Lymphocytes express a number of NAD-metabolizing ectoenzymes, including mono(ADP-ribosyl)transferases (ART) and ADP ribosylcyclases. These enzymes may regulate lymphocyte functions following the release of NAD in injured or inflammatory tissues We report here that extracellular NAD induces apoptosis in BALB/c splenic T cells with an IC50 of 3–5 μM. Annexin V staining of cells was observed already 10 min after treatment with NAD in the absence of any additional signal. Removal of GPI-anchored cell surface proteins by phosphatidylinositol-specific phospholipase C treatment rendered cells resistant to NAD-mediated apoptosis. RT-PCR analyses revealed that resting BALB/c T cells expressed the genes for GPI-anchored ART2.1 and ART2.2 but not ART1. ART2-specific antisera blocked radiolabeling of cell surface proteins with both [32P]NAD and NAD-mediated apoptosis. Further analyses revealed that natural knockout mice for Art2.a (C57BL/6) or Art2.b (NZW) were resistant to NAD-mediated apoptosis. Labeling with [32P]NAD revealed strong cell surface ART activity on T cells of C57BL/6 and little if any activity on cells of NZW mice. T cells of (C57BL/6 × NZW)F1 animals showed strong cell surface ART activity and were very sensitive to NAD-induced apoptosis. As in BALB/c T cells, ART2-specific antisera blocked cell surface ART activity and apoptosis in (C57BL/6 × NZW)F1 T cells. The fact that T cells of F1 animals are sensitive to rapid NAD-induced apoptosis suggests that this effect requires the complementation of (at least) two genetic components. We propose that one of these is cell surface ART2.2 activity (defective in the NZW parent), the other a downstream effector of ADP-ribosylation (defective in the C57BL/6 parent).

This is the first study reporting the inactivation of a member of the mouse gene family of toxin-related ecto-ADP-ribosyltransferases (ARTs). Transfer of the ADP-ribose moiety from NAD onto extracellular arginine residues on T-cell membrane proteins is mediated by glycosylphosphatidylinositol-linked cell surface ARTs. Exposure of T cells to ecto-NAD blocks T-cell activation and induces T-cell apoptosis. To determine a possible role of ecto-ART2.1 and ART2.2 in these processes, we generated ART2.1/ART2.2 double-knockout mice. ART2-deficient mice were healthy and fertile and showed normal development of lymphoid organs. ART2-deficient T cells showed a dramatically reduced capacity to ADP-ribosylate cell surface proteins, indicating that most if not all ART activity on the T-cell surface can be attributed to the ART2s. Moreover, ART2-deficient T cells were completely resistant to NAD-induced apoptosis and partially resistant to NADmediated suppression of proliferation. These results demonstrate that the ART2 ectoenzymes are an essential component in the regulation of T-cell functions by extracellular NAD, e.g., following release of NAD upon lysis of cells in tissue injury and inflammation.Posttranslational modification of proteins provides an important mechanism for regulating cellular functions. Protein phosphorylation mediated by protein kinases is well established as an essential component of intracellular signal transduction. Mounting evidence suggests that protein ADP-ribosylation mediated by ADP-ribosyltransferases (ARTs) may serve a similar regulatory function in the extracellular environment (10, 18, 32).We have previously reported the molecular cloning and biochemical characterization of a family of glycosylphosphatidylinositol (GPI)-anchored and secretory mammalian cell surface mono(ADP-ribosyl)transferases (ART1 to ART5) which are related in structure and function to bacterial ADP-ribosylating toxins (20,21). These enzymes catalyze the transfer of the ADP-ribose moiety of NAD onto a specific amino acid residue of a target protein, while the nicotinamide moiety is released.In the context of the immune system, treatment of lymphocytes with ART inhibitors or the ART substrate NAD profoundly affects cellular functions, including clustering of T-cell receptors, proliferation, cytotoxicity, and in vivo homing (26,29,36). Recently, we and others have shown that extracellular NAD can induce apoptosis of naïve T cells in vitro and in vivo (1, 25). NAD-induced T-cell apoptosis shares certain features with the classic death receptor-induced pathway of apoptosis, including exposure of phosphatidylserine on the outer leaflet of the plasma membrane, followed by fragmentation of DNA and, ultimately, failure to exclude the DNA-staining dye propidium iodide (1). However, in contrast to death receptorinduced apoptosis, NAD-induced apoptosis cannot be blocked with caspase inhibitors and affects naïve cells more severely than activated T cells. Thus, exposure to NAD may constitute an alternative or complementary mechanism t...

Extracellular nucleotides such as ATP and NAD can profoundly affect the functions of lymphocytes, macrophages, and other cells. We have recently shown that extracellular NAD induces rapid apoptosis in naive T cells by a mechanism involving the ADP-ribosylation of cell surface molecules. In the present paper, we describe that T cells of different developmental stages differ in their sensitivity to NAD-induced apoptosis. Thymocytes were less susceptible than peripheral lymph node T cells, and freshly activated cells were more resistant than resting cells. Sensitivity to NAD-induced apoptosis generally correlated with expression of the ADP-ribosyltransferase ART2.2, which is not expressed on thymocytes and shed from peripheral T cells upon activation. Our findings suggest that NAD-induced apoptosis does not play a role during thymic selection of T cells, but rather may play a role by preventing the activation of unwanted bystander T cells during an immune response, and thus may participate in the control of autoimmunity.