Differential Regulation of P2X₇ Receptor Activation by Extracellular Nicotinamide Adenine Dinucleotide and Ecto-ADP-Ribosyltransferases in Murine Macrophages and T Cells

Materials

Recombinant murine IFN-γ was from Boehringer Mannheim Biochemica and recombinant murine IFN-β was from US Biologicals. LPS (Escherichia coli serotype 01101:B4) was from List Biological Laboratories. ATP, NAD, etheno-NAD (εNAD), ADP-R, reduced glutathione (GSH), and TRIzol were from Sigma-Aldrich. Oligo(dT) primer was from Promega. Avian myeloblastosis virus reverse transcriptase was from Roche. Tag DNA polymerase was from New England Biolabs. The 1G4 mouse mAb (a generous gift from Dr. R. Santella, Columbia University, New York, NY) was prepared as previously described (36). Fura-2-AM, ethidium bromide, allophycocyanin-conjugated annexin V and YO-PRO-1 were from Molecular Probes. The anti-P2X₇R K1G Ab and mAbs directed against mouse ART2.1 or ART2.2 were generated and used as recently described (37). BALB/c, C57BL/6, and New Zealand White (NZW) mice were purchased from Taconic Farms. P2X₇R^−/− mice were originally provided by Pfizer Global Research and Development and then backcrossed into a pure C57BL/6 background for >12 generations from a P2X₇R^−/− mouse strain described previously (38). All experiments and procedures involving mice were approved by the Institutional Animal Use and Care Committees of Case Western Reserve University (Cleveland, OH) or Hamburg University Hospital (Hamburg, Germany).

Cell culture and animals

BMDM and splenocytes isolated from BALB/c, C57BL/6, NZW, or P2X₇R^−/− mice were prepared as previously described (36). Bac1.2F5 murine macrophages were cultured in DMEM (Sigma-Aldrich) supplemented with 25% L cell-conditioned medium, 15% calf serum (HyClone Laboratories), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) in the presence of 10% CO₂. RAW264.7 macrophages were cultured in DMEM supplemented with 10% calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in the presence of 10% CO₂. Where indicated, BMDM or the macrophage cell lines (Bac1.2F5 and RAW264.7) were primed for 24 h with either LPS (100 ng/ml), IFN-γ, (100 U/ml), or IFN-β (100 U/ml) to induce an inflammatory phenotype and the up-regulation of ART2.1 expression. Murine BW5147 T lymphoma cells were maintained in RPMI 1640 supplemented with 10% calf serum and 1% penicillin-streptomycin in the presence of 5% CO₂. Wild-type (WT) HEK293 cells were cultured in DMEM supplemented with 10% calf serum and 1% penicillin-streptomycin in the presence of 10% CO₂. HEK293 cells stably transfected with either the WT murine P2X₇R (HEK-mP2X₇ cells) or the mutant R276K P2X₇R (HEK-mP2X₇-R276K cells) were selected and maintained in DMEM supplemented with 400 μg/ml G418 or 10 μg/ml blasticidin. The ART2 plasmids were transiently transfected into HEK P2X₇R cells using cells seeded at 6 × 10⁵ per 35-mm dish 24 h before transient transfection. The cells were transfected using PolyFect reagent (Qiagen) with 2.5 μg of plasmid DNA per dish followed by incubation at 37°C for 36 h before experiments.

RT-PCR analyses

Total RNA was extracted using TRIzol; all primers and PCR conditions for ecto-ARTs, inducible NO synthase (iNOS), IFN-β, and GAPDH were prepared and used as previously described (36). The PCR amplicons were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining; the resulting fluorescence images were recorded with a BioRad Gel Doc 1000 system.

1G4 mAb-based assay of ART2 activity

ART activity in intact Bac1.2F5 macrophages, RAW264.7 macrophages, or BW5147 T lymphocytes was assayed using a Western blot protocol based on the 1G4 mAb as previously described (36). Briefly, intact cells were transferred to basic salt solution (BSS) containing 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂, 1 mM MgCl₂, 25 mM HEPES (pH 7.5), 5 mM glucose, and 0.1% BSA. The cells were incubated at 37°C for 15 min with 50 μM ε-NAD and 1 mM ADP-R in the presence of 1 mM DTT before extraction, SDS-PAGE, and Western blotting.

