CD38 Controls ADP-Ribosyltransferase-2-Catalyzed ADP-Ribosylation of T Cell Surface Proteins
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免疫学杂志 2005年第6期
Abstract
ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38high cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.
Introduction
Like phosphorylation, ADP-ribosylation is one of the posttranslational protein modifications. Mono-ADP-ribosyltransferases (ARTs)3 transfer the ADP-ribose moiety from NAD to specific amino acids in target proteins. Usually, this causes activation or inactivation of the target protein (1, 2). ADP-ribosylation is the mechanism by which several bacterial toxins, like cholera and pertussis toxins, cause pathology after translocating into mammalian host cells (3). Mammalian cells express toxin-related ecto-ARTs, designated ART1 to ART5. ART1 to ART4 are GPI-anchored cell surface ARTs, whereas ART5 is a secreted protein (4, 5). The ART2 gene is duplicated into two functional genes, designated Art2.1 and Art2.2, in the mouse, whereas it is inactivated by premature stop codons in the human. ART1 is prominently expressed in skeletal and cardiac muscle, ART2 by T cells, ART3 and ART5 in testis, and ART4 by erythrocytes and monocytes. LFA-1, CD8, CD27, CD43, CD44, CD45, and the P2X7 purinoceptor have been identified as target proteins of ADP-ribosylation on murine T cells (6, 7).
Treatment of T cells with the ART substrate, NAD+, affects cell proliferation, cytotoxicity, homing, TCR clustering, and survival (6, 7, 8, 9, 10). We have recently discovered that ADP-ribosylation activates the P2X7 purinoceptor (7). P2X7 is a member of the P2X family of ATP-gated ion channels and is widely expressed on blood cells (11, 12). P2X7 has sparked interest because of its peculiar ability to induce the formation of a large nonselective membrane pore (13). Activation of P2X7 with millimolar concentrations of ATP triggers calcium flux, phosphatidylserine (PS) exposure, shedding of CD62L, and apoptosis (14, 15). The same effects are triggered by NAD at micromolar concentrations via ADP-ribosylation of P2X7 (7). The effects are not observed in ART2-deficient T cells, demonstrating that activation of P2X7 by NAD is ART2 dependent (7, 16). Two commonly used strains of laboratory mice (BALB/c and C57BL/6) carry allelic variants of the P2x7 gene locus, which dramatically affects the susceptibility of T cells from these mice to ecto-NAD and ecto-ATP (8, 17). BALB/c T cells that express wild-type P2X7 are sensitive to NAD and ATP, whereas C57BL/6 T cells that express the P451L allelic variant of P2X7 are resistant (7).
The type II transmembrane protein CD38 is a potent ecto-NAD-glycohydrolase (ecto-NADase) (18, 19). CD38 is expressed by lymphocytes, macrophages, endothelial cells, dendritic cells, pancreatic islet cells, and several other cell types. A soluble form of CD38, presumably generated by proteolytic cleavage of its juxtamembrane stalk, has been found in human serum and other extracellular fluids (18). CD38-deficient mice show normal development of the major lymphocyte subsets, but show impaired humoral immune responses, neutrophil chemotaxis, and dendritic cell trafficking (20, 21, 22). The dramatic reduction in NADase activity in tissues of CD38–/– vs wild-type mice indicates that CD38 is the predominant NADase in most murine tissues, including lymph node, spleen, and bone marrow (21).
It has been proposed that the classic intracellular metabolites of energy metabolism, NAD and ATP, also play roles as signaling molecules in the extracellular environment (23, 24, 25). The plasma membrane of living cells is impermeable to these nucleotides, but they can be released from cells by lytic and nonlytic mechanisms (26, 27). The fate of extracellular signaling molecules (ligands, transmitters) is determined by the rate of their release, metabolism, reuptake by cells, and/or renal excretion. For example, the concentration of ATP and the duration of signaling via ATP in the extracellular compartment are controlled by CD39 and related ectonucleotidases, which hydrolyze ATP to ADP and/or AMP (28, 29). The purpose of this study was to determine whether the ecto-NADase CD38 similarly controls the signaling function of NAD by limiting the availability of NAD as a substrate for ART-catalyzed ADP-ribosylation of cell surface proteins. A recently described immunoassay for monitoring ADP-ribosylation of cell surface proteins on living cells by flow cytometry (30), CD38–/– and ART2–/– mice (16, 20), and the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide (araF-NAD) (31) provided useful tools to address this question. Our results show that CD38, indeed, controls the level of ART2-catalyzed ADP-ribosylation of cell surface proteins.
Materials and Methods
Chemicals and Abs
ADP-ribose, ATP, NAD, and 1,N6-ethenonicotinamide adenine dinucleotide (etheno-NAD) were obtained from Sigma-Aldrich (Fig. 1). PE- and FITC-conjugated mAbs and Annexin V were purchased from BD Pharmingen/BD Biosciences, including anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-5.8), anti-CD38 (90), and anti-CD45R/B220 (RA3-6B2). The anti-ART2 Ab (Ali) was raised in our lab by genetic immunization (32) and recognizes a common epitope on ART2.1 and ART2.2. For the sake of brevity, "ART2" is used instead of "ART2.1 and ART2.2" throughout this paper. Fluorochrome conjugation of Ali and the anti-ethenoadenosine Ab 1G4 (30) was performed using the Alexa Fluor 488 labeling kit from Molecular Probes. [32P]NAD was obtained from Amersham Biosciences.
FIGURE 1. Chemical structures of NAD and NAD analogs used in this study.
Animals and cells
BALB/c and C57BL/6 mice were obtained from The Jackson Laboratory or Charles River. Cd38–/– mice (20) and Art2–/– mice (16) were backcrossed onto the BALB/c and C57BL/6 backgrounds for 8–12 generations. The ART2–/– lines are deficient in both, ART2.1 and ART2.2 (16). Single-cell suspensions were prepared from lymph nodes and spleen of sacrificed animals in RPMI 1640 medium by passage through Nitex membrane (110-μm mesh; Tetko). CD38+ cells were depleted using anti-CD38PE and anti-PE MicroBeads with VarioMACS and AS columns according to the manufacturer’s instructions (Miltenyi Biotec).
Incubation of cells with etheno-NAD and staining with 1G4
For monitoring etheno-ADP-ribosylation of cell surface proteins on living cells (30), cells (106/100 μl of RPMI 1640 medium) were incubated with or without the indicated concentrations of etheno-NAD at 37°C, washed, and stained with 1G4Alexa488 (1 μg) for 30 min at 4°C. Cells were costained with saturating amounts of fluorochrome-conjugated Abs against other cell surface proteins as indicated in the figures. Where indicated, cells were preincubated with araF-NAD (Fig. 1) (31) before etheno-NAD treatment for 10 min at room temperature. Ab-stained cells were washed and resuspended in 250 μl of RPMI 1640 and 10 μl of propidium iodide (PI; 10 μg/ml; Molecular Probes) and analyzed by flow cytometry on a FACSCalibur (BD Biosciences) as described previously (32). Mean fluorescence intensity (MFI) of gated cells was calculated with CellQuest (BD Biosciences), and diagrams were produced with Excel (Microsoft).
Enzyme assays
A total of 2 x 106 cells was preincubated with araF-NAD (0–10 μM) in 10 μl of RPMI 1640 medium for 15 min at room temperature. An equal volume (10 μl) of RPMI 1640 medium containing 4 μM NAD, 2 μCi of [32P]NAD, and 8 mM agmatine was added to the reaction. Cells were incubated for 30 min at 37°C. Cells were pelleted by centrifugations, and cell supernatants were analyzed by TLC on polyethyleneimine cellulose (Merck) as described previously (33).
Assay for PS exposure and PI staining
Single-cell suspensions from lymph nodes were prepared, and B cells were depleted using magnetic cell separation with Dynabead-immobilized goat anti-mouse IgG (Dynal) as described (7). Purity of T cells was always >95% as verified by FACS analyses using PE-conjugated anti-B220 and FITC-conjugated anti-CD3. Following treatment with NAD or ATP for 45 min at 37°C, cells were washed in RPMI 1640 medium adjusted to 2 mM CaCl2, and were stained for 20 min on ice with FITC-conjugated Annexin V (1 μg/ml) (BD Biosciences) and PI (10 μg/ml) before flow cytometry.
Results
Expression of CD38 and ART2 is inversely correlated on murine lymphocytes
Murine lymphocytes have been shown to express at least two distinct NAD-metabolizing ectoenzymes, the GPI-anchored ecto-ADP-ribosyltransferase ART2 and the type II transmembrane bifunctional NADase/ADP-ribosylcylase CD38. Whereas ART2 reportedly is expressed by peripheral T cells and is shed upon T cell activation (33), CD38 is expressed by B cells and by activated T cells (20, 34). Fig. 2 shows FACS profiles of splenocytes and purified lymph node T cells from C57BL/6, BALB/c, and respective CD38–/– and ART2–/– mice after staining for ART2, CD38, CD3, and B220. As reported previously, CD38–/– and ART2–/– mice do not show any marked deviations from wild-type proportions of the major splenic lymphocyte subpopulations (16, 20). Also, in accord with previous reports, CD38 is expressed at high levels on B220+ cells (namely B cells), whereas most T cells are CD38low or CD38– (Fig. 2, A and C). Expression of ART2 is restricted to B220– cells (T cells), and only a small population of ART2+ cells coexpresses CD38. Note that ART2 is expressed at higher levels on C57BL/6 than BALB/c T cells, as observed previously (32).
