SEQUENTIAL INSULT ENHANCES LIVER MACROPHAGE-SIGNALED HEPATOCYTE DYSFUNCTION

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 mu g/ml) and hepatocyte dysfunction was assessed by determining secretory protein synthesis ([S-35]methionine labeling, trichloracetic acid precipitation, and SDS-PAGE) and energy metabolism [mitochondrial respiration using 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny 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 ([S-35]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, Coculture albumin synthesis, determined by immunoprecipitation, is significantly inhibited by sequential IL-6 followed by LPS compared to controls (P < 0.05) while synthesis of 23-, 38-, and 60-kDa proteins are maintained. Hepatocyte mitochondrial respiration is unaltered by IL-6, LPS, or sequential IL-6/LPS in the absence of liver macrophages; however, it is reduced significantly in sequential IL-6/LPS-stimulated cocultures (MTT OD: control, 0.461 +/- 0.2; LPS, 0.412 +/- 0.22; IL-6/LPS, 0.255 +/- 0.079; P < 0.05 vs control and IL-6 alone). These results indicate that liver macrophage-hepatocyte communication is altered following sequential IL-6/LPS compared to a single insult. Furthermore, they suggest that IL-6-induced hepatocyte products may enhance liver macrophage-signaled hepatocyte mitochondrial and albumin synthetic dysfunction following a second, infectious insult. (C) l994 Academic Press, Inc.