Department of Pediatrics, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima, Japan.
PLoS One. 2020 Apr 6;15(4):e0230665. doi: 10.1371/journal.pone.0230665. eCollection 2020.
Phagocytes in patients with chronic granulomatous disease (CGD) do not generate reactive oxidative species (ROS), whereas nitric oxide (NO) production is increased in response to the calcium ionophore A23187 in CGD phagocytes compared with healthy phagocytes. Recently, patients with X-linked CGD (X-CGD) have been reported to show higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production from phagocytes. We studied NOS3 and EDN1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs) in a co-culture system with neutrophils from X-CGD patients. HUVECs were co-cultured for 30 minutes with human neutrophils from X-CGD or healthy participants in response to A23187 without cell-to-cell contact. The expression of NOS3 and EDN1 mRNA in HUVECs was quantified by real-time polymerase chain reaction. Moreover, we demonstrated the protein expression of eNOS, ET-1, and NFκB p65, including phosphorylation at Ser1177 of eNOS and Ser536 of NFκB p65. Neutrophils from X-CGD patients showed significantly higher NO and lower H2O2 production in response to A23187 than healthy neutrophils in vitro. Compared with healthy neutrophils, X-CGD neutrophils under A23187 stimulation exhibited significantly increased NO and decreased H2O2, and promoted downregulated NOS3 and EDN1 expression in HUVECs. The total expression and phosphorylation at Ser1177 of eNOS and ET-1 expression were significantly decreased in HUVECs co-cultures with stimulated X-CGD neutrophils. Also, phosphorylation at Ser536 of NFκB p65 were significantly decreased. In conclusions, eNOS and ET-1 significantly down-regulated in co-culture with stimulated X-CGD neutrophils through their excessive NO and the lack of ROS production. These findings suggest that ROS generated from neutrophils may mediate arterial tone affecting eNOS and ET-1 expression via their NO and ROS production.
患有慢性肉芽肿病（CGD）的吞噬细胞不能产生活性氧（ROS），而 CGD 吞噬细胞在钙离子载体 A23187 的刺激下比健康吞噬细胞产生更多的一氧化氮（NO）。最近，报道称 X 连锁 CGD（X-CGD）患者表现出更高的血流介导扩张，这表明内皮细胞功能受到吞噬细胞产生的 NO 的影响。我们在与 X-CGD 患者的中性粒细胞共培养系统中研究了人类脐静脉内皮细胞（HUVEC）中的 NOS3 和 EDN1 mRNA 和蛋白表达。HUVEC 与 X-CGD 或健康参与者的人中性粒细胞在无细胞接触的情况下共培养 30 分钟，以响应 A23187。通过实时聚合酶链反应定量 HUVEC 中 NOS3 和 EDN1 mRNA 的表达。此外，我们还证明了 eNOS、ET-1 和 NFκB p65 的蛋白表达，包括 eNOS 的 Ser1177 和 NFκB p65 的 Ser536 的磷酸化。与健康的中性粒细胞相比，体外 X-CGD 中性粒细胞对 A23187 的反应表现出更高的 NO 和更低的 H2O2 产生。与健康中性粒细胞相比，A23187 刺激下的 X-CGD 中性粒细胞表现出明显增加的 NO 和减少的 H2O2，并且促进了 HUVEC 中 NOS3 和 EDN1 表达的下调。与刺激的 X-CGD 中性粒细胞共培养的 HUVEC 中 eNOS 和 ET-1 的总表达及其 Ser1177 的磷酸化显著降低，并且 NFκB p65 的 Ser536 的磷酸化显著降低。总之，通过其过度的 NO 和缺乏 ROS 产生，在与刺激的 X-CGD 中性粒细胞共培养中，eNOS 和 ET-1 显著下调。这些发现表明，来自中性粒细胞的 ROS 可能通过其 NO 和 ROS 产生来调节动脉张力，从而影响 eNOS 和 ET-1 的表达。
