阻断IgG-Fcγ受体结合的短肽对粒细胞与内皮细胞黏附及内皮细胞细胞间黏附分子1表达的影响

Modulation of the adhesion of neutrophils to endothelium and the expression of intercellular adhesion molecule I in endothelial cells by one small peptide interfering with IgG-FcγR interaction

目的观察阻断IgG-Fcγ结合的短肽tgl9320对粒细胞与内皮细胞黏附以及内皮细胞细胞间黏附分子1（ICAM-1）表达的影响，并探讨其作用机制。方法培养人脐静脉内皮细胞（HUVEC）。提纯活动期血管炎患者血清抗中性粒细胞胞质抗体（ANCA）IgG。以多肽固相合成tgl9320。分离健康人新鲜外周血中性粒细胞。分别以肿瘤坏死因子α（TNF-α）、健康人IgG、ANCA IgG 及ANCA IgG＋tgl9320处理HUVEC，用细胞直接计数法检测粒细胞与内皮细胞间的黏附率；应用Westem印迹及实时定量PCR方法检测HUVEC ICAM-1蛋白和mRNA表达；ELISA检测细胞培养上清液中可溶性ICAM-1（sICAM-1）的水平；Westem印迹检测HUVEC磷酸化核因子KB抑制物（p-IKB）的表达。结果ANCA IgG显著上调中性粒细胞与内皮细胞间的黏附率（P〈0.05），但ANCA IgG＋tgl9320组较ANCA IgG组黏附率显著降低（P〈0．05）。ANCA IgG组与健康人IgG组相比，HUVEC ICAM-1 mRNA及蛋白表达显著增加（P〈0．05）。tgl9320分别从mRNA和蛋白水平阻断ANCA对ICAM-1的作用（P〈0．05），并显著降低ANCAIgG引起的细胞培养上清液中sICAM水平增高（P〈0．05）。ANCA IgG增加HUVEC p-IκB表达，tgl9320显著降低p-IκB的表达。结论tgl9320通过抑制IκB磷酸化进而干预NF-κB活化；抑制ANCA IgG对内皮细胞与中性粒细胞间黏附的促进作用，并阻断ICAM-1上调表达。短肽tgl9320对原发性小血管炎具有体外保护作用。

Objective To investigate the effects of tg19320, a small peptide, interfering with IgG-FcγR interaction on the adhesion of neutrophils to endothelium and the expression of intercellular adhesion molecule 1 （ICAM-1）in endothelial cells and its possible mechanism. Methods Tg19320 was prepared by solid-phase peptide synthesis. ANCA lgG was isolated from the serum of active ANCA-associated systemic vasculitis （AASV） patients. When primary human umbilical vein endothelial cells （HUVEC） grew into confluence in cytokine-free conditions, the cells were stimulated with TNF-α, human normal IgG, ANCA IgG and ANCA IgG＋tg19320 respectively. HUVEC were pretreated with tg19320 for 45 minutes before being stimulated by ANCA IgG. Nonactivated neutrophils was added to treat HUVEC and adhesion was measured by cell count. The expression of ICAM-1 mRNA and protein was assessed by real-time PCR and Westem blot respectively.Soluble ICAM-1 （sICAM-1）was determined using ELISA technique.Phosphorylation of IκB-α was assessed by Westem blot. Results ANCA IgG significantly up-regulated the expression of ICAM-1 in HUVEC and promoted sICAM-1 release （P〈0.05）,and TNF-α enhanced the effect of ANCA.These effects were almost completely abolished by th19320 both at protein and mRNA level.Furthermore,ANCA IgG increased the IκB-α phosporylation in HUVEC and tg19320 could inhibit the effect. Conclusions ANCA IgG can modulate the expression of ICAM-1 and sICAM-1 release in endothelial cells.FcγR probably play a critical role in the ICAM-1 expression up-regulated by ANCA,which is mediated in part throuhg NF-κB signaling pathway.Tg19320 has protective effect on endothelivm in AASV in vitro.