The mechanism of hepatic cellular injury in sepsis: an in vitro study of the implications of cytokines and neutrophils in its pathogenesis.

Neutrophil-mediated injury to hepatocytes was evaluated in vitro. A new in vitro coculture system of neutrophils and a human hepatoblastoma cell line (HuH-6), instead of normal hepatocytes, was established. Recombinant human tumor necrosis factor (TNF) activated neutrophils to release neutrophil elastase and showed the significant cytotoxicity for HuH-6 cells, which was determined by measuring the release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), from HuH-6 cells. The concentration of neutrophil elastase from zymosan-primed/TNF (1.0 ng/ml)-stimulated neutrophils cocultured with HuH-6 cells reached to the level of 1.59 +/- 0.18 micrograms/10(6) cells in 24 hr. The release of LDH from HuH-6 cells in this coculture system was 84.8 +/- 17.8 units/liter after 24 hr incubation. Purified human neutrophil elastase also increased LDH release from HuH-6 cells. When HuH-6 cells were cocultured with zymosan-primed/TNF (1.0 ng/ml)-stimulated neutrophils, the secretion of the negative acute phase reactant (APR), alpha-fetoprotein, from HuH-6 cells was significantly decreased, and the production of the positive APR, pancreatic secretory trypsin inhibitor, was decreased in response to the stimulation of interleukin 6. Urinary trypsin inhibitor, the inhibitor of neutrophil elastase, decreased the release of LDH from HuH-6 cells cocultured with stimulated neutrophils, while superoxide scavenger did not. These results show that human neutrophils activated by TNF injure hepatocytes, thus causing hepatic dysfunction, through the release of neutrophil elastase.