The P2X7-nonmuscle myosin membrane complex regulates phagocytosis of nonopsonized particles and bacteria by a pathway attenuated by extracellular ATP

Abstract

Phagocytosis of nonopsonized bacteria is central to innate immunity, but its regulation is less defined. We show that overexpression of the P2X 7 receptor greatly augments the phagocytosis of nonopsonized beads and heat-killed bacteria by transfected HEK-293 cells, whereas blocking P2X 7 expression by siRNA significantly reduces the phagocytic ability of human monocytic cells. An intact P2X 7 -nonmuscle myosin complex is required for phagocytosis of nonopsonized beads because activation of P2X 7 receptors by adenosine triphosphate (ATP), which dissociates myosin IIA from the P2X 7 complex, inhibits this phagocytic pathway. Fresh human monocytes rapidly phagocytosed live and heat-killed Staphylococcus aureus and Escherichia coli in the absence of serum, but the uptake was reduced by prior incubation with ATP, or P2X 7 monoclonal antibody, or recombinant P2X 7 extracellular domain. Injection of beads or bacteria into the peritoneal cavity of mice resulted in their brisk phagocytosis by macrophages, but injection of ATP before particles markedly decreased this uptake. These data demonstrate a novel pathway of phagocytosis of nonopsonized particles and bacteria, which operate in vivo and require an intact P2X 7 -nonmuscle myosin IIA membrane complex. The inhibitory effect of ATP on particle uptake by the macrophage is regulated by the P2X 7 receptor and defines this phagocytic pathway. IntroductionPhagocytosis is a fundamental function of innate immune cells, and many receptors, including Fc receptors, complement receptors, various integrins, and scavenger receptors, have been shown to mediate phagocytosis of opsonized microbes. 1 Several scavenger receptors have also been identified that directly interact with nonopsonized particles, and these are broadly classified as class A (macrophage receptor with collagenous structure) and class B (2 transmembrane receptors), 2 whereas among others is the C-type lectin-like receptor, dectin-1. [3][4][5][6][7][8][9] Assays for these scavenger receptors use nonselective inhibitors, polyinosinic acid, liposomes containing phosphatidylserine, oxidized low-density lipoprotein, or fucoidan, to inhibit this pathway. Phagocytosis of particles is accompanied by major rearrangements in the actin-myosin cytoskeleton, but there is little information on how scavenger receptors interact with proteins of the cytoskeleton. Nevertheless, cytochalasin D (CytD), a classic inhibitor of F-actin polymerization, is able to block particle uptake mediated by all these receptors.The P2X 7 receptor is a ligand-gated cation channel highly expressed on monocytes and macrophages and exists within the plasma membrane as a large multimolecular complex containing components of the cytoskeleton, including ␤-actin 10 and nonmuscle myosin. 11 Activation of P2X 7 mediates actin reorganization and membrane blebbing in mouse macrophages, 12-14 suggesting a close functional connection between the P2X 7 receptor and the actin-myosin cytoskeleton. The P2X 7 receptor has 2 transmembrane domains with intracellular ami...

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

“…Exposure of wild type P2X7 +/+ macrophages to ATP, either in vitro or by intraperitoneal injection of ATP in vivo reduced the subsequent uptake of beads by these mononuclear phagocytes when the beads were injected 30 min after the ATP. In contrast, exposure of P2X7 −/− peritoneal macrophages to ATP had no effect on the subsequent uptake of beads by macrophages either in vitro or after instillation of beads into the peritoneal cavity [24]. 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].…”

“…A reliable method for establishing the phagocytic role of a receptor is to express it on a nonphagocytic cell type (such as HEK-293, an epithelial cell line) and show it confers phagocytic capacity on the recipient cell. Transfection of HEK-293 cells with P2X7-DsRed constructs conferred a brisk uptake of beads by those cells expressing high amounts of P2X7-DsRed, whereas cells with low P2X7 expression took up few or no beads [24]. Moreover, preincubation of the P2X7-transfected cells with mAb (clone L4) to the ectodomain of P2X7 inhibited the uptake of the fluorescent beads down to the level observed with CytD [24].…”

“…Confocal microscopy has shown that internal membranes within the cell fuse with plasma membrane during the course of particle ingestion, and that recycling endosomes are the primary source of membrane for enlargement of phagocytic cup [22]. Flow cytometric assessment of particle engulfment has to some extent replaced microscopic observation [23] particularly as the kinetics of uptake of fluorescent targets by phagocytes can be followed by instruments capable of time-resolved measurements in a stirred cuvette at 37°C [24]. These assays of phagocytosis by flow cytometry usually include measurements of fluorescence particle uptake by cells pre-incubated with cytochalasin D (CytD), an inhibitor of F-actin polymerization and phagocytic cup formation which allows the assay to distinguish engulfment of particles from adhesion.…”

“…Transfection of HEK-293 cells with P2X7-DsRed constructs conferred a brisk uptake of beads by those cells expressing high amounts of P2X7-DsRed, whereas cells with low P2X7 expression took up few or no beads [24]. Moreover, preincubation of the P2X7-transfected cells with mAb (clone L4) to the ectodomain of P2X7 inhibited the uptake of the fluorescent beads down to the level observed with CytD [24]. A second approach to the phagocytic role of P2X7 involved peritoneal macrophages from C57BL/6 mice with and without deletion of the P2X7 gene [26], which confirmed that attenuation of phagocytosis by ATP was mediated via the P2X7 receptor.…”

“…[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]. Numerous receptors have been shown to mediate bacterial uptake by phagocytes and the contribution of P2X7-mediated phagocytosis to the uptake of bacteria may be limited to serum-free compartments of the body such as the central nervous system.…”