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J. Biol. Chem., Vol. 280, Issue 7, 5843-5853, February 18, 2005

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Distinct Signaling Pathways Are Involved in Leukosialin (CD43) Down-regulation, Membrane Blebbing, and Phospholipid Scrambling during Neutrophil Apoptosis^*

Patrick Nusbaum, Claudianne Lainé, Mohamed Bouaouina, Stéphanie Seveau, Elisabeth M. Cramer¶, Jean Marc Masse¶, Philippe Lesavre, and Lise Halbwachs-Mecarelli||

From the INSERM U507, Hôpital Necker and the Unité Intéractions Bactéries-Cellules, Institut Pasteur, 75015 Paris, France and the ^¶Institut Cochin, 75014 Paris, France

Although leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Membrane blebbing is a transient event, hardly appreciated during the asynchronous, spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15 °C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 °C. CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. 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. Phosphatidylserine externalization required caspases and protein kinase C (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, whereas CD43 down-regulation involved caspases but neither PKC nor MLCK. Furthermore, CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultracentrifugation of apoptotic cell supernatants made it possible to recover <1 µm microparticles, which contained the entire CD43 molecule. These microparticles expressed neutrophil membrane markers such as CD11b, CD66b, and CD63, together with CD43. In conclusion, we show that the three early membrane events of apoptosis, namely blebbing, phosphatidylserine externalization, and CD43 down-regulation, result from different signaling pathways and can occur independently from one another. CD43 down-regulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.

Received for publication, November 29, 2004

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material in the form of Videos 1-3 on bleb formation.

^|| To whom correspondence should be addressed: INSERM U 507, Hôpital Necker, 161 Rue de Sèvres, 75015 Paris, France. Tel.: 33-144-49-5232; Fax: 33-145-66-5133; E-mail: mecarelli{at}necker.fr.

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