New trends in polycystic ovary syndrome (PCOS)
CD43 downregulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing, which result from different signaling pathways and can occur independently from one another.
Abstract
Background: Leukosialin (CD43) membrane expression decreases during neutrophil apoptosis but CD43 is neither proteolysed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Methods: Membrane blebbing is a transient event, hardly appreciated during asynchronous spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15°C allowed to observe numerous blebbing neutrophils for a short 1-hour period at 37°C. Blebs detaching from the cell body were observed by time-lapse fluorescence microscopy and the release of bleb-derived vesicles was followed by flow cytometry. Results: CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Phosphatidylserine externalization required caspases and PKC but not the myosin light chain kinase MLCK. By contrast, bleb formation and release was caspase and PKC-independent but required an active MLCK, while CD43 down-regulation involved caspases but neither PKC nor MLCK. CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultra centrifugation of apoptotic cells supernatant allowed to recover <1 micron microparticles, which contained the entire CD43 molecule.These microparticles expressed neutrophil membrane markers such as CD11b, CD66b and CD63, together with CD43. Conclusion: Blebbing, phosphatidylserine externalization and CD43 down-regulation result from different signaling pathways and can occur independently from one another. CD43 downregulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.