Rapid Induction of Naive T Cell Apoptosis by Ecto-Nicotinamide Adenine Dinucleotide: Requirement for Mono(ADP-Ribosyl)Transferase 2 and a Downstream Effector

Abstract

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).

Unstable FoxP3 + T regulatory cells in NZW mice

“…We found no difference in Mcl1, which encodes an antiapoptotic molecule shown to promote Treg survival (49), or any difference in transcripts from the Sle1a lupus susceptibility region, which the Morel group have found to regulate Treg proportions (50), albeit by an indirect mechanism involving Tconv and dendritic cells, hence different from the intrinsic defect tracked here. The defective expression of Art2b in NZW Tregs is consistent with previous reports of Art2b inactivity in NZW (51) and is also shared by Tconv cells (www. immgen.org).…”

NAD+-Consuming Enzymes in the Regulation of Lung Immune Responses

“…As previously mentioned, NAD+ was shown to induce T cell apoptosis [5]. This was demonstrated to be an ART2-dependent mechanism [33,36]. Indeed two crucial findings confirmed ART2 as a necessary mediator of NICD.…”

“…Yet ART2.1 is active only in the presence of reducing agent such as dithiothreitol (DTT) or cystein while ART2.2 appears to be constitutively active [32]. The ART2 protein has been shown to play a potentially important role in the control of immune responses by regulating T cell function [33], however its role in the lung immunity is not yet known.…”

Basal and inducible expression of the thiol-sensitive ART2.1 ecto-ADP-ribosyltransferase in myeloid and lymphoid leukocytes

“…This inhibited state of ART2.1 is readily reversed in the presence of thiol reductants, such as exogenous dithiothreitol or endogenous cysteine or glutathione, that accumulate in the extracellular compartments of inflamed or hypoxically stressed tissues [11,12]. The additional layer of allosteric regulation for ART2.1, but not ART2.2, indicates that these isoforms are not simply redundant gene products; this is also consistent with their differential expression in various inbred mouse strains [13][14][15]. These differences in allosteric regulation and effect of genetic background further suggest that ART2.1 versus ART2.2 may be selectively utilized for signaling by different subpopulations of leukocytes, or during particular inflammatory/immune responses that occur in the context of hypoxia and ischemia.…”