Labeling and immunoprecipitation of ADP-ribosylated P2X₇R

HEK293 cells were harvested by trypsinization at 20 h after cotransfection with P2X₇R and either ART2.1 or ART2.2. ART2/P2X₇R-coexpressing HEK cells were then incubated at 37°C for 15 min with 50 μM ³²P-NAD and 1 mM ADP-R in the presence or absence of 1 mM DTT. Washed cells were lysed in PBS, 1% Triton-X100, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Sigma-Aldrich) for 20 min at 4°C. Insoluble material was pelleted by high-speed centrifugation (for 15 min at 13,000 × g). K1G Ab at 3 μg/ml (39) was added into the lysate and incubated for 2 h at 4°C, and lysates were further incubated with protein G-Sepharose beads (20 μl beads/lysates from 10⁶ cells) for 60 min at 4°C. Immunoprecipitates were washed three times using Triton X-100 buffer. The final product was eluted into 30 μl of 2× SDS binding buffer and boiled for 5 min. The samples were loaded onto 15% SDS-polyacrylamide gels and proteins were detected by autoradiography. P2X₇ was detected with a rabbit anti-P2X₇ C-terminal peptide Ab (1/1000) (Alomone Labs) and peroxidase-conjugated anti-rabbit IgG (1/5000) using the ECL system (Amersham-GE Healthcare).

FACS analysis

Staining for the P2X₇R was performed with Alexa Fluor 488-conjugated K1G Ab for 30 min at 4°C (39). Stained cells were washed and analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). Gating was performed on living cells on the basis of propidium iodide exclusion.

Measurement of P2X₇R-mediated Ca²⁺ influx or nucleotide-induced Ca²⁺ mobilization

Macrophages (primary BMDM or macrophage cell lines) were collected in DMEM by gently scraping the monolayers and transferring the detached cells into 50-ml tubes. HEK293 cells were detached by trypsinization. The detached macrophages or HEK293 cells were centrifuged and the cell pellets were washed twice with BSS. Primary spleen lymphocytes or BW5147 T cell suspensions were directly pelleted from growth medium before washing. Washed cells were resuspended in BSS supplemented with 1 μM fura-2-AM and incubated at 37°C for 40 min. The fura-2-loaded cells were then washed, resuspended in BSS (10⁶/ml), and then stored on ice for up to 3 h during measurements. For each measurement, a 1.5-ml aliquot of cell suspension was stirred at 37°C in a quartz cuvette for measurement of fura-2-AM fluorescence (339 nm excitation/500 nm emission) and calibration as previously described (40). Murine macrophages (41, 42) and HEK293 cells (43, 44) also express several subtypes of G protein-coupled, Ca²⁺-mobilizing P2Y receptors. Thus, the macrophages or HEK293 cells were first treated with a mixture of 50 μM ADP and 50 μM UTP to activate and desensitize these P2Y receptors before P2X₇R stimulation by the indicated concentrations of ATP or NAD (45). In some experiments, P2Y receptor-dependent Ca²⁺ mobilization in Bac1.2F5 macrophages or BW5147 T lymphocytes was directly assayed by stimulating the cells with 30 μM ADP, UTP, or UDP. Where indicated, the macrophages or HEK293 cells were treated in the presence or absence of 1 mM DTT (plus 1 mM ADP-R) for 1–5 min before stimulation with 10–100 μM NAD.

Measurement of P2X₇R-mediated YO-PRO-1 or ethidium dye uptake

HEK293 cells transfected with ART2.1 and the hyperresponsive R276K P2X₇R variant (21) were gently trypsinized and incubated in the absence or presence of NAD or ATP in 10 mM HEPES (pH 7.5), 140 mM NaCl, 5 mM KCl, 10 mM glucose, and 1 μM YO-PRO-1 (Molecular Probes) for 60 min at 37°C. Cells were subsequently washed, resuspended in annexin V binding buffer (BD Biosciences), and stained with annexin V-allophycocyanin (BD Biosciences) before analysis by flow cytometry. Spleen cells from BALB/c or NZW mice were incubated with YO-PRO-1 as described above, stained with Abs against the surface markers CD3 and B220, and analyzed by flow cytometry. Cells staining positive for CD3 were analyzed for the expression of P2X₇R, ART2.1, ART2.2, and the uptake of YO-PRO-1. Control or IFN-γ-primed Bac1.2F5 macrophages were collected in DMEM by gently scraping of the monolayers and transfer into 50-ml tubes. The cells were centrifuged, washed, and resuspended in BSS. Aliquots (1.5 ml) of cell suspension were transferred to the stirred measuring cuvette and preincubated for 3 min at 37°C. Ethidium bromide (2.5 μM) was added and baseline fluorescence (360 nm excitation/580 nm emission) was recorded before stimulation of the cells with various concentrations of NAD and/or ATP as described in the Fig. 10 legend. All ethidium⁺ fluorescence increases were corrected for background dye fluorescence. The P2X₇R-mediated increases in ethidium⁺ accumulation were expressed as percentages of the maximal fluorescence observed when the cells were permeabilized with 0.003% digitonin.