FIGURE 2. ART2 expression and ecto-ART activity are restricted to CD38– or CD38low cells. A and C, Splenocytes (A) and purified lymph node T cells (C) of C57BL/6, BALB/c wild-type, and respective CD38–/–, or ART2–/– mice were double-stained with fluorochrome-conjugated Abs for expression of CD38, ART2, CD3, and B220, and then analyzed by FACS. B and D, Splenocytes (B) and purified T cells (D) of the indicated mice were incubated with 10 μM etheno-NAD for 20 min and stained with anti-B220PE and 1G4Alexa488 before flow cytometry. Results are representative of three independent experiments.
CD38–/– lymphocytes exhibit higher levels of cell surface etheno-ADP-ribosylation than CD38 wild-type cells
Many extracellular ligands such as ATP or acetylcholine are degraded hydrolytically by specific enzymes, which thereby limit the availability of the ligand for its receptor. Because CD38 is known to possess potent NADase activity, we reasoned that CD38 might influence the substrate availability for NAD-dependent ADP-ribosyltransferase ART2. To address this question, we compared cell surface protein ADP-ribosylation of lymphocytes from wild-type, CD38–/–, and ART2–/– mice. To this end, we treated splenocytes (Fig. 2B) and purified lymph node T cells (Fig. 2D) with etheno-NAD, which is an efficient substrate for CD38 and ARTs (30, 35), and then detected etheno-ADP-ribosylated proteins with etheno-adenosine-specific mAb 1G4. In accord with the expression pattern of ART2, cell surface etheno-ADP-ribosylation is restricted to B220– cells (Fig. 2B). Interestingly, cells from CD38-deficient mice show enhanced cell surface etheno-ADP-ribosylation in comparison with their wild-type counterparts (Fig. 2, B and D). Cells from ART2-deficient mice do not show any etheno-ADP-ribosylation, indicating that ART2 is the only ART activity on splenic and lymph node T cells. Dose-response analyses further demonstrate the striking differences in apparent cell surface ART activity between CD38–/– and wild-type cells (Fig. 3). Following incubation of cells with etheno-NAD, both CD4+ and CD8+ T cell subsets show dose-dependent staining with 1G4 (Fig. 3B). In contrast, CD4– and CD8– cells show little if any staining with 1G4. CD8+ cells exhibit brighter 1G4 staining than CD4+ cells, in accord with the higher level of ART2 expression by CD8+ vs CD4+ cells (Fig. 3A) (32). Strikingly, at low concentrations of exogenous NAD (<50 μM), cells from wild-type mice show much lower apparent ART activity than cells from CD38-deficient mice. At high levels of exogenous NAD (>50 μM), wild-type cells show similar if not slightly stronger levels of cells surface etheno-ADP-ribosylation.
FIGURE 3. Comparative analysis of cell surface protein etheno-ADP-ribosylation by splenocytes of CD38wt and CD38–/– mice. A, Splenocytes from C57BL/6 wild-type or C57BL/6 CD38–/– mice were incubated for 20 min in the absence or presence of the indicated concentrations of etheno-NAD. Cells were washed and stained with 1G4Alexa488, CD4Cy3, and CD8PE before FACS analysis. For control, cells were also stained with ART2Alexa488, CD4Cy3, and CD8PE (left panels). B, Cells were stained as in A, and gating was performed on CD8+ cells or on CD4+ cells. The MFI of 1G4 staining of gated cells was determined and plotted as log MFI vs concentration of etheno-NAD. Results are representative of three independent experiments.
Depletion of CD38+ cells results in enhanced levels of cell surface etheno-ADP-ribosylation on CD38– T cells
Because T cells express only low levels of CD38, but B cells express very high levels of CD38 (Fig. 2), the differences in cell surface ADP-ribosylation of CD38–/– vs wild-type cells (Fig. 3) could, in principle, be explained by low levels of CD38 acting in cis on the T cell surface and/or by high levels of CD38 acting in trans on the B cell surface. To investigate whether ADP-ribosylation of cell surface proteins can be influenced in trans by the presence of cells expressing CD38, we removed CD38+ cells by magnetic cell separation. CD38-depleted and total splenocytes were then incubated with or without etheno-NAD and stained with fluorochrome-conjugated 1G4 and anti-CD38 Abs (Fig. 4A). Double staining of total splenocytes revealed prominent 1G4 staining of CD38low and CD38– cells, but little if any 1G4 staining of CD38high cells (the latter correspond to ART2– B cells; see Fig. 2). Moreover, depletion of CD38high cells, indeed, resulted in strongly enhanced 1G4 staining of CD38– and CD38low cells. Similar results were obtained with splenocytes prepared from C57BL/6 and BALB/c mice (Fig. 4, B and C). Note, however, that BALB/c cells exhibit lower levels of etheno-ADP-ribosylation than C57BL/6 cells (Fig. 4, C vs B), in accord with the lower level of ART2 expression by BALB/c cells (Fig. 2A).
FIGURE 4. Etheno-ADP-ribosylation of splenocyte cell surface proteins in the presence or absence of CD38+ cells. A, Splenocytes obtained from C57BL/6 mice were stained with anti-CD38PE, and CD38+ cells were depleted by magnetic depletion. Total splenocytes and CD38-depleted splenocytes (106/100 μl of RPMI 1640 medium) were incubated for 20 min with 2 or 20 μM etheno-NAD. Cells were then washed and stained with anti-etheno-adenosine mAb 1G4Alexa488 and anti-CD38PE before FACS analysis. B and C, Total and CD38-depleted splenocytes were prepared from C57BL/6 mice (B) or BALB/c mice (C) as described above. Cells were incubated for the indicated time (0–120 min) with 2 or 20 μM etheno-NAD and were then washed and stained with 1G4Alexa488 and anti-CD38PE before FACS analysis. Gating was performed on CD38– cells (as indicated by the gate in A). The MFI of 1G4 staining of CD38– cells was determined and plotted as log MFI vs time of incubation in the presence of etheno-NAD. Results are representative of three independent experiments.
CD38 decreases ART2-catalyzed ADP-ribosylation in trans by reducing the amount of ART substrate NAD
The results obtained so far show that the presence of CD38 can influence cell surface protein ADP-ribosylation on a distinct cell population. To determine whether CD38+ cells inhibit ADP-ribosylation in trans by limiting substrate availability, we adapted our 1G4-based FACS assay as a bioassay for assessing ecto-etheno-NAD concentrations available for ADP-ribosylation. To this end, supernatants were harvested from wild-type or CD38–/– splenocytes that had been incubated with etheno-NAD for 20 min, and these supernatants were used as a source of etheno-NAD for cells that had not been exposed to etheno-NAD (Fig. 5). By comparing 1G4 staining of these cells in FACS analyses, it was possible to estimate the reduction in etheno-NAD levels following a 20-min incubation of cells with etheno-NAD. Fig. 5A shows comparative FACS analyses of splenocytes following a 20-min incubation either with 12.5 μM etheno-NAD or with the supernatant harvested from cells after a 20-min incubation with 12.5 μM etheno-NAD. The results show that incubation of wild-type cells with etheno-NAD for 20 min dramatically reduces the level of substrate available for subsequent etheno-ADP-ribosylation, as reflected by the much lower level of 1G4 staining of cells incubated with supernatant of etheno-NAD-treated cells than of cells incubated with fresh etheno-NAD (Fig. 5A, panel 3 vs panel 2). In striking contrast, incubation of CD38–/– cells with etheno-NAD for 20 min had little if any effect on substrate available for subsequent etheno-ADP-ribosylation as reflected in similar 1G4-staining levels of cells incubated with supernatant and with fresh etheno-NAD (Fig. 5A, panel 6 vs panel 5). Dose-response analyses confirm that wild-type splenocytes catabolize etheno-NAD in a dose-dependent manner (Fig. 5B, left panel). Low levels of ecto-etheno-NAD (<5 μM) are completely catabolized by wild-type splenocytes within 20 min. Wild-type cells quite efficiently catabolize even much higher concentrations of etheno-NAD (50% hydrolysis of 50 μM etheno-NAD within 20 min). In contrast, CD38–/– splenocytes show little if any capacity to metabolize even low levels of etheno-NAD (Fig. 5B, right panel).
FIGURE 5. Comparative analysis of etheno-NAD consumption by CD38 wild-type and CD38–/– cells. A, Splenocytes from C57BL/6 wild-type mice (panels 1–3) or C57BL/6 CD38–/– mice (panels 4–6) were incubated for 20 min in the absence (panels 1 and 4) or presence of 12.5 μM etheno-NAD (panels 2 and 5). Cells were then pelleted by centrifugation and the supernatants were collected. Fresh cells of the same animal were incubated for 20 min with these supernatants (panels 3 and 6). Cells were washed and stained with 1G4Alexa488, CD4Cy3, and CD8PE before FACS analysis. B, Cells were incubated for 20 min with the indicated concentrations of etheno-NAD and processed for FACS analysis as in A. Gating was performed on CD8+ cells (corresponding to gate R2 in A). The MFI of 1G4 staining of CD8+ cells was determined and plotted vs concentration of etheno-NAD. The panel on the left shows etheno-ADP-ribosylation by CD8+ cells from CD38 wild-type mice following incubation either with fresh etheno-NAD () or with the supernatants of cells after a 20-min incubation with the indicated concentrations of etheno-NAD (). The panel on the right shows a similar analysis for cells from CD38–/– mice. Results are representative of three independent experiments.
