title: Sequential insult enhances liver macrophage-signaled hepatocyte dysfunction.
abstract: Injury results in altered hepatocyte protein synthesis including the production of acute-phase reactants. Evidence suggests that these hepatocyte products regulate macrophage function; however, their role in liver macrophage-mediated hepatocyte dysfunction following a second insult is poorly characterized. We hypothesize that IL-6-stimulated hepatocyte products alter liver macrophage responses to lipopolysaccharide, contributing to enhanced hepatocyte dysfunction. To test this hypothesis, hepatocytes, obtained by liver collagenase digestion, were treated with rIL-6 (murine, 300 units/ml) for 24 hr, and then liver macrophages, obtained by perfusion and pronase digestion, were added to establish cocultures. Cocultures were then stimulated with endotoxin (LPS, Escherichia coli O111:B4, 10 micrograms/ml) and hepatocyte dysfunction was assessed by determining secretory protein synthesis ([35S]methionine labeling, trichloracetic acid precipitation, and SDS-PAGE) and energy metabolism [mitochondrial respiration using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye]. Cultures of hepatocytes alone stimulated with IL-6, LPS, or sequential IL-6 followed by LPS demonstrate no difference in total secretory protein synthesis or mitochondrial respiration. In contrast, hepatocyte-liver macrophage cocultures demonstrate significantly reduced total secretory protein synthesis following sequential IL-6 followed by LPS ([35S]methionine cpm x 10(3): control, 33.8 +/- 8.5; LPS, 25.8 +/- 6.3; IL-6/LPS, 15.7 +/- 6.4; P < 0.05 vs control). This effect is specific to IL-6 since sequential TNF-alpha followed by LPS did not result in significant suppression of secretory protein synthesis. One-dimensional SDS-PAGE of labeled coculture secretory proteins demonstrates qualitative changes following sequential insult in vitro compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Surgery, University of Texas, Southwestern Medical Center, Dallas 75235-9031.
authors.forename: P E
authors.initials: PE
authors.lastname: Bankey
authors.forename: S
authors.initials: S
authors.lastname: Hill
authors.forename: D
authors.initials: D
authors.lastname: Geldon
mesh_terms: D000818:Animals; D002450:Cell Communication; D002478:Cells, Cultured; D015850:Interleukin-6 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000008:administration & dosage* / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000473:pathology / Q000503:physiopathology*; D008264:Macrophages / Q000502:physiology*; D008930:Mitochondria, Liver / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D014176:Protein Biosynthesis; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D015850:Interleukin-6; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha