The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Z.-X.
Right arrow Articles by Dennert, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Z.-X.
Right arrow Articles by Dennert, G.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
The Journal of Immunology, 2001, 167: 4942-4947.
Copyright © 2001 by The American Association of Immunologists

Extracellular Nicotinamide Adenine Dinucleotide Induces T Cell Apoptosis In Vivo and In Vitro1

Zhang-Xu Liu, Olga Azhipa, Shigefumi Okamoto, Sugantha Govindarajan and Gunther Dennert2

Department of Molecular Microbiology and Immunology, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine at University of Southern California, Los Angeles, CA 90089


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3+, CD4+, and CD8+ cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Posttranslational modification of proteins involved in signaling constitutes an important mechanism in cell regulation. While this is well established for transmission of intracellular signals, it has yet to be clearly documented for signals received on the cell surface. Evidence for existence of such a mechanism has emerged from the demonstration of mono-ADP ribosyltransferases (ADPRT)3 on mammalian cells (1). ADPRTs introduce posttranslational modifications into proteins by transferring ADP ribose from nicotinamide adenine dinucleotide (NAD) to arginine or cysteine residues. Five ADPRTs (ART1–5) have been cloned from mouse, rat, rabbit, and human tissues and shown to be 25- to 40-kDa, GPI-anchored proteins, with exception of ART5 (2, 3, 4). In the rat, there exist two ART2 alleles, previously known as T cell differentiation marker RT6. Interestingly, T cells expressing RT6 (ART2) were reported to induce resistance to autoimmune diabetes in diabetes-prone BB/W rats (5, 6). In the mouse, two ART2 genes, ART2a and ART2b, have been described, besides ART1, ART3, ART4, and ART5, all of which may be expressed on lymphocytes, with exception of ART3 and ART5 (3, 4, 7, 8, 9). There is therefore a large family of enzymes capable of ADP ribosylating proteins on various cell types, including lymphocytes. This emerging picture is complicated by the fact that there are strain variations in expression of these enzymes. Thus, ART2a is not expressed in C57BL/6 mice, and ART2b is not expressed in NZW mice (10, 11). Reminiscent of observations in rats, in mice a correlation between ART2 transcript levels in diabetes-prone nonobese diabetic/Lt mice and control nonobese nondiabetic/Lt mice has been reported, pointing to a role of ADPRT in regulation of autoimmune diabetes (12).

Because of the many genes coding for ADPRTs (3, 13, 14, 15) and lack of Abs specific for these molecules, much work with lymphocytes has been accomplished by using enzyme assays to identify respective molecules and to study their function. Results showed that ADPRT activity is expressed on T cells, but not B cells (16, 17). Upon incubation with NAD, cell surface proteins on T cells are ADP ribosylated, and responses to activation signals, transmitted by the TCR, are inhibited (18, 19, 20). These observations then raise the question as to how precisely ADP ribosylation modulates T cell functions. Experiments revealed that the enzyme attaches ADP ribose to arginine residues of CD45, CD44, CD43, CD8, and LFA-1 (18, 19), in turn posing the question as to how modification of these molecules regulates TCR signaling. Using T cell lines, expressing ADPRT, evidence was obtained that ADP ribosylation of cell surface molecules inhibits association of receptors into a signal-transmitting cluster (19, 20). The speculation was hence put forward that ADP ribosylation of coreceptors inhibits TCR signaling by altering receptor association (19, 20). Such changes in receptor interaction could have various effects. Receptors could fail to transmit stimulatory signals or generate negative signals, leading to anergy or cell death.

In this communication, we report that treatment of mouse T cells with NAD before injection into animals results in their inability to mediate immune functions and to reconstitute recipients. Moreover, injection of NAD into animals causes accumulation of apoptotic T cells in the liver. It is shown that incubation of naive, but not activated T cells with NAD induces cell death in vitro, explaining the effects NAD exerts in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Assays for graft-vs-host disease (GVHD), marrow stem cell proliferation, T cell proliferation, and homing

To induce GVHD, spleen cells from C57BL/6 (H-2b) mice were injected i.v. into B6D2F1 (H-2dxb) mice. All mice were obtained from The Jackson Laboratory (Bar Harbor, ME). After 10 days, spleens were harvested, weights determined, and spleen indexes, i.e., spleen weight increases over controls, calculated. Spleen cells were assayed for presence of CTL by cytotoxicity assay on P815 targets (H-2d) or cultured with C3H (H-2k) spleen stimulator cells for 5 days, followed by cytotoxicity assay on C1.18.4 targets (H-2k).

Bone marrow was assayed for the number of CFU spleen (CFUS) (21) after treatment with NAD. Marrow was harvested from long bones and incubated in RPMI 1640 medium, with or without 1 mM NAD, at 37°C for 3 h. After washing, aliquots of 106 cells were injected i.v. into lethally irradiated syngeneic recipients After 8 days, spleens were harvested and stained with Bouin’s solution, and colonies were counted (21).

