CD177在ANCA相关小血管炎发病机制中的作用

The pathogenesis of ANCA-associated systemic vasculitis (AAV) has not been fully demonstrated. It has been descripted that ANCA antigens are brought to the neutrophil surface after priming by inflammatory cytokines, which are recognized by ANCA. Then, the oxidative spices are released, which directly damage the vessel wall immediately adjacent. The pathological feature of AAV is necrosis of endothelial cells of small-to-medium sized vessels where adhesion and transmigration may take place, highlighting the importance of these processes in disease development. Proteinase 3, one of the ANCA antigens, is presented on the cell surface by CD177, which is a glycoprotein. CD177 may interact with PECAM1 on endothelial cells and play a role as an adhesion molecule. Based on recent findings on this subject, we hypothesize that 1) neutrophils expressing CD177 are more efficient in adhesion and transmigration; 2) PR3 are released during CD177-PECAM1 interaction, and may cause extra damage to the vessel wall when ANCA in presence.

ANCA相关小血管炎的发病机制尚不明确。目前认为ANCA自身抗原在炎症状态下表达于中性粒细胞膜表面，被ANCA识别并引起中性粒细胞呼吸爆发，导致临近血管内皮损伤，组织病理表现为坏死性血管炎。活性氧自由基的非特异性损伤局限于中-小血管，提示中性粒细胞粘附、游出血管内皮的过程是发病的重要阶段。蛋白酶3（Proteinase 3, PR3）是ANCA自身抗原，以CD177为受体表达于中性粒细胞膜表面。CD177属糖蛋白（PECAM1配体），具有粘附分子功能。我们的前期工作证实CD177在AAV患者中表达增加。根据CD177与PECAM1的配体关系，我们推测1）CD177+中性粒细胞在炎症发生时，粘附、游出血管内皮更迅速；2）在中性粒细胞游出过程中，CD177与PECAM-1的结合，导致其配体PR3的释放，并在ANCA作用下，加重内皮细胞损伤。

在AAV中，PR3是ANCA主要的抗原之一。CD177为PR3在中性粒细胞表面受体，而且CD177被证实可以和PECAM-1结合。本研究是为了探究PECAM-1与中性粒细胞表面分子CD177的相互作用对中性粒表面PR3表达的影响，进而探究在AAV中其相互作用对于ANCA导致的中性粒细胞的活化和内皮细胞损伤的影响。将提取的PMN与PECAM-1孵育，用流式细胞术检测PECAM-1与CD177的结合对细胞膜PR3表达量的影响，用ELISA检测上清中游离PR3的含量。PECAM-1与中性粒细胞预孵育后，加入PR3-ANCA阳性IgG，以DHR实验检测细胞内活性氧释放水平来反应呼吸爆发程度，用ELISA检测上清中乳铁蛋白水平来评估中性粒细胞脱颗粒水平。对中性粒细胞进行磁珠筛选得到CD177阴性的中性粒细胞，评价PECAM-1对由PR3-ANCA诱导的中性粒细胞脱颗粒的抑制作用。用ICAM-1含量反应肾小球内皮细胞的活化和损伤水平。将与PECAM-1预孵育的中性粒细胞与原代肾小球内皮细胞共孵育，并且加PR3-ANCA阳性IgG刺激，用ELISA检测上清中ICAM-1含量，以反应PECMA-1对PR3-ANCA介导的中性粒细胞活化和内皮损伤的作用。CD177与PECAM-1的结合水平随着CD177水平增加而升高。PECAM-1与中性粒细胞孵育之后，中性粒细胞膜上PR3表达量降低，并且存在剂量依赖性。PECAM-1对中性粒细胞预孵育可以显著可以减轻显著降低中性粒细胞呼吸爆发和脱颗粒水平。PECAM-1预孵育对PR3-ANCA阳性IgGs诱导的中性粒细胞呼吸爆发在CD177阳性细胞中具有保护作用。PR3-ANCA阳性IgG和中性粒细胞与内皮细胞作用可导致上清中ICAM-1水平显著升高，而PECAM-1与中性粒细胞的预孵育可以明显降低ICAM-1在上清中的水平。PECAM-1与CD177相互作用可以降低膜PR3水平，从而减轻PR3-ANCA导致的中性粒细胞的活化和内皮细胞的损伤，这对于PR3-ANCA阳性的AAV治疗可能具有临床意义。