Differential stimulation of monocytic cells results in distinct populations of microparticles

Abstract

Summary Background Microparticles (MPs), small vesicles shed from stimulated cells, permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known. Methods We studied the characteristics of MPs produced from monocytic THP-1 cells upon stimulation with lipopolysaccharide or a soluble P-selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy. Results Utilizing a novel criterion of calcein-AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte-associated immunoglobin-like-receptor (LAIR)-1, which was found only upon stimulation with the soluble P-selectin chimera. Conclusion We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.

Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro

“…However, whether the elevated level of SD MVs with selectively enriched proteins is induced via these mechanisms has yet to be studied. Although it was reported that stimulation of cells did not result in changes in the size distribution of MVs [9] , we observed that SD resulted in a different size distribution of MVs compared with non-SD conditions. We found that MVs with diameters below 0.5 μm significantly increased in number after SD treatment, whereas MVs larger than 0.5 μm decreased in number.…”

“…Upon cleavage by cytosolic esterases, calcein binds to calcium, becomes fluorescent, and is retained within MVs. Thus, only intact MVs will fluoresce, and concomitant staining of debris is avoided [9] . The data suggested that our MV sample collected following the differential centrifugation protocol had a purity of 70% ( Figure 2F), and cell debris or mass-molecule compounds were observed as calcein-negative events in the FACS analysis.…”

“…Gating and analysis were determined by a reported method [9] . Flow cytometry data were analyzed using FlowJo software, version 7.6 (Tree Star Inc, Ashland, OR, USA).…”

“…MVs were isolated from the conditioned medium of myeloma cells by continuous differential centrifugation, as described [9] . Briefly, myeloma cells harvested in the log growth phase were reseeded at 5×10 5 cells/mL in RPMI-1640 with 10% FBS or 1% FBS (we also removed FBS-derived MVs by the method of differential centrifugation before use).…”

“…These vesicles were originally considered inert cellular debris; however, it is now known that both their cytoplasmic fragments and their membranes contain a variable spectrum of molecules whose pattern specifically reflects the donor cell that secretes these MVs [9] . Release of MVs is a process that occurs in a spectrum of cell types and, more frequently, in tumor cells.…”