Ectosomes of polymorphonuclear neutrophils activate multiple signaling pathways in macrophages

Ectosomes are vesicles shed directly from the cell surface. Human polymorphonuclear neutrophils release ectosomes (PMN-Ect) upon their activation. PMN-Ect expose phosphatidylserine (PS) on the outer leaflet of the plasma membrane, and down-modulate the inflammatory response of human macrophages and dendritic cells exposed to TLR-2 and -4 ligands. This down-modulation is mediated by PS via the engagement and activation of the Mer receptor tyrosine kinase (MerTK).In the present study, we demonstrate that exposure of macrophages to PMN-Ect activates directly 2 additional pathways, an immediate Ca2+ flux and a rapid release of TGF-β1. As expected, the Ca2+ flux was necessary for the activation of TLR-2 pathway with the release of cytokines. However, MerTK blockade with antibodies did not modify the Ca2+ flux, indicating an independent activation of Ca2+ by PMN-Ect. Striking was that the rapid release of TGF-β1 was independent of the MerTK pathway and did not require a Ca2+ flux. TGF-β1 was present in cytosolic storage pools, which were depleted after exposure of the macrophages to PMN-Ect, and no increase in TGF-β1 mRNA could be detected in the 3 first hours when maximal release had occurred. The release of TGF-β1 by macrophages was seen only for PMN-Ect and not for PS-liposomes or erythrocyte ectosomes, which express PS. However, blocking the PS of PMN-Ect inhibited TGF-β1 release, suggesting that PS expression was necessary although not sufficient for this release. Interestingly, the effects of PMN-Ect pre-exposure were lasting for 24 h with the macrophages being less receptive to TLR-2 activation and TGF-β1 stores remaining low.In sum, PMN-Ect induce several signaling pathways in resting and stimulated macrophages, which include independently the MerTK pathway, Ca2+ flux and the release of stored TGF-β1, and each might influence the immunomodulatory effects of macrophages.