Ballieux B E, Hiemstra P S, Klar-Mohamad N, Hagen E C, van Es L A, van der Woude F J, Daha M R
Department of Nephrology, University Hospital, Leiden, The Netherlands.
Eur J Immunol. 1994 Dec;24(12):3211-5. doi: 10.1002/eji.1830241245.
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 51Cr-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 1PI 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.
韦格纳肉芽肿病（WG）患者血管中多形核白细胞（PMN）的激活和脱颗粒以及蛋白水解酶如蛋白酶3（PR3）和弹性蛋白酶的释放，被认为在血管内皮细胞损伤中起重要作用。我们研究了高度纯化、具有酶活性的PR3和弹性蛋白酶诱导的51Cr标记脐静脉内皮细胞（HUVEC）的脱离和细胞溶解。将融合的HUVEC单层在37℃下与100 mU/ml的PR3孵育3小时，通常导致20%的脱离和30%的细胞溶解。弹性蛋白酶（350 mU/ml）诱导约40%的脱离和15%的细胞溶解。PR3介导和弹性蛋白酶介导的脱离和细胞溶解均被α1-蛋白酶抑制剂（α1PI）完全抑制，而抗白细胞蛋白酶（ALP）仅抑制弹性蛋白酶介导的内皮损伤。通过用α1PI或ALP选择性抑制嗜天青颗粒提取物，我们计算出PR3占提取物诱导的总脱离和细胞溶解的23%。弹性蛋白酶占脱离的60%和细胞溶解的19%。从WG患者的c-ANCA阳性血清中纯化的五种IgG制剂中有三种抑制了PR3诱导的脱离。每种c-ANCA+IgG制剂均抑制PR3介导的细胞溶解，对照IgG也有一定程度的抑制，提示对内皮细胞有部分非特异性稳定作用。这些研究提供了证据，表明除弹性蛋白酶外，PR3在PMN介导的内皮细胞损伤中也起重要作用。
