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NAD⁺ and ATP Released from Injured Cells Induce P2X₇-Dependent Shedding of CD62L and Externalization of Phosphatidylserine by Murine T Cells¹

Felix Scheuplein^2,*,, Nicole Schwarz^2,*, Sahil Adriouch^*,, Christian Krebs^*, Peter Bannas^*, Björn Rissiek^*, Michel Seman, Friedrich Haag^* and Friedrich Koch-Nolte^3,*

^* Institute of Immunology, University Hospital, Hamburg, Germany; The Jackson Laboratory, Bar Harbor, ME, 04609, and Inserm U905 and Faculté de médicine et de pharmacie, Université de Rouen, Rouen, France

Extracellular NAD⁺ and ATP trigger the shedding of CD62L and the externalization of phosphatidylserine on murine T cells. These events depend on the P2X₇ ion channel. Although ATP acts as a soluble ligand to activate P2X₇, gating of P2X₇ by NAD⁺ requires ecto-ADP-ribosyltransferase ART2.2-catalyzed transfer of the ADP-ribose moiety from NAD⁺ onto Arg125 of P2X₇. Steady-state concentrations of NAD⁺ and ATP in extracellular compartments are highly regulated and usually are well below the threshold required for activating P2X₇. 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 P2X₇. Dilution of erythrocyte lysates or incubation of lysates at 37°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 P2X₇, even at 4°C. Gating of P2X₇ occurs when T cells are returned to 37°C, rapidly inducing CD62L-shedding and PS-externalization by a substantial fraction of the cells. The "spontaneous" activation of P2X₇ 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Deutsche Forschungsgemeinschaft Grant No310/6 (to F.K.-N. and F.H.) and by stipends from the Werner Otto Foundation (to C.K. and P.B.) and from the Fondation pour la Recherche Medical (to S.A.).

² F.S. and N.S. contributed equally to this study.

³ Address correspondence and reprint requests to Dr. Friedrich Koch-Nolte, Institute of Immunology, University Hospital, Hamburg, Germany. E-mail address: nolte{at}uke.uni-hamburg.de

⁴ Abbreviations used in this paper: ART, ADP-ribosyltransferase; etheno-NAD⁺, 1,N6-Ethenonicotinamide adenine dinucleotide; DC, dendritic cell; PS, phosphatidylserine; WT, wild type; PI, propidium iodide; sdAb, single domain Ab.