To assay the proliferative activity of naive T cells, splenic T cells were isolated by nylon wool adherence from C57BL/6 Thy-1.2 mice. Purified T cells were incubated in RPMI 1640 medium, with or without 1 mM NAD for 3 h at 37°C. After washing, 107 T cells per mouse were injected i.v. into T cell-deficient recipients. The recipients were adult-thymectomized C57BL/6 Thy-1.1 mice (The Jackson Laboratory) that had been lethally irradiated and reconstituted with 107 bone marrow cells, depleted of T cells by treatment with anti-Thy-1 Ab and complement, 1 day before T cell injection. Four weeks later, spleens were harvested and assayed for Thy-1.2-staining cells by FACS analysis.

To assay homing of T cells (20), nylon wool-purified splenic T cells were incubated in RPMI 1640 medium, with or without 1 mM NAD, at 37°C for 3 h, labeled with 51Cr, and injected i.v. into syngeneic recipients at a dose of 5 x 106 cells per recipient. After 1 h, animals were sacrificed, spleens and livers were harvested, and radioactivity was determined in a gamma counter. The percentage of radioactivity recovered from organs was plotted, setting results from untreated cells as 100%.

Histology

To visualize apoptotic lymphocytes in the liver, liver tissue was fixed in 10% neutral buffered Formalin and embedded in paraffin. Five-micrometer sections were affixed to slides, deparaffinized. Apoptotic cells were visualized by the TUNEL assay, using the in situ cell death detection kit purchased from Boehringer Mannheim (Indianapolis, IN).

Lymphocyte purification and fluorometric analysis and tissue culture procedures

T cells were purified from spleen cells by nylon wool adherence (20). B cells were isolated from spleen cells by panning spleen cells on plates coated with anti-Ig Abs (21). The resulting cell populations were assayed for purity by fluorometric staining. For FACS analysis, 106 cells were stained with mAbs at a concentration of 1 µg per 100 µl PBS containing 0.2% BSA (Boehringer Mannheim) and 0.05% sodium azide for 30 min on ice (17). The following Abs were used: FITC-conjugated anti-Thy-1.2, anti-CD62L, PE-conjugated anti-CD3, anti-CD4, anti-CD8, and CyChrome-conjugated anti-CD69, all purchased from BD PharMingen (San Diego, CA). Apoptotic cells were detected by the annexin V fluos staining kit (Roche, Indianapolis, IN) (22). In all analyses, dead cells were gated out using propidium iodide (PI) staining; therefore, all data represent results from live cells. FACS analysis was performed on a FACStarPlus (BD Biosciences, Mountain View, CA).

To assay CTL responses, spleen cells were harvested 10 days after induction of GVHD and cultured with irradiated C3H stimulator cells in complete RPMI 1640 medium, containing 10% FCS for 5 days in 24-well plates (20). To prepare C1.18.4 (H-2k) targets, cells were labeled with 100 µCi Na2[51Cr]O4 (DuPont, Boston, MA) in 5% FCS RPMI 1640 for 120 min at 37°C. Ex vivo CTL assays on H-2d target P815 were performed with spleen cells from mice undergoing GVHD.

To stimulate T cells in vitro, nylon wool-purified T cells were cultured on anti-CD3-coated tissue culture plates. Plates were incubated with a 1/1000 dilution of anti-CD3 Ab (500AA2 ascites) overnight to absorb the Ab. After washing the plates, T cells were added in complete RPMI 1640 medium and incubated for 24 h. Cells were harvested and then cultured in the presence or absence of 1 mM NAD for 24 h and analyzed for activation by CD69 staining and for cell death by annexin V staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spleen cells treated with NAD lose ability to induce GVHD

Previous experiments had shown that incubating T cells, expressing ADPRT, with NAD leads to ADP ribosylation of cell surface molecules (16, 17, 18, 23, 24) and concomitant failure to respond to activation signals (19, 20). These findings raised the question as to what are the long-term effects of cell surface ADP ribosylation on in vivo T cell responses. To examine this, spleen cells from B6 (H-2b) mice were incubated with NAD and then injected into B6D2F1 (H-2bxd) recipients to assay ability to induce GVHD. Fig. 1GoA shows that infusion of untreated B6 spleen cells into B6D2F1 mice induces cell dose-dependent symptoms of GVHD, demonstrable by increased spleen weights. Preincubation of donor cells with NAD causes almost complete inhibition of ability to induce splenomegaly (Fig. 1GoA).



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 1. NAD treatment of donor T cells inhibits induction of GVHD in F1 hybrid mice. A, B6 spleen cells were cultured with or without 1 mM NAD for 3 h and then injected i.v. into groups of four BDF1 recipients at the cell concentrations indicated. On day 10, animals were sacrificed, spleen weights determined, and indexes calculated. B, Animals were prepared as in A, and spleen cells were harvested on day 10, at which time they were assayed for cytolytic activity on P815 (H-2d) targets at the E:T ratios indicated. C, Animals were prepared as in A. Spleen cells were harvested on day 10 and cultured with C3H stimulator cells for 5 days, at which time cytolytic activity was assayed on H-2k target C1.18.4 at the E:T ratios indicated. Results presented are from one of three repeat experiments.

 
A hallmark of GVHD is the induction of donor CTL with specificity for recipient MHC Ags that are not shared between the two. Thus, spleen cells from F1 mice, undergoing GVHD, contain B6 CTL able to lyse H-2d targets (Fig. 1GoB). Consistent with results in Fig. 1GoA, spleen cells from mice, injected with NAD-treated B6 cells, are not able to lyse H-2d targets (Fig. 1GoB). Therefore, NAD-treated B6 T cells fail to generate CTL specific for H-2d targets when injected into F1 recipients. A prediction therefore is that in animals, infused with NAD-treated B6 cells, the function of recipient T cells should be preserved. Indeed, Fig. 1GoC shows that spleen cells from F1 mice injected with NAD-treated B6 cells mount a perfectly normal CTL response to third-party H-2k stimulator cells in vitro. In contrast, spleen cells from mice injected with untreated B6 cells do not respond, because recipient T cells had been inactivated by action of B6 donor CTL, recognizing H-2d Ags. These results show that spleen cells from F1 mice, infused with NAD-treated parental B6 T cells, retain immune responsiveness.

NAD-incubated T cells are not able to reconstitute T cell-deficient mice

The finding that NAD-incubated T cells fail to induce GVHD points to the possibility that NAD inhibits T cell proliferation. To examine this, NAD-treated T cells were adoptively transferred into T cell-deficient syngeneic recipients to assay their survival and expansion. B6 Thy-1.1+ mice were thymectomized, lethally irradiated, and reconstituted with syngeneic bone marrow. Twenty-four hours later, animals were adoptively transferred with 107 purified, control, or NAD-treated B6 Thy-1.2+ T cells. Mice were sacrificed 4 wk later, and spleens were analyzed for Thy-1.2+ T cells by FACS analysis. Results in Fig. 2GoA show that mice that had been injected with NAD-treated T cells do not contain a detectable number of Thy-1.2+ T cells. In contrast, spleen cells from animals reconstituted with untreated T cells contain 3.5% Thy-1.2-staining T cells. Therefore, incubation of T cells with NAD before adoptive transfer into T cell-deficient mice interferes with their ability to reconstitute the T cell compartment of these mice.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 2. NAD inhibits T cells, but not bone marrow stem cells. A, Groups of four, adult-thymectomized, B6 Thy-1.1 mice were lethally irradiated and reconstituted with syngeneic T cell-free bone marrow. One day later, purified splenic T cells from B6 Thy-1.2 mice were incubated with 1 mM NAD for 3 h at 37°C, before i.v. injection at a dose of 107 T cells per mouse, as indicated. Four weeks later, animals were sacrificed and spleens were assayed for the percentage of Thy-1.2+ cells by FACS. The percentage of Thy-1.2-staining cells per spleen is plotted from one of two similar experiments. B, B6 bone marrow was treated or not treated with 1 mM NAD for 3 h and injected i.v. at a dose of 106 cells per mouse into groups of three lethally irradiated animals. CFUS were determined on day 8. Results from two experiments are shown.

 
The inhibitory effect of NAD on T cells could be due to toxicity, independent of expression of ADPRT. To examine this, effects of NAD on cells, known to lack ADPRT, were investigated. Assay of bone marrow from B6 mice for presence of ADPRT activity, using ADPRT enzyme assays, as well as labeling with radioactive NAD (16), failed to demonstrate any ADPRT activity on these cells (data not shown). Therefore, bone marrow cells appeared to be suitable to examine effects of NAD on cells not expressing ADPRT. To this end, bone marrow cells were incubated with NAD and then injected into syngeneic, lethally irradiated recipients. Results in Fig. 2GoB show that NAD-treated marrow generates the same number of CFUS as marrow not treated with NAD. Therefore, the inhibitory effect of NAD on T cells is not seen in bone marrow stem cells, devoid of ADPRT.

Injection of NAD-treated T cells leads to accumulation of apoptotic lymphocytes in the liver

The observation that NAD-treated T cells do not induce GVHD and are not able to repopulate spleens of T cell-depleted mice raises the possibility that NAD induces T cell anergy or apoptosis. Interestingly, our previous experiments had shown that NAD-treated T cells, when injected into mice, fail to home to lymphoid organs (20). This pointed to the possibility that aberrant homing is responsible for failure of T cells to respond to and to thrive in recipient animals. An important question, therefore, is where do NAD-treated T cells migrate? Results in Fig. 3GoA show that incubating purified T cells with increasing concentrations of NAD, before injection, leads to a gradual decrease of T cell homing to the spleen and a reciprocal increase of cells in the liver. Therefore, the inability of NAD-treated T cells to induce GVHD could be due to sequestration of T cells to the liver, which in turn raises the question as to why T cells migrate to this organ.



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 3. NAD induces accumulation of T cells and T cell death in the liver. A, B6 T were cultured with or without 1 mM NAD for 3 h and labeled with 51Cr for the last 60 min of the incubation. After washing, cells were injected i.v. into sets of three B6 recipients at a dose of 5 x 106 cells per animal. After 1 h, mice were sacrificed, and spleens and livers were recovered for determination of radioactivity. Radioactivity in organs of mice injected with untreated cells was set at 100%. Shown are data from one of two similar experiments. B, Injection of NAD-treated T cells leads to an increase of apoptotic cells in the liver. B6 T cells were incubated with or without 1 mM NAD for 3 h and then injected i.v. at a dose of 107 cells per mouse into groups of three B6 recipients. After 1, 2, and 12 h, mice were sacrificed, and liver sections were prepared and assayed for TUNEL-staining lymphocytes. Plotted are the means of TUNEL-staining lymphocytes per section in a x20 field from multiple sections. The data shown are from one of two similar experiments. C, NAD injection induces lymphocyte death in the liver. Groups of three B6 mice were injected i.v. with PBS (control) or 1 mg NAD in PBS. At the times indicated, livers were harvested and sections assayed for TUNEL-staining lymphocytes. The number of TUNEL-staining lymphocytes per x20 field is plotted, as well as SD values derived from multiple sections. The data shown are from one of two similar experiments. D, NAD injection induces an increase of T cells in the liver. Groups of three B6 mice were injected i.v. with PBS (No NAD) or 1 mg NAD in PBS, and livers were harvested at 6 and 12 h. Mononuclear cells were isolated from pooled livers from these groups; stained for CD3, CD4, and CD8; and analyzed by FACS. The calculated number of cells per liver is plotted. The data shown are from one of two similar experiments. E, NAD injection induces death of T cell in the liver. Groups of three B6 mice were injected i.v. with PBS (No NAD) or 1 mg NAD in PBS, and livers were harvested after 12 h. Mononuclear cells were isolated from pooled livers from these groups, stained for T cell markers as well as annexin V, and analyzed by FACS. The data shown are from one of two similar experiments.

 
Published data have shown that T cells, undergoing cell death, are captured in the liver (25). Therefore, the observation that NAD-treated T cells accumulate in this organ could indicate that they undergo apoptosis. To examine this, purified T cells were incubated with NAD, injected into mice and liver sections assayed by TUNEL staining for apoptotic lymphocytes. Results in Fig. 3GoB show that livers from mice injected with untreated T cells contain 5–7 TUNEL-staining cells per field at all time points. In contrast, liver sections from mice injected with NAD-treated T cells contain a mean of 37 staining cells at the 1-h time point, 27 at 2 h, but only 3 at the 12-h time point. These data suggest that NAD-treated T cells die in the liver or, less likely, induce cell death in companion lymphocytes.

NAD injection induces accumulation of apoptotic T cells in the liver

The observation that in vitro treatment of T cells with NAD leads to accumulation of apoptotic lymphocytes in the liver raises the question as to whether NAD has similar effects in vivo. To find out, mice were injected with NAD, and liver sections were assayed for apoptotic lymphocytes at various times thereafter. Results in Fig. 3GoC show that livers from NAD-injected mice contain a significantly higher number of TUNEL-staining lymphocytes at the 6-, 12-, and 24-h time points than PBS-injected controls.

To examine the phenotype of the apoptotic cells, liver mononuclear cells from a pool of three livers were isolated, stained with Abs, and analyzed by FACS. Fig. 3GoD shows that NAD injection causes a small increase in the total number of CD3+ cells per liver at the 6-h time point and a more significant increase of CD3+, CD4+, and CD8+ cells at 12 h. To examine whether this increase consists of apoptotic cells, lymphocytes were stained for annexin V. Results in Fig. 3GoE show that the majority of annexin V-staining cells at the 12-h time point are T cells, both CD4+ and CD8+ cells. Therefore, injection of NAD into naive animals induces rapid accumulation of apoptotic CD4+ and CD8+ T cells in the liver.

NAD induces apoptosis of T cells, but not B cells in vitro

The finding that NAD-treated T cells do not reconstitute T cell-deficient mice and that injection of NAD induces T cell death in vivo suggests that NAD induces T cell apoptosis. To directly show this, purified, splenic T cells were incubated with various amounts of NAD for various times and assayed for apoptosis by annexin V staining. Results in Fig. 4GoA reveal a small increase of annexin V-staining cells as early as 3 h after addition of 1000 µM NAD, reaching a substantial level by 24 h. Titration of NAD for the 24-h time point reveals a very small increase of cell death at 10 µM NAD, an intermediate effect at 100 µM NAD, and a high effect at 1000 µM NAD (Fig. 4GoC). In contrast to the results with T cells, purified, splenic B cells, previously shown to lack detectable cell surface ADPRT (20), when incubated with 1000 µM NAD, show no increase in annexin V-staining cells (Fig. 4GoB).



