Neutrophil Proteinase 3 (PR3) Enhances Vascular Endothelial Cell Barrier Function

Abstract 2108

Proteinase 3 (PR3) is a 29 kDa serine protease that is released from neutrophils (PMN) upon cellular activation. Following its release, PR3 can rebind to the neutrophil expressed cell surface protein NB1 (CD177). NB1 has recently been demonstrated to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Therefore NB1/PECAM-1 interactions have the potential to localize PR3 to cell junctions, where it might play a role in facilitating neutrophil transendothelial migration, possibly by regulating vascular permeability.

To test the hypothesis that PR3 regulates endothelial cell barrier function, human umbilical vein endothelial cells (HUVEC) were cultured on transwell membranes, stimulated with IL-1β, and then incubated with NB1+ or NB1- PMN. Flow cytometric analysis revealed that transmigration alone resulted in a significant increase in PR3 expression on NB1+ but not NB1- PMN. Interestingly, NB1+/PR3+ PMNs promoted increased HUVEC barrier function compared to the NB1-/PR3- PMNs, as determined by electric cell substrate impedance sensing. Incubation with PR3 alone also resulted in an increase in HUVEC barrier function similar to a PAR2 peptide agonist (SLIGKV), but this was not observed in HUVEC incubated with the closely related neutrophil proteases HNE or cathepsin G. Both PR3 and SLIGKV dramatically inhibited RhoA phosphorylation, induced calcium signaling, and were able to significantly inhibit the ability of a PAR1 peptide agonist (SFLLR) to induce endothelial cell monolayer permeability.

These results demonstrate that the neutrophil expressed serine protease PR3 can play a role in enhancing endothelial cell vascular integrity and can antagonize vascular permeability induced by PAR1, possibly through regulation of RhoA activity.