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Human neutrophil elastase increases permeability of cultured pulmonary endothelial cell monolayers
1Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany.
Abstract:
Polymorphonuclear leukocytes (PMN) contribute to increased pulmonary vascular permeability in inflammatory lung injury, but the mechanism of their action is complex. In the present study we examined possible effects of PMN-derived proteases on the permeability of pulmonary endothelial cell monolayers grown on polycarbonate filter membranes and exposed continuously to a hydrostatic pressure of 10 cm H2O. Cell- and serum free PMN-supernatants (human PMN, stimulated with 30 ng/ml phorbol-myristate acetate for 30 min, presence of catalase, were centrifuged, the supernatants were passed through a 0.45 micron filter) dose-dependently (calculated PMN: endothelial cell ratio of 2:1 and more) increased hydraulic conductivity of endothelial cell monolayers ten- to twentyfold within 20-70 min. At the same time the dextran reflection coefficient decreased from 0.8 to 0.1. Phase contrast and scanning electronmicroscopy showed a widening of intercellular gaps. The effects of the postsecretory PMN-supernatant were blocked dose-dependently by inhibitors of human neutrophil elastase (HNE) but not of cathepsin G. On quantitative grounds highly purified HNE was similarly active as postsecretory PMN supernatant. The effects of HNE were inhibited by pretreatment with eglin-c or heat, but not with heparin. The data suggest that HNE is an effective and sufficient neutrophil-derived mediator to increase endothelial permeability. HNE appears to act primarily enzymatically and not as a cationic protein.
Insights
Human neutrophil elastase (HNE) significantly increases pulmonary endothelial cell permeability by widening intercellular gaps. This protease acts enzymatically, making it a key mediator in inflammatory lung injury.
Area of Science:
- Pulmonary medicine
- Cell biology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMN) are implicated in inflammatory lung injury.
- The precise mechanisms by which PMNs increase pulmonary vascular permeability are complex.
- Understanding PMN-derived mediators is crucial for addressing lung injury.
Purpose of the Study:
- To investigate the role of PMN-derived proteases in regulating pulmonary endothelial cell permeability.
- To identify specific proteases responsible for increased vascular permeability.
- To elucidate the mechanism of action of these proteases.
Main Methods:
- Cultured pulmonary endothelial cell monolayers on filter membranes under hydrostatic pressure.
- Application of cell- and serum-free PMN supernatants.
- Measurement of hydraulic conductivity and dextran reflection coefficient.
- Phase contrast and scanning electron microscopy.
- Inhibition studies using specific protease inhibitors (HNE, cathepsin G) and other agents.
Main Results:
- PMN supernatants dose-dependently increased endothelial hydraulic conductivity tenfold to twentyfold.
- Dextran reflection coefficient significantly decreased, indicating increased permeability.
- Intercellular gaps widened, visualized by microscopy.
- Inhibitors of human neutrophil elastase (HNE) blocked the effects, while cathepsin G inhibitors did not.
- Purified HNE mimicked the effects of PMN supernatants.
- HNE's effects were inhibited by eglin-c and heat, suggesting enzymatic activity.
Conclusions:
- Human neutrophil elastase (HNE) is an effective and sufficient mediator released by neutrophils to increase pulmonary endothelial permeability.
- HNE primarily acts through its enzymatic function rather than as a cationic protein.
- These findings highlight HNE as a critical target for therapeutic interventions in inflammatory lung injury.