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

Abstract

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.

Early Development in the Peritoneal Cavity of CD49dhigh Th1 Memory Phenotype CD4+ T Cells with Enhanced B Cell Helper Activity

“…5). + T cells might be susceptible to extracellular ATP or NAD + generated during in vitro cell preparation (21). Upon adding 100 mM ATP for 15 min in a shortterm culture, we observed significantly greater downregulation of CD27 in the peritoneal CD49d (Fig.…”

Reducing Resistance to Acid Hemolysis by Iron-Contained Drug Increases the Level of Hemoglobin in the Erythrocytes of Aging Animals

“…рис.4,а), але не в самих еритроцитах (див. рис.4,б) у старих щурів за дії урфугему, внаслідок можливого звільнен-ня при їх гемолізі АТФ, який діє на ці фер-менти синтезу NO через свої пуринорецеп-тори на плазматичних мембранах лейкоцитів [17]. У разі дії препарату більш ніж у 3 рази підвищувався індекс спряження сNOS в плаз-мі крові старих тварин (див.…”

Nucleotide-Induced Membrane-Proximal Proteolysis Controls the Substrate Specificity of T Cell Ecto–ADP-Ribosyltransferase ARTC2.2

“…Previous studies have shown that gating of the P2X7 ion channel by extracellular ATP or by NAD + -dependent ADP-ribosylation induces ectodomain shedding of CD62L (4,5). To assess whether the observed downmodulation of ARTC2.2 is also mediated by ARTC2.2-catalyzed ADP-ribosylation of P2X7, we tested whether available Abs against ARTC2.2 or P2X7 could block this phenomenon (Fig.…”

“…These nucleotides are released from cells during inflammation and tissue damage and modulate T cell functions (10)(11)(12)(13)(14). Gating of P2X7 induces externalization of phosphatidylserine, shedding of metalloprotease-sensitive membrane proteins, blebbing, and pore formation (5,(15)(16)(17)(18). P2X7 is gated either directly by high concentrations of ATP acting as a soluble ligand, or indirectly by NAD + -dependent ADP-ribosylation, a covalent modification catalyzed by ecto-ADP-ribosyltransferase ARTC2.2 (5,19).…”

“…A potent physiological stimulus of ectodomain shedding is gating of the P2X7 ion channel by extracellular ATP and by NAD + -dependent ADPribosylation (3)(4)(5)(6).…”