SEQUENTIAL INSULT ENHANCES LIVER MACROPHAGE-SIGNALED HEPATOCYTE DYSFUNCTION

Injury results in altered hepatocyte protein synthesis including the p roduction of acute-phase reactants. Evidence suggests that these hepat ocyte products regulate macrophage function; however, their role in li ver macrophage-mediated hepatocyte dysfunction following a second insu lt is poorly characterized. We hypothesize that IL-6-stimulated hepato cyte products alter liver macrophage responses to lipopolysaccharide, contributing to enhanced hepatocyte dysfunction, To test this hypothes is, hepatocytes, obtained by liver collagenase digestion, were treated with rIL-6 (murine, 300 units/ml) for 24 hr, and then liver macrophag es, obtained by perfusion and pronase digestion, were added to establi sh cocultures. Cocultures were then stimulated with endotoxin (LPS, Es cherichia coli O111:B4, 10 mu g/ml) and hepatocyte dysfunction was ass essed by determining secretory protein synthesis ([S-35]methionine lab eling, trichloracetic acid precipitation, and SDS-PAGE) and energy met abolism [mitochondrial respiration using 3-(4,5-dimethylthiazol-2-yl)- 2,5-dipheny tetrazolium bromide (MTT) dye]. Cultures of hepatocytes al one stimulated with IL-6, LPS, or sequential IL-6 followed by LPS demo nstrate no difference in total secretory protein synthesis or mitochon drial respiration. In contrast, hepatocyte-liver macrophage cocultures demonstrate significantly reduced total secretory protein synthesis f ollowing 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 T NF-alpha followed by LPS did not result in significant suppression of secretory protein synthesis. One-dimensional SDS-PAGE of labeled cocul ture secretory proteins demonstrates qualitative changes following seq uential insult in vitro compared to controls, Coculture albumin synthe sis, 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 sign ificantly in sequential IL-6/LPS-stimulated cocultures (MTT OD: contro l, 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 mac rophage-hepatocyte communication is altered following sequential IL-6/ LPS compared to a single insult. Furthermore, they suggest that IL-6-i nduced hepatocyte products may enhance liver macrophage-signaled hepat ocyte mitochondrial and albumin synthetic dysfunction following a seco nd, infectious insult. (C) l994 Academic Press, Inc.