Renal Division, Department of Medicine, Peking University First Hospital, Peking University Institute of Nephrology, Beijing, 100034, China.
Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, 100034, China.
Arthritis Res Ther. 2018 Sep 20;20(1):213. doi: 10.1186/s13075-018-1710-0.
A recent study found that CD177 served as a receptor of membrane-bound proteinase-3 (mPR3) in a subset of neutrophils. Furthermore, CD177 has been identified as a high-affinity heterophilic binding partner for the endothelial cell platelet endothelial cell adhesion molecule-1 (PECAM-1). The current study aimed to investigate whether the interaction between PECAM-1 and CD177 could influence mPR3 expression as well as PR3-antineutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation and glomerular endothelial cell (GEnC) injury.
The effect of interaction between CD177 and PECAM-1 on mPR3 expression was explored by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The effect of PECAM-1 on neutrophil activation and GEnC injury induced by PR3-ANCA-positive immunoglobulin (Ig)Gs was evaluated by dihydrorhodamine (DHR) assay and ELISA. CD177-negative neutrophils were selected by magnetic cell sorting (MACS), and the inhibitory effect of PECAM-1 on CD177-negative and mixed neutrophils was explored by measuring neutrophil degranulation.
The level of specific interaction between CD177 and PECAM-1 was elevated with increasing CD177 concentration. The expression of mPR3 significantly decreased in neutrophils preincubated with PECAM-1 in a dose-dependent manner. Consistently, the levels of respiratory burst and degranulation induced by PR3-ANCA-positive IgGs in recombinant human tumor necrosis factor-alpha (TNF-α)-primed neutrophils was significantly reduced by preincubation with PECAM-1 (440.6 ± 123.0 vs. 511.4 ± 95.5, p < 0.05; and 3155.0 ± 1733.0 ng/ml vs. 5903.0 ± 717.5 ng/ml, p < 0.05, respectively). In CD177-negative neutrophils incubated with PR3-ANCA-positive IgGs, the level of degranulation was not significantly changed by preincubation with PECAM-1. However, in mixed neutrophils, PECAM-1 significantly decreased the level of degranulation induced by PR3-ANCA-positive IgGs (1015.9 ± 229.2% vs. 1725.2 ± 412.4%, p < 0.01). Furthermore, with preincubation of TNF-α-primed neutrophils with PECAM-1, the level of soluble intercellular cell adhesion molecule-1 (sICAM-1), a marker of endothelial cell activation and injury, in the supernatant of GEnCs treated with primed neutrophils plus PR3-ANCA-positive IgGs was significantly attenuated (112.7 ± 24.2 pg/ml vs. 167.5 ± 27.7 pg/ml, p < 0.05).
PECAM-1 can decrease the level of mPR3 expression on neutrophils, resulting in attenuation of neutrophil activation and subsequent GEnC injury induced by PR3-ANCA-positive IgGs.
最近的一项研究发现，CD177 作为细胞膜结合蛋白酶 3（mPR3）在中性粒细胞亚群中的受体。此外，CD177 已被鉴定为内皮细胞血小板内皮细胞黏附分子-1（PECAM-1）的高亲和力异源结合伴侣。本研究旨在探讨 PECAM-1 与 CD177 之间的相互作用是否会影响 mPR3 表达以及 PR3-抗中性粒细胞胞质抗体（ANCA）诱导的中性粒细胞活化和肾小球内皮细胞（GEnC）损伤。
通过酶联免疫吸附试验（ELISA）和流式细胞术探讨 CD177 与 PECAM-1 之间相互作用对 mPR3 表达的影响。通过二氢罗丹明（DHR）试验和 ELISA 评估 PECAM-1 对 PR3-ANCA 阳性免疫球蛋白（IgG）诱导的中性粒细胞活化和 GEnC 损伤的影响。通过磁细胞分选（MACS）选择 CD177 阴性中性粒细胞，通过测量中性粒细胞脱颗粒来探讨 PECAM-1 对 CD177 阴性和混合中性粒细胞的抑制作用。
随着 CD177 浓度的增加，CD177 与 PECAM-1 之间特异性相互作用的水平升高。用 PECAM-1 进行剂量依赖性预孵育后，中性粒细胞 mPR3 的表达显著降低。一致地，用 PECAM-1 预孵育可显著降低重组人肿瘤坏死因子-α（TNF-α）-预刺激中性粒细胞中 PR3-ANCA 阳性 IgG 诱导的呼吸爆发和脱颗粒水平（440.6±123.0 比 511.4±95.5，p<0.05；和 3155.0±1733.0 ng/ml 比 5903.0±717.5 ng/ml，p<0.05）。在与 PR3-ANCA 阳性 IgG 孵育的 CD177 阴性中性粒细胞中，用 PECAM-1 预孵育并不显著改变脱颗粒水平。然而，在混合中性粒细胞中，PECAM-1 显著降低 PR3-ANCA 阳性 IgG 诱导的脱颗粒水平（1015.9±229.2%比 1725.2±412.4%，p<0.01）。此外，用 PECAM-1 预孵育 TNF-α 预刺激的中性粒细胞后，用预刺激中性粒细胞加 PR3-ANCA 阳性 IgG 处理的肾小球内皮细胞上清液中可溶性细胞间黏附分子-1（sICAM-1）的水平，一种内皮细胞活化和损伤的标志物，显著降低（112.7±24.2 pg/ml 比 167.5±27.7 pg/ml，p<0.05）。
PECAM-1 可降低中性粒细胞上 mPR3 的表达水平，从而减弱 PR3-ANCA 阳性 IgG 诱导的中性粒细胞活化和随后的 GEnC 损伤。
