Excessive Nitric Oxide Production of CGD Neutrophils Induces the Down-Regulation of NOS3 and EDN1 Expression in Human Endothelial Cells

Introduction: Chronic granulomatous disease (CGD) is an inherited immunodeficiency due to a mutation in genes that encode the subunits of NADPH oxidase of phagocytes. Phagocytes of CGD can not generate the reactive oxidative species (ROS), whereas nitric oxide (NO) production of CGD phagocytes is increased in response to calcium ionophore, A23187, compared with that of phagocytes from healthy subjects. Recently, X-linked CGD (X-CGD) patients showed lower oxidative stress and higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production of phagocytes. We studied the effects of NO on the regulation of endothelial gene expression related to dilator and constrictive effect of blood vessels, such as NOS3 and EDN1 using neutrophils from X-CGD patients.

Methods: Eighteen X-CGD patients and 18 age-matched healthy male subjects were enrolled in this study from 2009 to 2013. NO production of phagocytes was assessed by flow cytometry using DAF2/DA fluorescent probe. Human umbilical vein endothelial cells (HUVECs) were co-cultured with human neutrophils of X-CGD or healthy subjects in response to calcium ionophore,A23187 without cell to cell contact using the Transwell-permeable support systems. The expression of endothelial NOS3 and EDN1 mRNA gene in HUVECs were quantified by real-time PCR.

Results: Neutrophils of X-CGD patients showed significantly higher production of NO in response to A23187 for 30 to 60 minutes than those of normal subjects detected by flow cytometric analyses. NO concentration artificially generated by NO donor was significantly decreased by ROS produced by xanthine and xanthine oxidase. Similarly, neutrophils from healthy subjects, but not from X-CGD stimulated with A23187 induced the decrease in the concentration of NO. These results strongly suggest that the lack of ROS generation in CGD neutrophils leads to the increase of NO production in response to A23187. HUVECs were incubated with NO generation system, ROS generation system alone or with both systems. The expression of NOS3 and EDN1 was significantly decreased depending on the concentration of extracellular NO. In contrast, both NOS3 and EDN1 expression was significantly up-regulated under the ROS generation system without NO. Next, HUVECs were incubated by X-CGD neutrophils or by control neutrophils with or without A23187 for 30 minutes using the wells divided by membrane (0.45μm) to prevent cell to cell contact. Both NOS3 and EDN1 expression of HUVECs incubated with X-CGD neutrophils was significantly down-regulated under A23187 stimulation compared with normal neutrophils (112±117 vs. 34.5±39.9, n=8, p<0.05, 0.94± 0.24 vs. 0.61±0.24, n=5, p<0.05, respectively).

Conclusion: This study demonstrated that the stimulated X-CGD neutrophils induced the decreased endothelial NOS3 and EDN1 gene expression through the excessive generation of NO due to the lack of ROS production. These findings suggest that ROS generated by phagocytes may modulate arterial tone affecting the amount of NO, a potent vasodilator molecule produced by endothelial cells.