HPLC analysis of extracellular NAD metabolism

Monolayers of Bac1.2F5 macrophages (10⁶ cells/well in 6-well dishes) were incubated in 1 ml of BSS at 37°C supplemented with 100 μM NAD in the presence or absence of 1 mM ADP-R. At selected times (0–60 min), 100-μl aliquots of the extracellular medium were removed, boiled for 5 min, and centrifuged to sediment any precipitated protein. NAD and its principle metabolite, ADP-R, were separated and quantified using a reverse-phase HPLC protocol. Briefly, 50-μl aliquots were injected onto an Alltech C18 Adsorbosphere column that was isocratically eluted at 1.3 ml/min with a running buffer of 0.1 M KH₂PO₄ and 5% methanol (pH 6). NAD (elution time 8.2 min) and ADP-R (elution time 4 min) were detected by absorbance at 254 nm.

Statistics

All experiments were repeated 2–6 times using different preparations of primary leukocytes isolated from different mice or with leukocyte cell lines from separate cultures. All data, unless otherwise stated, represent mean ± SEM. A two-tailed, one-variable Student’s t test was used to analyze these data with statistical significance defined as p < 0.05.

NAD induces activation of P2X₇R in murine lymphocytes but not murine macrophages

We confirmed the ability of extracellular NAD to induce P2X₇R activation (as assayed by Ca²⁺ influx) in freshly isolated splenic lymphocytes from BALB/c or C57BL/6 mice (Fig. 1⇓, A and B), but not in lymphocytes from P2X₇^−/− mice (Fig. 1⇓C). (Although splenic lymphocytes include both T cells and B cells, P2X₇R is not expressed by murine B cells; see Ref. 46). We tested lymphocytes from the BALB/c and C57BL/6 mouse strains because they express polymorphic variants of the P2X₇R (P451 for BALB/c and L451 for C57BL/6) (47). Additionally, T lymphocytes from BALB/c mice express both ART2.1 and ART2.2 as functional enzymes, whereas leukocytes from C57BL/6 mice express only ART2.2 due to a premature stop codon in ART2.1 mRNA that prevents translation of a functional protein (34, 35).

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FIGURE 1.

NAD induces the activation of P2X₇R in murine lymphocytes but not murine macrophages. Changes in cytosolic Ca²⁺ in response to ATP (3 mM) or NAD (100 μM) were measured in fura-2-loaded suspensions of spleen lymphocytes or BMDM as described in Materials and Methods. Macrophage suspensions were pretreated with a mixture of 50 μM ADP and 50 μM UTP to activate and desensitize P2Y receptors 5 min before P2X₇R stimulation by ATP or NAD. Where indicated, 1 mM DTT was included in the assay medium to support the activity of the thiol-sensitive ART2.1 ectoenzyme. All cells were permeabilized with digitonin (Dig) to release fura-2 for subsequent calibration. All traces are representative of observations from three to six experiments with the leukocyte preparations isolated from different mice of the noted strains. A, Freshly isolated spleen lymphocytes from BALB/c mice. B, Freshly isolated spleen lymphocytes from C57BL/6 mice. C, Freshly isolated spleen lymphocytes from the spleen of P2X₇R-knockout mice (C57BL/6 background). D, Naive BMDM from BALB/c mice. E, Naive BMDM from C57BL/6 mice. F, Naive BMDM from P2X₇R-knockout mice (C57BL/6 background). G, BMDM from BALB/c mice were primed with 100 U/ml IFN-γ for 24 h to induce ART2.1 expression before the Ca²⁺ assay. H, BMDM from C57BL/6 mice were primed with 100 ng/ml LPS plus 10 μM U0126 for 24 h to induce proinflammatory gene expression before the Ca²⁺ assay. I, BMDM from P2X₇R-knockout mice (C57BL/6 background) were primed with 100 ng/ml LPS plus 10 μM U0126 for 24 h to induce proinflammatory gene expression before the Ca²⁺ assay.

