Pancreatitis-Associated Ascitic Fluid Induces Hepatocyte Death Independent of Local Cytokines

Abstract

Introduction. Kupffer-cell-derived cytokines mediate liver injury, yet macrophage pacification does not abolish hepatocyte injury. We undertook this study to examine the role of pancreatitis-associated ascitic fluid (PAAF) in liver injury. Methods. Pathogen-free PAAF was perfused into healthy rat livers in situ for 60 min ( n = 5, sham = 5, LPS = 5). AST, ALT, LDH, and TNF were measured in the effluent. Primary cultures of rat Kupffer cells or hepatocytes were treated with PAAF; AST, ALT, LDH, and TNF were measured and cell proliferation was determined by MTT assay. A hepatocyte human cell line (CCL-13) was treated with PAAF and apoptosis was measured by flow cytometry. Results. Liver perfusion with PAAF induced a >15-fold increase in AST/ALT/LDH ( P < 0.001 PAAF vs sham), but not in TNF. In vitro, Kupffer cell viability was sharply reduced by PAAF in a dose-dependent manner; however, 5% PAAF (50% viability) did not induce TNF production from Kupffer cells. PAAF induced a multifold increase in AST/ALT/LDH from fresh hepatocytes ( P < 0.001 vs control), which was not attenuated by a protease inhibitor. The CCL-13 cell population was reduced to 15 ± 2% of baseline by PAAF ( P < 0.001 vs control), whereas elastase, trypsin, or TNF had no effect. PAAF increased the percentage of nonviable CCL-13 cells (78 ± 4% vs 28 ± 1%, P < 0.001 vs control). Neither protease inhibitor nor heat inactivation of PAAF altered this pattern of hepatocyte death. Conclusion. PAAF induces direct hepatocyte injury and death by heat-stable factors other than pancreatic enzymes but not via local production of Kupffer-cell-derived cytokines.