Microparticles released by ectocytosis from human neutrophils : characterisation, properties and functions

The field of microparticles (MPs) has gained growing interest over the last decade. Numbers of papers have come out recently describing molecular or functional characteristics of MPs derived from various cells, suggesting in vivo roles for MPs other than being inert side-products of cellular activation.
The properties and characteristics of MPs released from the surface of activated human polymorphonuclear neutrophils (PMN), called ectosomes, will be discussed in Part I. The functional implications of these characteristics with regards to cellular interaction of PMN-ectosomes, in particular with macrophages, and their circulation in blood will be addressed in Part II and III, respectively. As presented in Part I, many neutrophil-derived membrane proteins were translocated to the surface of ectosomes. There was no positive or negative selection with regards to transmembrane type versus glycophosphatidylinositol-linked type of proteins. Indeed, both types had representatives present and absent on the surface of ectosomes. In addition, ectosomes exposed several active enzymes on their surface, such as proteinase-3, myeloperoxidase, elastase and matrix metalloproteinase-9. The fact that ectosomes are unable to maintain the asymmetry of their membrane bilayer was illustrated by the presence of phosphatidylserine on their outer membrane leaflet. Ectosomes were also found to bind the first component of the classical pathway of complement, C1q; an additional finding that has been looked at more in detail in Part III.
Binding assays revealed affinity of ectosomes to endothelial cells as well as macrophages. Further analyses of the interaction between PMN ectosomes and human monocyte derived macrophages (HMDM) presented in Part II provided data suggesting that ectosomes do not
only bind, but are subsequently phagocytosed by HMDM. The sole binding of ectosomes, however, was sufficient to induce an anti-inflammatory reprogramming of HMDM. In particular, ectosomes dose-dependently counteracted the pro-inflammatory response of HMDM to stimuli such as zymosan and LPS. This effect comprised an early increase in the release of the anti-inflammatory cytokine TGFβ1 and subsequent downregulation of IL-8, IL-10 and TNFα secretion. Data obtained using neutralising anti-TGFβ antibodies suggested that both phenomena might be causally linked, at least partially.
As alluded to above, ectosomes bind C1q. As presented in Part III, ectosomes were also found to activate and fix complement. C3- and C4-fragments were detected on the surface of ectosomes after incubation with normal human serum. Using C1q- and C2-deficient serum, the ectosome-induced activation of complement could be mainly attributed to the classical pathway. Finally, the opsonisation of ectosomes by complement induced their immune adherence to erythrocytes. These data suggest that blood-borne ectosomes might be sequestered on erythrocytes, a mechanism that might drive their clearance from circulation, similarly to immune complexes.