effects of intraportal Escherichia coli (EC) bacteremia and secondary constant-flow hypoxia (Po2, approximately 46 Torr for 30 min) on IL-1 alpha and IL-1 beta gene expression in ex situ buffer-perfused rat livers over 180 min (n = 67). At t = 0, normoxic EC and normal saline (NS) controls received 10(9) live EC serotype 055:B5 and 0.9% NaCl, respectively; in livers subjected to EC+hypoxia-reoxygenation (H/R) and NS+H/R, hypoxia began 0.5 h after EC or NS and was followed by 120 min of reoxygenation. Portal and hepatic venous perfusates were serially analyzed for bacterial colony-forming units, O2 uptake, and aspartate aminotransferase. At 60 min (peak hypoxia) and 180 min, cDNAs for IL-1 alpha and IL-1 beta were hybridized to whole liver RNA, and IL-1 beta protein levels in venous perfusates were assessed. Intrahepatic levels of reduced glutathione (GSH) were measured as an index of oxidative stress. Compared with normoxic EC, IL-1 alpha transcripts decreased at 180 min in EC+H/R livers (P < 0.0001) as did IL-1 beta mRNA (P < 0.05), despite similar EC clearance, GSH levels, posthypoxic O2 uptake, and aspartate aminotransferase release. Hepatic secretion of IL-1 beta likewise fell in EC+H/R vs. EC controls (P < 0.005). Prostaglandin H synthase-2 (COX-2) message accumulation was not enhanced by H/R, and indomethacin did not reverse H/R-mediated suppression of IL-1 production. In contrast, H/R-related falls in EC-induced IL-1S expression were reversed by allopurinol or catalase. Thus brief hypoxic stress of the liver causing neither GSH depletion nor functional impairment downregulates postbacteremic IL-1 expression by a mechanism involving O2 radicals but not cyclooxygenase metabolites.

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

“…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.…”

“…Supplemental NaHCO3 was added as needed to maintain arterial pH at 7.36 -7.44. Ex situ perfusions were performed in the recirculating mode in a temperature-controlled apparatus (37.0 Ϯ 0.5°C) at a flow rate of 3.75 ml ⅐ min Ϫ1 ⅐ g liver Ϫ1 (19,22), which was maintained for the remainder of experiments. Experimental protocol.…”

“…Sodium taurocholate (50 M) was given every 30 min to replace bile acids. The hepatic V O2 (in mol ⅐ min Ϫ1 ⅐ g Ϫ1 ) was calculated as previously described (22). The number of circulating EC (in cfu/ml) in hepatic venous perfusates was determined at each time point by duplicate nutrient agar streak plates.…”