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4. NAD induces apoptosis in T cells, but not B cells. A, B6 T cells were cultured for the times indicated in 1 mM NAD at 37°C in complete medium. At the times indicated, cells were harvested and assayed for annexin V staining by FACS. The mean percentage of annexin V-staining cells is plotted from a total of nine independent experiments, including SD values. B, B6 B cells were cultured with NAD and assayed as in A. The data shown are means from three independent experiments, including SD values. C, B6 T cells were cultured with various concentrations of NAD, as in A. After 24 h, cells were harvested and assayed for annexin V staining by FACS. The mean percentage of annexin V-staining cells from three independent experiments is plotted, including SD values.

 
NAD induces apoptosis of naive, but not activated T cells

The finding that NAD-treated B6 T cells when injected into F1 mice do not respond (Fig. 1Go) suggests that naive T cells are sensitive to NAD-induced cell death. Splenic T cells from unprimed mice express CD62L, but not CD69, and are therefore predominantly naive T cells (26, 27) (Fig. 5GoA). Incubation of these cells with NAD causes an increase of annexin V-staining cells in the PI-negative population from 5% to 33% (Fig. 5GoB). The annexin V-staining cells are contained in the CD3+ cell population, which increases from 11% to 36% in the presence of NAD (Fig. 5GoC). Therefore, cell death is induced by NAD in the naive CD62L+CD69- T cell population. To examine whether cell death is induced in both the CD4+ and CD8+ cell population, double staining for CD4, CD8, and annexin V was done. Fig. 5GoC shows that annexin V staining in the CD4+ population increases from 7 to 18%, and in the CD8+ population from 3 to 15%. Therefore, both CD4+ and CD8+ cells are induced for cell death in the presence of NAD.



View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 5. NAD induces apoptosis in naive T cells. A, Purified splenic B6 T cells were analyzed by FACS for expression of CD62L and CD69. The dotted lines represent nonstained control cells. B, The cells from A were cultured for 24 h with or without 1 mM NAD in complete medium, stained with PI and annexin V, and analyzed by FACS. Analysis of the live cell population is shown. C, B6 T cells were cultured for 24 h with or without 1 mM NAD in complete medium. Cells were then stained for CD3, CD4, CD8, and annexin V, and analyzed by FACS. D, B6 T cells were first cultured for 24 h on anti-CD3-coated tissue culture plates. Cells were harvested and then recultured for 24 h with or without 1 mM NAD in complete medium. Cells were stained for CD69 and annexin V and analyzed by FACS. Data shown in B, C, and D are from one of three independent and similar experiments.

 
Previous experiments had shown that activation of T cells by TCR cross-linking leads to a rapid release of ADPRT from the T cell surface (17, 24). One would therefore predict that activated T cells are resistant to NAD-induced apoptosis. To examine this, T cells were first incubated on anti-CD3-coated tissue culture plates, followed by incubation with NAD. Results in Fig. 5GoD show that T cells stimulated on anti-CD3-coated plates for 24 h contain 78% CD69+ cells. Among these, 47% are apoptotic and stain with annexin V after a 24-h incubation without NAD. Inclusion of NAD into the culture medium does not increase the percentage of annexin V-staining cells in the CD69+ population. Therefore, activated, CD69+ cells do not undergo increased cell death when incubated with NAD.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results presented in this communication show that T cells treated with NAD accumulate in the liver as early as 1 h after injection, and TUNEL staining suggests that the injected cells undergo apoptosis in the liver. Because the liver is known as a graveyard for dying T cells (25), these results support the notion that in vitro treatment of T cells with NAD induces an apoptotic signal, resulting in accumulation of dying T cells in the liver. In agreement, functional assays show that NAD-treated T cells do not induce GVHD and, when injected into T cell-deficient mice, fail to expand.

The striking efficiency with which NAD inactivates T cells in vitro raised the question as to whether NAD can exert similar effects in vivo. We show that NAD concentrations in vitro, as low as 1–10 µM, may exert effects on T cell homing in vivo (20). It was therefore possible that injection of NAD leads to transient NAD levels able to induce demonstrable effects in vivo. Indeed, we show that injection of NAD causes a significant increase of TUNEL-staining lymphocytes in the liver. FACS analysis of mononuclear cells, isolated from livers of NAD-injected mice, revealed that apoptotic cells are in both the CD4+ and CD8+ cell populations. Therefore, injection of NAD can lead to concentrations, capable of inducing sequestration of T cells, undergoing apoptosis, in the liver.

