P2X7 Is a Scavenger Receptor for Apoptotic Cells in the Absence of Its Ligand, Extracellular ATP

Abstract

Phagocytosis of apoptotic cells is essential during development and tissue remodeling. Our previous study has shown that the P2X7 receptor regulates phagocytosis of nonopsonized particles and bacteria. In this study, we demonstrate that P2X7 also mediates phagocytosis of apoptotic lymphocytes and neuronal cells by human monocyte-derived macrophages under serum-free conditions. ATP inhibited this process to a similar extent as observed with cytochalasin D. P2X7-transfected HEK-293 cells acquired the ability to phagocytose apoptotic lymphocytes. Injection of apoptotic thymocytes into the peritoneal cavity of wild-type mice resulted in their phagocytosis by macrophages, but injection of ATP prior to thymocytes markedly decreased this uptake. In contrast, ATP failed to inhibit phagocytosis of apoptotic thymocytes in vivo by P2X7-deficient peritoneal macrophages. The surface expression of P2X7 on phagocytes increased significantly during phagocytosis of either beads or apoptotic cells. A peptide screen library containing 24 biotin-conjugated peptides mimicking the extracellular domain of P2X7 was used to evaluate the binding profile to beads, bacteria, and apoptotic cells. One peptide showed binding to all particles and cell membrane lipids. Three other cysteine-containing peptides uniquely bound the surface of apoptotic cells but not viable cells, whereas substitution of alanine for cysteine abolished peptide binding. Several thiol-reactive compounds including N-acetyl-L-cysteine abolished phagocytosis of apoptotic SH-SY5Y cells by macrophages. These data suggest that the P2X7 receptor in its unactivated state acts like a scavenger receptor, and its extracellular disulphide bonds play an important role in direct recognition and engulfment of apoptotic cells.

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

“…This engulfment process for apoptotic cells is slower than for beads, and incubations of 2-3 h are required to demonstrate substantial uptake of apoptotic material. As found for beads and bacterial targets, exposure of macrophages to ATP added either in vitro or by intraperitoneal injection into mice in vivo inhibited the subsequent uptake of apoptotic lymphocytes by human or murine macrophages, respectively [29]. This effect of ATP Annexin V Fig.…”

“…It should be noted that the total phagocytic ability of peritoneal macrophages was similar for wild type and P2X7 −/− animals, presumably due to upregulation of other scavenger receptors, an effect which has also been observed in CD204 (class A scavenger receptor type I, II)-deficient mouse and CD36 knockout mouse [27,28]. [29] These data are useful in showing that the inhibitory effect of extracellular ATP on particle uptake by phagocytes can be used to define the contribution made by the P2X7 uptake pathway. Both live and heat-killed bacteria can be phagocytosed by cells expressing P2X7, although the uptake process is slower and can only be demonstrated in the absence of ambient ATP and serum [24].…”

“…However, P2X7 receptors may play a role in linking innate and adaptive immunity after phagocytosis has occurred. Confocal microscopy shows P2X7 in the phagosome membrane surrounding engulfed particles or bacteria [29,30] and recent reports suggest that P2X7 together with pannexin-1 may facilitate the delivery of bacterial products from phagosome lumen into the cytosol, where they can activate inflammasome assembly [31,32]. Moreover P2X7 has a role in facilitating the fusion of phagosome with lysosome by a process involving stimulation of phospholipase D activity presumably on the phagolysosomal membrane [33,34].…”

“…All events were gated on the low 7-AAD population to exclude necrotic cells. From Gu et al [29] % of Max CFSE intensity on gated CMTMR + cells fixed Basal (untreated) Basal (Annexin V pretreated) Fig. 3 Pretreatment of apoptotic lymphocytes with purified Annexin V does not inhibit their phagocytosis by autologous monocyte-derived macrophages.…”

“…After two washes, 500 μL of SuperSignal West Pico was added and the chemiluminescence was measured by a Glo 20/20 luminometer (Promega). From Gu et al [29] with and without annexin V before they were seeded onto the adherent macrophage layer. After 3 h incubation, the macrophages were analyzed by flow cytometry which showed these cells had engulfed substantial numbers of apoptotic lymphocytes and that annexin V had little or no inhibitory effect (Fig.…”