NAD(+) and ATP Released from Injured Cells Induce P2X(7)-Dependent Shedding of CD62L and Externalization of Phosphatidylserine by Murine T Cells

Institute of Immunology, University Hospital, Hamburg, Germany.
The Journal of Immunology (Impact Factor: 4.92). 04/2009; 182(5):2898-908. DOI: 10.4049/jimmunol.0801711
Source: PubMed


Extracellular NAD(+) and ATP trigger the shedding of CD62L and the externalization of phosphatidylserine on murine T cells. These events depend on the P2X(7) ion channel. Although ATP acts as a soluble ligand to activate P2X(7), gating of P2X(7) by NAD(+) requires ecto-ADP-ribosyltransferase ART2.2-catalyzed transfer of the ADP-ribose moiety from NAD(+) onto Arg125 of P2X(7). Steady-state concentrations of NAD(+) and ATP in extracellular compartments are highly regulated and usually are well below the threshold required for activating P2X(7). The goal of this study was to identify possible endogenous sources of these nucleotides. We show that lysis of erythrocytes releases sufficient levels of NAD(+) and ATP to induce activation of P2X(7). Dilution of erythrocyte lysates or incubation of lysates at 37 degrees C revealed that signaling by ATP fades more rapidly than that by NAD(+). We further show that the routine preparation of primary lymph node and spleen cells induces the release of NAD(+) in sufficient concentrations for ART2.2 to ADP-ribosylate P2X(7), even at 4 degrees C. Gating of P2X(7) occurs when T cells are returned to 37 degrees C, rapidly inducing CD62L-shedding and PS-externalization by a substantial fraction of the cells. The "spontaneous" activation of P2X(7) during preparation of primary T cells could be prevented by i.v. injection of either the surrogate ART substrate etheno-NAD or ART2.2-inhibitory single domain Abs 10 min before sacrificing mice.

Download full-text


Available from: Sahil Adriouch, Sep 15, 2015
  • Source
    • "Beta-nicotinamide adenine dinucleotide (b-NAD+) is present in mammalian serum at around 100 nM [1], and can be released extracellularly from cells by lytic and nonlytic mechanisms [2] [3] [4] [5] [6]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of the study was to identify a signalling pathway allowing NAADP-induced intracellular NAADP increase and involving the P2Y11-like receptor. P2Y11-like and β-adrenergic receptors may play important regulatory roles within the cardiovascular system. Both receptors have been shown to be involved in triggering myocardial preconditioning. Using a Langendorff model we report a positive inotropic response induced by extracellular NAADP via P2Y11-like receptor stimulation. In cardiomyocyte cultures, P2Y11-like receptor stimulation by extracellular NAADP ([NAADP]e) increased intracellular cADP-ribose and NAADP concentration as evidenced by direct measurements. NF546, a new selective P2Y11 receptor agonist, increased intracellular cAMP, cADP-ribose and NAADP concentration confirming the involvement of the P2Y11-like receptor in this signalling pathway. NF157, a P2Y11 receptor antagonist, suppressed the increase in intracellular cADPr, NAADP and NAAD induced by either [NAADP]e or NF546. The response profile for intracellular cADP-ribose and NAADP concentration following P2Y11-like stimulation with NF546 was similar to reported data relating β-adrenergic stimulation with isoprenaline. This response represents the signature of the Gs/ADP-ribosyl cyclase activity. Moreover, this study provides a signalling pathway: intracellular NAADP increase induced by extracellular NAADP via metabotropic activity of P2Y11-like receptor. This pathway implying P2Y11-like could take part in the intracellular calcium rise reported for extracellular NAADP.
    Biochemical and Biophysical Research Communications 05/2013; 436(2). DOI:10.1016/j.bbrc.2013.04.110 · 2.30 Impact Factor
  • Source
    • "Naive T cells and in particular CD4+CD25+Foxp3+ regulatory T cells are highly sensitive to gating of P2X7 by ADP-ribosylation even at low micromolar concentrations of extracelluar NAD+ [14], [15]. This allows influx of Ca2+ and efflux of K+, and induces a cascade of prominent downstream reactions, including the rapid externalization of phosphatidylserine, ADAM-metalloprotease mediated shedding of L-selectin/CD62L, formation of a membrane pore permeable to large molecules (<900 Da) including DNA-staining dyes, and ultimately results in T cell death [16], [17]. A commonly used strain of mice, C57BL/6, carries an allelic variant of P2X7 that encodes a single point mutation (P451L) located in the long cytosolic domain of P2X7. "
    [Show abstract] [Hide abstract]
    ABSTRACT: P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.
    PLoS ONE 07/2012; 7(7):e41269. DOI:10.1371/journal.pone.0041269 · 3.23 Impact Factor
  • Source
    • "The concentration of NAD + in human plasma ranges between 10 and 50 nM [5], depending on the balance between the opposing processes of dinucleotide release from cells and its enzymatic degradation [5]. However, NAD + levels in specific tissue districts may be significantly higher than those in plasma, especially during inflammation [6] [7] [8]. Outside cells, NAD + may work as a cytokine, eliciting rapid functional responses through binding to specific purinergic type 2 receptors. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nicotinamide adenine dinucleotide (NAD(+)) is an essential co-enzyme that can be released in the extracellular milieu. Here, it may elicit signals through binding purinergic receptors. Alternatively, NAD(+) may be dismantled to adenosine, up-taken by cells and transformed to reconstitute the intracellular nucleotide pool. An articulated ecto-enzyme network is responsible for the nucleotide-nucleoside conversion. CD38 is the main mammalian enzyme that hydrolyzes NAD(+), generating Ca(2+)-active metabolites. Evidence suggests that this extracellular network may be altered or used by tumor cells to (i) nestle in protected areas, and (ii) evade the immune response. We have exploited chronic lymphocytic leukemia as a model to test the role of the ecto-enzyme network, starting by analyzing the individual elements that make up the whole picture.
    FEBS letters 06/2011; 585(11):1514-20. DOI:10.1016/j.febslet.2011.04.036 · 3.17 Impact Factor
Show more