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*ADENOSINE 5'-PHOSPHATE
The Journal of Immunology, 2001, 167: 2049-2059.
Copyright © 2001 by The American Association of Immunologists

Nicotinamide Adenine Dinucleotide (NAD) and Its Metabolites Inhibit T Lymphocyte Proliferation: Role of Cell Surface NAD Glycohydrolase and Pyrophosphatase Activities1

Rita Bortell2,*, Joel Moss{dagger}, Robert C. McKenna*, Mark R. Rigby*, Dena Niedzwiecki*, Linda A. Stevens{dagger}, Walter A. Patton{dagger}, John P. Mordes*, Dale L. Greiner* and Aldo A. Rossini*

* Diabetes Division, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655; and {dagger} Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2- T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nicotinamide adenine dinucleotide and its metabolites are important regulators of numerous intracellular processes. The relatively recent identification of extracellular membrane-bound NAD-metabolizing enzymes led to the suggestion that ecto-NAD or its metabolites, which may be present at sites of inflammation and cell lysis, could have regulatory activities (1). Two closely related families of extracellular NAD-metabolizing enzymes are the NAD glycohydrolases (NADases)3 and ADP-ribosyltransferases (ARTs) (2, 3, 4, 5). Both cleave NAD at the adenosine diphosphoribosyl-nicotinamide linkage. NADases generate free ADP-ribose, whereas ARTs catalyze Sn2-like transfer of ADP-ribose to an acceptor protein. Free ADP-ribose, e.g., as generated by NADases, has also been shown to attach covalently to proteins, although at much lower efficiency than the linkage mediated by ARTs (6).

Pyrophosphatases, a second family of extracellular enzymes, can metabolize either NAD (or ADP-ribose) directly to AMP and nicotinamide mononucleotide (or ribosylphosphate) (7). Nucleotide pyrophosphatase activity is attributed, in part, to the extracellular phosphodiesterase/pyrophosphatases PC-1, PD-1{alpha}, and PD-1{beta} (7). AMP generated by these enzymes may participate in signaling through purinergic receptors or can be cleaved by 5'-nucleotidases (e.g., CD73) to generate adenosine, which can also signal through purinergic receptors or can be recycled by the cell through salvage pathways (8).

Although membrane-bound NAD-metabolizing enzymes have been identified in a variety of immune cells, the role that these proteins play in immune function is not clear. ART activity has been demonstrated on mouse lymphoma and thymoma cells (9). GPI-linked and apparently non-GPI-linked ART (designated ART1 and ART5, respectively) cDNAs have been cloned from mouse T cell lymphoma cells (10, 11). However, comparable transferase activity or ART1/ART5-like clones has not been reported in rat cells. CD38, a type II membrane protein expressed on certain subsets of T and B lymphocytes in humans and mice, has NADase and cyclic ADP-ribose synthase activities (12, 13, 14).

An ART family member whose expression on rat T cells has been well characterized is ART2 (15, 16, 17). ART2 is a GPI-linked membrane protein with two alleles, ART2a and ART2b.4 ART2a protein exists as both a nonglycosylated and a variably glycosylated protein, whereas ART2b protein exists only in nonglycosylated form (18, 19). ART2 is expressed on a rat T lymphocyte subset that modulates expression of autoimmune diabetes mellitus (20, 21, 22, 23). Both recombinant ART2a and ART2b exhibit NADase activity, metabolizing NAD to nicotinamide and free ADP-ribose (24, 25); however, it is unclear what role the NADase activity of ART2 plays in the immunoregulation mediated by ART2+ T cells. Interestingly, the NAD metabolite, nicotinamide, has been shown to lower the incidence of diabetes in the BioBreeding diabetes-prone rat if administered orally (26), although parental administration appears to be ineffective (27).

Exogenous NAD, but not ADP-ribose or nicotinamide, inhibited the proliferation and suppressed the activity of mouse CTLs (28), consistent with the involvement of an ART rather than an NADase. Phosphatidylinositol-specific phospholipase C (PI-PLC) abolished the inhibition, suggesting that a GPI-linked ART mediated the inhibitory effects of ecto-NAD (29). Several ADP-ribosylated proteins have been identified, one of which may modulate tyrosine phosphorylation (30). Although ART2b can undergo auto-ADP-ribosylation, ADP-ribosylation of exogenous acceptors (e.g., histone) has not been demonstrated (24, 25, 31). However, posttranslational auto-ADP-ribosylation may affect transferase and NADase activities and thus serve a regulatory role (32). Based on these studies, it appears that NAD may affect cellular pathways through the formation of adenine nucleotides that can act via purinergic receptors and also by serving as a substrate for ART.

