A Loss-of-Function Polymorphism in the Human P2X7 Receptor Abolishes ATP-Mediated Killing of Mycobacteria

Saunders, Bernadette M.; Fernando, Suran L.; Sluyter, Ronald; Britton, Warwick J.; Wiley, James S.

doi:10.4049/jimmunol.171.10.5442

Cited by 114 publications

(113 citation statements)

References 31 publications

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“…Three loss-of function SNPs (T357S, E496A, and I568N) and one gain-of-function SNP (Q460R) are located in the long intracellular domain [54][55][56]58]. These loss-of-function polymorphisms lead not only to reduced P2X 7 pore function but also to impaired ATP-induced mycobacterial killing by macrophages [56,60,61,81,82]. Recent studies have found that the E496A polymorphism does not affect the electrophysiological phenotype of the P2X 7 channel in transfected oocytes and HEK293 cells [83].…”

Section: Discussionmentioning

confidence: 99%

“…Another putative gain-of-function polymorphism described recently, the Q460R substitution, appears to enhance pore activity of the P2X 7 receptor [58]. Increasing evidence from genetic association studies have demonstrated that the polymorphisms of the P2X 7 gene are implicated in various diseases such as chronic lymphocytic leukemia, tuberculosis, bipolar affective disorders, and diabetes [60][61][62][63][64][65][66][67]. It has also been reported that the polymorphisms are related to clinical outcome in allogeneic stem cell transplantation and to fracture risk and the efficacy of hormone replacement therapy [28,68].…”

Section: Introductionmentioning

confidence: 99%

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Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Sun

Chu

Singh

et al. 2009

Purinergic Signalling

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The P2X 7 receptor exhibits significant allelic polymorphism in humans, with both loss and gain of function variants potentially impacting on a variety of infectious and inflammatory disorders. At least five loss-offunction polymorphisms (G150R, R307Q, T357S, E496A, and I568N) and two gain-of-function polymorphisms (H155Y and Q460R) have been identified and characterized to date. In this study, we used RT-PCR cloning to isolate and characterize P2X 7 cDNA clones from human PBMCs and THP-1 cells. A previously unreported variant with substitutions of V80M and A166G was identified. When expressed in HEK293 cells, this variant exhibited heightened sensitivity to the P2X 7 agonist (BzATP) relative to the most frequent allele, as shown by pore formation measured by fluorescent dye uptake into cells. Mutational analyses showed that A166G alteration was critical for the gain-offunction change, while V80M was not. Full-length variants with multiple previously identified nonsynonymous SNPs (H155Y, H270R, A348T, and E496A) were also identified. Distinct functional phenotypes of the P2X 7 variants or mutants constructed with multiple polymorphisms were observed. Gain-of-function variations (A166G or H155Y) could not rescue the loss-of-function E496A polymorphism. Synergistic effects of the gain-of-function variations were also observed. We also identified the A348T alteration as a weak gain-of-function variant. Thus, these results identify the new gain-of-function variant A166G and demonstrate that multiple-gene polymorphisms contribute to functional phenotypes of the human P2X 7 receptor.Furthermore, the results demonstrate that the C-terminal of the cysteine-rich domain 1 of P2X 7 is critical for regulation of P2X 7 -mediated pore formation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Sun

Chu

Singh

et al. 2009

Purinergic Signalling

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“…P2X 7 surface expression on homozygous monocytes was significantly lower than P2X 7 surface expression on monocytes from wild-type subjects (mean fluorescent intensity of 20.676.3, n ¼ 4 vs 47.078.1, n ¼ 6 respectively, Po0.05; Figure 2c). We have previously shown that P2X 7 expression is approximately 11-fold lower on monocyte-derived macrophages homozygous for the Glu 496 Ala polymorphism than on wild-type macrophages, 11,14 however P2X 7 surface expression does not differ on lymphocytes of either genotype, 11 suggesting that this polymorphism, in addition to loss-offunction, may affect the upregulation or retention of this receptor on the cell surface of monocytes upon activation and/or differentiation to macrophages.…”

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confidence: 93%

P2X7 receptor polymorphism impairs extracellular adenosine 5′-triphosphate-induced interleukin-18 release from human monocytes

2004

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Interleukin (IL)-18 is an important proinflammatory cytokine processed and released from cells of the monocyte lineage by activation of the P2X 7 receptor by extracellular adenosine 5 0 -triphosphate (ATP). We examined if a loss-of-function polymorphism of the human P2X 7 receptor (glutamic acid-496 to alanine) impairs this process. Using a whole blood-based assay, ATP-induced release of IL-18 from homozygous subjects after 120 min incubation with ATP was 42% of that from wildtype subjects. Moreover, the level of ATP-induced IL-18 release from lipopolysaccharide (LPS)-primed monocytes of homozygous subjects after 30 and 60 min incubation with ATP was 21 and 44%, respectively, of that from wild-type monocytes. Nigericin, a K þ ionophore, induced a similar release of IL-18 from monocytes of either genotype. ATP-induced ethidium þ uptake in LPS-primed, monocytes of homozygous subjects was only 11% of that in wild-type monocytes, while P2X 7 surface expression on LPS-primed, homozygous monocytes was 44% of that on wild-type monocytes.

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“…[26][27][28] Certain genetic variations in the P2X 7 molecule reduce or abolish the ability of ATP to activate the receptor as well as the capacity of macrophages for ATP-induced killing of pathogens, and this evidence, together with the effect of P2X 7 antagonists, point to the bactericidal effects of ATP working via the P2X 7 receptor. 26,29,30 It is noteworthy that these studies of P2X 7 -mediated killing of pathogens have generally examined the effect of ATP on viability of microbes engulfed within macrophages, and this study is the first to explore a function of the P2X 7 molecule in the absence of extracellular ATP.…”

Section: Introductionmentioning

confidence: 99%

“…As a sequel to this second phase, the activation of cell surface P2X 7 receptors by ATP drives a process of phagosome-lysosome fusion, elevation of cytosolic calcium, and killing of engulfed bacteria. 26,27,29 P2X 7 regulated phagocytosis represents a new biologic function for this membrane receptor, which has clearly separate features from the well-described P2X 7 cation-selective channel. 50 Thus, CytD, a potent phagocytosis inhibitor, has no effect on P2X 7 pore formation measured by ATP-induced ethidium ϩ uptake.…”

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confidence: 99%

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

Saunders

Jursik

et al. 2010

Blood

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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...

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A Loss-of-Function Polymorphism in the Human P2X7 Receptor Abolishes ATP-Mediated Killing of Mycobacteria

Cited by 114 publications

References 31 publications

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

P2X7 receptor polymorphism impairs extracellular adenosine 5′-triphosphate-induced interleukin-18 release from human monocytes

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

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