raF-NAD enhances etheno-ADP-ribosylation by wild-type but not by CD38–/– splenocytes
The results shown so far indicate that CD38 influences ART2-mediated ADP-ribosylation of cell surface proteins by catabolizing the substrate NAD. If so, specifically inhibiting the NAD-metabolizing activity of CD38 should increase the substrate availability and thereby indirectly enhance ART2-catalyzed cell surface ADP-ribosylation. araF-NAD, in which the ribose group adjacent to the nicotinamide moiety of NAD is replaced by fluoroarabinoside (see Fig. 1), has been described as a very potent, nonhydrolyzable inhibitor of splenocyte ecto-NADases (31). However, it was not known whether araF-NAD affects ARTs. To assess the effects of araF-NAD on CD38 and ART2, we preincubated wild-type or CD38–/– splenocytes with araF-NAD for 20 min before exposure to etheno-NAD and then measured cell surface etheno-ADP-ribosylation using the 1G4-based FACS assay. In these experiments, we used mAb B220 to stain B cells and then analyzed ADP-ribosylation on T cells by gating on cells lacking B220. The results shown in Fig. 6A reveal a dose-dependent enhancement of cell surface protein etheno-ADP-ribosylation by araF-NAD in case of wild-type B220– splenocytes, whereas araF-NAD had little if any influence on cell surface etheno-ADP-ribosylation by B220– splenocytes from CD38–/– mice. Kinetic analyses confirmed the potent dose-dependent stimulation of cell surface etheno-ADP-ribosylation following preincubation with araF-NAD for 20 min in the case of wild-type but not CD38–/– splenocytes (Fig. 6B). These results indicate that the apparent stimulatory effect of araF-NAD on cell surface ADP-ribosylation is mediated indirectly by its inhibition of CD38 rather than by a direct action on ART2.
FIGURE 6. Comparative analyses of cell surface ADP-ribosylation by CD38 wild-type and CD38–/– splenocytes in the presence of araF-NAD. A, Splenocytes obtained from C57BL/6 wild-type () and CD38–/– () mice were preincubated with the indicated concentrations of araF-NAD (0–2 μM) for 20 min before addition of 2 μM etheno-NAD and further incubation in the presence of araF-NAD and etheno-NAD for 20 min. Cells were washed and stained with 1G4Alexa488 and anti-B220PE before analysis by flow cytometry. Analysis of 1G4 staining was performed on cells gated for lack of B220 expression. B, Splenocytes obtained from C57BL/6 wild-type and CD38 knockout mice were preincubated without (), with 0.1 μM (), or with 1.0 μM araF-NAD () for 15 min before addition of 2 μM etheno-NAD and further incubation for the indicated times. Cells were washed and stained with 1G4Alexa488 and anti-B220PE before analysis by flow cytometry. Analysis of 1G4 staining on gated cells (B220–) was performed as in A. Results are representative of three independent experiments.
To further assess the effects of araF-NAD, we analyzed ADP-ribosylation of agmatine—a soluble arginine analog and efficient ART2 substrate (36)—in the presence or absence of CD38 or ART2. Following the incubation of wild-type, CD38–/–, or ART2–/– splenocytes with radiolabeled NAD and agmatine, the cell supernatants were analyzed by TLC. Soluble Neurospora crassa NADase and recombinant murine ART2.2 were used as controls. The former converted almost the entire input NAD (2 μM) to ADP-ribose but did not show any detectable ADP-ribosylation of agmatine (Fig. 7, lane 2), whereas the latter converted nearly the entire input NAD to agmatine-ADP-ribose but showed little if any NAD-hydrolyzing activity (lane 3). The results shown in Fig. 7 further reveal that ART2–/– splenocytes (lane 4) metabolized almost the entire input NAD (2 μM) to ADP-ribose within 20 min. In contrast, CD38–/– splenocytes showed only background levels of NAD-hydrolysis to ADP-ribose; but these cells did show prominent ADP-ribosylation of agmatine (lane 5). Wild-type cells exhibited potent NAD-hydrolysis activity as well as detectable, albeit very low levels of agmatine-ADP-ribosylation (lane 6). Preincubation of wild-type cells with araF-NAD inhibited NAD-hydrolysis in a dose-dependent manner (lanes 6, 10, and 14). Similar effects were observed for ART2–/– cells (lanes 4, 8, and 12). Note that preincubation with 1.6 μM araF-NAD blocked NAD-hydrolysis by ART2–/– cells almost completely, whereas—expectedly—these cells did not show any detectable agmatine ADP-ribosylation (lane 12 vs lane 4). In the case of wild-type cells, pretreatment with 1.6 μM araF-NAD effectively blocked NAD-hydrolysis and enhanced ADP-ribosylation of agmatine (lane 14 vs lane 6). As in the case of cell surface protein etheno-ADP-ribosylation (Fig. 6), araF-NAD had no detectable effect on CD38–/– cells (Fig. 7, lanes 5, 9, and 13), supporting the conclusion that the effects of araF-NAD on ADP-ribosylation are mediated indirectly via its inhibition of CD38.
FIGURE 7. Comparative analysis of NAD-glycohydrolysis and agmatine ADP-ribosylation by splenocytes of wild-type, CD38–/–, and ART2–/– mice in the absence and presence of araF-NAD. Splenocytes from BALB/c wild-type, CD38–/–, and ART2–/– mice were preincubated for 15 min without and with 0.1 or 1.6 μM araF-NAD, before addition of 2 μM [32P]NAD and 4 mM agmatine and further incubation for 20 min. Cells were pelleted by centrifugation and aliquots of the supernatants were analyzed by TLC. Chromatograms were subjected to autoradiography for 14 h at –80°C. Control incubations were performed in the presence of H2O, N. crassa NADase, or purified recombinant murine ART2.2 (lanes 1–3, 7, and 11). Results are representative of three independent experiments.
T cells from CD38–/– show higher cell surface levels of PS and lower levels of CD62L than wild-type T cells
ADP-ribosylation of T cell surface proteins upon incubation of cells with ecto-NAD activates the P2X7 purinoceptor (7). As in the case of P2X7 activation by high doses of ATP, this induces shedding of CD62L, exposure of PS, and staining with PI. Cells lacking ART2 are resistant to NAD-induced apoptosis but still undergo apoptosis in response to ATP provided that the P2X7 receptor is functional. Cells expressing the P451L variant of the P2X7 receptor are resistant to both NAD- and ATP-mediated activation of P2X7. Remarkably, even in the absence of exogenously added NAD or ATP, a small but distinct subpopulation of lymph node T cells from wild-type mice but not from ART2–/– mice exposed PS and lacked CD62L. We hypothesized that this spontaneous exposure of PS and loss of CD62L might reflect ADP-ribosylation consequential to the release of endogenous NAD from cells in situ and/or during the preparation of lymph node cells (7, 16). If so, we reasoned that lymph node cells from CD38–/– mice, which lack the major
NAD-hydrolyzing activity, should encounter higher levels of endogenous ecto-NAD under these conditions, and therefore, should show higher levels of PS and lower levels of CD62L on the cell surface. To test this hypothesis, we analyzed Annexin V/PI and CD62L staining of lymph node T cells from BALB/c wild-type and CD38–/– mice in the absence of exogenously NAD or ATP (Fig. 8A). Indeed, a much larger subpopulation of cells from CD38–/– mice than from wild-type or ART2–/– mice showed spontaneous staining by Annexin V (39%) and PI (28%). Moreover, a large fraction (38 of 67 = 58%) of CD3+ cells from these mice did not stain for CD62L (vs 43% of wild-type T cells; Fig. 8B). The fact that cells from ART2-deficient mice contain little if any cells exposing PS (8%) (Fig. 8A) and only a small fraction of cells lacking CD62L (15%) (B) argues that these effects are mediated by ART2-catalyzed endogenous ADP-ribosylation.
FIGURE 8. Comparative analysis of NAD- and ATP-induced apoptosis (A) and CD62L shedding (B) by T cells of wild-type and CD38–/– mice. Purified lymph node T cells from BALB/c wild-type, CD38–/–, and ART2–/– mice were incubated without or with 20 μM NAD or 200 μM ATP for 40 min. Cells were washed and stained with Annexin VFITC and PI (A) or with anti-CD62LPE and anti-CD3FITC (B) before FACS analysis. Numbers indicate the percentage of cells in each quadrant. Results are representative of five independent experiments.
Upon addition of exogenous NAD or ATP, cells from both BALB/c CD38–/– and wild-type mice responded by exposing PS and shedding CD62L (Fig. 8), even though much higher concentrations of ATP than NAD were needed to elicit an effect. Saturation responses were obtained with 30 μM NAD and 1000 μM ATP (not shown). In case of ART2–/– mice, even high concentrations of exogenously added NAD failed to induce PS exposure or CD62L shedding, whereas these effects were readily triggered by high concentrations of exogenously added ATP. These findings imply that the spontaneous PS exposure and loss of CD62L observed in CD38–/– and wild-type mice are induced by endogenously released NAD rather than ATP. Cells derived from the C57BL/6 background, which express the P451L variant of the P2X7 receptor (17), showed strongly diminished responses to both NAD and ATP (not shown).
Discussion
Lymphocytes express a flurry of nucleotide-metabolizing ectoenzymes, including ENTPDases (CD39), pyrophosphatases (CD203), 5'-nucleotidase (CD73), NADases (CD38, CD157), and ARTs (37, 38), the function of which has puzzled investigators. One hypothesis holds that these enzymes catabolize ATP, NAD, and other nucleotides released from dying cells in inflamed tissues to recycle nucleotides by providing precursors for uptake by proliferating lymphocytes (37). Another hypothesis proposes that these enzymes control the availability of ATP and its metabolites ADP, AMP, and adenosine as ligands for specific receptors (P2X and P2Y family of purinergic receptors, P1 family of adenosine receptors) (24, 28, 29). In this study, we explore the hypothesis that CD38 may similarly affect the availability of NAD as an extracellular signaling molecule by controlling the concentration of NAD and the duration of its presence as a substrate for ART2-catalyzed ADP-ribosylation of cell surface proteins.
Our results show that CD38, indeed, controls the level of ART2-catalyzed cell surface protein ADP-ribosylation and indicate that CD38 exerts this control not only in cis on the same cell surface as ART2, but also in trans on the surface of other cells. Thus, transfection of ART2 into lymphoma cells lacking CD38 results in much higher levels of cell surface ADP-ribosylation than transfection into cells expressing CD38 (results not shown). Moreover, purified T cells from CD38–/– mice show stronger ADP-ribosylation of cell surface proteins than cells from wild-type mice, especially at lower and more physiological concentrations of ecto-NAD (Fig. 2D). The effect of CD38 on ADP-ribosylation in cis is moderate, reflecting the low level of CD38 on T cells. A stronger effect of CD38 in trans becomes apparent when comparing levels of cell surface ADP-ribosylation by wild-type vs CD38–/– T cells in total splenocyte populations (Figs. 2B and 3). In this situation, wild-type T cells are in the company of cells expressing high levels of CD38 (wild-type B cells), whereas CD38–/– T cells are in the company of cells lacking CD38. Consistently, removal of CD38+ cells from suspensions of wild-type splenocytes markedly enhances the degree of cell surface ADP-ribosylation by the remaining cells (Fig. 4).
The finding that araF-NAD, a specific inhibitor of CD38-mediated NAD-hydrolysis, enhances ADP-ribosylation by wild-type but not by CD38–/– T cells (Figs. 6 and 7) further supports the conclusion that CD38 controls ART2-catalyzed protein ADP-ribosylation. This finding is of import also to studies aimed at developing inhibitors of CD38 for experimental and therapeutic modulation of immune functions (39). Our results demonstrate that such inhibitors can indirectly effect ADP-ribosylation reactions catalyzed by ecto-ARTs by increasing the levels of ecto-NAD. Our results may also be relevant to other studies using CD38–/– mice as some of the immune phenotypes reported for these mice may be due to enhanced ADP-ribosylation of target proteins by ARTs consequential to the inefficient removal of ecto-NAD in these mice (20, 22, 40, 41, 42). Note that all of the studies published to date have been conducted using CD38-deficient mice on the C57BL/6 background expressing the functionally impaired P451L variant of P2X7.
Thus, if ARTs affected the immune phenotypes in these mice, it is more likely that this is mediated by ADP-ribosylation of LFA-1 and other targets rather than by ART-mediated activation of P2X7.
The rapid consumption of exogenously added etheno-NAD and [32P]NAD by CD38-expressing splenocytes but not by CD38–/– cells (Figs. 4 and 6) implies that local concentrations of NAD may vary dramatically in vivo, depending on the presence of CD38-expressing cells and/or soluble CD38. Considering that cells in the experimental setting are suspended in a much larger volume of extracellular medium (107 cells/ml) than in vivo (2 x 108 splenocytes in a total volume of 250 μl) suggests that ecto-NAD is metabolized even faster in the surroundings of CD38-expressing cells in vivo than in the experimental situation illustrated in Fig. 5. Given the strikingly different patterns of expression of CD38 and ART2, areas rich in B cells that express very high levels of CD38 but not ART2 (Fig. 2) can be predicted to be essentially ecto-NAD-free zones, because extracellular NAD will be rapidly degraded by CD38. In contrast, local levels of ecto-NAD may be sustained longer and at higher levels in areas rich in T cells, most of
which express ART2 but little if any CD38 (Fig. 2).
Our results further illustrate that endogenous sources of ecto-NAD can affect T cell functions. Thus, a substantial fraction of freshly prepared T cells from CD38–/– but not from ART2–/– mice exposes PS on the cell surface (Fig. 8A) and lacks cell surface CD62L (B), whereas wild-type T cells show an intermediate phenotype. ART2-dependent PS exposure and shedding of CD62L both result from the activation of the P2X7 purinoceptor by ADP-ribosylation (7). C57BL/6 cells express a variant P2X7 receptor carrying the P451L mutation in the cytoplasmic domain, which strongly impairs P2X7-mediated signaling (17). Consistently, C57BL/6 T cells exhibit little if any spontaneous PS exposure or loss of CD62L (not shown). Considering that PS exposure and shedding of CD62L both occur within minutes after exposure of cells to NAD (7), it is possible that PS exposure and loss of CD62L by freshly prepared cells is induced by NAD released from cells lysed during cell preparation. Indeed, fresh preparations of lymph node cells consistently contain a small but distinct population of dead cells characterized by bright PI staining and small forward scatter, and these cells conceivably could have released their intracellular pool of NAD during cell preparation. Alternatively, T cells may have experienced NAD exposure in situ before sacrifice of the animal. In either case, the presence or absence of CD38 evidently profoundly affects the effective ecto-NAD concentration (Fig. 8). By analogy, NAD released from lysed cells during acute inflammation or tissue damage would be expected to exert strikingly different effects on T cells in vivo, depending on the presence or absence of CD38.
Only little is known about physiological and pathological release of NAD in vivo. Intracellular concentrations of NAD may reach millimolar levels, whereas steady-state serum levels of ecto-NAD are in the submicromolar range (23, 43). A substantial body of evidence indicates that nucleotides can be released from cells by nonlytic mechanisms, e.g., following mechanical shear forces or stimulation (23, 24, 27). Moreover, connexin 43 hemichannels may function as channels for the release of ATP and NAD (26). During infection, killed bacteria, yeast, and protozoa as well as lysed host cells represent additional potential sources of ecto-NAD. Of note, certain bacterial pathogens, e.g., Haemophilus influenzae, are known to lack endogenous NAD-synthesizing machinery and depend on ecto-NAD (44). Thus, relatively high levels of ecto-NAD can be predicted in the surroundings of lysed cells, especially in tissues lacking CD38 or expressing only low levels of CD38.
On the basis of our results, we speculate that elevated or sustained levels of ecto-NAD, as predicted to occur in CD38–/– mice under conditions of infection or tissue damage, lead to enhanced T cell death by triggering NAD-induced cell death via the ART2/P2X7 pathway and, subsequently, to an increased compensatory homeostatic proliferation of ART2-negative cells. Combined with a predisposing genetic background, enhanced homeostatic proliferation of T cells can precipitate autoimmune disease, as recently demonstrated in the case of the NOD mouse model for insulin-dependent diabetes mellitus (45). It should, therefore, be of great interest to determine whether CD38 deficiency affects T cell homeostasis in physiological and pathophysiological settings, and to determine whether the presence or absence of CD38 and/or ART2 affects the incidence or progression of autoimmune disease in NOD mice, which carry the wild-type P2X7 receptor (17).
Given the striking effect of CD38 on ART2-catalyzed ADP-ribosylation on T cells, it is not unlikely that CD38 also influences ADP-ribosylation by other members of the ART family. Cells in other tissues expressing high levels of CD38 can be expected to mediate locally low levels of ecto-NAD. CD38 is expressed in tissues, such as heart and reproductive organs, known to express other members of the ART family (5, 46, 47).
It has been proposed that nucleotides released from cells may function as signaling molecules (24). Our results support this notion and indicate that NAD itself may play a signaling role. On the basis of our findings, it is tempting to speculate that ART2 acts as a sensor of ecto-NAD levels, which translates the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins. The duration and intensity of exposure of cells to ecto-NAD in turn are determined by the local levels of CD38 on the cell surface or in the extracellular medium. In this scenario, the NAD-metabolizing ectoenzymes CD38 and ART2 together convey information about the presence of the signaling molecule ecto-NAD to lymphocytes and thereby may help to fine-tune immune responses.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
This work represents the partial fulfillment of the requirements for the graduate thesis of C. Krebs. C. Krebs thanks E. H. Leiter and his staff for the hospitality during C. Krebs’s stay as a visiting scientist in his lab. We thank Dunja Freese and Gudrun Dubberke (University Hospital, Hamburg, Germany) for excellent technical assistance. F. Koch-Nolte, F. Haag, E. H. Leiter, F. E. Lund, and M. Seman designed and supervised the study. F. E. Lund provided the CD38–/– mice, and N. Oppenheimer provided araF-NAD. C. Krebs and F. Braasch performed the experiments shown in Figs. 3–7, and S. Adriouch performed those shown in Figs. 2 and 8. W. Koestner performed the cotransfection studies. C. Krebs and F. Koch-Nolte wrote the paper. We thank B. Fleischer, S. Rothenburg, P. Bannas, and F. Scheuplein (University Hospital, Hamburg, Germany) for critical reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 545/B9 and No310/6 (to F.K.-N. and F.H.), by stipends from the Werner Otto Foundation and the Boehringer Ingelheim Fonds (to C.K.), by a stipend from the Fondation pour la Recherche Medical (to S.A.), by National Institutes of Health Grant AI-057996 (to F.E.L.), and by National Institutes of Health Grants DK27722 and DK36175 (to E.H.L.).
2 Address correspondence and reprint requests to Dr. Friedrich Koch-Nolte, Institute for Immunology, University Hospital, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail address: nolte{at}uke.uni-hamburg.de
3 Abbreviations used in this paper: ART, ADP-ribosyltransferase; NADase, NAD-glycohydrolase; araF-NAD, nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide; MFI, mean fluorescence intensity; PS, phosphatidylserine; PI, propidium iodide; etheno-NAD, nicotinamide 1,N6-ethenoadenine dinucleotide.
Received for publication November 24, 2004. Accepted for publication December 21, 2004.
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ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38high cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.
Introduction
Like phosphorylation, ADP-ribosylation is one of the posttranslational protein modifications. Mono-ADP-ribosyltransferases (ARTs)3 transfer the ADP-ribose moiety from NAD to specific amino acids in target proteins. Usually, this causes activation or inactivation of the target protein (1, 2). ADP-ribosylation is the mechanism by which several bacterial toxins, like cholera and pertussis toxins, cause pathology after translocating into mammalian host cells (3). Mammalian cells express toxin-related ecto-ARTs, designated ART1 to ART5. ART1 to ART4 are GPI-anchored cell surface ARTs, whereas ART5 is a secreted protein (4, 5). The ART2 gene is duplicated into two functional genes, designated Art2.1 and Art2.2, in the mouse, whereas it is inactivated by premature stop codons in the human. ART1 is prominently expressed in skeletal and cardiac muscle, ART2 by T cells, ART3 and ART5 in testis, and ART4 by erythrocytes and monocytes. LFA-1, CD8, CD27, CD43, CD44, CD45, and the P2X7 purinoceptor have been identified as target proteins of ADP-ribosylation on murine T cells (6, 7).
Treatment of T cells with the ART substrate, NAD+, affects cell proliferation, cytotoxicity, homing, TCR clustering, and survival (6, 7, 8, 9, 10). We have recently discovered that ADP-ribosylation activates the P2X7 purinoceptor (7). P2X7 is a member of the P2X family of ATP-gated ion channels and is widely expressed on blood cells (11, 12). P2X7 has sparked interest because of its peculiar ability to induce the formation of a large nonselective membrane pore (13). Activation of P2X7 with millimolar concentrations of ATP triggers calcium flux, phosphatidylserine (PS) exposure, shedding of CD62L, and apoptosis (14, 15). The same effects are triggered by NAD at micromolar concentrations via ADP-ribosylation of P2X7 (7). The effects are not observed in ART2-deficient T cells, demonstrating that activation of P2X7 by NAD is ART2 dependent (7, 16). Two commonly used strains of laboratory mice (BALB/c and C57BL/6) carry allelic variants of the P2x7 gene locus, which dramatically affects the susceptibility of T cells from these mice to ecto-NAD and ecto-ATP (8, 17). BALB/c T cells that express wild-type P2X7 are sensitive to NAD and ATP, whereas C57BL/6 T cells that express the P451L allelic variant of P2X7 are resistant (7).
The type II transmembrane protein CD38 is a potent ecto-NAD-glycohydrolase (ecto-NADase) (18, 19). CD38 is expressed by lymphocytes, macrophages, endothelial cells, dendritic cells, pancreatic islet cells, and several other cell types. A soluble form of CD38, presumably generated by proteolytic cleavage of its juxtamembrane stalk, has been found in human serum and other extracellular fluids (18). CD38-deficient mice show normal development of the major lymphocyte subsets, but show impaired humoral immune responses, neutrophil chemotaxis, and dendritic cell trafficking (20, 21, 22). The dramatic reduction in NADase activity in tissues of CD38–/– vs wild-type mice indicates that CD38 is the predominant NADase in most murine tissues, including lymph node, spleen, and bone marrow (21).
It has been proposed that the classic intracellular metabolites of energy metabolism, NAD and ATP, also play roles as signaling molecules in the extracellular environment (23, 24, 25). The plasma membrane of living cells is impermeable to these nucleotides, but they can be released from cells by lytic and nonlytic mechanisms (26, 27). The fate of extracellular signaling molecules (ligands, transmitters) is determined by the rate of their release, metabolism, reuptake by cells, and/or renal excretion. For example, the concentration of ATP and the duration of signaling via ATP in the extracellular compartment are controlled by CD39 and related ectonucleotidases, which hydrolyze ATP to ADP and/or AMP (28, 29). The purpose of this study was to determine whether the ecto-NADase CD38 similarly controls the signaling function of NAD by limiting the availability of NAD as a substrate for ART-catalyzed ADP-ribosylation of cell surface proteins. A recently described immunoassay for monitoring ADP-ribosylation of cell surface proteins on living cells by flow cytometry (30), CD38–/– and ART2–/– mice (16, 20), and the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide (araF-NAD) (31) provided useful tools to address this question. Our results show that CD38, indeed, controls the level of ART2-catalyzed ADP-ribosylation of cell surface proteins.
Materials and Methods
Chemicals and Abs
ADP-ribose, ATP, NAD, and 1,N6-ethenonicotinamide adenine dinucleotide (etheno-NAD) were obtained from Sigma-Aldrich (Fig. 1). PE- and FITC-conjugated mAbs and Annexin V were purchased from BD Pharmingen/BD Biosciences, including anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-5.8), anti-CD38 (90), and anti-CD45R/B220 (RA3-6B2). The anti-ART2 Ab (Ali) was raised in our lab by genetic immunization (32) and recognizes a common epitope on ART2.1 and ART2.2. For the sake of brevity, "ART2" is used instead of "ART2.1 and ART2.2" throughout this paper. Fluorochrome conjugation of Ali and the anti-ethenoadenosine Ab 1G4 (30) was performed using the Alexa Fluor 488 labeling kit from Molecular Probes. [32P]NAD was obtained from Amersham Biosciences.
FIGURE 1. Chemical structures of NAD and NAD analogs used in this study.
Animals and cells
BALB/c and C57BL/6 mice were obtained from The Jackson Laboratory or Charles River. Cd38–/– mice (20) and Art2–/– mice (16) were backcrossed onto the BALB/c and C57BL/6 backgrounds for 8–12 generations. The ART2–/– lines are deficient in both, ART2.1 and ART2.2 (16). Single-cell suspensions were prepared from lymph nodes and spleen of sacrificed animals in RPMI 1640 medium by passage through Nitex membrane (110-μm mesh; Tetko). CD38+ cells were depleted using anti-CD38PE and anti-PE MicroBeads with VarioMACS and AS columns according to the manufacturer’s instructions (Miltenyi Biotec).
Incubation of cells with etheno-NAD and staining with 1G4
For monitoring etheno-ADP-ribosylation of cell surface proteins on living cells (30), cells (106/100 μl of RPMI 1640 medium) were incubated with or without the indicated concentrations of etheno-NAD at 37°C, washed, and stained with 1G4Alexa488 (1 μg) for 30 min at 4°C. Cells were costained with saturating amounts of fluorochrome-conjugated Abs against other cell surface proteins as indicated in the figures. Where indicated, cells were preincubated with araF-NAD (Fig. 1) (31) before etheno-NAD treatment for 10 min at room temperature. Ab-stained cells were washed and resuspended in 250 μl of RPMI 1640 and 10 μl of propidium iodide (PI; 10 μg/ml; Molecular Probes) and analyzed by flow cytometry on a FACSCalibur (BD Biosciences) as described previously (32). Mean fluorescence intensity (MFI) of gated cells was calculated with CellQuest (BD Biosciences), and diagrams were produced with Excel (Microsoft).
Enzyme assays
A total of 2 x 106 cells was preincubated with araF-NAD (0–10 μM) in 10 μl of RPMI 1640 medium for 15 min at room temperature. An equal volume (10 μl) of RPMI 1640 medium containing 4 μM NAD, 2 μCi of [32P]NAD, and 8 mM agmatine was added to the reaction. Cells were incubated for 30 min at 37°C. Cells were pelleted by centrifugations, and cell supernatants were analyzed by TLC on polyethyleneimine cellulose (Merck) as described previously (33).
Assay for PS exposure and PI staining
Single-cell suspensions from lymph nodes were prepared, and B cells were depleted using magnetic cell separation with Dynabead-immobilized goat anti-mouse IgG (Dynal) as described (7). Purity of T cells was always >95% as verified by FACS analyses using PE-conjugated anti-B220 and FITC-conjugated anti-CD3. Following treatment with NAD or ATP for 45 min at 37°C, cells were washed in RPMI 1640 medium adjusted to 2 mM CaCl2, and were stained for 20 min on ice with FITC-conjugated Annexin V (1 μg/ml) (BD Biosciences) and PI (10 μg/ml) before flow cytometry.
Results
Expression of CD38 and ART2 is inversely correlated on murine lymphocytes
Murine lymphocytes have been shown to express at least two distinct NAD-metabolizing ectoenzymes, the GPI-anchored ecto-ADP-ribosyltransferase ART2 and the type II transmembrane bifunctional NADase/ADP-ribosylcylase CD38. Whereas ART2 reportedly is expressed by peripheral T cells and is shed upon T cell activation (33), CD38 is expressed by B cells and by activated T cells (20, 34). Fig. 2 shows FACS profiles of splenocytes and purified lymph node T cells from C57BL/6, BALB/c, and respective CD38–/– and ART2–/– mice after staining for ART2, CD38, CD3, and B220. As reported previously, CD38–/– and ART2–/– mice do not show any marked deviations from wild-type proportions of the major splenic lymphocyte subpopulations (16, 20). Also, in accord with previous reports, CD38 is expressed at high levels on B220+ cells (namely B cells), whereas most T cells are CD38low or CD38– (Fig. 2, A and C). Expression of ART2 is restricted to B220– cells (T cells), and only a small population of ART2+ cells coexpresses CD38. Note that ART2 is expressed at higher levels on C57BL/6 than BALB/c T cells, as observed previously (32).