An interesting question then is whether NAD has effects on both naive and activated T cells. We show that NAD-treated naive T cells fail to induce GVHD in F1 hybrid mice and, when injected into T cell-depleted syngeneic mice, do not reconstitute these mice. These data are consistent with the notion that naive T cells are inactivated by NAD incubation, a conclusion that is also supported by in vitro data. Incubation of naive T cells with NAD leads to death in the CD62L+CD69- cell population. In contrast, no death was demonstrable in CD69+ cells, activated by TCR cross-linking. It therefore appears that naive, but not activated T cells are sensitive to the NAD-induced apoptosis mechanism. This result is not unexpected, as activation of T cells had been shown to cause release of ADPRT from the cell surface, thereby depriving cells of the enzyme, responsible for modification of cell surface molecules (17, 18, 19, 20, 24). Our experiments showing that T cells, but not B cells and hemopoietic stem cells, are sensitive to NAD-induced apoptosis are consistent with this finding, as neither B cells nor bone marrow cells express ADPRT (20, 23 and unpublished results). Therefore, there is a correlation between expression of ADPRT and sensitivity to NAD-induced cell death.

A molecule that is also expressed on lymphoid cells and utilizes NAD as substrate is CD38. CD38 mediates NADase and ADP ribosecyclase activity, and is commonly expressed on B cells, hemopoietic stem cells, and activated, but not naive T cells (28, 29, 30). Given this distribution, it is highly unlikely that CD38 causes NAD-induced apoptosis, as no cell death is demonstrable in B cells, hemopoietic stem cells, and activated T cells. It is possible, however, that CD38 counteracts the action of ADPRT by removing NAD substrate from the extracellular space via its NADase activity.

An intriguing question is how ADP ribosylation of cell surface molecules, mediated by ADPRT, induces apoptosis. We had previously reported that in T cells in which cell surface molecules are ADP ribosylated, Ab-induced receptor capping and transmembrane signaling are inhibited (19, 20). It is therefore possible that changes in receptor association, caused by the modification, initiate the apoptotic signal. Recently, results were reported suggesting that ADP ribosylation of CD38 can induce cell death in various cell types (30). The demonstration in this study that NAD induces cell death in naive T cells that do not express CD38 (data not shown) makes this mechanism of ADPRT action unlikely.

The finding that NAD induces cell death in vitro and in vivo raises the more general question as to whether this pathway is utilized by the immune system to limit T cell responses. Published data have shown that elimination of RT6 (ART2)-expressing T cells in BB-DR rats induces diabetes (5), pointing to an immunoregulatory function of RT6. An unresolved question then is how does intracellular NAD reach an enzyme expressed on the cell surface? NAD is present not only in nucleated cells, but also in erythrocytes in concentrations approaching 1 mM (31). Hence, trauma and inflammation, associated with cell lysis, could result in levels of ecto-NAD, able to suppress T cell functions. Under normal conditions, NAD released from lysing cells is removed from the extracellular space by cell-bound and free NADases, of which CD38 is one. The steady state concentration of NAD in the serum is 0.1 µM (32), which is below the Km of ADPRTs. NAD released from lysing cells, exceeding this base level during inflammation, could therefore serve to limit the destructive action of autoreactive T cells, infiltrating the site of tissue injury.


   Footnotes
 
1 This work was supported by Public Health Service Grants AI 43954 and AI 40038. Back

2 Address correspondence and reprint requests to Dr. Gunther Dennert, University of Southern California Keck School of Medicine, Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, M/S #73, Los Angeles, CA 90089-9176. E-mail address: dennert{at}hsc.usc.edu Back

3 Abbreviations used in this paper: ADPRT, ADP ribosyltransferase; CFUS, CFU spleen; GVHD, graft-vs-host disease; NAD, nicotinamide adenine dinucleotide; PI, propidium iodide. Back

