Taekema-Roelvink M E, van Kooten C, Janssens M C, Heemskerk E, Daha M R
Department of Nephrology, University Hospital Leiden, The Netherlands.
Scand J Immunol. 1998 Jul;48(1):37-43. doi: 10.1046/j.1365-3083.1998.00348.x.
Wegener's granulomatosis is characterized by crescentic necrotizing glomerulonephritis and systemic vasculitis. Both proteinase 3 (PR3) and anti-neutrophil cytoplasmic antibodies (ANCA), directed against this enzyme, are thought to play a pathogenic role. PR3 has been shown to cause detachment and cytolysis of human umbilical vein endothelial cells (HUVEC) in vitro and to induce apoptosis of bovine pulmonary artery endothelial cells. In the present study we investigated the effect of PR3 and ANCA on the induction of apoptosis of human endothelial cells in vitro. HUVEC were cultured in the absence or presence of varying concentrations of PR3 for different time periods and apoptosis was assessed by three different methods. Staining of the cells with Hoechst 33258 and assessment of nuclear morphology by ultraviolet (UV) light microscopy revealed a dose-dependent induction of apoptosis, as determined by cell counts. A concentration of 8 microg/ml PR3 was found to induce 16% apoptosis after 16 h incubation. Analysis of apoptosis by flow cytometry using the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) method also demonstrated a dose-dependent induction of apoptosis by PR3. DNA fragmentation was confirmed by agarose gel electrophoresis. To investigate the effect of ANCA on PR3-mediated apoptosis, HUVEC were exposed to immunoglobulin G (IgG) from patients with Wegener's granulomatosis or systemic vasculitis, and from normal controls, in the presence or absence of PR3. Enhancement of PR3-mediated apoptosis was found in two of 10 IgG samples with anti-PR3 activity, whereas a reduction in apoptosis was observed in two others. Anti-MPO (myeloperoxidase)-positive IgG, six additional anti-PR3 positive IgG samples and control IgG samples did not have any detectable effect on apoptosis. These studies suggest that ANCA may modulate the relative degree of injury in some cases of Wegener's granulomatosis.
韦格纳肉芽肿病的特征是新月形坏死性肾小球肾炎和系统性血管炎。蛋白酶3（PR3）和针对该酶的抗中性粒细胞胞浆抗体（ANCA）都被认为起致病作用。PR3已被证明在体外可导致人脐静脉内皮细胞（HUVEC）脱离和细胞溶解，并诱导牛肺动脉内皮细胞凋亡。在本研究中，我们调查了PR3和ANCA对体外诱导人内皮细胞凋亡的影响。将HUVEC在有无不同浓度PR3的情况下培养不同时间段，并用三种不同方法评估细胞凋亡。用Hoechst 33258对细胞染色并通过紫外（UV）光学显微镜评估核形态，通过细胞计数确定凋亡呈剂量依赖性诱导。发现8μg/ml的PR3浓度在孵育16小时后可诱导16%的细胞凋亡。使用末端脱氧核苷酸转移酶介导的dUTP-荧光素缺口末端标记（TUNEL）方法通过流式细胞术分析细胞凋亡也证明PR3可诱导凋亡呈剂量依赖性。DNA片段化通过琼脂糖凝胶电泳得以证实。为了研究ANCA对PR3介导的细胞凋亡的影响，将HUVEC在有或无PR3的情况下暴露于来自韦格纳肉芽肿病或系统性血管炎患者以及正常对照的免疫球蛋白G（IgG）。在10份具有抗PR3活性的IgG样本中有2份发现增强了PR3介导的细胞凋亡，而在另外2份样本中观察到细胞凋亡减少。抗MPO（髓过氧化物酶）阳性IgG、另外6份抗PR3阳性IgG样本和对照IgG样本对细胞凋亡没有任何可检测到的影响。这些研究表明，在某些韦格纳肉芽肿病病例中，ANCA可能调节损伤的相对程度。