FIGURE 2. ART2 expression and ecto-ART activity are restricted to CD38– or CD38low cells. A and C, Splenocytes (A) and purified lymph node T cells (C) of C57BL/6, BALB/c wild-type, and respective CD38–/–, or ART2–/– mice were double-stained with fluorochrome-conjugated Abs for expression of CD38, ART2, CD3, and B220, and then analyzed by FACS. B and D, Splenocytes (B) and purified T cells (D) of the indicated mice were incubated with 10 μM etheno-NAD for 20 min and stained with anti-B220PE and 1G4Alexa488 before flow cytometry. Results are representative of three independent experiments.
CD38–/– lymphocytes exhibit higher levels of cell surface etheno-ADP-ribosylation than CD38 wild-type cells
Many extracellular ligands such as ATP or acetylcholine are degraded hydrolytically by specific enzymes, which thereby limit the availability of the ligand for its receptor. Because CD38 is known to possess potent NADase activity, we reasoned that CD38 might influence the substrate availability for NAD-dependent ADP-ribosyltransferase ART2. To address this question, we compared cell surface protein ADP-ribosylation of lymphocytes from wild-type, CD38–/–, and ART2–/– mice. To this end, we treated splenocytes (Fig. 2B) and purified lymph node T cells (Fig. 2D) with etheno-NAD, which is an efficient substrate for CD38 and ARTs (30, 35), and then detected etheno-ADP-ribosylated proteins with etheno-adenosine-specific mAb 1G4. In accord with the expression pattern of ART2, cell surface etheno-ADP-ribosylation is restricted to B220– cells (Fig. 2B). Interestingly, cells from CD38-deficient mice show enhanced cell surface etheno-ADP-ribosylation in comparison with their wild-type counterparts (Fig. 2, B and D). Cells from ART2-deficient mice do not show any etheno-ADP-ribosylation, indicating that ART2 is the only ART activity on splenic and lymph node T cells. Dose-response analyses further demonstrate the striking differences in apparent cell surface ART activity between CD38–/– and wild-type cells (Fig. 3). Following incubation of cells with etheno-NAD, both CD4+ and CD8+ T cell subsets show dose-dependent staining with 1G4 (Fig. 3B). In contrast, CD4– and CD8– cells show little if any staining with 1G4. CD8+ cells exhibit brighter 1G4 staining than CD4+ cells, in accord with the higher level of ART2 expression by CD8+ vs CD4+ cells (Fig. 3A) (32). Strikingly, at low concentrations of exogenous NAD (<50 μM), cells from wild-type mice show much lower apparent ART activity than cells from CD38-deficient mice. At high levels of exogenous NAD (>50 μM), wild-type cells show similar if not slightly stronger levels of cells surface etheno-ADP-ribosylation.
FIGURE 3. Comparative analysis of cell surface protein etheno-ADP-ribosylation by splenocytes of CD38wt and CD38–/– mice. A, Splenocytes from C57BL/6 wild-type or C57BL/6 CD38–/– mice were incubated for 20 min in the absence or presence of the indicated concentrations of etheno-NAD. Cells were washed and stained with 1G4Alexa488, CD4Cy3, and CD8PE before FACS analysis. For control, cells were also stained with ART2Alexa488, CD4Cy3, and CD8PE (left panels). B, Cells were stained as in A, and gating was performed on CD8+ cells or on CD4+ cells. The MFI of 1G4 staining of gated cells was determined and plotted as log MFI vs concentration of etheno-NAD. Results are representative of three independent experiments.
Depletion of CD38+ cells results in enhanced levels of cell surface etheno-ADP-ribosylation on CD38– T cells
Because T cells express only low levels of CD38, but B cells express very high levels of CD38 (Fig. 2), the differences in cell surface ADP-ribosylation of CD38–/– vs wild-type cells (Fig. 3) could, in principle, be explained by low levels of CD38 acting in cis on the T cell surface and/or by high levels of CD38 acting in trans on the B cell surface. To investigate whether ADP-ribosylation of cell surface proteins can be influenced in trans by the presence of cells expressing CD38, we removed CD38+ cells by magnetic cell separation. CD38-depleted and total splenocytes were then incubated with or without etheno-NAD and stained with fluorochrome-conjugated 1G4 and anti-CD38 Abs (Fig. 4A). Double staining of total splenocytes revealed prominent 1G4 staining of CD38low and CD38– cells, but little if any 1G4 staining of CD38high cells (the latter correspond to ART2– B cells; see Fig. 2). Moreover, depletion of CD38high cells, indeed, resulted in strongly enhanced 1G4 staining of CD38– and CD38low cells. Similar results were obtained with splenocytes prepared from C57BL/6 and BALB/c mice (Fig. 4, B and C). Note, however, that BALB/c cells exhibit lower levels of etheno-ADP-ribosylation than C57BL/6 cells (Fig. 4, C vs B), in accord with the lower level of ART2 expression by BALB/c cells (Fig. 2A).
FIGURE 4. Etheno-ADP-ribosylation of splenocyte cell surface proteins in the presence or absence of CD38+ cells. A, Splenocytes obtained from C57BL/6 mice were stained with anti-CD38PE, and CD38+ cells were depleted by magnetic depletion. Total splenocytes and CD38-depleted splenocytes (106/100 μl of RPMI 1640 medium) were incubated for 20 min with 2 or 20 μM etheno-NAD. Cells were then washed and stained with anti-etheno-adenosine mAb 1G4Alexa488 and anti-CD38PE before FACS analysis. B and C, Total and CD38-depleted splenocytes were prepared from C57BL/6 mice (B) or BALB/c mice (C) as described above. Cells were incubated for the indicated time (0–120 min) with 2 or 20 μM etheno-NAD and were then washed and stained with 1G4Alexa488 and anti-CD38PE before FACS analysis. Gating was performed on CD38– cells (as indicated by the gate in A). The MFI of 1G4 staining of CD38– cells was determined and plotted as log MFI vs time of incubation in the presence of etheno-NAD. Results are representative of three independent experiments.
CD38 decreases ART2-catalyzed ADP-ribosylation in trans by reducing the amount of ART substrate NAD
The results obtained so far show that the presence of CD38 can influence cell surface protein ADP-ribosylation on a distinct cell population. To determine whether CD38+ cells inhibit ADP-ribosylation in trans by limiting substrate availability, we adapted our 1G4-based FACS assay as a bioassay for assessing ecto-etheno-NAD concentrations available for ADP-ribosylation. To this end, supernatants were harvested from wild-type or CD38–/– splenocytes that had been incubated with etheno-NAD for 20 min, and these supernatants were used as a source of etheno-NAD for cells that had not been exposed to etheno-NAD (Fig. 5). By comparing 1G4 staining of these cells in FACS analyses, it was possible to estimate the reduction in etheno-NAD levels following a 20-min incubation of cells with etheno-NAD. Fig. 5A shows comparative FACS analyses of splenocytes following a 20-min incubation either with 12.5 μM etheno-NAD or with the supernatant harvested from cells after a 20-min incubation with 12.5 μM etheno-NAD. The results show that incubation of wild-type cells with etheno-NAD for 20 min dramatically reduces the level of substrate available for subsequent etheno-ADP-ribosylation, as reflected by the much lower level of 1G4 staining of cells incubated with supernatant of etheno-NAD-treated cells than of cells incubated with fresh etheno-NAD (Fig. 5A, panel 3 vs panel 2). In striking contrast, incubation of CD38–/– cells with etheno-NAD for 20 min had little if any effect on substrate available for subsequent etheno-ADP-ribosylation as reflected in similar 1G4-staining levels of cells incubated with supernatant and with fresh etheno-NAD (Fig. 5A, panel 6 vs panel 5). Dose-response analyses confirm that wild-type splenocytes catabolize etheno-NAD in a dose-dependent manner (Fig. 5B, left panel). Low levels of ecto-etheno-NAD (<5 μM) are completely catabolized by wild-type splenocytes within 20 min. Wild-type cells quite efficiently catabolize even much higher concentrations of etheno-NAD (50% hydrolysis of 50 μM etheno-NAD within 20 min). In contrast, CD38–/– splenocytes show little if any capacity to metabolize even low levels of etheno-NAD (Fig. 5B, right panel).
FIGURE 5. Comparative analysis of etheno-NAD consumption by CD38 wild-type and CD38–/– cells. A, Splenocytes from C57BL/6 wild-type mice (panels 1–3) or C57BL/6 CD38–/– mice (panels 4–6) were incubated for 20 min in the absence (panels 1 and 4) or presence of 12.5 μM etheno-NAD (panels 2 and 5). Cells were then pelleted by centrifugation and the supernatants were collected. Fresh cells of the same animal were incubated for 20 min with these supernatants (panels 3 and 6). Cells were washed and stained with 1G4Alexa488, CD4Cy3, and CD8PE before FACS analysis. B, Cells were incubated for 20 min with the indicated concentrations of etheno-NAD and processed for FACS analysis as in A. Gating was performed on CD8+ cells (corresponding to gate R2 in A). The MFI of 1G4 staining of CD8+ cells was determined and plotted vs concentration of etheno-NAD. The panel on the left shows etheno-ADP-ribosylation by CD8+ cells from CD38 wild-type mice following incubation either with fresh etheno-NAD () or with the supernatants of cells after a 20-min incubation with the indicated concentrations of etheno-NAD (). The panel on the right shows a similar analysis for cells from CD38–/– mice. Results are representative of three independent experiments.
