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Activated neutrophils from rat injured isolated hepatocytes
P E Ganey1, M B Bailie, S VanCise
1Department of Pharmacology and Toxicology, Michigan State University, East Lansing.
Background:
Activated neutrophils (PMNs) release cytotoxic agents that can damage surrounding tissue. These studies were performed to determine whether activated PMNs from rat could injure isolated, rat hepatic parenchymal cells (HCs) in vitro.
Experimental Design:
HCs were cocultured with unstimulated rat PMNs or with PMNs activated with either f-met-leu-phe (FMLP) or phorbol myristate acetate (PMA), that stimulate predominantly degranulation or superoxide production, respectively. Toxicity to HCs was evaluated from release of alanine aminotransferase into the medium.
Results:
Alanine aminotransferase release was greater in HCs cocultured with FMLP- or PMA-stimulated PMNs compared with unstimulated PMNs. Toxicity was observed by 16 hours after stimulation of PMNs. To test the possible involvement of a soluble mediator released by activated PMNs, HCs were incubated with conditioned medium from PMNs. Compared with unstimulated PMNs, toxicity to HCs was greater in the presence of conditioned medium from FMLP-stimulated PMNs, but not conditioned medium from PMA-activated PMNs. Reactive oxygen species do not appear to be involved in the mechanism by which activated PMNs damage HCs since superoxide dismutase, catalase, superoxide dismutase+catalase, or desferrioxamine failed to prevent the injury. Furthermore, less superoxide anion was detected in PMA-stimulated PMNs when either HCs or HC-conditioned medium was present. Proteolytic enzymes released by stimulated PMNs may play a role in HC damage since an inhibitor of proteases diminished injury due to PMNs activated by either FMLP or PMA.
Conclusions:
These results indicate that activated, rat PMNs damage HCs in culture. The data suggest that reactive oxygen species are not involved in the mechanism, but that release of proteolytic enzymes may play a role in the toxic response.
Insights
Activated neutrophils (PMNs) can damage rat liver cells (HCs) in vitro. Proteolytic enzymes, not reactive oxygen species, appear to mediate this liver injury.
Area of Science:
- Immunology
- Hepatology
- Cellular Toxicology
Background:
- Activated neutrophils (PMNs) release cytotoxic agents, potentially damaging surrounding tissues.
- Investigating the in vitro cytotoxic effects of activated rat PMNs on isolated rat hepatic parenchymal cells (HCs).
Purpose of the Study:
- To determine if activated rat neutrophils (PMNs) can injure isolated rat hepatic parenchymal cells (HCs) in vitro.
- To elucidate the mechanisms involved in PMN-induced hepatic cell damage.
Main Methods:
- Co-culturing hepatic cells (HCs) with unstimulated or activated rat neutrophils (PMNs) stimulated by f-met-leu-phe (FMLP) or phorbol myristate acetate (PMA).
- Evaluating toxicity via alanine aminotransferase release.
- Assessing the role of soluble mediators and reactive oxygen species (ROS) using conditioned media and scavengers.
- Investigating the involvement of proteolytic enzymes using protease inhibitors.
Main Results:
- Activated PMNs (FMLP or PMA stimulated) caused greater alanine aminotransferase release from HCs compared to unstimulated PMNs.
- Toxicity was evident by 16 hours post-PMN stimulation.
- Proteolytic enzyme release, not reactive oxygen species, was implicated in PMN-induced HC damage, as protease inhibitors reduced injury.
Conclusions:
- Activated rat neutrophils demonstrably damage rat hepatic cells in culture.
- The mechanism of injury does not appear to involve reactive oxygen species.
- Proteolytic enzymes released by activated PMNs likely play a significant role in the observed hepatic cell toxicity.