Berger S P, Seelen M A, Hiemstra P S, Gerritsma J S, Heemskerk E, van der Woude F J, Daha M R
Department of Nephrology, University Hospital Leiden, Leiden, The Netherlands.
J Am Soc Nephrol. 1996 May;7(5):694-701. doi: 10.1681/ASN.V75694.
Proteinase 3 is the major target antigen of antineutrophil cytoplasmic autoantibodies (ANCA) in Wegener's granulomatosis and is contained in the azurophilic granules of polymorphonuclear neutrophils, the dominant cell type in vascular lesions during the early stages of systemic vasculitis. This study questioned whether neutrophil lysosomal enzymes, once released at the site of inflammation, are able to potentiate the influx of additional neutrophils by enhancing the production of the chemotactic cytokine interleukin-8 (IL-8) by endothelial cells. Therefore, human umbilical vein endothelial cells in culture were incubated with varying concentrations of highly purified proteinase 3, human neutrophil elastase, and cathepsin G for different time periods. The supernatants were subsequently assessed for IL-8 antigen by using a sandwich ELISA. The presence of both proteinase 3 and elastase resulted in an increased production of IL-8, up to 15.6- and 4.2-fold, respectively, in a dose- and time-dependent fashion. Cathepsin G did not influence IL-8 production. Although the addition of an alpha 1-proteinase inhibitor completely abrogated elastase-mediated IL-8 production, it did not significantly influence the effect of proteinase 3. Both proteinase 3-and elastase-mediated production of IL-8 was inhibited by cycloheximide, indicating de novo synthesis. This was supported by the finding of increased IL-8 mRNA levels in proteinase 3-treated human umbilical vein endothelial cells by using Northern blot analysis. Taken together, the neutrophil lysosomal enzymes proteinase 3 and human neutrophil elastase may contribute to a self-perpetuating process of neutrophil recruitment in acute inflammation by increasing de novo synthesis of IL-8 by endothelial cells. The studies presented here also show that proteinase 3 mediates its effect independently of its enzymatic activity, indicating a hitherto unknown mode of action on endothelial cells.
蛋白酶3是韦格纳肉芽肿中抗中性粒细胞胞浆自身抗体（ANCA）的主要靶抗原，存在于多形核中性粒细胞的嗜天青颗粒中，多形核中性粒细胞是系统性血管炎早期血管病变中的主要细胞类型。本研究质疑中性粒细胞溶酶体酶一旦在炎症部位释放，是否能够通过增强内皮细胞趋化细胞因子白细胞介素-8（IL-8）的产生来促进更多中性粒细胞的流入。因此，将培养的人脐静脉内皮细胞与不同浓度的高度纯化的蛋白酶3、人中性粒细胞弹性蛋白酶和组织蛋白酶G孵育不同时间段。随后使用夹心ELISA法评估上清液中的IL-8抗原。蛋白酶3和弹性蛋白酶的存在均导致IL-8产生增加，分别以剂量和时间依赖性方式增加至15.6倍和4.2倍。组织蛋白酶G不影响IL-8的产生。虽然添加α1-蛋白酶抑制剂完全消除了弹性蛋白酶介导的IL-8产生，但它对蛋白酶3的作用没有显著影响。蛋白酶3和弹性蛋白酶介导的IL-8产生均被放线菌酮抑制，表明是从头合成。通过Northern印迹分析发现在蛋白酶3处理的人脐静脉内皮细胞中IL-8 mRNA水平增加，支持了这一点。综上所述，中性粒细胞溶酶体酶蛋白酶3和人中性粒细胞弹性蛋白酶可能通过增加内皮细胞IL-8的从头合成，在急性炎症中促成中性粒细胞募集的自我延续过程。此处呈现的研究还表明，蛋白酶3独立于其酶活性介导其作用，表明其对内皮细胞有一种迄今未知的作用方式。
