In order to verify whether the cytokine-mediated MV release is peculiar of this cell line or, alternatively, represents a general phenomenon, the same experiments were carried out by using the murine microglia cell line BV2. Long exposures (24 h) to IFN-γ and IL-4 enhanced the release of MVs also from human blood monocytes (p: 0.0003 and p: 0.0081) (Figure 3E). The effect of different cytokines on the release of MVs was tested by western blotting: similarly to CHME-5, BV2 cells respond to IFN-γ and IL-4 by releasing MVs. Also IL-13, IL-23, TGF-β, TNF-α, and IL-6 were found to share this property; on the other hand, IL-5 and IL-27 were not effective (Figure 3F). Previous reports had already demonstrated a strong proliferative effect of IFN-γ and IL-4 on T (17–19) and B lymphocytes (20); in contrast, the contribution of the two cytokines in macrophage cell expansion is contradictory, mainly as far as IL-4 is concerned. IFN-γ potently reduces myeloid cell proliferation (21) and protects cells from apoptosis by inducing p21-waf1 (22). p21-waf1 induction and cell cycle arrest have also been reported in IL-4-treated macrophages (23); however, an opposite observation has also been published (24). Therefore, we checked whether IFN-γ and IL-4 increase the number of microglia cells by accelerating cell cycle progression: in this case, the high levels of MVs induced by such stimuli would be simply due to a cell number higher than that of unstimulated controls. Upon IFN-γ or IL-4 treatment (for 12 and 24 h), microglia cells (BV2 and CHME-5) were stained with Ki67, a protein expressed in the nucleus of proliferating cells, and with DAPI for nuclei labeling. Cells were imaged and the fraction of Ki67-positive nuclei, as well as the total cell number, was calculated. Figure S3 in Supplementary Material shows that, at both 12 and 24 h, the percentage of proliferating (Ki67+) IFN-γ stimulated cells is significantly lower than that of IL-4 treated or untreated controls. The total cell number decreased after both IFN-γ and IL-4 treatment, confirming the observation previously reported by others (21–23). Therefore, the higher amounts of MV released by cytokines are not due to increased cell proliferation. Apoptosis has been excluded by MTT assays performed on cells treated with IFN-γ or IL-4 for 24 h (Figures S3A–F in Supplementary Material). Moreover, by western blotting we cannot detect the activated form of caspase-3 in the cells treated with ATP or in those exposed to cytokines (Figure S3G in Supplementary Material). IFN-γ and IL-4 trigger the production of MVs from myeloid cells in overlapping amounts and with similar kinetics but, as expected from our previous observations (10), with a different molecular signature. In the context of inflammation, one of the best characterized cargo of MVs is IL-1β, a cytokine with strong pro-inflammatory effects. It has been already described the ability of exATP to sustain the release of MVs loaded with IL-1β from microglia cells primed with LPS (25), a potent trigger of inflammation. Hence, we wondered whether MVs deriving from IFN-γ and IL-4 stimulation are characterized by different levels of IL-1-β.