We have shown that exogenous NAD inhibits the proliferation of mitogen-activated rat T cells (31, 33) and report here that ADP-ribose, a metabolite of NAD, is a potent inhibitor of rat T cell proliferation, whereas AMP and adenosine are much less effective. Thus, NAD-dependent ADP-ribosylation, catalyzed by surface transferases, does not appear to be required for the effects of ecto-NAD. Furthermore, our data suggest that extracellular pyrophosphatases may in part regulate the generation of NAD and ADP-ribose metabolites and thus affect the rate of T cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Wistar-Furth (WF; ART2b) rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Viral Ab-free, BioBreeding diabetes-resistant (BBDR; ART2a) rats were obtained from Biomedical Research Models (Worcester, MA). All rats were certified as free of Sendai virus, pneumonia virus of mice, sialodacryoadenitis virus, rat corona virus, Kilham rat virus, H1 (Toolan’s virus), GD7, Reo-3, Mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse adenovirus, Hantaan virus, and Encephalitozoon cuniculi. Animals of either sex were used when 7–12 wk old. They were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996) and the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.

Thymectomy

Five- to 6-wk-old BBDR rats were anesthetized with Metofane (Schering-Plough, Union, NJ) and thymectomized by suction. Animals were allowed to recover from surgery for at least 2 wk and then injected with anti-ART2a mAb to in vivo deplete ART2+ cells as described below. Absence of the thymus was confirmed by gross examination at necropsy. Data obtained from rats with residual thymic tissue were excluded from these studies.

In vivo depletion of ART2+ T cells

For certain experiments, populations of ART2- cells for study were obtained by in vivo depletion methodology. Thymectomized and euthymic BBDR rats (7–12 wk of age) were injected i.p. with 2 ml of anti-ART2a (clone DS4.23) mAb daily for 3 consecutive days. On the fourth day, rats were killed and purified T cell populations were prepared as described below. Flow cytometric analysis for cell expression of ART2 was performed to document the extent of depletion of ART2+ cells. This in vivo depletion method was used because it is known to generate populations of ART2- cells that are capable of inducing autoimmune diabetes in the BBDR rat (34). In other experiments, ART2- cells were obtained by in vitro procedures described below.

T cell isolation and culture

Rats were killed by exposure to an atmosphere of 100% CO2. Cervical and mesenteric lymph nodes were collected, and lymphocytes were prepared by extrusion through metal sieves. T lymphocytes were purified on a nylon-wool column (31).

Rat T lymphocytes (2.5 x 105 cells/well) were incubated in 96-well culture plates (Fisher Scientific, Malvern, PA) in 200 µl of complete medium (RPMI 1640, plus 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME) (Life Technologies, Grand Island, NY) or serum-free medium (AIMV, plus 20 µM 2-ME) (Life Technologies). T cells were activated using plate-bound anti-rat CD3 mAb (BD PharMingen, San Diego, CA), 0.5 µg/well, Con A (Sigma, St. Louis, MO), 5 µg/ml, or PMA, 5 ng/ml, plus ionomycin (Sigma), 375 ng/ml. NAD, ADP-ribose, ADP, AMP, and adenosine were obtained from Sigma and nicotinamide from Fisher Scientific. The compounds were dissolved in serum-free RPMI 1640 or AIMV. Although FBS itself exhibited some NADase activity, no differences between FBS-supplemented and serum-free media were observed when comparing the effects of NAD, ADP-ribose, or nicotinamide on rat T cell activation (data not shown).

T cell proliferation assay

T cell proliferation, following mitogen stimulation, was quantified by the addition of 1 µCi of [3H]thymidine (Amersham, Arlington Heights, IL) to each well during the last 16 h of culture (31), followed by collection of cells with a CellHarvester (Skatron, Lier, Norway) and radioassay using a BetaScope (Betagen, Waltham, MA).

Viability assays

Cell viability was determined using the method of trypan blue dye and enumeration of dye-excluding cells using a hemocytometer or by addition of propidium iodide and quantification of dye-excluding cells by flow cytometric analysis (35).

Antibodies

Anti-ART2a mAb (clone DS4.23) is maintained in our laboratory. The secondary Ab was FITC-conjugated goat anti-rat IgG (Cappel, Durham, NC). In cell sorting experiments, anti-ART2a mAb was conjugated to biotin. The developing reagent was FITC-conjugated streptavidin (BD PharMingen). Anti-rat {alpha}{beta} TCR (R73) mAb and isotype control mouse IgG1 were directly conjugated to PE (BD PharMingen).

Flow cytometry

The percentage of T cells and ART2+ cells present in nylon-wool purified T cell preparations was determined by flow cytometry. Cells (1 x 106 cells per well) were incubated with the appropriate Ab using standard methods (36). Cells were analyzed using a FACScan instrument (BD Biosciences, Mountain View, CA).

To obtain populations of sorted cells, freshly isolated lymph node cells were reacted with biotin-conjugated DS4.23 anti-ART2a mAb, FITC-conjugated streptavidin, and PE-conjugated R73 anti-{alpha}{beta} TCR mAb. Cells were sorted by FACScan. In all cases, purity of sorted populations was >95% as determined by flow cytometry.

PI-PLC treatment

Aliquots of purified rat T cells (2 x 106) were incubated with 1 U of PI-PLC (Sigma) in 1 ml of PBS for 1 h at 37°C.