Received for publication June 18, 2001. Accepted for publication August 24, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zolkiewska, A., M. S. Nightingale, J. Moss. 1992. Molecular characterization of NAD:arginine ADP-ribosyltransferase from rabbit skeletal muscle. Proc. Natl. Acad. Sci. USA 89:11352.[Abstract/Free Full Text]
  2. Koch-Nolte, F., F. Haag. 1997. Mono(ADP-ribosyl)transferases and related enzymes in animal tissues: emerging gene families. Adv. Exp. Med. Biol. 419:1.
  3. Yu, Y., S. Okamoto, E. Nemoto, G. Dennert. 1997. Molecular cloning of a functional murine arginine-specific mono-ADP-ribosyltransferase and its expression in lymphoid cells. DNA Cell Biol. 16:235.[Medline]
  4. Okazaki, I. J., J. Moss. 1998. Glycosylphosphatidylinositol-anchored and secretory isoforms of mono-ADP-ribosyltransferases. J. Biol. Chem. 273:23617.[Free Full Text]
  5. Greiner, D. L., J. P. Mordes, E. S. Handler, M. Angelillo, N. Nakamura, A. A. Rossini. 1987. Depletion of RT6.1+ T lymphocytes induces diabetes in resistant biobreeding/Worcester (BB/W) rats. J. Exp. Med. 166:461.[Abstract/Free Full Text]
  6. Bortell, R., T. Kanaitsuka, L. A. Stevens, J. Moss, J. P. Mordes, A. A. Rossini, D. L. Greiner. 1999. The RT6 (Art2) family of ADP-ribosyltransferases in rat and mouse. Mol. Cell. Biochem. 193:61.[Medline]
  7. Hollmann, C., F. Haag, M. Schlott, A. Damaske, H. Bertuleit, M. Matthes, M. Kuhl, H. G. Thiele, F. Koch-Nolte. 1996. Molecular characterization of mouse T-cell ecto-ADP-ribosyltransferase Rt6: cloning of a second functional gene and identification of the Rt6 gene products. Mol. Immunol. 33:807.[Medline]
  8. Okazaki, I. J., H. J. Kim, J. Moss. 1996. Cloning and characterization of a novel membrane-associated lymphocyte NAD:arginine ADP-ribosyltransferase. J. Biol. Chem. 271:22052.[Abstract/Free Full Text]
  9. Prochazka, M., H. R. Gaskins, E. H. Leiter, F. Koch-Nolte, F. Haag, H. G. Thiele. 1991. Chromosomal localization, DNA polymorphism, and expression of Rt-6, the mouse homologue of rat T-lymphocyte differentiation marker RT6. Immunogenetics 33:152.[Medline]
  10. Kanaitsuka, T., R. Bortell, L. A. Stevens, J. Moss, D. Sardinha, T. V. Rajan, D. Zipris, J. P. Mordes, D. L. Greiner, A. A. Rossini. 1997. Expression in BALB/c and C57BL/6 mice of Rt6-1 and Rt6-2 ADP-ribosyltransferases that differ in enzymatic activity: C57BL/6 Rt6-1 is a natural transferase knockout. J. Immunol. 159:2741.[Abstract]
  11. Koch-Nolte, F., J. Klein, C. Hollmann, M. Kuhl, F. Haag, H. R. Gaskins, E. Leiter, H. G. Thiele. 1995. Defects in the structure and expression of the genes for the T cell marker Rt6 in NZW and (NZB x NZW)F1 mice. Int. Immunol. 7:883.[Abstract/Free Full Text]
  12. Prochazka, M., D. V. Serreze, S. M. Worthen, E. H. Leiter. 1989. Genetic control of diabetogenesis in NOD/Lt mice: development and analysis of congenic stocks. Diabetes 38:1446.[Abstract]
  13. Haag, F., V. Andresen, S. Karsten, F. Koch-Nolte, H. G. Thiele. 1995. Both allelic forms of the rat T cell differentiation marker RT6 display nicotinamide adenine dinucleotide (NAD)-glycohydrolase activity, yet only RT6.2 is capable of automodification upon incubation with NAD. Eur. J. Immunol. 25:2355.[Medline]
  14. Maehama, T., H. Nishina, S. Hoshino, Y. Kanaho, T. Katada. 1995. NAD+-dependent ADP-ribosylation of T lymphocyte alloantigen RT6.1 reversibly proceeding in intact rat lymphocytes. J. Biol. Chem. 270:22747.[Abstract/Free Full Text]
  15. Koch-Nolte, F., D. Petersen, S. Balasubramanian, F. Haag, D. Kahlke, T. Willer, R. Kastelein, F. Bazan, H. G. Thiele. 1996. Mouse T cell membrane proteins Rt6-1 and Rt6-2 are arginine/protein mono(ADPribosyl)transferases and share secondary structure motifs with ADP-ribosylating bacterial toxins. J. Biol. Chem. 271:7686.[Abstract/Free Full Text]
  16. Wang, J., E. Nemoto, A. Y. Kots, H. R. Kaslow, G. Dennert. 1994. Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchored/arginine ADP-ribosyltransferase. J. Immunol. 153:4048.[Abstract]
  17. Nemoto, E., S. Stohlman, G. Dennert. 1996. Release of a glycosyl-phosphatidylinositol-anchored ADP-ribosyltransferase from cytotoxic T cells upon activation. J. Immunol. 156:85.[Abstract]
  18. Nemoto, E., Y. Yu, G. Dennert. 1996. Cell surface ADP-ribosyltransferase regulates LFA-1 function in T cells. J. Immunol. 157:3341.[Abstract]
  19. Liu, Z. X., Y. Yu, G. Dennert. 1999. A cell surface ADP-ribosyltransferase modulates T cell receptor association and signaling. J. Biol. Chem. 274:17399.[Abstract/Free Full Text]
  20. Okamoto, S., O. Azhipa, Y. Yu, E. Russo, G. Dennert. 1998. Expression of ADP-ribosyltransferase on normal T lymphocytes and effects of nicotinamide adenine dinucleotide on their function. J. Immunol. 160:4190.[Abstract/Free Full Text]