In contrast to its actions on T cells, NAD did not trigger Ca²⁺ influx in naive BMDM isolated from either BALB/c or C57BL/6 mice (Fig. 1⇑, D and E) even though ATP stimulated robust increases in Ca²⁺ in both WT BMDM populations, but not in BMDMs from P2X₇R-deficient mice (Fig. 1⇑F). This absence of an NAD effect in naive macrophages was consistent with our previous findings that murine macrophages express only low levels of ecto-ARTs in the absence of proinflammatory activation by IFNs or LPS. Likewise, C57BL/6 BMDM were unresponsive to NAD even after proinflammatory activation by LPS (Fig. 1⇑H) or IFNs (not shown); this is consistent with the general lack of ART2.2 expression in murine myeloid leukocytes and the specific lack of ART2.1 expression in C57BL/6 myeloid leukocytes (35). As expected, neither NAD nor ATP elicited a Ca²⁺ influx response in P2X₇-knockout BMDM (Fig. 1⇑I). Surprisingly, however, inflammatory BALB/c BMDM primed with IFN-γ (Fig. 1⇑G) to up-regulate ART2.1 were also unresponsive to extracellular NAD but retained a robust response to ATP. We have previously described the use of anti-ART2.1, anti-ART2.2, and anti-εADP- mAbs in FACS analyses to confirm that LPS and IFN-γ induced the specific up-regulation of ART2.1 protein and ADP-ribosyltransferase activity in BMDM from WT BALB/c mice (36), but not in BMDM from an ART2.1 knockout BALB/c strain (data not shown). Consistent with the thiol dependence of ART2.1 enzyme activity, the inclusion of DTT markedly increases ADP-ribosylation of cell surface proteins in intact BALB/c BMDM (36). However, the inclusion of extracellular DTT did not facilitate NAD-induced Ca²⁺ influx in these IFN-primed BMDM (Fig. 1⇑G).

The murine P2X₇R is a substrate for ADP-ribosylation and gating by the thiol-sensitive ART2.1 ectoenzyme

The ability of extracellular NAD to activate the P2X₇R in BALB/c T cells that express ART2.1 and ART2.2, but not in BALB/c macrophages that express only ART2.1, raised the critical question of whether ART2.1 (similarly as ART2.2) can recognize the P2X₇R as a substrate. To test this, the murine P2X₇R was coexpressed with ART2.1 or ART2.2 in HEK293 cells. The ART2-expressing cells were briefly incubated with [³²P]NAD in the presence or absence of DTT before extraction, immunoprecipitation of the P2X₇R, SDS-PAGE, and detection of [³²P]ADP-ribosylated P2X₇R by autoradiography. Fig. 2⇓A illustrates the extracellular domain of the mP2X₇R including the relative positions of seven (of 18 total) arginine residues within this domain. Previous studies identified R125 and R133 as the critical sites for ART2.2-catalyzed ADP-ribosylation of the P2X₇R (21). Thus, we compared WT P2X₇R vs a P2X₇R construct (R125K/R133K) wherein the two target arginines for ART2.2 within the cysteine-rich loop were replaced by lysine and, as a consequence, cannot function as an acceptor for ADP-ribosylation by ART2.2. The expression of ART2.1 in HEK293 cells facilitated the robust ADP-ribosylation of the WT P2X₇R, but not the double arginine mutant variant as assayed by incorporation of [³²P]ADP-R (Fig. 2⇓B). Notably, the ability of ART2.1 to ADP-ribosylate the WT P2X₇R was highly dependent on exogenously added DTT. In contrast, HEK293 cells coexpressing ART2.2 and WT P2X₇R showed strong and equivalent [³²P]ADP-ribosylation of P2X₇R in the absence or presence of DTT; mutants of P2X₇ in which the R125 and R133 residues were changed to lysine did not function as a substrate for ART2.2 regardless of the presence or absence of DTT (data not shown). Importantly, we used the K1G anti-P2X₇ Ab and FACS analyses to confirm similar cell surface expression levels of all WT and mutant P2X₇R constructs (Fig. 2⇓).

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FIGURE 2.

The murine P2X₇R is a substrate for ADP-ribosylation and gating by both the thiol-sensitive ART2.1 and the thiol-insensitive ART2.2. A, Schematic of murine P2X₇R showing the relative distribution of important arginine residues (diamonds), disulfide-bonded cysteine residues (circles), and N-linked glycosylation sites (arrowheads) in the extracellular ligand-binding domain, as well as the natural allelic 451L variant in the cytosolic domain of P2X₇R. B, HEK cells were cotransfected with WT murine P2X₇R plus murine ART2.1 or with an R125K/R133K double mutant of the murine P2X₇R (R125K133K) plus murine ART2.1. Aliquots of the control and transfected cells were stained with the anti-P2X₇ K1G Ab for FACS analysis as described in Materials and Methods. Parallel aliquots of the control (mock-tranfected) or cotransfected cells were incubated for 15 min with 50 μM [³²P]NAD and 1 mM ADP-R in the presence of the indicated concentrations of DTT before lysis and immunoprecipitation with the K1G Ab; immunoprecipitated products were resolved by SDS-PAGE and detected by autography. Results are representative of observations from two or more independent experiments. C, FACS analysis of splenocytes from BALB/c and NZW mice. CD3-expressing T lymphocytes (left panel) were analyzed for surface expression of P2X₇R, ART2.1, and ART2.2 (right panels). D, Splenocytes were incubated with the fluorescent dye YO-PRO-1 and NAD and DTT as indicated. The mean fluorescence intensity (MFI) in the YO-PRO-1 channel of cells gated for CD3 expression as in C was measured as an indication of pore formation in T lymphocytes. Data bars show the mean ± SE of the MFI values from three independent experiments for each T cell type with p < 0.05 for the indicated comparisons in the NZW data sets.

We next asked whether ADP-ribosylation of P2X₇R by ART2.1 also induces the gating of P2X₇R. To this end, we examined NAD-induced and P2X₇R-dependent pore formation in T lymphocytes from BALB/c mice, which express both ART2.1 and ART2.2, or corresponding cells from NZW mice, which lack ART2.2 and express only ART2.1 (Fig. 2⇑C). The formation of membrane pores that allow the incorporation of DNA-staining dyes like YO-PRO-1 is considered to be a typical hallmark of P2X₇ activation. Fig. 2⇑D shows that T lymphocytes from BALB/c mice, which express high levels of ART2.2, incorporate YO-PRO-1 in response to micromolar NAD regardless of the presence or absence of DTT. In contrast, NAD-induced YO-PRO-1 uptake into T lymphocytes from NZW mice, which express only ART2.1, is strictly dependent on DTT and also requires higher concentrations of NAD.

NAD potentiates ATP-induced P2X₇R activation in HEK293 cells coexpressing ART2 and P2X₇R

The data in Fig. 2⇑ verified the ability of NAD to effectively ADP-ribosylate the WT murine P2X₇R when heterologously expressed with ART2.1 in the HEK293 cell background. However, we have reported that this covalent modification of the WT P2X₇R expressed in HEK293 cells is not sufficient for functional activation of the receptor (21). Fig. 3⇓A shows this by comparing NAD with ATP as stimuli for Ca²⁺ influx in a HEK293 line (HEK-mP2X₇ cells) stably transfected with murine P2X₇R cDNA before transient transfection with an ART2.2 expression plasmid. Western blot analysis confirmed the expression of functional ART activity in ART2.2-transfected, but not parental, HEK293 cells (Fig. 3⇓C). This assay involves incubation of intact cells with εNAD (an NAD analog) to covalently ε-ADP-ribosylate cell surface proteins followed by cell extraction, SDS-PAGE, and probing with the anti-ε-ADP-R 1G4 mAb. Fig. 3⇓C also shows that inclusion of extracellular ADP-R potentiated the accumulation of ADP-ribosylated proteins by attenuating the metabolism of εNAD (or NAD) by ectonucleotidases. Despite the robust ART activity in the cotransfected HEK-mP2X₇ cells, extracellular NAD did not mimic the ability of ATP to trigger P2X₇R-dependent Ca²⁺ influx.

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FIGURE 3.

Differential effects of NAD/ART2 on activation of the murine P2X₇R vs R276K-mutated P2X₇R in HEK293 cells. A and B, HEK293 cells were stably transfected with either WT murine P2X₇R (HEK-mP2X₇ cells) in A or the R276K mutant of murine P2X₇R (HEK-mP2X₇-R276K cells) in B. The stably transfected lines were transiently cotransfected with murine ART2.2 24 h before an analysis of P2X₇R-dependent Ca²⁺ influx was conducted. Cytosolic Ca²⁺ was measured in fura-2-loaded HEK cell suspensions as described in Materials and Methods and Fig. 1. The cell suspensions were pretreated with a mixture of 50 μM ADP and 50 μM UTP to activate and desensitize P2Y receptors 5 min before P2X₇R stimulation by the indicated concentrations of ATP or NAD. NAD was added together with 1 mM ADP-R to attenuate metabolism of the NAD. These traces are representative of observations from three to four experiments with each cell line. Digi, Digitonin. C, HEK293 cells were transiently mock transfected (left lane) or transfected with ART2.2 (middle and right lanes). After 36 h, the intact cells were acutely incubated for 15 min with 50 μM ε-NAD with or without 1 mM ADP-R before extraction, SDS-PAGE, and Western blotting with the 1G4 mAb that detects ε-ADP-ribosylated proteins. IB, Immunoblotting. D, HEK cells were transiently cotransfected with murine ART2.1 and the indicated murine P2X₇R mutants (R276K single mutant or R125K/R133K/R276K (R276K125K133K) triple mutant) before experiments. At 20 h posttransfection, the cells were harvested by trypsinization without further treatment (control) or following 10-min incubations with 1 mM ADP-R, 50 μM NAD, or 500 μM ATP separately in the presence of 1 mM DTT. The cells were then treated with 1 μM YO-PRO-1 for 1 h. Aliquots of the cotransfected cells were stained with allophycocyanin-conjugated annexin V before FACS analysis. All results are representative of observations from two or more independent experiments. The numbers in each panel represent the percentages of cells in the respective quadrants.

