Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function

Joseph Khoory,Nathan I Shapiro,Itaru Hamachi,Abdallah Elkhal,Mark I Melhorn,Yasutaka Kurishita,Asmae Lazaar,Ben Illigens,Ionita C Ghiran,Alexander R Ivanov,Abigail Brodsky,Sergey Shevkoplyas,Jessica Estanislau,Ming Dao

doi:10.1371/journal.pone.0141206

Abstract

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.

Highlights

In humans, during normal conditions, low concentrations of complement-opsonized circulating inflammatory particles are bound to red blood cells (RBCs) via complement receptor 1 (CR1) and delivered to resident sinusoidal macrophages in the liver and spleen, a process known as immune-adherence clearance [1,2]
Our results show that glycophorin A (GPA) ligation induces a NADPH oxidase-dependent increase in the intracellular concentration of reactive oxygen species (ROS), which, in turn, trigger multiple downstream cascades leading to caspase-3 activation, increased band 3 phosphorylation, and significant RBC ATP release
We investigated the deleterious effects of RBC ROS production promoted by GPA ligation using both in vitro and ex vivo approaches

Summary

Introduction

In humans, during normal conditions, low concentrations of complement-opsonized circulating inflammatory particles are bound to red blood cells (RBCs) via complement receptor 1 (CR1) and delivered to resident sinusoidal macrophages in the liver and spleen, a process known as immune-adherence clearance [1,2]. Genetic or acquired conditions associated with decreased ROS scavenging capabilities can cause extensive membrane damage leading to reduced RBC half-life and intravascular hemolysis [13]. Acute and chronic inflammatory conditions associated with complement activation promote a sustained increase in total blood ROS content that parallels disease severity. Mice lacking either P2Y2 or A3 receptors showed reduced organ damage and mortality following septic shock [18] These data strongly argue that ATP and its by-products, acting on their cognate receptors, are essential for the disproportionate PMN and T cell inflammatory host responses that characterize acute and chronic inflammatory conditions such as sepsis. Our results show that GPA ligation induces a NADPH oxidase-dependent increase in the intracellular concentration of ROS, which, in turn, trigger multiple downstream cascades leading to caspase-3 activation, increased band 3 phosphorylation, and significant RBC ATP release. Overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, and microcirculation impairment

Experimental Procedures

Results

Discussion