Yang J J, Kettritz R, Falk R J, Jennette J C, Gaido M L
Department of Medicine, University of North Carolina at Chapel Hill 27599-7155, USA.
Am J Pathol. 1996 Nov;149(5):1617-26.
The pathogenesis of vasculitis associated with anti-neutrophil cytoplasmic antibodies is not established. The anti-neutrophil cytoplasmic antibody autoanigens proteinase 3 (PR3) and elastase induce detachment and cytolysis of endothelial cells in vitro. We investigated whether PR3 and elastase trigger endothelial cell apoptosis. Primary bovine pulmonary artery endothelial cells were treated with either PR3, elastase, or myeloperoxidase (MPO) and apoptosis assessed by four different methods. By the cell death detection enzyme-linked immunosorbent assay, DNA fragmentation increased to 208 +/- 84% or 153 +/- 27% of control with 1 micrograms/ml PR3 or elastase at 24 hours. By ultraviolet light microscopy, the percentage of apoptotic cells significantly increased (P < 0.05) with 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase at 6, 12, or 24 hours. Values at the 24-hour time point are 15.3 +/- 6.4% or 25.8 +/- 6.6% for 5 or 10 micrograms/ml PR3 and 13.9 +/- 3.6% or 20.7 +/- 1.8% for 25 or 50 micrograms/ml elastase compared with 2.2 +/- 1.2% for control. Similarly, with flow cytometry, 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase for 6, 12, or 24 hours demonstrated increasing apoptosis in a dose- and time-dependent manner with the highest values achieved at 24 hours (23.4 +/- 4.0% and 35.6% for 5 and 10 micrograms/ml PR3 and 31.8 +/- 4.0% and 47.8% for 25 and 50 micrograms/ml elastase compared with 7.9 +/- 2.2% in control). Typical DNA laddering was apparent from 6 to 24 hours at 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase. Myeloperoxidase did not induce cell apoptosis. Release of PR3 and elastase by activated neutrophils during acute inflammation, including anti-neutrophil cytoplasmic antibody-associated vasculitis, may result in vascular damage by endothelial cell apoptosis.
抗中性粒细胞胞浆抗体相关血管炎的发病机制尚未明确。抗中性粒细胞胞浆抗体自身抗原蛋白酶3（PR3）和弹性蛋白酶在体外可诱导内皮细胞脱离和溶解。我们研究了PR3和弹性蛋白酶是否会引发内皮细胞凋亡。用PR3、弹性蛋白酶或髓过氧化物酶（MPO）处理原代牛肺动脉内皮细胞，并通过四种不同方法评估细胞凋亡情况。通过细胞死亡检测酶联免疫吸附测定法，在24小时时，1微克/毫升PR3或弹性蛋白酶处理组的DNA片段化增加至对照组的208±84%或153±27%。通过紫外线显微镜观察，在6、12或24小时时，5或10微克/毫升PR3以及25或50微克/毫升弹性蛋白酶处理组的凋亡细胞百分比显著增加（P<0.05）。在24小时时间点，5或10微克/毫升PR3处理组的值分别为15.3±6.4%或25.8±6.6%，25或50微克/毫升弹性蛋白酶处理组的值分别为13.9±3.6%或20.7±1.8%，而对照组为2.2±1.2%。同样，通过流式细胞术检测，5或10微克/毫升PR3以及25或50微克/毫升弹性蛋白酶处理6、12或24小时后，凋亡呈剂量和时间依赖性增加，在24小时时达到最高值（5和10微克/毫升PR3分别为23.4±4.0%和35.6%，25和50微克/毫升弹性蛋白酶分别为31.8±4.0%和47.8%，而对照组为7.9±2.2%）。在5或10微克/毫升PR3以及25或50微克/毫升弹性蛋白酶处理6至24小时期间，可见典型的DNA梯状条带。髓过氧化物酶未诱导细胞凋亡。在急性炎症（包括抗中性粒细胞胞浆抗体相关血管炎）期间，活化的中性粒细胞释放PR3和弹性蛋白酶，可能通过内皮细胞凋亡导致血管损伤。