We have described another P2X₇R arginine residue (R276) that is not an ecto-ADP-ribosylation site but is a critical modulator of ATP potency (21). Fig. 3⇑B shows that the HEK293 line (HEK-mP2X₇-R276K), stably transfected with the R276K gain of function mutant of P2X₇R, exhibited a maximal Ca²⁺ influx response to 50 μM ATP, which is a subthreshold concentration in cells expressing WT P2X₇R (see Fig. 4⇓A). Notably, this R276K mutation also facilitated the gating of P2X₇R channel activity in response to NAD in HEK293 cells cotransfected with ART2.2 (Fig. 3⇑B).

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FIGURE 4.

NAD potentiates ATP-induced P2X₇R activation in HEK293 cells coexpressing ART2 and P2X₇R. HEK293 cells stably transfected with WT murine P2X₇R (HEK-mP2X₇ cells) were used in all experiments. In experiments for A–D, the cells were transiently cotransfected with murine ART2.2 24 h before analysis of P2X₇R-dependent Ca²⁺ influx. In experiments for E and F, the HEK-mP2X₇ cells that lack ART2.2 expression were directly assayed. Cytosolic Ca²⁺ was measured in fura-2-loaded HEK cell suspensions as described in Fig. 3. The cell suspensions were pretreated with a mixture of 50 μM ADP and 50 μM UTP to activate and desensitize P2Y receptors 5 min before P2X₇R stimulation by the indicated concentrations of ATP; where indicated (as in the traces shown in B), 100 μM NAD (plus ADP-R) was also added 5 min prior to the addition of ATP. In each assay, the cells were permeabilized with digitonin (Digi) for the calibration of Ca²⁺-dependent fura-2 fluorescence. A and B, ATP-induced Ca²⁺ influx without (A) or with (B) NAD pretreatment for 5 min. These traces are representative of observations from four experiments. C and D, Changes in cytosolic Ca²⁺ in ART2.2-transfected cells were quantified at 30 s (C) or 3 min (D) after the addition of the indicated concentrations of ATP with or without NAD pretreatment. Data bars represent the mean ± SE from four experiments. E and F, Identical experiments as in C and D, but with HEK-mP2X₇ cells not transfected with ART2.2. Data bars represent the mean ± SE from three experiments.

Fig. 3⇑D demonstrates that ART2.1 also mediates the NAD-dependent activation of the hypersensitive R276K mutant P2X₇R in transiently cotransfected HEK293 cells. These experiments used two FACS-based readouts of P2X₇R function: 1) the transfer of PS to the external leaflet of the plasma membrane bilayer (“PS-flip”) as measured by increased binding of fluorochrome-conjugated annexin V; and 2) induction of the nonselective permeability pore as measured by the influx of YO-PRO-1 dye. In the absence of either ATP or NAD stimuli, the cells exhibited little if any surface annexin V staining or YO-PRO-1 accumulation. When stimulated by ATP, the majority of the ART2.1-mP2X₇-R276K-cotransfected HEK cells showed strongly increased annexin V binding and YO-PRO-1 uptake. Treatment with NAD in the presence of DTT caused a similar stimulation of annexin V binding but a somewhat lower induction of YO-PRO-1 uptake. In contrast, cells treated with ADP-R plus DTT exhibited control levels of annexin binding and dye accumulation. Notably, these PS-flip and YO-PRO-1 influx responses to NAD were absent when the R276K mutation was combined with the double R125K/R133K substitutions that eliminate the P2X₇ ADP-ribosylation sites targeted by ART2.1. In contrast, and in accord with our previous report (21), the triple R276K/R125K/R133K mutant of P2X₇ retained robust responses to ATP.

