DETACHMENT AND CYTOLYSIS OF HUMAN ENDOTHELIAL-CELLS BY PROTEINASE-3

Activation and degranulation of polymorphonuclear leukocytes (PMN) with release of proteolytic enzymes, such as proteinase 3 (PR3) and elastase, in the vessels of patients with Wegener's granulomatosis (WG) is thought to play an important role in the vascular endothelial cell damage. We have investigated the detachment and cytolysis of Cr-51-labeled umbilical vein endothelial cells (HUVEC) induced by highly purified, enzymatically active, PR3 and elastase. Incubation of confluent monolayers of HUVEC with 100 mU/ml of PR3 for 3 h at 37 degrees C generally resulted in 20 % detachment and 30 % cytolysis. Elastase (350 mU/ml) induced approximately 40 % detachment and 15 % cytolysis. Both PR3-mediated and elastase-mediated detachment and cytolysis were fully inhibited by alpha-1-proteinase inhibitor (alpha(1)PI), while anti-leukoprotease ((ALP) only inhibited elastase-mediated endothelial damage. By selective inhibition of an azurophilic granule extract with either alpha(1)PI or ALP we calculated that PR3 is responsible for 23 % of the total detachment and cytolysis induced by the extract. Elastase was responsible for 60 % of the detachment and 19 % of the cytolysis. Detachment induced by PR3 was inhibited by three out of five IgG preparations purified from c-ANCA-positive sera of WG patients. PR3-mediated cytolysis was inhibited by each of the c-ANCA+ IgG preparations and also to a limited extent by control IgG, suggesting a partial nonspecific stabilization of the endothelial cells. These studies provide evidence that besides elastase, PR3 also plays an important role in the PMN-mediated endothelial cell damage.