Vesicles Generated during Storage of Red Blood Cells Enhance the Generation of Radical Oxygen Speciesin Activated Neutrophils

Herbert Jank,Ulrich Salzer

doi:10.1100/tsw.2011.25

Abstract

Erythrocytes are known to shed vesicles in vivo, under various conditions in vitro, and, with impact for transfusion medicine, during storage of red blood cell concentrates (Vsto vesicles). Vsto vesicles of blood transfusions have been shown to deliver glycosylphosphatidylinositol-linked proteins to recipient erythrocytes, to display prothrombotic activity, and to have an inhibitory effect on macrophages. The interaction of Vsto vesicles with and their effect on neutrophilic granulocytes has not yet been studied in detail. Fluorescentlylabeled Vsto and calcium-induced vesicles were preparedin order to study the uptake of labeled vesicular components by neutrophils as compared to the process of phagocytosis of zymosan using flow cytometry and confocal microscopy. The activating effect of Vsto vesicles on neutrophils was addressed by a luminometric assay for stimulated radical oxygen species (ROS) generation. Coincubation of vesicles and neutrophils results in a transfer of vesicular components to the cells. This uptake is different from a phagocytotic process and is enhanced upon interference with the cellular actin cytoskeleton. Preincubation of neutrophils with Vsto vesicles results in an enhanced ROS generation by neutrophils, which is further increased upon fMLP stimulation and during zymosan phagocytosis. The activating effect of Vsto vesicles on neutrophils might be due to the specific accumulation of lysophospholipidsin Vsto vesicles and should be considered as a possible contributor to the pathogenesis of transfusion-related acute lung injury.

Highlights

Erythrocytes have the ability to shed hemoglobin-rich vesicles and they lose about 20% of their surface area during their life span via vesiculation[1]
A variety of conditions are known that trigger the vesiculation process in vitro: (a) incubation of erythrocytes with calcium and the ionophore A23187 leads to a sequence of changes at the erythrocyte membrane and to the concomitant formation of exovesicles[2,3]; (b) exhaustion of the cellular energy reserve (ATP depletion)[4], incubation with (c) anionic or zwitterionic amphiphiles, and (d) lysophosphatidic acid (LPA)[5] result in vesicle shedding from erythrocytes; (e) erythrocyte concentrates in blood transfusion units have long been known to contain increasing amounts of vesicles dependent on the duration of storage[6,7]
To study the interaction of erythrocyte-derived vesicles with neutrophils, we used two different types of vesicles: the well-characterized vesicles obtained by the calcium/ionophore method (Vca) and the clinically important vesicles that accumulate in stored red blood cell concentrates (Vsto)

Summary

Introduction

Erythrocytes have the ability to shed hemoglobin-rich vesicles and they lose about 20% of their surface area during their life span via vesiculation[1]. The formation of these vesicles is an inherent property of the erythrocyte plasma membrane. A general characteristic of the erythrocyte vesicles is the strong depletion or even the absence of cytoskeletal components, and the relative enrichment of glycosylphosphatidylinositol (GPI)–linked proteins. There are considerable variations in the exact membrane composition of the various vesicle types that probably reflect the different mechanisms of vesicle generation; the difference in the mobility, the intrinsic curvature, and cytoskeletal attachment of the membrane components that lead to different component sorting during the budding process[9,10]

Methods

Results

Conclusion