The differential ability of NAD/ART2 to activate the R276K-mutated P2X₇R (Fig. 3⇑, B and D), but not the WT P2X₇R (Fig. 3⇑A), in an HEK293 background is similar to the observations in Fig. 1⇑ regarding the differential ability of NAD to activate P2X₇R in murine T lymphocytes, but not in murine macrophages. These differences indicate that the consequences of ADP-ribosylation on P2X₇R function are cell type specific, perhaps due to the differential expression of cell-specific accessory signaling molecules or of variant forms of P2X₇R. We tested the hypothesis that ADP-ribosylation of WT P2X₇R in HEK293 cells, although insufficient to activate the receptor per se, might modulate the ATP activation threshold. HEK293 cells coexpressing ART2.2 and WT P2X₇R were prestimulated with or without 100 μM NAD for 3–5 min before being challenged with increasing concentrations of ATP to activate P2X₇R-mediated Ca²⁺ influx. The NAD pretreatment decreased the threshold concentration of ATP required to stimulate P2X₇R in cells that coexpressed ART2 (Fig. 4⇑, A and B). In Fig. 4⇑, C–F compare the quantification of ATP-induced changes in Ca²⁺ (with or without NAD pretreatment) at 30 s (Fig. 4⇑, C and E) and 3 min (Fig. 4⇑, D and F) in ART2-transfected or -nontransfected HEK-mP2X₇ cells. The NAD-induced shift in ATP sensitivity was most apparent with [ATP] in the 200–600 μM range. NAD did not further increase the Ca²⁺ influx response to [ATP] ≥ 1 mM, which maximally activates P2X₇R function. Notably, NAD pretreatment did not shift the ATP dose-response relationship in the HEK293 cells expressing P2X₇R but not ART2.2 (Fig. 4⇑, B, E, and F).

These experiments focused on defining changes in P2X₇R activation by [ATP] in the threshold-to-EC₅₀ range because we (16) and others (2, 15) have noted that the conventional analysis of ATP concentration-response relationships for recombinant or native P2X₇R is complicated by the following: 1) the unusually high ATP EC₅₀ (∼1 mM); 2) the allosteric effects of extracellular Mg²⁺ and Ca²⁺ on P2X₇R activity; and 3) the strong chelation of divalent cations by ATP (a divalent anion at physiological pH and ionic strength), such that the extracellular concentrations of free Mg²⁺ and free Ca²⁺ are decreased as the concentration of added ATP is increased to supramillimolar levels. The assay of Ca²⁺ influx as a sensitive and convenient readout of P2X₇R channel gating is further convoluted by the decreased extracellular [Ca²⁺] and the consequent reduction in the chemical driving force at the supramillimolar [ATP]. Because the contribution of these various complicating factors is minimized at submillimolar [ATP], we assayed changes in threshold ATP concentrations rather than changes in the ATP EC₅₀ as the simplest index of increased ATP potency at the ADP-ribosylated P2X₇R.

Differential effects of NAD/ART2 on P2X₇R function in established murine macrophage and T lymphocyte cell lines

The observation that NAD treatment decreased the threshold ATP concentration for heterologously expressed P2X₇R in HEK293 suggested that NAD/ART2.1 might similarly regulate natively expressed P2X₇R in murine macrophages. To facilitate these studies, we first determined that established murine macrophage and T cell lines were appropriate models for mechanistic analysis of the differential effects of NAD/ART2 on P2X₇R function in primary murine macrophages vs T cells. We tested two murine macrophage cell lines (Bac1.2F5 and RAW264.7) and a murine T lymphoma cell line (BW5147). As in primary BMDM (36), the Bac1.2F5 macrophages (Fig. 5⇓A) and RAW264.7 macrophages (data not shown) lacked basal expression of any ecto-ART subtypes at the mRNA level (heart or spleen extracts from BALB/c mice were used as positive control sources of ART1, ART2, ART3, ART4, and ART5 transcripts). However, Bac1.2F5 cells stimulated with 100 ng/ml LPS for 2–24 h selectively accumulated ART2.1 mRNA (Fig. 5⇓C), and this up-regulation of ART2.1 correlated with increased expression of iNOS and IFN-β, two other LPS-inducible gene products. Thiol-dependent ART enzyme activity in intact Bac1.2F5 macrophages was detected using the 1G4 mAb-based Western blot assay (Fig. 5⇓D). Multiple cell surface proteins were ADP-ribosylated in a strictly DTT-dependent manner in Bac1.2F5 macrophages primed by LPS, IFN-γ, or IFN-β to up-regulate ART2.1 expression (Fig. 5⇓D). Extracellular GSH, a physiological thiol reductant, also supported the ADP-ribosylation of multiple surface proteins in the IFN-primed macrophages (Fig. 5⇓E). Similarly as primary spleen T cells from BALB/c mice, BW5147 T lymphoma cells constitutively expressed both ART2.1 and ART2.2 mRNA (Fig. 5⇓, A and B) and also expressed functional ART2 activity as detected by the 1G4 mAb assay (data not shown). Splenic T cells from C57BL/6 mice expressed ART2.2, but not ART2.1, at significant levels. Notably, the BW5147 T cells also exhibited robust Ca²⁺ influx responses to either ATP or NAD (Fig. 5⇓F) as observed in primary splenic lymphocytes (Fig. 1⇑, A and B). In contrast, IFN-γ-primed Bac1.2F5 macrophages responded to exogenous ATP, but not to NAD alone (Fig. 5⇓G), similarly as IFN- or LPS-primed primary BMDM (Fig. 1⇑D).

