Microparticles released by human neutrophils adhere to erythrocytes in the presence of complement

Abstract

The release of cell surface-derived microparticles, or ectosomes, has now been described for many different cell types. In various diseases characterized by systemic inflammation, the numbers of ectosomes released from specific cell-types are found increased manifold in the circulation. Their pro-inflammatory and pro-coagulant functions make them potentially important actors in disease establishment and/or progression. Until now, ectosomes have been believed to be free in the circulation. Herein, we provide evidence for sequestration of ectosomes derived from human polymorphonuclear neutrophils to erythrocytes, similarly to immune complexes. We show that ectosomes activate and bind complement in vitro. In whole blood, opsonization of ectosomes by complement mediated their immune adherence to erythrocytes through complement receptor 1. Taken together, our data suggest an important role for complement and erythrocytes in the sequestration, and possibly clearance, of blood-borne ectosomes stemming from neutrophils. The immune adherence described here may modify the biological activity and function of ectosomes.

Introduction

When challenging polymorphonuclear leukocytes (PMN) with sublytic amounts of complement, Stein and Luzio observed the release of vesicles that pinched off directly from the cell membrane (ectocytosis) [1]. A similar phenomenon occurs in many different cell types in response to a variety of stimuli. Ectocytosis occurs immediately after stimulation and is therefore timely and mechanistically distinct from apoptosis. Beside PMN, endothelial cells, monocytes, platelets, and erythrocytes are known to release microparticles by ectocytosis in vitro and in vivo [2], [3], [4], [5], [6]. For clarity, we will refer to “ectosomes” when describing such microparticles. A number of different functions have been attributed to ectosomes. For example, they allow the rapid shedding of stored interleukin-1β, released by activated THP-1 cells [7]. Ectosomes serve as intercellular protein carriers for tissue factor and the chemokine receptor CCR5 [8], [9]. As far as platelet ectosomes are concerned, their most prominent feature is a pronounced procoagulant activity. Interestingly, cell-derived adhesion molecules exposed by ectosomes of various origins are known to mediate several functions of ectosomes, e.g., hematopoietic recovery after transplantation, thrombus formation, and platelet–vessel wall interactions [10], [11], [12], [13]. The functions of ectosomes described until now are largely favoring inflammation and coagulation. In contrast, we recently described PMN ectosomes to be so far the only ectosomes having anti-inflammatory properties. Indeed, we found that PMN ectosomes downregulate the pro-inflammatory response of macrophages to stimuli such as zymosan and LPS [14].

Microparticles of diverse cellular origin are present in blood of normal individuals [15], [16], [17]. Although most such microparticles correspond probably to ectosomes originating from different cells, it is possible that some are apoptotic bodies or exosomes [16], [17], [18]. Thus we will refer here to “microparticles” when their origin has not been defined. Quantitative and qualitative changes, i.e., increased numbers of microparticles derived from different origins, were described in many diseases including sepsis [19], severe trauma [20], paroxysmal nocturnal hemoglobinuria [21], diabetes [22], [23], acute coronary syndromes [24], and lupus [2]. However, even in sepsis the number of PMN-derived microparticles is surprisingly low, when compared to the high numbers of ectosomes released by activated PMN in vitro.

Here we present evidence that most ectosomes of PMN might not circulate freely, as assumed until now. Indeed, ectosomes released by human PMN activate the classical pathway of complement and fix C4 and C3 fragments. These opsonized ectosomes bind in turn to erythrocytes via the complement receptor 1 (CD35/CR1).