Quantification of NAD metabolites

Thin-layer chromatography. Purified rat T cells (2 x 106) were incubated at 37°C with 100 µM [adenylate-32P]NAD (0.1 µCi/assay; New England Nuclear, Boston, MA) in a total volume of 50 µl of PBS. In some experiments, purified rat T cells (2.5 x 105) were incubated with 200 µM [32P]NAD (0.1 µCi) in a total volume of 200 µl of serum-free AIMV or complete RPMI 1640 medium and activating mitogens (as described above). Samples (2 µl) from these reactions were removed at designated times and applied to 20 x 20 cm polyethyleneimine cellulose F TLC plates (EM Sciences, Gibbstown, NJ), which were then placed upright in a TLC chamber containing 100 ml of 0.3 M LiCl (Fisher Scientific). After the solvent had migrated 15–16 cm, the plate was removed and air-dried. Kodak X-OMAT film (Rochester, NY) was then exposed to the TLC plates using an amplifying screen at -70°C for 12–15 h for autoradiography. Positions of radiolabeled NAD and ADP-ribose were verified with NAD and ADP-ribose standards.

HPLC analysis. T cells were pelleted by centrifugation and suspended in PBS. Approximately 7.7 x 106 cells in a volume of 125 µl were added to 300 µl of PBS containing OVA (0.1 mg/ml), 20 mM DTT, 10 mM MgCl2, and 100 µM [adenosine-14C]NAD (0.05 µCi) (Amersham), with or without 5 mM ADP-ribose. After incubation for 1 h at 37°C, cells were sedimented by centrifugation (500 x g), and the supernatants were further clarified by centrifugation at 14,000 x g. Each supernatant (200 µl) was subjected to ion-exchange HPLC on a Zorbax SAX column (4.6 x 250 mm; DuPont, Wilmington, DE) using 20 mM potassium phosphate, pH 4.5 (buffer A), for 20 min, followed by a linear gradient of a 0–1 M NaCl in the same buffer (buffer B) for 25 min and 100% buffer B for 30 min at a flow rate of 1 ml/min.

NADase activity

NADase activity was quantified in 50 mM potassium phosphate (pH 7.5) containing 0.1 mM [carbonyl-14C]NAD (0.05 µCi) (Amersham). Following incubation at 30°C for 1 h (final volume 300 µl), samples (100 µl) were applied to columns (0.5 x 4 cm) of Dowex AG1-X2 (Bio-Rad, Melville, NY), and [carbonyl-14C]nicotinamide was eluted with 5 ml of water for scintillation counting.

Statistical analysis

Effects of mitogens and metabolites were evaluated using ANOVA for a factorial design. In the presence of significant interaction effects between mitogen and metabolite, pairwise comparisons between metabolites for each mitogen were evaluated using Tukey’s honestly significant difference test (37). To compensate for the additive type I error due to multiple comparisons, a Bonferroni adjustment (37) was applied to the p values from the Tukey’s honestly significant difference tests to adjust for the number of mitogens evaluated. All analyses were performed using SPSS7.5 for Windows NT (38).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NAD and ADP-ribose inhibit mitogen-activated rat T cell proliferation to a similar degree

To distinguish among effects due to different ecto-enzymes, we compared the ability of NAD, free ADP-ribose, and nicotinamide to inhibit mitogen-induced rat T cell proliferation. T cells from BBDR (ART2a+) and WF (ART2b+) rats were cultured without mitogen (negative control) or with plate-bound anti-CD3 mAb (Fig. 1GoA), Con A (Fig. 1GoB), or PMA plus ionomycin (Fig. 1GoC) without or with 200 µM NAD, ADP-ribose, or nicotinamide. To determine whether there were interactions among NAD and its metabolites, we also compared combinations of these nucleotides on T cell proliferation.



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  		FIGURE 1. Effects of NAD, ADP-ribose, or nicotinamide on [3H]thymidine incorporation by mitogen-stimulated T cells. BBDR or WF rat T cells were cultured without additions or with 200 µM NAD, 200 µM ADP-ribose (ADPR), 200 µM nicotinamide (Nic), or combinations of each at 200 µM, in the presence of (A) anti-CD3, (B) Con A, or (C) PMA plus ionomycin. [3H]Thymidine incorporation into cells was measured at day 2 (left) or day 3 (right). Incorporation with each mitogen in the absence of NAD/ADP-ribose/nicotinamide is 100%. Data are means ± SD of values from four experiments, each with triplicate determinations. *, p < 0.05 vs both mitogen control (no treatment) and mitogen + 200 µM nicotinamide (Nic). Values in cpm at day 2 of a representative experiment for BBDR/WF stimulation were: anti-CD3, 55,669/69,997; Con A, 44,670/42,793; and PMA plus ionomycin, 195,573/139,402.

 
[3H]Thymidine incorporation by mitogen-activated BBDR or WF T lymphocytes was assayed as a measure of cell proliferation. The data for day 2 (Fig. 1Go, left) demonstrate that ADP-ribose inhibited proliferation of both BBDR and WF T cells to a degree comparable to that observed with NAD. The extent of inhibition was mitogen dependent. Inhibition of cells activated by anti-CD3 mAb was greater than inhibition of Con A-activated cells. Inhibition of both anti-CD3 mAb- and Con A-activated cell cultures was greater than that of cultures activated by PMA plus ionomycin. Nicotinamide had no significant inhibitory effect on any of the mitogen-activated cells. The inhibitory effect of NAD or ADP-ribose alone was largely overcome by day 3, whereas cultures incubated in the presence of NAD plus ADP-ribose still showed inhibition at day 3 (Fig. 1Go, right).

