Transmigration of neutrophils through epithelium

我们评价了来自内皮细胞的一氧化氮(NO)在中性粒细胞跨内皮细胞迁移(TEM)中的作用。用一氧化氮合酶抑制剂N-硝基-L-精氨酸甲酯盐酸盐(L-NAME)或N-硝基-L-精氨酸单甲基-精氨酸(L-N-甲基精氨酸)处理人脐静脉内皮细胞，可增强中性粒细胞的透射电子显微镜。在NO清除剂2-(4-carboxyphenyl)-4，4，5，5-tetramethylimidazoline-1-oxyl-3-oxide(羧基PTIO)的存在下，也观察到了类似的透射电子显微镜增强。经L或L处理的人脐静脉内皮细胞的中性粒细胞透射电子显微镜可被NO供体持续供给NO所抑制。这些发现支持内皮细胞持续产生NO抑制中性粒细胞透射电子显微镜的说法。流式细胞仪分析显示，中性粒细胞与NO产生的HUVECs共培养后，中性粒细胞中NO积聚。与未处理的人脐静脉内皮细胞共培养的中性粒细胞相比，与L作用的人脐静脉内皮细胞共培养的中性粒细胞中的NO含量降低。可溶性鸟苷酸环化酶(SGC)是中性粒细胞NO的重要靶点之一。SGC激活剂3-(53-羟甲基-23-呋喃)-1-苯基吲唑(YC-1)能抑制L诱导的中性粒细胞透射电子显微镜。有趣的是，1-H[1，2，4-]恶二唑并[4，3-a]喹恶灵-1-1(ODQ)对中性粒细胞sGC的抑制足以增强透射电子显微镜。这些结果表明，来自HUVECs的NO至少部分通过激活sGC作用于中性粒细胞以抑制透射电子显微镜。我们的发现暗示，在生理条件下，HUVECs结构性产生的NO在保护过度的中性粒细胞外溢和不必要的组织损伤方面起到了作用。

We evaluated the roles of nitric oxide(NO) derived from endothelial cells in neutrophil transendothelial migration(TEM). Pretreatment of human umbilical vein endothelial cells (HUVECs) with NG-nitro-L-arginine methyl ester hydrochloride(L-NAME) or NG-monomethyl L-arginine (L-NMMA), which are inhibitors of NO synthases, enhanced neutrophil TEM. Similar augmentation of TEM was observed in the presence of an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy PTIO). Neutrophil TEM across L-NAME- or L-NMMA-treated HUVECs was inhibited by continuous NO supply by NO donors. These findings support the suggestion that continuous production of NO by endothelial cells suppresses neutrophil TEM. Flow cytometric analyses revealed that NO accumulates in neutrophils co-cultured with NO-producing HUVECs. A decreased amount of NO was detected in neutrophils co-cultured with L-NAME-treated HUVECs compared with neutrophils co-cultured with untreated HUVECs. Soluble guanylyl cyclase (sGC) is known as one of the most important targets of NO in neutrophils. 3-(53-Hydroxymethyl-23furyl)-1-benzyl indazole (YC-1), an activator of sGC, inhibited L-NAME-induced neutrophil TEM. It was interesting that inhibition of neutrophil sGC with 1-H[1,2,4-]oxadiazolo[4,3-a]quinoxalin-1-1 (ODQ) was sufficient to enhance TEM. These results suggest that NO derived from HUVECs acts on neutrophils to inhibit TEM, at least in part by activating sGC. Our findings imply the role of NO constitutively generated by HUVECs in protection against excessive neutrophil extravasation and unnecessary tissue damage under physiological conditions.