gms | German Medical Science

Background: Extracellular vesicles (EVs) can be released from nearly all mammalian cells after cellular activation or apoptosis induction. To date different subtypes of EVs have been described. Microvesicles represent large EVs (LEVs) released from the cellular surface, while exosomes are small EVs (SEVs) released from an intracellular compartment. Most likely different stimuli promote the release of distinct EV populations. Therefore, we characterized EVs releasedfrom human T- lymphocytes considering the respective release stimuli, namely activation and induction of apoptosis. Moreover, we wanted to investigate the role of distinct EV subtypes in the regulation of immune responses. A dysregulation of apoptosis and clearance is a key feature of SLE pathogenesis and we show here, that apoptosis induction causes a substantial release of LEVs. Thus, it seems likely that distinct EVs populations might play a role in the pathogenesis of SLE. In fact, we have previously shown, that microveicles released from apoptotic cells can induce an alternative maturation of dendritic cells, which is disturbed in SLE patients. However, a systematic characterization of distinct EV populations released after cellular activation or apoptosis induction is very important to further understand these phenomena.

Methods: EV subpopulations were isolated from activated and apoptozing T-cellsby differential Ultracentrifugation (100.000 g for SEVs; 10.000 g for LEVs). The morphology of these isolated EVs was then analyzed by transmission electron microscopy andnano particle tracking analysis. Protein content of isolated EVs was analyzed by 2D-gel electrophoresis and subsequent mass spectrometry. Further, proteins were detected by western blot analysis.

Results: We could clearly separate two EV populations. SEVs showed an average diameter <200 nm, while LEVs were larger vesicles with a diameter range between 200 and 1000 nm. Apoptosis induction caused a massive release of LEVs, while activated cells released SEVs and LEVs in considerably lower amounts. LEVs contained signaling proteins and proteins of the actin-myosin cytoskeleton. SEVs carried cytoplasmic/endosomal proteins like the 70-kDa heat shock protein (HSP70) or tumor susceptibility 101 (TSG101), microtubule-associated proteins, and ubiquitinated proteins. The protein expression profile of SEVs and LEVs changed substantially after the induction of apoptosis. Interestingly, after apoptosis induction HSP70 and TSG101 (often used as exosome markers) were highly expressed within LEVs. In contrast to HSP70 and TSG101, gelsolin and eps15 homology domain-containing protein 3 (EHD3) turned out to be specific for SEVs irrespective of the stimulus causing the EV release. Finally, we detected several subunits of the proteasome (PSMB9, PSMB10) as well as the danger signal HMGB1 exclusively within apoptotic cell-released LEVs. Thus, we were able to identify new marker proteins that can be useful to discriminate between distinct LEV subpopulations.

Conclusion: In this present study we focussed on the characterization of the protein cargo of distinct EV subpopulations released either after cellular activation or after the induction of apoptosis. We clearly demonstrated that apoptosis induction causes a substantial release of a distinct EVs subpopulation, namely LEVs (also referred to as microvesicles orapoptotic bodies). These LEVs carry a specific protein cargo. Moreover, we present here a set of new marker proteins which can be used to discriminate between distinct EV populations. We have previously shown that EVs released from apoptotic cells induce an alternative maturation of dendritic cells. The phenotype of these dendritic cells was interestingly characterized by a downregulation of MHC class II molecules, which was absent in SLE patients. Our data suggest that EVs, namely large EVs play a role in the pathogenesis of SLE. Currently, we are investigating the protein profile of distinct EV subpopulations released from T-cells obtained from SLE patients and normal healthy individuals to further understand how distinct EVs can mediate intercellular communication in healthy individuals and SLE patients.

This work was supported by a "DGRh-Forschungsförderung".