raF-NAD enhances etheno-ADP-ribosylation by wild-type but not by CD38–/– splenocytes
The results shown so far indicate that CD38 influences ART2-mediated ADP-ribosylation of cell surface proteins by catabolizing the substrate NAD. If so, specifically inhibiting the NAD-metabolizing activity of CD38 should increase the substrate availability and thereby indirectly enhance ART2-catalyzed cell surface ADP-ribosylation. araF-NAD, in which the ribose group adjacent to the nicotinamide moiety of NAD is replaced by fluoroarabinoside (see Fig. 1), has been described as a very potent, nonhydrolyzable inhibitor of splenocyte ecto-NADases (31). However, it was not known whether araF-NAD affects ARTs. To assess the effects of araF-NAD on CD38 and ART2, we preincubated wild-type or CD38–/– splenocytes with araF-NAD for 20 min before exposure to etheno-NAD and then measured cell surface etheno-ADP-ribosylation using the 1G4-based FACS assay. In these experiments, we used mAb B220 to stain B cells and then analyzed ADP-ribosylation on T cells by gating on cells lacking B220. The results shown in Fig. 6A reveal a dose-dependent enhancement of cell surface protein etheno-ADP-ribosylation by araF-NAD in case of wild-type B220– splenocytes, whereas araF-NAD had little if any influence on cell surface etheno-ADP-ribosylation by B220– splenocytes from CD38–/– mice. Kinetic analyses confirmed the potent dose-dependent stimulation of cell surface etheno-ADP-ribosylation following preincubation with araF-NAD for 20 min in the case of wild-type but not CD38–/– splenocytes (Fig. 6B). These results indicate that the apparent stimulatory effect of araF-NAD on cell surface ADP-ribosylation is mediated indirectly by its inhibition of CD38 rather than by a direct action on ART2.
FIGURE 6. Comparative analyses of cell surface ADP-ribosylation by CD38 wild-type and CD38–/– splenocytes in the presence of araF-NAD. A, Splenocytes obtained from C57BL/6 wild-type () and CD38–/– () mice were preincubated with the indicated concentrations of araF-NAD (0–2 μM) for 20 min before addition of 2 μM etheno-NAD and further incubation in the presence of araF-NAD and etheno-NAD for 20 min. Cells were washed and stained with 1G4Alexa488 and anti-B220PE before analysis by flow cytometry. Analysis of 1G4 staining was performed on cells gated for lack of B220 expression. B, Splenocytes obtained from C57BL/6 wild-type and CD38 knockout mice were preincubated without (), with 0.1 μM (), or with 1.0 μM araF-NAD () for 15 min before addition of 2 μM etheno-NAD and further incubation for the indicated times. Cells were washed and stained with 1G4Alexa488 and anti-B220PE before analysis by flow cytometry. Analysis of 1G4 staining on gated cells (B220–) was performed as in A. Results are representative of three independent experiments.
To further assess the effects of araF-NAD, we analyzed ADP-ribosylation of agmatine—a soluble arginine analog and efficient ART2 substrate (36)—in the presence or absence of CD38 or ART2. Following the incubation of wild-type, CD38–/–, or ART2–/– splenocytes with radiolabeled NAD and agmatine, the cell supernatants were analyzed by TLC. Soluble Neurospora crassa NADase and recombinant murine ART2.2 were used as controls. The former converted almost the entire input NAD (2 μM) to ADP-ribose but did not show any detectable ADP-ribosylation of agmatine (Fig. 7, lane 2), whereas the latter converted nearly the entire input NAD to agmatine-ADP-ribose but showed little if any NAD-hydrolyzing activity (lane 3). The results shown in Fig. 7 further reveal that ART2–/– splenocytes (lane 4) metabolized almost the entire input NAD (2 μM) to ADP-ribose within 20 min. In contrast, CD38–/– splenocytes showed only background levels of NAD-hydrolysis to ADP-ribose; but these cells did show prominent ADP-ribosylation of agmatine (lane 5). Wild-type cells exhibited potent NAD-hydrolysis activity as well as detectable, albeit very low levels of agmatine-ADP-ribosylation (lane 6). Preincubation of wild-type cells with araF-NAD inhibited NAD-hydrolysis in a dose-dependent manner (lanes 6, 10, and 14). Similar effects were observed for ART2–/– cells (lanes 4, 8, and 12). Note that preincubation with 1.6 μM araF-NAD blocked NAD-hydrolysis by ART2–/– cells almost completely, whereas—expectedly—these cells did not show any detectable agmatine ADP-ribosylation (lane 12 vs lane 4). In the case of wild-type cells, pretreatment with 1.6 μM araF-NAD effectively blocked NAD-hydrolysis and enhanced ADP-ribosylation of agmatine (lane 14 vs lane 6). As in the case of cell surface protein etheno-ADP-ribosylation (Fig. 6), araF-NAD had no detectable effect on CD38–/– cells (Fig. 7, lanes 5, 9, and 13), supporting the conclusion that the effects of araF-NAD on ADP-ribosylation are mediated indirectly via its inhibition of CD38.
FIGURE 7. Comparative analysis of NAD-glycohydrolysis and agmatine ADP-ribosylation by splenocytes of wild-type, CD38–/–, and ART2–/– mice in the absence and presence of araF-NAD. Splenocytes from BALB/c wild-type, CD38–/–, and ART2–/– mice were preincubated for 15 min without and with 0.1 or 1.6 μM araF-NAD, before addition of 2 μM [32P]NAD and 4 mM agmatine and further incubation for 20 min. Cells were pelleted by centrifugation and aliquots of the supernatants were analyzed by TLC. Chromatograms were subjected to autoradiography for 14 h at –80°C. Control incubations were performed in the presence of H2O, N. crassa NADase, or purified recombinant murine ART2.2 (lanes 1–3, 7, and 11). Results are representative of three independent experiments.
T cells from CD38–/– show higher cell surface levels of PS and lower levels of CD62L than wild-type T cells
ADP-ribosylation of T cell surface proteins upon incubation of cells with ecto-NAD activates the P2X7 purinoceptor (7). As in the case of P2X7 activation by high doses of ATP, this induces shedding of CD62L, exposure of PS, and staining with PI. Cells lacking ART2 are resistant to NAD-induced apoptosis but still undergo apoptosis in response to ATP provided that the P2X7 receptor is functional. Cells expressing the P451L variant of the P2X7 receptor are resistant to both NAD- and ATP-mediated activation of P2X7. Remarkably, even in the absence of exogenously added NAD or ATP, a small but distinct subpopulation of lymph node T cells from wild-type mice but not from ART2–/– mice exposed PS and lacked CD62L. We hypothesized that this spontaneous exposure of PS and loss of CD62L might reflect ADP-ribosylation consequential to the release of endogenous NAD from cells in situ and/or during the preparation of lymph node cells (7, 16). If so, we reasoned that lymph node cells from CD38–/– mice, which lack the major
NAD-hydrolyzing activity, should encounter higher levels of endogenous ecto-NAD under these conditions, and therefore, should show higher levels of PS and lower levels of CD62L on the cell surface. To test this hypothesis, we analyzed Annexin V/PI and CD62L staining of lymph node T cells from BALB/c wild-type and CD38–/– mice in the absence of exogenously NAD or ATP (Fig. 8A). Indeed, a much larger subpopulation of cells from CD38–/– mice than from wild-type or ART2–/– mice showed spontaneous staining by Annexin V (39%) and PI (28%). Moreover, a large fraction (38 of 67 = 58%) of CD3+ cells from these mice did not stain for CD62L (vs 43% of wild-type T cells; Fig. 8B). The fact that cells from ART2-deficient mice contain little if any cells exposing PS (8%) (Fig. 8A) and only a small fraction of cells lacking CD62L (15%) (B) argues that these effects are mediated by ART2-catalyzed endogenous ADP-ribosylation.
FIGURE 8. Comparative analysis of NAD- and ATP-induced apoptosis (A) and CD62L shedding (B) by T cells of wild-type and CD38–/– mice. Purified lymph node T cells from BALB/c wild-type, CD38–/–, and ART2–/– mice were incubated without or with 20 μM NAD or 200 μM ATP for 40 min. Cells were washed and stained with Annexin VFITC and PI (A) or with anti-CD62LPE and anti-CD3FITC (B) before FACS analysis. Numbers indicate the percentage of cells in each quadrant. Results are representative of five independent experiments.
Upon addition of exogenous NAD or ATP, cells from both BALB/c CD38–/– and wild-type mice responded by exposing PS and shedding CD62L (Fig. 8), even though much higher concentrations of ATP than NAD were needed to elicit an effect. Saturation responses were obtained with 30 μM NAD and 1000 μM ATP (not shown). In case of ART2–/– mice, even high concentrations of exogenously added NAD failed to induce PS exposure or CD62L shedding, whereas these effects were readily triggered by high concentrations of exogenously added ATP. These findings imply that the spontaneous PS exposure and loss of CD62L observed in CD38–/– and wild-type mice are induced by endogenously released NAD rather than ATP. Cells derived from the C57BL/6 background, which express the P451L variant of the P2X7 receptor (17), showed strongly diminished responses to both NAD and ATP (not shown).
Discussion
Lymphocytes express a flurry of nucleotide-metabolizing ectoenzymes, including ENTPDases (CD39), pyrophosphatases (CD203), 5'-nucleotidase (CD73), NADases (CD38, CD157), and ARTs (37, 38), the function of which has puzzled investigators. One hypothesis holds that these enzymes catabolize ATP, NAD, and other nucleotides released from dying cells in inflamed tissues to recycle nucleotides by providing precursors for uptake by proliferating lymphocytes (37). Another hypothesis proposes that these enzymes control the availability of ATP and its metabolites ADP, AMP, and adenosine as ligands for specific receptors (P2X and P2Y family of purinergic receptors, P1 family of adenosine receptors) (24, 28, 29). In this study, we explore the hypothesis that CD38 may similarly affect the availability of NAD as an extracellular signaling molecule by controlling the concentration of NAD and the duration of its presence as a substrate for ART2-catalyzed ADP-ribosylation of cell surface proteins.