  21. Takeda, K., M. W. Moore, G. Dennert. 1994. Acute rejection of marrow grafts in mice: dependence on and independence of functional TCR in the rejection process. J. Immunol. 152:4407.[Abstract]
  22. Liu, Z. X., S. Govindarajan, S. Okamoto, G. Dennert. 2001. Fas-mediated apoptosis causes elimination of virus-specific cytotoxic T cells in the virus-infected liver. J. Immunol. 166:3035.[Abstract/Free Full Text]
  23. Koch-Nolte, F., T. Duffy, M. Nissen, S. Kahl, N. Killeen, V. Ablamunits, F. Haag, E. H. Leiter. 1999. A new monoclonal antibody detects a developmentally regulated mouse ecto-ADP-ribosyltransferase on T cells: subset distribution, inbred strain variation, and modulation upon T cell activation. J. Immunol. 163:6014.[Abstract/Free Full Text]
  24. Kahl, S., M. Nissen, R. Girisch, T. Duffy, E. H. Leiter, F. Haag, F. Koch-Nolte. 2000. Metalloprotease-mediated shedding of enzymatically active mouse ecto-ADP-ribosyltransferase ART2.2 upon T cell activation. J. Immunol. 165:4463.[Abstract/Free Full Text]
  25. Crispe, I. N., W. Z. Mehal. 1996. Strange brew: T cells in the liver. Immunol. Today 17:522.[Medline]
  26. Sprent, J., D. F. Tough. 1994. Lymphocyte life-span and memory. Science 265:1395.[Abstract/Free Full Text]
  27. Ziegler, S. F., F. Ramsdell, M. R. Alderson. 1994. The activation antigen CD69. Stem Cells 12:456.[Medline]
  28. Lund, F., N. Solvason, J. C. Grimaldi, R. M. Parkhouse, M. Howard. 1995. Murine CD38: an immunoregulatory ectoenzyme. Immunol. Today 16:469.[Medline]
  29. Hirata, Y., N. Kimura, K. Sato, Y. Ohsugi, S. Takasawa, H. Okamoto, K. Ishikawa, T. Hirano. 1994. ADP-ribosecyclase activity of a novel bone marrow stromal cell surface molecule BST.1. FEBS Lett. 356:244.[Medline]
  30. Han, M. K., Y. S. Cho, Y. S. Kim, C. Y. Yim, U. H. Kim. 2000. Interaction of two classes of ADP-ribose transfer reactions in immune signaling. J. Biol. Chem. 275:20799.[Abstract/Free Full Text]
  31. Jacobson, E. L., V. Nunbhakdi-Craig, D. G. Smith, H. Y. Chen, B. L. Wasson, M. K. Jacobson. 1992. ADP-ribose polymer metabolism: implication for human nutrition. G. G. Proivier, and P. Moreau, eds. ADP-Ribosylation Reactions 153.-162. Springer Verlag, Heidelberg, Germany.
  32. Kim, U. H., M. K. Kim, J. S. Kim, M. K. Han, B. H. Park, H. R. Kim. 1993. Purification and characterization of NAD glycohydrolase from rabbit erythrocytes. Arch. Biochem. Biophys. 305:147.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Adriouch, S. Hubert, S. Pechberty, F. Koch-Nolte, F. Haag, and M. Seman
NAD+ Released during Inflammation Participates in T Cell Homeostasis by Inducing ART2-Mediated Death of Naive T Cells In Vivo
J. Immunol., July 1, 2007; 179(1): 186 - 194.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Kawamura, F. Aswad, M. Minagawa, S. Govindarajan, and G. Dennert
P2X7 Receptors Regulate NKT Cells in Autoimmune Hepatitis
J. Immunol., February 15, 2006; 176(4): 2152 - 2160.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
A. Zolkiewska
Ecto-ADP-ribose Transferases: Cell-Surface Response to Local Tissue Injury
Physiology, December 1, 2005; 20(6): 374 - 381.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Aswad, H. Kawamura, and G. Dennert
High Sensitivity of CD4+CD25+ Regulatory T Cells to Extracellular Metabolites Nicotinamide Adenine Dinucleotide and ATP: A Role for P2X7 Receptors
J. Immunol., September 1, 2005; 175(5): 3075 - 3083.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Krebs, S. Adriouch, F. Braasch, W. Koestner, E. H. Leiter, M. Seman, F. E. Lund, N. Oppenheimer, F. Haag, and F. Koch-Nolte
CD38 Controls ADP-Ribosyltransferase-2-Catalyzed ADP-Ribosylation of T Cell Surface Proteins
J. Immunol., March 15, 2005; 174(6): 3298 - 3305.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Kawamura, F. Aswad, M. Minagawa, K. Malone, H. Kaslow, F. Koch-Nolte, W. H. Schott, E. H. Leiter, and G. Dennert
P2X7 Receptor-Dependent and -Independent T Cell Death Is Induced by Nicotinamide Adenine Dinucleotide
J. Immunol., February 15, 2005; 174(4): 1971 - 1979.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
W. Ohlrogge, F. Haag, J. Lohler, M. Seman, D. R. Littman, N. Killeen, and F. Koch-Nolte
Generation and Characterization of Ecto-ADP-Ribosyltransferase ART2.1/ART2.2-Deficient Mice
Mol. Cell. Biol., November 1, 2002; 22(21): 7535 - 7542.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Z.-X.
Right arrow Articles by Dennert, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Z.-X.
Right arrow Articles by Dennert, G.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS