Abstract
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.
Highlights
Chronic granulomatous disease (CGD) is a rare, heterogenous, and inherited disorder that affects approximately 1 in 250,000 births [1]
We hypothesized that the gp91phox subunit of NADPH oxidase derived from neutrophils could have a significant effect on endothelial function. To investigate this potential effect, we focused on the effect of nitric oxide (NO) and H2O2 from neutrophils obtained from patients with X-linked CGD (X-CGD) on the expression of NOS3 and EDN1 mRNA in human umbilical vein endothelial cells (HUVECs)
NOS3 mRNA and the protein expression of phosphorylated endothelial NOS (eNOS) at Ser1177 position in HUVECs co-cultured with X-CGD neutrophils are shown in Figs 1A and 2
Summary
Chronic granulomatous disease (CGD) is a rare, heterogenous, and inherited disorder that affects approximately 1 in 250,000 births [1]. It has been reported that X-linked CGD occurs in approximately 70% of patients with CGD and is due to the mutation of CYBB encoding gp91phox, which is located at Xp21.1 [2, 3]. In 2009, Violi et al reported that lower oxidative stress and enhanced arterial dilatation as assessed by flow-mediated dilatation (FMD) testing were detected in X-CGD patients, reflecting increased bioavailability or higher levels of NO [11,12,13]. Their findings suggested that oxidative stress derived from neutrophils may have a pivotal role in modulating endothelial function [14,15]. The precise interactions between the NO and ROS produced by neutrophils in particular, and their effects on endothelial function, remain to be elucidated
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