Our results show that CD38, indeed, controls the level of ART2-catalyzed cell surface protein ADP-ribosylation and indicate that CD38 exerts this control not only in cis on the same cell surface as ART2, but also in trans on the surface of other cells. Thus, transfection of ART2 into lymphoma cells lacking CD38 results in much higher levels of cell surface ADP-ribosylation than transfection into cells expressing CD38 (results not shown). Moreover, purified T cells from CD38–/– mice show stronger ADP-ribosylation of cell surface proteins than cells from wild-type mice, especially at lower and more physiological concentrations of ecto-NAD (Fig. 2D). The effect of CD38 on ADP-ribosylation in cis is moderate, reflecting the low level of CD38 on T cells. A stronger effect of CD38 in trans becomes apparent when comparing levels of cell surface ADP-ribosylation by wild-type vs CD38–/– T cells in total splenocyte populations (Figs. 2B and 3). In this situation, wild-type T cells are in the company of cells expressing high levels of CD38 (wild-type B cells), whereas CD38–/– T cells are in the company of cells lacking CD38. Consistently, removal of CD38+ cells from suspensions of wild-type splenocytes markedly enhances the degree of cell surface ADP-ribosylation by the remaining cells (Fig. 4).
The finding that araF-NAD, a specific inhibitor of CD38-mediated NAD-hydrolysis, enhances ADP-ribosylation by wild-type but not by CD38–/– T cells (Figs. 6 and 7) further supports the conclusion that CD38 controls ART2-catalyzed protein ADP-ribosylation. This finding is of import also to studies aimed at developing inhibitors of CD38 for experimental and therapeutic modulation of immune functions (39). Our results demonstrate that such inhibitors can indirectly effect ADP-ribosylation reactions catalyzed by ecto-ARTs by increasing the levels of ecto-NAD. Our results may also be relevant to other studies using CD38–/– mice as some of the immune phenotypes reported for these mice may be due to enhanced ADP-ribosylation of target proteins by ARTs consequential to the inefficient removal of ecto-NAD in these mice (20, 22, 40, 41, 42). Note that all of the studies published to date have been conducted using CD38-deficient mice on the C57BL/6 background expressing the functionally impaired P451L variant of P2X7.
Thus, if ARTs affected the immune phenotypes in these mice, it is more likely that this is mediated by ADP-ribosylation of LFA-1 and other targets rather than by ART-mediated activation of P2X7.
The rapid consumption of exogenously added etheno-NAD and [32P]NAD by CD38-expressing splenocytes but not by CD38–/– cells (Figs. 4 and 6) implies that local concentrations of NAD may vary dramatically in vivo, depending on the presence of CD38-expressing cells and/or soluble CD38. Considering that cells in the experimental setting are suspended in a much larger volume of extracellular medium (107 cells/ml) than in vivo (2 x 108 splenocytes in a total volume of 250 μl) suggests that ecto-NAD is metabolized even faster in the surroundings of CD38-expressing cells in vivo than in the experimental situation illustrated in Fig. 5. Given the strikingly different patterns of expression of CD38 and ART2, areas rich in B cells that express very high levels of CD38 but not ART2 (Fig. 2) can be predicted to be essentially ecto-NAD-free zones, because extracellular NAD will be rapidly degraded by CD38. In contrast, local levels of ecto-NAD may be sustained longer and at higher levels in areas rich in T cells, most of
which express ART2 but little if any CD38 (Fig. 2).
Our results further illustrate that endogenous sources of ecto-NAD can affect T cell functions. Thus, a substantial fraction of freshly prepared T cells from CD38–/– but not from ART2–/– mice exposes PS on the cell surface (Fig. 8A) and lacks cell surface CD62L (B), whereas wild-type T cells show an intermediate phenotype. ART2-dependent PS exposure and shedding of CD62L both result from the activation of the P2X7 purinoceptor by ADP-ribosylation (7). C57BL/6 cells express a variant P2X7 receptor carrying the P451L mutation in the cytoplasmic domain, which strongly impairs P2X7-mediated signaling (17). Consistently, C57BL/6 T cells exhibit little if any spontaneous PS exposure or loss of CD62L (not shown). Considering that PS exposure and shedding of CD62L both occur within minutes after exposure of cells to NAD (7), it is possible that PS exposure and loss of CD62L by freshly prepared cells is induced by NAD released from cells lysed during cell preparation. Indeed, fresh preparations of lymph node cells consistently contain a small but distinct population of dead cells characterized by bright PI staining and small forward scatter, and these cells conceivably could have released their intracellular pool of NAD during cell preparation. Alternatively, T cells may have experienced NAD exposure in situ before sacrifice of the animal. In either case, the presence or absence of CD38 evidently profoundly affects the effective ecto-NAD concentration (Fig. 8). By analogy, NAD released from lysed cells during acute inflammation or tissue damage would be expected to exert strikingly different effects on T cells in vivo, depending on the presence or absence of CD38.
Only little is known about physiological and pathological release of NAD in vivo. Intracellular concentrations of NAD may reach millimolar levels, whereas steady-state serum levels of ecto-NAD are in the submicromolar range (23, 43). A substantial body of evidence indicates that nucleotides can be released from cells by nonlytic mechanisms, e.g., following mechanical shear forces or stimulation (23, 24, 27). Moreover, connexin 43 hemichannels may function as channels for the release of ATP and NAD (26). During infection, killed bacteria, yeast, and protozoa as well as lysed host cells represent additional potential sources of ecto-NAD. Of note, certain bacterial pathogens, e.g., Haemophilus influenzae, are known to lack endogenous NAD-synthesizing machinery and depend on ecto-NAD (44). Thus, relatively high levels of ecto-NAD can be predicted in the surroundings of lysed cells, especially in tissues lacking CD38 or expressing only low levels of CD38.
On the basis of our results, we speculate that elevated or sustained levels of ecto-NAD, as predicted to occur in CD38–/– mice under conditions of infection or tissue damage, lead to enhanced T cell death by triggering NAD-induced cell death via the ART2/P2X7 pathway and, subsequently, to an increased compensatory homeostatic proliferation of ART2-negative cells. Combined with a predisposing genetic background, enhanced homeostatic proliferation of T cells can precipitate autoimmune disease, as recently demonstrated in the case of the NOD mouse model for insulin-dependent diabetes mellitus (45). It should, therefore, be of great interest to determine whether CD38 deficiency affects T cell homeostasis in physiological and pathophysiological settings, and to determine whether the presence or absence of CD38 and/or ART2 affects the incidence or progression of autoimmune disease in NOD mice, which carry the wild-type P2X7 receptor (17).
Given the striking effect of CD38 on ART2-catalyzed ADP-ribosylation on T cells, it is not unlikely that CD38 also influences ADP-ribosylation by other members of the ART family. Cells in other tissues expressing high levels of CD38 can be expected to mediate locally low levels of ecto-NAD. CD38 is expressed in tissues, such as heart and reproductive organs, known to express other members of the ART family (5, 46, 47).
It has been proposed that nucleotides released from cells may function as signaling molecules (24). Our results support this notion and indicate that NAD itself may play a signaling role. On the basis of our findings, it is tempting to speculate that ART2 acts as a sensor of ecto-NAD levels, which translates the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins. The duration and intensity of exposure of cells to ecto-NAD in turn are determined by the local levels of CD38 on the cell surface or in the extracellular medium. In this scenario, the NAD-metabolizing ectoenzymes CD38 and ART2 together convey information about the presence of the signaling molecule ecto-NAD to lymphocytes and thereby may help to fine-tune immune responses.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
This work represents the partial fulfillment of the requirements for the graduate thesis of C. Krebs. C. Krebs thanks E. H. Leiter and his staff for the hospitality during C. Krebs’s stay as a visiting scientist in his lab. We thank Dunja Freese and Gudrun Dubberke (University Hospital, Hamburg, Germany) for excellent technical assistance. F. Koch-Nolte, F. Haag, E. H. Leiter, F. E. Lund, and M. Seman designed and supervised the study. F. E. Lund provided the CD38–/– mice, and N. Oppenheimer provided araF-NAD. C. Krebs and F. Braasch performed the experiments shown in Figs. 3–7, and S. Adriouch performed those shown in Figs. 2 and 8. W. Koestner performed the cotransfection studies. C. Krebs and F. Koch-Nolte wrote the paper. We thank B. Fleischer, S. Rothenburg, P. Bannas, and F. Scheuplein (University Hospital, Hamburg, Germany) for critical reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 545/B9 and No310/6 (to F.K.-N. and F.H.), by stipends from the Werner Otto Foundation and the Boehringer Ingelheim Fonds (to C.K.), by a stipend from the Fondation pour la Recherche Medical (to S.A.), by National Institutes of Health Grant AI-057996 (to F.E.L.), and by National Institutes of Health Grants DK27722 and DK36175 (to E.H.L.).
2 Address correspondence and reprint requests to Dr. Friedrich Koch-Nolte, Institute for Immunology, University Hospital, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail address: nolte{at}uke.uni-hamburg.de
3 Abbreviations used in this paper: ART, ADP-ribosyltransferase; NADase, NAD-glycohydrolase; araF-NAD, nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide; MFI, mean fluorescence intensity; PS, phosphatidylserine; PI, propidium iodide; etheno-NAD, nicotinamide 1,N6-ethenoadenine dinucleotide.
Received for publication November 24, 2004. Accepted for publication December 21, 2004.
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