Inhibition of proliferation did not appear to be due to cell death resulting from toxic effects of NAD or its metabolites. The viability of cells in culture on day 2 was determined using both propidium iodide and trypan blue dye exclusion. In cultures stimulated without NAD, cell viability ranged from 67 to 81%. In cultures stimulated in the presence of 200 µM NAD, cell viability ranged from 73 to 91%.

On day 2 or 3, only very low levels of proliferation were observed in control cultures of T cells not stimulated with mitogen. This low proliferation rate was unaffected by the addition of any of the nucleotides or nucleotide combinations (data not shown).

Inhibition of T cell proliferation is greater with NAD, ADP-ribose, and ADP than with AMP and adenosine

NAD and its metabolites may potentially act as ligands for purinergic-like receptors on rat T cells. Receptors that bind purine analogs have been demonstrated on human and murine lymphocytes (39, 40). Based on this observation, the inhibitory effects of NAD and ADP-ribose were compared with several known purinergic receptor ligands including ADP (P2 ligand), as well as AMP and adenosine (P1 ligands). Proliferation of T cells activated with anti-CD3 mAb, Con A, or PMA plus ionomycin in the presence of different nucleotide concentrations was quantified by incorporation of [3H]thymidine (Fig. 2Go). For each mitogen, the inhibitory concentrations of NAD, ADP-ribose, and ADP were similar. AMP and adenosine were less effective inhibitors up to 200 or 500 µM, respectively. T cell proliferation at concentrations of up to 1 mM nicotinamide was 100 ± 20% of control (data not shown).



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  		FIGURE 2. Effects of NAD, ADP-ribose, ADP, AMP, or adenosine concentrations on [3H]thymidine incorporation by mitogen-stimulated T cells. WF rat T cells were incubated with the indicated concentrations of NAD, ADP-ribose (ADPR), ADP, AMP, or adenosine (ADE) and activating concentrations of (A) anti-CD3, (B) Con A, or (C) PMA plus ionomycin. [3H]Thymidine incorporation into cells was measured at day 2 (left) or day 3 (right). Incorporation with each mitogen in the absence of exogenous nucleotide (no Tx.) is 100%. Data are means of triplicate determinations from one of two similar experiments.

 
Effect of pertussis toxin (PT) and cholera toxin (CT) on inhibition of T cell proliferation by adenine nucleotides

CT and PT ADP-ribosylate Gs or Go and Gi proteins, respectively, resulting in irreversible activation or inactivation of their respective signals (41). CT causes an increase in cellular cAMP. PT, by uncoupling the modified G protein from its receptor, blocks Gi inhibition of adenylyl cyclase and agonist-mediated effects on ion channels or phospholipids. To determine whether the inhibition of rat T cell proliferation by NAD or ADP-ribose was mediated through G protein-coupled receptors and cAMP, CT and PT were added to the mitogen-stimulated cultures with or without addition of exogenous NAD or ADP-ribose.

Activation of T cells by anti-CD3 mAb or Con A was suppressed by CT; PT was less inhibitory (Fig. 3Go, A and B). Nonreceptor-based stimuli leading to elevation of intracellular cAMP (e.g., forskolin, dibutyryl-cAMP) also inhibited T cell proliferation following anti-CD3 mAb or Con A stimulation. CT, PT, or forskolin did not inhibit proliferation of T cells activated by PMA plus ionomycin (Fig. 3GoC). PT did not block the effects of NAD or ADP-ribose (Fig. 3Go) on rat T cell proliferation, consistent with the hypothesis that they are not dependent on Gi or Go.



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  		FIGURE 3. Effects of inducers of increased intracellular cAMP with or without addition of NAD or ADP-ribose on [3H]thymidine incorporation by mitogen-stimulated T cells. WF rat T cells were incubated with CT (0.5 µg/ml) or PT (0.2 µg/ml) for 3 h before addition of mitogens and nucleotides as indicated. Other samples of T cells were incubated with forskolin (5 µM) or dibutyryl-cAMP (db-cAMP; 1 mM) for 30 min before addition of mitogens and nucleotides. Incorporation with each mitogen in the absence of toxin or nucleotide (no Tx.) is 100%. [3H]Thymidine incorporation was measured after 2 days of incubation with cAMP-generating agents alone or plus 200 µM NAD or 200 µM ADP-ribose (ADPR) and stimulated with (A) anti-CD3 (CD3), (B) Con A, or (C) PMA plus ionomycin (P/I). Data are the means ± SD of values from three experiments, each with duplicate determinations.

 
BBDR and WF T cells metabolize [32P]NAD to [32P]ADP-ribose in vitro

To confirm the presence of cell surface NADase activity and to compare the endogenous NADase activities of rat strains expressing distinct allelic forms of ART2, intact T cells (2 x 106/50 µl) from BBDR (ART2a+) or WF (ART2b+) animals were incubated with [32P]NAD for 3–20 min. Conversion of [32P]NAD to [32P]ADP-ribose was quantified by TLC and autoradiography. Time-dependent increases in ADP-ribose accumulation were similar in both BBDR (ART2a+) and WF (ART2b+) cell cultures (Fig. 4Go).



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  		FIGURE 4. TLC of NAD metabolites in medium from T cell cultures. Nylon wool-purified WF or BBDR T cells (2 x 106) were incubated in PBS with 100 µM [32P]NAD (0.1 µCi) at 37°C for 3 to 20 min. At the indicated times, samples of medium were removed for TLC, followed by autoradiography. [32P]NAD in PBS alone (no cells) was used as a control for nonenzymatic hydrolysis of NAD (left). Arrows mark the migration of [32P]NAD (top) and [32P]ADP-ribose (middle). Data shown are representative of those from two independent experiments.

 
ADP-ribose is the major metabolite produced by incubation of NAD with cultured quiescent or activated rat T cells

To determine the effects of activating mitogens on NAD metabolism, intact T cells (2.5 x 105/200 µl) from BBDR rats were incubated with [32P]NAD in the absence or presence of activating mitogens. BBDR T cells metabolized NAD in a time-dependent manner (Fig. 5Go, A–D, left), with most of the 200 µM NAD degraded within 20 h (Fig. 5GoD). At all times, ADP-ribose was the major metabolite; additional minor metabolites were seen with increasing time of incubation (Fig. 5Go). Similar results were obtained using WF T cells (data not shown). NAD was also hydrolyzed at a slow rate by complete medium containing FBS, in the absence of T cells, but not by serum-free medium (Fig. 5Go, A–D, right). Overall, the metabolism of NAD by rat T cells was not significantly altered by any of the three mitogens that induced proliferation. Furthermore, stimulation of T cells up to 20 h did not induce changes in the level of cell surface ART2 or frequency of ART2+ cells (data not shown).



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  		FIGURE 5. Time course of NAD metabolism in the presence of quiescent or mitogen-stimulated T cells. Serum-free medium (SFM) or complete medium (CM) alone (no cells, right) or with BBDR rat T cells (2.5 x 105 in 200 µl, + cells, left) was incubated at 37°C with 200 µM [32P]NAD (0.1 µCi) without mitogen (control) or with anti-CD3 (A-CD3), Con A, or PMA plus ionomycin (PMA+Iono) for 5 min (A), 90 min (B), 5 h (C), or 20 h (D). Samples of culture supernatants were removed for separation of NAD and its metabolites by TLC, followed by autoradiography. Arrows mark the positions of [32P]NAD, [32P]ADP-ribose, and a minor metabolite (unlabeled arrow).

 
Rat T cells incubated with NAD produce multiple purine metabolites

To identify metabolites other than ADP-ribose, samples of medium from intact rat T cells incubated with [adenosine-14C]NAD were analyzed by HPLC. A significant amount of [adenosine-14C]AMP was observed in addition to [adenosine-14C]ADP-ribose (Fig. 6Go, C and D). The generation of AMP from ecto-NAD is consistent with the presence of an ecto-pyrophosphatase on the surface of rat T cells. Adenosine was also generated, consistent with the further hydrolysis of AMP to adenosine (Fig. 6Go, C and D). In the presence of 5 mM unlabeled ADP-ribose, accumulation of radiolabeled AMP and adenosine was reduced (Fig. 6GoF).



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  		FIGURE 6. HPLC separation of NAD and its metabolites following incubation of radiolabeled NAD with T cells. WF rat T cells (7.7 x 106) in 125 µl were added to reaction mixtures that contained 100 µM [adenosine-14C]NAD (0.05 µCi), PBS, OVA (0.1 mg/ml), 20 mM DTT, and 10 mM MgCl2 with or without 5 mM ADP-ribose (final concentrations, volume 300 µl). After incubation for 1 h at 37°C, cells were pelleted by centrifugation (500 x g), and the supernatants were further clarified by centrifugation at 14,000 x g. A sample (200 µl) of each supernatant was subjected to ion-exchange HPLC analysis. Elution profiles of the reaction mix (A and B), the reaction mix plus cells (C and D), and the reaction mix plus cells and 5 mM ADP-ribose (E and F) are shown as absorbance at A260, in arbitrary units (A, C, and E), and radioactivity in 0.5 ml fractions (0.5 min each, B, D, and F). Elution times of standards are noted in the upper panels by arrows. Data shown are representative of those from triplicate reactions.

 
NADase and pyrophosphatase activities are present on ART2+ and ART2- T lymphocytes

ART2 is known to have NADase activity (24). To determine whether NADase activity was present on cells deficient in ART2, we isolated T lymphocytes from BBDR rats depleted of their ART2-expressing cells by in vivo treatment with a cytolytic mAb to ART2a (42). To reduce the potential of recent thymic emigrants that express ART2 to contaminate the ART2-deficient population after anti-ART2 treatment in vivo, some BBDR rats were thymectomized before Ab treatment. NAD metabolism by T cells isolated from untreated or ART2-depleted rats was compared. HPLC analysis demonstrated significant formation of ADP-ribose following incubation of NAD with either ART2+ or ART2- T cells; small quantities of AMP and/or adenosine were also seen on the chromatogram (Fig. 7Go). Flow cytometry analyses confirmed that the percentage of ART2+ T cells was reduced from ~80% in purified populations of T cells in control (euthymic or thymectomized) BBDR rats to <10% following in vivo anti-ART2 mAb treatment (data not shown).



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  		FIGURE 7. HPLC analysis of NAD metabolites from T cell cultures. The separation of [adenosine-14C]NAD hydrolysis products, as described in Materials and Methods, was performed on three separate reactions for each experimental group. A representative chromatogram is shown. The analyses of ART2+ T cell suspensions from euthymic BBDR rats (I) and thymectomized BBDR rats (III) are shown along with ART2- cells from ART2-depleted euthymic (II) or thymectomized BBDR rats (IV). In this experiment, the analyses in III and IV were performed with 10 and 50%, respectively, of the cells in I and II. The elution times of adenosine (Ade), NAD, AMP, and ADP-ribose (ADPR) standards are shown by the bars in each panel. In samples identified as generating AMP, a fourth peak elutes between NAD and ADPR. The elution of this peak can vary slightly from sample to sample, perhaps due to varied levels of lipid in the sample.

 
NADase activity of ART2- T cells is not released by PI-PLC treatment

Many ARTs and NADases, including ART2, are anchored to the cell membrane by a GPI linkage (43). As noted in Fig. 8Go, NAD was also metabolized by PI-PLC-treated rat T cells, suggesting that at least some of the total NADase activity was not GPI anchored. Alternatively, acylation of the GPI oligosaccharide may reduce the ability of PI-PLC to cleave the anchor. To confirm release of GPI-anchored proteins, flow cytometric analysis was performed on control and PI-PLC-treated cells using ART2 as a marker for release of GPI-anchored proteins. The number of ART2+ cells was reduced by 80% with this treatment (data not shown).



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  		FIGURE 8. TLC of NAD metabolites in medium from T cells incubated in the presence or absence of PI-PLC. Purified BBDR rat T cells (2 x 106) were incubated in the presence (+PIPLC) or absence (-PIPLC) of PI-PLC in 1 ml of PBS for 1 h at 37°C. Cells were washed twice, resuspended in PBS, and incubated with 100 µM [32P]NAD (0.1 µCi) at 37°C for 5 or 10 min. At the indicated times, samples of medium were removed for TLC, followed by autoradiography. [32P]NAD in PBS alone (no cells) was used as a control for nonenzymatic hydrolysis of NAD (left). Arrows mark the migration of [32P]NAD (top) and [32P]ADP-ribose (middle). Data shown are representative of those from two independent experiments.

 
PI-PLC treatment of ART2+ T cells from euthymic or thymectomized BBDR rats released NADase activity into the supernatant (Table IGo). Conversely, even following PI-PLC treatment, no difference in NADase activity was observed in supernatants from T cells isolated from either euthymic or thymectomized ART2-depleted BBDR rats.


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  		Table I. NADase activity in supernatants of BBDR rat T cell culturesa

 
To confirm these results and to exclude the possibility of artifact induced by in vivo depletion methodology, the experiment was repeated using ART2+ and ART2- cells obtained by conventional in vitro sorting procedures. PI-PLC treatment of sorted ART2+ TCR+ cells obtained from untreated BBDR rats released NADase activity into the supernatant (Table IIGo). However, ART2- TCR+ cells derived from the same BBDR rats showed no difference in NADase activity in the supernatant following PI-PLC treatment. Unsorted lymph node cells (Table IIGo, groups 3 and 4) demonstrate that neither Ab staining nor the cell sorting procedure appreciably affected either the ability of GPI-linked proteins to be released or the activity of the NADase(s) in the supernatant. Furthermore, the unsorted lymph node cells demonstrate that the bulk of the GPI-releasable NADase activity resides in the ART2+ TCR+ cells.


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  		Table II. NADase activity in supernatants of ART2+ and ART2- T cell culturesa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report demonstrates that exogenous NAD and many of the purine nucleotide metabolites of NAD inhibit rat T cell proliferation. The potency of inhibition depends on the activating mitogen and likely involves interaction of NAD and its purine metabolites with P2-like purinergic receptors. ADP-ribose is the major metabolite, indicating that the T cells have surface-associated NADase activities. The generation of AMP from NAD and ADP-ribose by both ART2+ and ART2- T cells is consistent with the presence of a cell surface pyrophosphatase.

