The dynamics of ATP- and cytokine-mediated shedding are very different (few minutes versus 24 h, respectively), making also a quantitative comparison difficult, although cytokines in general appear less potent than ATP. The relatively long time required by cytokines made us hypothesize that their ability to induce MV release is related to the transcription of genes controlled by the same cytokines. A subtoxic concentration of actD, a potent inhibitor of transcription, completely abolished MV production in the presence of cytokines but not of ATP. Even more interestingly, in the presence of the inhibitor the number of MVs produced was lower than that of untreated cells. The latter differential results obtained with cytokines and ATP strongly suggest that: (i) the factor/s involved in cytokine-enhanced MV generation is/are the same required for the constitutive MV release and, as a consequence (ii) the promoting effects of cytokines on MV release may represent an increase of the basal shedding activity present at the plasma membrane of all cells. In contrast, the antagonists of P2X7 signaling pathway did not reduce significantly the constitutive release of MVs, as previously described (7). Since cytokines induce transcription of specific gene networks, future work will be aimed at the identification of the genes involved in vesicle generation. In our hands, the inhibition of three out of the eleven transcriptional factors we found to be strongly associated to the genes regulated by both IFN-γ and IL-4 were ineffective in reducing the release of MV after cytokine treatment. In order to clarify the contribution of exATP in the generation of the myeloid cell-derived MVs released during neuroinflammation, we extended the work to a disease model. Mice affected by EAE, a model of human multiple sclerosis, were treated with imipramine, which changed neither the amount of MVs released to the CSF in the chronic phase of the disease, nor the neurological traits of the diseased animals. Therefore, it seems that an ASMase-independent mechanism is responsible for the enhanced release of MVs in mice with EAE, perhaps controlled by cytokine signaling. Although this conclusion is encouraging, two considerations about the role of exATP should be clarified: (i) cytokines and purinergic signaling are linked by the fact that the P2X7 receptor is upregulated by the former as observed by us and others (44–46). This may be indicative of the fact that cytokines-primed cells are somehow sensitized to the exATP released by surrounding cells; whether this upregulation is sufficient to contribute to plasma membrane shedding in vivo has to be proven. In addition, agonists of TRPV1channels have been also recently shown to acutely enhance MV production from microglia through a pathway involving p38 MAP kinase, an enzyme which is also activated downstream P2X7 (47). (ii) The role of P2X7-dependent signaling in EAE is controversial: in fact, treating EAE mice with oxATP or BBG reduced the severity of the disease, with a decrease of neuronal loss and increase of oligodendroglia survival (45), whereas P2X7 knockout mice displayed a more severe pathology (48). However, another study based on a similar knockout model reported opposite results (49). Anyway, whether the positive role of P2X7 antagonists on the pathology depends on modulation of MV release, is not known.