Engulfment of apoptotic cells involves the redistribution of membrane phosphatidylserine on phagocyte and prey

Abstract

he clearance of cells that are dying by apoptosis is essential to the control of tissue homeostasis 1 . In contrast to classical models of phagocytosis, the molecular dissection of engulfment of apoptotic cells is still preliminary 1 . Genetic approaches in Caenorhabditis elegans have yielded several mutants, characterized by the persistence of apoptotic corpses 2-5 . In mammals, in vitro studies have mostly shed light on the macrophage receptors engaged in the recognition of apoptotic surfaces 1,6 . Both of these approaches highlighted the importance of an ATP-binding cassette (ABC)-type transporter, named CED-7 in C. elegans and ABC1 in mouse 4,7 . The requirement for ABC1 on the macrophage surface is specific to the engulfment 7 . Here we assess the distinctive features of engulfment in comparison with other forms of phagocytosis, and find that engulfment exhibits a unique sensitivity to the ABC1 blocker glyburide 8 . In addition, glyburide, as well as antimycin and oligomycin, affects membrane behaviour in both partners of engulfment: when applied to either phagocyte or prey, these drugs impair the Ca 2+ -induced transmembrane randomization of phospholipids 9 . Our results show that engulfment requires a symmetrical transbilayer movement of lipids across the membranes of both the phagocyte and the prey.To differentiate the engulfment from classical forms of phagocytosis, we analysed the effect of pretreatment of mouse macrophages with classical inhibitors of phagocytosis 10 . The phagocytosis of opsonized sheep red blood cells and of apoptotic thymocytes exhibited similar sensitivity to cytochalasin D, lysosomotropic agents, cytosolic acidification, inhibitors of protein kinase C, and nocodazole ( Fig. 1A).However, sensitivity to glyburide 8 was correlated to the particle to be ingested (Fig. 1B). The uptake of yeast or opsonized sheep red blood cells was unaffected by the drug (yeast, 97 ± 8% of control; opsonized cells, 98.8 ± 3% of control) whereas the engulfment of apoptotic thymocytes was reduced to 56 ± 8% of control. As the larger size of the apoptotic thymocytes could influence the mechanics of engulfment, we checked that the ingestion of opsonized apoptotic thymocytes was insensitive to glyburide (116 ± 11% of control). Finally, we showed that the engulfment of fixed and unfixed apoptotic cells was equally efficient and sensitive to glyburide (fixed, 66 ± 3% of control; unfixed, 56 ± 8% of control), thus ruling out the possibility that glyburide acts by blocking the ABC1-mediated transport of a substrate released from the cell.Glyburide has been reported to decrease secretion of interleukin-1β (IL-1β) 11 , and IL-1β can potentiate engulfment 12 ; however, engulfment by macrophages from IL-1β-deficient mice was similarly inhibited by the drug (46 ± 4% of control).As the phosphatidylserine exposure by the apoptotic prey is the key to recognition by macrophages 6 , we extended our analysis to other phosphatidylserine-exposing particles, that is, cells rendered necrotic by heat 13 and human red blood cel...

Treatment with annexin V increases immunogenicity of apoptotic human T-cells in Balb/c mice

“…6 Furthermore, it was demonstrated that redistribution of PS both on phagocyte and prey is involved in the engulfment of apoptotic cells. 7 This paper confirmed the inhibition by annexin V of apoptotic cell uptake. 7 In addition, annexin V was shown to decelerate apoptosis in CEM cells by its Ca 2+ channel activity.…”

“…7 This paper confirmed the inhibition by annexin V of apoptotic cell uptake. 7 In addition, annexin V was shown to decelerate apoptosis in CEM cells by its Ca 2+ channel activity. 8 Usually engulfment of apoptotic cells induces neither inflammation 5 nor an immune response.…”

“…24 In recent publications it was shown that AxV generally leads to a marked inhibition of the uptake of apoptotic cells by both activated and unactivated macrophages. 6,7 Treatment of apoptotic cells with AxV increased their immunogenicity, suggesting that they were able to encounter a pathway leading to both antigen presentation and delivery of costimulatory signals. We speculate that AxV interfered with the PS recognition and the highly efficient engulfment of apoptotic cells by macrophages.…”

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways

“…Neutrophils stimulated with chemotactic peptides, such as formylated Met-Leu-Phe (fMLP), exhibit transient cell surface PS expression [10]. Macrophages per se undergo membrane PS flip during their recognition and phagocytosis of dead cells [11][12][13][14]. Similarly, rapid changes in cell surface PS triggered by the natively expressed P2X7R in various leukocyte types have been associated with physiological signal transduction.…”

UV irradiation inhibits ABC transporters via generation of ADP-ribose by concerted action of poly(ADP-ribose) polymerase-1 and glycohydrolase

“…6 Furthermore, ABC1 was shown to be involved in engulfment of apoptotic cells. 7,8 One of the physiological roles of ABC transporters is detoxification by extrusion of various toxic compounds from the cytoplasm. [9][10][11] Detoxification is particularly crucial when cell injury occurs.…”