Monocytic THP-1 cells stimulated by normal human serum (NHS) release cytokine-bearing plasma membrane-derived vesicles (PMVs), and can be inhibited by methyl-beta-cyclodextrin, calpeptin and Rho-kinase inhibitor, Y-27632 (98.27)

Abstract

Abstract Stimulation of human acute monocytic cells with sublytic complement leads to an increase of intracellular calcium resulting in the release of two forms of microvesicles, one derived from the surface membrane (between 0.1-1 μm), which we term plasma membrane-derived vesicles (PMVs) and exosomes (50-100nm) released by exocytosis. Much work has been documented on the release of PMVs, using various stimuli, and the roles they play in disease, but comparatively little is available on inhibition of PMV release. Although upon stimulation cells release exosomes in addition to PMVs, little has been reported on ways of differentiating between these two microvesicle types. Our aim was therefore to identify inhibitors of PMV release and to effectively differentiate between PMVs and exosomes. Methyl-β -cyclodextrin, an inhibitor of lipid rafts, calpeptin, which inhibits calpain and Rho-kinase inhibitor, Y-27632 were found to inhibit effectively the release of PMVs. Exosomes separated from PMVs by sucrose gradient were confirmed by electron microscopy and compared to PMVs were found to express less phosphatidylserine, but more CD63. Of the various cytokines carried by PMVs and exosomes, macrophage migration inhibition factor was found at a four-fold higher level in PMVs. Source of Research Support: Royal Society, London.