The proinflammatory actions of mast cell tryptase on human endothelial cells
The proinflammatory actions of mast cell tryptase on human endothelial cells
Investigations into cytokine release revealed a selective increase of IL-8 in the supernatants from tryptase treated endothelial cells. Maximal release of IL-8 occurred at 50 mU/ml tryptase. Time course studies revealed maximum IL-8 release at 24 hr which was inhibited by the protease inhibitors leupeptin and benzamidine. Heat inactivation and depletion of tryptase from samples using an anti-tryptase monoclonal antibody also reversed the observed increases in IL-8 release. RT-PCR established that tryptase was eliciting an increase in levels of mRNA for IL-8 suggesting de novo synthesis of this cytokine. Further investigations revealed tryptase had no effect on expression of mRNA for GM-CSF, IL-6, IL-15 and RANTES, but could upregulate mRNA for IL-1β. However, IL-1β was not detectable in the lysates or supernatants of endothelial cells treated with tryptase. Flow cytometry experiments indicated that tryptase had no effect on ICAM-1, VCAM-1 and E-selection expression over the range of concentrations and times studied.
The role of tryptase in altering endothelial cell monolayer permeability and the release of neutrophil chemotactic activity from endothelial cells was also investigated. Tryptase added over a range of concentrations to endothelial cell monolayers had no effect on the rate of flux of 125I-human serum albumin either in the presence or absence of heparin. However, addition of tryptase did stimulate a dose dependent increase in neutrophil chemotactic activity in endothelial cell supernatants as assessed by the transmigration of 51Cr labelled neutrophils through Transwell filters. Maximal neturophil transmigration was induced with 10 mU/ml tryptase following a 24 hr incubation. This chemotactic activity was derived from the endothelial cells as tryptase alone was not chemotactic for neturophils. The release of chemotactic activity from endothelial cells was inhibited by preincubation with the protease inhibitor, leupeptin, suggesting a dependency on an active catalytic site.
In conclusion, tryptase may through its actions on endothelial cells facilitate the recruitment of leukocytes to sites of mast cell activation, and thus contribute in an important way to the genesis of inflammatory responses.
University of Southampton
1998
Compton, Steven John
(1998)
The proinflammatory actions of mast cell tryptase on human endothelial cells.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Investigations into cytokine release revealed a selective increase of IL-8 in the supernatants from tryptase treated endothelial cells. Maximal release of IL-8 occurred at 50 mU/ml tryptase. Time course studies revealed maximum IL-8 release at 24 hr which was inhibited by the protease inhibitors leupeptin and benzamidine. Heat inactivation and depletion of tryptase from samples using an anti-tryptase monoclonal antibody also reversed the observed increases in IL-8 release. RT-PCR established that tryptase was eliciting an increase in levels of mRNA for IL-8 suggesting de novo synthesis of this cytokine. Further investigations revealed tryptase had no effect on expression of mRNA for GM-CSF, IL-6, IL-15 and RANTES, but could upregulate mRNA for IL-1β. However, IL-1β was not detectable in the lysates or supernatants of endothelial cells treated with tryptase. Flow cytometry experiments indicated that tryptase had no effect on ICAM-1, VCAM-1 and E-selection expression over the range of concentrations and times studied.
The role of tryptase in altering endothelial cell monolayer permeability and the release of neutrophil chemotactic activity from endothelial cells was also investigated. Tryptase added over a range of concentrations to endothelial cell monolayers had no effect on the rate of flux of 125I-human serum albumin either in the presence or absence of heparin. However, addition of tryptase did stimulate a dose dependent increase in neutrophil chemotactic activity in endothelial cell supernatants as assessed by the transmigration of 51Cr labelled neutrophils through Transwell filters. Maximal neturophil transmigration was induced with 10 mU/ml tryptase following a 24 hr incubation. This chemotactic activity was derived from the endothelial cells as tryptase alone was not chemotactic for neturophils. The release of chemotactic activity from endothelial cells was inhibited by preincubation with the protease inhibitor, leupeptin, suggesting a dependency on an active catalytic site.
In conclusion, tryptase may through its actions on endothelial cells facilitate the recruitment of leukocytes to sites of mast cell activation, and thus contribute in an important way to the genesis of inflammatory responses.
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Published date: 1998
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Local EPrints ID: 463273
URI: http://eprints.soton.ac.uk/id/eprint/463273
PURE UUID: 40909a58-1679-4baa-8bd9-c1e0fd2e4bdc
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Date deposited: 04 Jul 2022 20:48
Last modified: 04 Jul 2022 20:48
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Author:
Steven John Compton
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