The GPI-anchored rat T cell protein, ART2, has previously been reported to have NADase activity (24). In agreement, treatment of T cells with PI-PLC removes much of the total NADase activity. This was true for cells from both euthymic and thymectomized rats. Analysis of ART2- T cell subsets from both thymectomized and euthymic rats also revealed the presence of other NADase activities that are not releasable by PI-PLC. This was demonstrated for both 1) ART2a- cells obtained from rats treated in vivo with anti-ART2 mAb and 2) ART2- T cells obtained by in vitro sorting of cells from untreated DR rats. Interestingly, thymectomy appeared to enhance NADase activity. We attribute this observation to the differences that exist between populations comprised exclusively of mature cells vs populations comprised of a mixture of immature and mature peripheral T cells. Several maturational changes occur over time in thymic emigrants, among them the loss of expression of Thy-1.1 and increased expression of ART2 (15).

Our results suggest that NAD-metabolizing enzymes may mediate the inhibitory effects of NAD on T cell proliferation by multiple pathways. In one case, NAD or its metabolites may engage purinergic receptors and directly modulate immune function (39, 40, 44, 45, 46, 47). Purinergic receptors have been identified on numerous immune cells, including macrophages, neutrophils, platelets, and thymocytes, where they are known to mediate signaling events leading to selection of thymocytes for apoptosis (46). Purinergic receptors have also been described on mouse and human T cells where they modulate T cell proliferation (39, 40). In vivo, extracellular concentrations of NAD and related compounds may be increased in cases of extensive cell death, as might occur at sites of inflammation. In our studies, both NAD and ADP-ribose inhibited proliferation of rat T cells following anti-CD3 mAb or Con A activation. Inhibition by NAD and ADP-ribose was similar to that mediated by P2-type purinergic ligands such as ADP. P1 ligands, such as AMP and adenosine, were less potent. These data support a regulatory role for pyrophosphatase in hydrolyzing NAD and ATP to less potent catabolites. Finally, nicotinamide, which is produced by NAD hydrolysis, had little effect on T cell proliferation. Conceivably, NAD may be translocated across membranes; however, the active factor is unlikely to be a catabolite such as AMP or adenosine, because both were less active than NAD.

Because these data were consistent with a mechanism whereby NAD or its purine metabolites act as ligands at purinergic receptors that may interact with G proteins, we also investigated whether CT or PT abrogated the effects of these nucleotides on T cell proliferation. PT, which had little effect on T cell proliferation, did not block the inhibitory action of NAD or ADP-ribose, consistent with the hypothesis that the inhibitory activity of NAD and ADP-ribose was not dependent on receptors coupled to Gi or Go, although the involvement of other pertussis-insensitive G proteins cannot be excluded. CT, forskolin, and dibutyryl-cAMP, agents that increase intracellular cAMP (41), all had inhibitory effects on anti-CD3 mAb- or Con A-stimulated T cell proliferation similar to observations with exogenous NAD. Therefore, potential release of endogenous NAD at sites of inflammation and cell lysis may play a role in regulating cellular responses. In renal epithelial cells, released endogenous nucleotides, e.g., ATP, modulate the "basal" activity of signaling pathways via P2 receptor activation, leading to elevated levels of intracellular cAMP (48).

NAD might also act in an alternative pathway as an ADP-ribose donor in transferase reactions. In support of this possibility, mouse ART1 and ART5, both transferases, have been cloned from T cell lines (10, 11). In both mice and rats, ART2 mRNA has been identified in lymphoid tissues (49, 50). However, the mouse and rat ART2 isoforms differ in their enzymatic activities, with the recombinant rat ART2 protein exhibiting predominantly NADase activity and the recombinant mouse ART2 protein exhibiting predominantly transferase activity (25, 31, 51, 52). It should be noted that the NADase and ART families of NAD-metabolizing enzymes are structurally related, with a high degree of amino acid identity. Indeed, a single amino acid replacement at the active site (Glu for Gln at amino acid 200) resulted in generation of transferase activity in recombinant rat ART2. Conversely, mutation of the mouse ART2 sequence (Glu to Gln at amino acid 210) caused loss of transferase activity with the retention of NADase activity (52, 53). This redundancy and preservation of enzymatic activity between species suggests that these cell surface enzymes are likely to have important functions in immune cells.

We observed earlier that exogenous NAD inhibited the proliferation of WF rat T cells, and this inhibition was concentration and mitogen dependent (31). Because rat T cells express ART2, a known NADase (24, 25), we hypothesized that reported differences in the enzymatic activities of ART2 allelic proteins (25, 54) might have a role in mediating the inhibitory effects of NAD on rat T cell proliferation. However, our data documented no difference in the inhibition of BBDR (ART2a+) or WF (ART2b+) T cell proliferation by NAD. Furthermore, BBDR and WF T cells were equivalent in their ability to generate [32P]ADP-ribose following incubation with [32P]NAD.

The presence of NADase activity in both ART2+ and ART2- cells is consistent with the presence of multiple NADase isoforms. Our analyses indicated that the NADase activity in ART2- T cells was not releasable with PI-PLC treatment. A candidate NADase known to be an integral membrane protein is CD38 (12, 13, 14), although we found no detectable formation of cyclic ADP-ribose (a product of the CD38-catalyzed reaction) in our HPLC analyses. However, CD38 is a bifunctional enzyme that produces cyclic ADP-ribose as well as catalyzing its hydrolysis (12, 13). Therefore, the steady-state levels of cyclic ADP-ribose may have been below detectable levels. Consequently, we were unable to verify CD38 as the source of the rat T cell NADase activity.

