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,Lise Halbwachs-Mecarelli

doi:10.1074/jbc.m413405200

Patrick Nusbaum, Claudianne Lainé + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m413405200

Copy DOI

Abstract

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 degrees C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 degrees 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 microM 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.

Highlights

The most obvious initial stages of neutrophil apoptosis occur at the plasma membrane as follows. (i) Cytoskeleton move
Leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized
Modulation of Membrane Receptors and Phosphatidylserine Exposure on Apoptotic Neutrophils—As reported previously [7], Fig. 1A shows that CD43 membrane expression decreases during neutrophil incubation for 20 h at 37 °C as compared with cells maintained at 4 °C

Summary

Introduction

The most obvious initial stages of neutrophil apoptosis occur at the plasma membrane as follows. (i) Cytoskeleton move-. (iii) Membrane expression of various receptors decreases [3]. The consequence of decreased expression of functional receptors such as adhesion molecules, phagocytic receptors, formyl-methionyl-leucyl-phenylalanine receptors, or TNF receptors is probably the turning off of neutrophil cellular functions [2, 3, 6, 7]. The consequence of membrane blebbing is not known. It has been shown in some cases to result in a decreased expression of membrane receptors via the release of bleb-derived vesicles bearing these receptors [8]. CD43 expression decreases but, as shown here and unlike what had been postulated [3, 7], without proteolysis or internalization of the molecule. We investigated the release of membrane vesicles during neutrophil apoptosis in relation to the decreased expression of CD43. We show that CD43 down-regulation parallels but does not result from membrane blebbing, with CD43 being released on microvesicles distinct from membrane blebs.

Objectives

Methods

Results

Conclusion