Uriarte Silvia M, McLeish Kenneth R, Ward Richard A
Department of Medicine, University of Louisville, 615 S. Preston Street, Louisville, KY 40202-1718, USA.
Nephrol Dial Transplant. 2009 Apr;24(4):1150-7. doi: 10.1093/ndt/gfn580. Epub 2008 Oct 24.
Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are postulated to injure vascular endothelium by inducing cytokine-primed neutrophils to release proteolytic enzymes and generate reactive oxygen species. Anti-PR3 induce exocytosis, and since priming is associated with upregulation of plasma membrane proteins by exocytosis of intracellular granules, we tested the hypothesis that anti-PR3 prime neutrophils in the absence of cytokines.
Isolated human neutrophils were incubated with or without anti-PR3. Superoxide release was determined by measuring the reduction of ferricytochrome C. Exocytosis of secretory vesicles and specific granules was determined by measuring the expression of CD35 and CD66b, respectively, using flow cytometry.
Anti-PR3 (15 mug/mL) directly stimulated superoxide production and enhanced FMLP-stimulated superoxide production. Anti-PR3 (0.5 mug/mL) did not stimulate superoxide production but did enhance FMLP-stimulated superoxide production. Incubation of neutrophils with anti-PR3 resulted in time-dependent exocytosis of secretory vesicles and specific granules. Anti-PR3-induced exocytosis, but not superoxide production, was dependent on p38 mitogen-activated protein kinase. Conclusions. These data demonstrate that anti-PR3 can directly stimulate production of reactive oxygen species by neutrophils without cytokine priming, and that anti-PR3 prime neutrophils for increased FMLP-stimulated reactive oxygen species production. Anti-PR3 also induce exocytosis via a mechanism separate from their effect on reactive oxygen species production. These findings suggest that anti-PR3 ANCA may activate neutrophils and cause endothelial cell injury by multiple pathways, including some that are independent of priming by a second agent.
抗蛋白酶3（PR3）的抗中性粒细胞胞浆抗体（ANCA）被认为可通过诱导细胞因子预激活的中性粒细胞释放蛋白水解酶并产生活性氧来损伤血管内皮。抗PR3可诱导胞吐作用，由于预激活与细胞内颗粒通过胞吐作用上调质膜蛋白有关，我们测试了抗PR3在无细胞因子情况下预激活中性粒细胞的假说。
将分离的人中性粒细胞与抗PR3一起孵育或不孵育。通过测量高铁细胞色素C的还原率来测定超氧化物释放。分别使用流式细胞术通过测量CD35和CD66b的表达来测定分泌囊泡和特异性颗粒的胞吐作用。
抗PR3（15μg/mL）直接刺激超氧化物生成并增强FMLP刺激的超氧化物生成。抗PR3（0.5μg/mL）不刺激超氧化物生成，但确实增强FMLP刺激的超氧化物生成。中性粒细胞与抗PR3孵育导致分泌囊泡和特异性颗粒的时间依赖性胞吐作用。抗PR3诱导的胞吐作用而非超氧化物生成依赖于p38丝裂原活化蛋白激酶。结论。这些数据表明，抗PR3可在无细胞因子预激活的情况下直接刺激中性粒细胞产生活性氧，并且抗PR3可预激活中性粒细胞以增加FMLP刺激的活性氧生成。抗PR3还通过与其对活性氧生成的作用分开的机制诱导胞吐作用。这些发现表明，抗PR3 ANCA可能通过多种途径激活中性粒细胞并导致内皮细胞损伤，包括一些独立于第二种因子预激活的途径。