Our data demonstrate that NADase activity is not the only NAD-metabolizing enzyme on the surface of rat T cells. Our HPLC analysis of NAD metabolism by intact rat T cells demonstrated the generation of ADP-ribose, AMP, and adenosine in the medium. These data confirm the presence of NADase activity (generation of free ADP-ribose) previously described for intact rat T cells and for recombinant ART2, a GPI-anchored protein normally expressed on rat T cells (24, 25, 55). The generation of AMP also demonstrates the presence of a rat T cell surface pyrophosphatase. Stimulation of human T cells by PMA, PHA, or compounds that specifically increase intracellular cAMP enhanced cell surface expression of pyrophosphatase (PC-1), NADase (CD38), and 5'-nucleotidase activities (56). Activities analogous to PC-1 pyrophosphatase in rat T cells could generate AMP, as observed in our studies. AMP was further converted to adenosine, consistent with the presence of CD73 or another 5'-nucleotidase similar to those identified on human and mouse T lymphocytes (47, 57).

ADP-ribose, the primary metabolite of NAD in our studies, inhibited the proliferation of activated rat T cells as potently as did NAD. These data are in contrast to those reported for mice by Wang et al. (28), who observed that NAD, but not free ADP-ribose or nicotinamide, inhibited CTL proliferation and cytotoxic activity. Our demonstration that ADP-ribose had an inhibitory effect on rat T cell function suggests that NAD-mediated inhibition does not require transferase activity.

AMP was less potent than either NAD or ADP-ribose in its inhibitory effects on proliferation, suggesting that pyrophosphatase activity, which may be up-regulated in activated T cells (56), could modulate the inhibitory effects of NAD. Conversely, because the inhibitory activity of ADP-ribose is comparable to that of NAD, NADase activity does not have an obvious role in modulation of NAD-mediated inhibitory activity. However, it is tempting to speculate that if ADP-ribose is a more effective substrate for pyrophosphatase activity than NAD, the conversion of NAD to ADP-ribose by NADase would play a role in modulating the inhibitory effects of NAD.

Furthermore, it is interesting that one of the primary metabolites of NAD, nicotinamide, has been proposed to modulate development of autoimmunity (58). A number of clinical studies are currently underway to determine whether oral nicotinamide may prevent autoimmune diabetes in potentially susceptible children (59, 60). These clinical trials are based on numerous studies in animal models of autoimmune diabetes, including the biobreeding rat. Nicotinamide given orally induced tolerance in the biobreeding rat (26), although parenteral administration was ineffective (27). Oral administration of nicotinamide, which has been shown to increase intracellular levels of NAD (61, 62), may act indirectly to inhibit autoreactive cells by increasing the NAD levels available at sites of inflammation. Although speculative, this may explain the disparate results in nicotinamide-induced tolerance depending on the route of administration; e.g., nicotinamide absorbed in the gut increases intracellular NAD levels and provides additional substrate for ART2 and other NAD-metabolizing enzymes, thereby enhancing immune tolerance.

In their aggregate, these studies are consistent with a role for extracellular NAD and other purine metabolites in immune regulation. Our data demonstrate that exogenous NAD and other purine compounds inhibit T cell proliferation. Likely situations for the in vivo participation of NAD and its metabolites in immune modulation would include sites of inflammation, where dead and dying cells release their intracellular NAD (and other purine metabolites) into the extracellular space. The effect appears to be modulated by cell surface expression of multiple NAD-metabolizing enzymes, including NADase, pyrophosphatase, and nucleotidase. In vivo, these enzymes may alter the balance of inhibitory or benign NAD metabolites to modulate the local immune microenvironment.


   Footnotes
 
1 This work was supported in part by Grants DK25306 (to A.A.R.), DK36024 (to D.L.G.), and DK41235 (to J.P.M.) from the National Institutes of Health, an institutional Diabetes and Endocrinology Research Center Grant from the National Institutes of Health (DK32520), and by Research Grants from the Juvenile Diabetes Foundation International. R.B. is the recipient of a Career Development Award from the Iacocca Foundation. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Rita Bortell, Diabetes Division, 373 Plantation Street, Suite 218, University of Massachusetts Medical School, Worcester, MA 01655. E-mail address: rita.bortell{at}umassmed.edu Back

3 Abbreviations used in this paper: NADase, NAD glycohydrolase; ART, ADP-ribosyltransferase; BBDR, biobreeding diabetes-resistant; PI-PLC, phosphatidylinositol-specific phospholipase C; WF, Wistar-Furth; CT , cholera toxin; PT, pertussis toxin. Back

4 A new nomenclature for the rat has been adopted. ART2a and ART2b now replace RT6.1 and RT6.2 allelic proteins, respectively (15 ). Back

Received for publication April 5, 2001. Accepted for publication June 6, 2001.


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 Results
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