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Mechanisms involved in the induction of human endothelial cell necrosis
J H Wang1, H P Redmond, R W Watson
1Department of Surgery, The Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin.
Abstract:
The effects of the inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF) and unstimulated and activated neutrophils (PMNs) on endothelial cell (EC) necrosis were studied using the cultured human EC line (ECV-304) and human PMNs in vitro. LPS and TNF alone or their combination failed to induce EC necrosis. Activated PMNs, as evidenced by augmentations in CD11b expression and respiratory burst, induced significant EC necrosis commencing at 12 hr of coculture, which was strongly dependent on the ratio of PMN:ECs and the duration of PMN:EC coculture. In contrast, unstimulated PMNs induced no significant increases in EC necrosis. To examine the mechanisms of activated PMN-mediated EC necrosis, the oxygen radical scavengers superoxide dismutase (SOD) and catalase, as well as the protease inhibitors phenylmethylsulfonyl fluoride (PMSF), alpha 1-antitrypsin (alpha 1-AT), soybean trypsin-chymotrypsin inhibitor (TCI), and aprotinin, were studied in coculture experiments. EC necrosis induced by activated PMNs could be markedly attenuated by SOD, PMSF, alpha 1-AT, TCI, aprotinin, or their combinations. Although aprotinin enhanced respiratory burst, this agent inhibited necrosis by downregulating PMN CD11b and PMN-EC adhesion. These results demonstrate that the inflammatory mediators LPS and TNF and quiescent PMNs fail to induce EC necrosis. However, PMNs activated by inflammatory mediators can induce EC necrosis through oxidative and nonoxidative mechanisms and this process is dependent on PMN-EC adhesion.
Insights
Activated neutrophils, but not inflammatory mediators alone, cause endothelial cell necrosis. This cell death involves oxidative and nonoxidative mechanisms and depends on neutrophil adhesion, highlighting a key aspect of inflammatory responses.
Area of Science:
- Immunology
- Cell Biology
- Vascular Biology
Background:
- Endothelial cells (ECs) are crucial for vascular integrity.
- Inflammatory mediators like lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF) can activate immune cells.
- Neutrophils (PMNs) are key players in inflammatory responses and can damage tissues.
Purpose of the Study:
- To investigate the direct effects of inflammatory mediators and neutrophils on endothelial cell necrosis.
- To elucidate the mechanisms by which activated neutrophils induce endothelial cell death.
- To determine the role of oxidative and nonoxidative pathways in this process.
Main Methods:
- Utilized cultured human ECV-304 cells and human PMNs in vitro.
- Assessed endothelial cell necrosis following co-culture with LPS, TNF, and/or PMNs.
- Evaluated the impact of neutrophil activation markers (CD11b, respiratory burst) and PMN:EC ratios.
- Tested the efficacy of oxygen radical scavengers and protease inhibitors in preventing necrosis.
Main Results:
- LPS and TNF alone did not induce EC necrosis.
- Activated PMNs, but not unstimulated PMNs, induced significant EC necrosis.
- Necrosis was dependent on PMN:EC ratio and co-culture duration.
- Superoxide dismutase, catalase, and protease inhibitors (PMSF, alpha 1-AT, TCI, aprotinin) attenuated PMN-mediated EC necrosis.
- Aprotinin inhibited necrosis by downregulating PMN CD11b and PMN-EC adhesion.
Conclusions:
- Inflammatory mediators LPS and TNF do not directly cause EC necrosis.
- Activated PMNs are potent inducers of EC necrosis via both oxidative and nonoxidative mechanisms.
- PMN-EC adhesion is critical for the induction of EC necrosis by activated PMNs.