Mast cell tryptase as a stimulus for up-regulation of adhesion molecule expression and cytokine release from endothelial cells

Khedr, M.E.M.S., Abdelmotelb, A.M., Lau, L., Arno, M., Zhou, X. and Walls, A.F. (2012) Mast cell tryptase as a stimulus for up-regulation of adhesion molecule expression and cytokine release from endothelial cells. Journal of Allergy and Clinical Immunology, 129 (2, Supplment), AB122. (doi:10.1016/j.jaci.2011.12.586).

Abstract

Rationale
Mast cell tryptase can be released in substantial quantities at sites of allergic inflammation. Pro-inflammatory actions mediated through protease activated receptor 2 (PAR-2) have been proposed, but the precise contribution of tryptase to disease processes remains unclear. We have investigated alterations in the whole genome expression profile of endothelial cells in response to tryptase.

Methods
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical vein tissue and grown to confluence. Following addition of tryptase or other agents, quantitative polymerase chain reactions (qPCR) followed by whole genome microarray analysis were performed. Translation of certain genes was investigated by immunocytochemistry or specific ELISA. In parallel studies, calcium flux was measured using a fluorescence based microplate procedure.

Results
Microarray analysis indicated that the genes whose expression was most up-regulated following tryptase treatment of endothelial cells were those for the adhesion molecules ICAM-1, VCAM-1, EPCAM and ITGAL, and the cytokines IL-2, IL-3, IL-6 and CXCL10. Increased expression was seen also for genes for the cell signalling molecules TNFAIP3, TLL1 and SMAD2. None of these were up-regulated in response to a peptide agonist (SLIGKV-NH2) of PAR-2. Microarray data was confirmed by separate qPCR experiments, immunocytochemistry and by the measurement of cytokines in cell supernatants. The actions of tryptase were inhibited using selective protease inhibitors indicating a requirement for an intact catalytic site. Addition of tryptase to endothelial cells stimulated concentration-dependent increases in intracellular calcium.

Conclusions
The pro-inflammatory actions of tryptase may be mediated through increased expression of adhesion molecules and production of inflammatory cytokines in endothelial cells.

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Contributors

Author: M.E.M.S. Khedr

Author: A.F. Walls

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