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FIGURE 5.

Differential effects of NAD/ART2 on P2X₇R function in established murine macrophage and T lymphocyte cell lines. A, Expression of ART1, ART2, ART3, ART4, or ART5 mRNA was assayed by RT-PCR in BW5147 lymphocytes and Bac1.2F5 macrophages, as well as in freshly isolated extracts of heart or spleen from a BALB/c mouse. B, RT-PCR analysis for total ART2, ART2.1, and ART2.2 in BW5147 cells and freshly isolated spleen tissues from BALB/c or C57BL/6 mice. C, Bac1.2F5 macrophages were transferred to M-CSF-free medium and stimulated with LPS (100 ng/ml) for the indicated times before extraction and RT-PCR analysis for ART2, ART2.1, ART2.2, inducible NO synthase (iNOS), IL-1β, or GAPDH content. D, Bac1.2F5 macrophages were transferred to M-CSF-free medium and stimulated with or without IFN-β (100 U/ml), IFN-γ (100 U/ml), or LPS (100 ng/ml) for 24 h (priming incubation). The primed cells were then stimulated with 50 μM ε-NAD and 1 mM ADP-R in the presence or absence of 2 mM DTT at 37°C for 15 min (test incubation) before extraction for SDS-PAGE and Western blot analysis of 1G4-reactive, εADP-ribosylated proteins. IB, Immunoblotting. E, Bac1.2F5 macrophages were stimulated with or without IFN-γ (100 U/ml) for 24 h. The primed cells were then stimulated with 50 μM ε-NAD and 1 mM ADP-R in the presence or absence of the indicated concentrations of GSH or DTT at 37°C for 15 min. F and G, ATP- or NAD-induced changes in Ca²⁺ were assayed in fura-2-loaded suspensions of untreated BW5147 T lymphocytes (F) or Bac1.2F5 macrophages primed for 24 h with IFN-γ (G). For Bac1 cells, the 100 μM NAD was added together 1 mM ADP-R and 1 mM DTT. These results are representative of observations from three or more experiments.

Recent studies have indicated that extracellular NAD also activates some subtypes of G protein-coupled P2Y nucleotide receptors that can trigger mobilization of intracellular Ca²⁺ stores (48). The Ca²⁺ influx-based assay used to monitor P2X₇R activity required prestimulation of the macrophages with a mixture of ADP and UTP to first activate and desensitize the Ca²⁺-mobilizing P2Y1, P2Y2, and P2Y6 receptors expressed in these cells (41, 42). We tested the possibility that this protocol also desensitized NAD-reactive P2Y receptors that might be expressed in T cells and macrophages. BW5147 and Bac1.2F5 macrophages were stimulated with only single-nucleotide agonists with no prior desensitization incubation. Although murine T cells have been reported to express mRNA for various P2Y receptor subtypes (14), we observed no Ca²⁺-mobilizing responses to micromolar concentrations of ADP (P2Y1 agonist), UTP (P2Y2/P2Y4 agonist), or UDP (P2Y6 agonist) in the BW5147 T cells (Fig. 6⇓A). Moreover, primary T cells from P2X₇R-knockout mice showed no Ca²⁺ response to ATP or NAD (Fig. 1⇑). Thus, naive murine T cells do not express functionally significant levels of Ca²⁺-mobilizing P2Y receptors that can be activated by ATP, ADP, UTP, UDP, or NAD. In contrast, Bac1 macrophages, like primary murine BMDM (36), exhibited strong Ca²⁺ mobilization responses to ADP, UTP, and UDP, but not to NAD (Fig. 6⇓B). Thus, the differential responses of T cells vs macrophages to NAD do not involve obvious roles for NAD-sensitive P2Y receptor subtypes.

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FIGURE 6.

P2Y receptor-based Ca²⁺ signaling and extracellular NAD metabolism in murine macrophage and T lymphocyte cell lines. A and B, Cytosolic Ca²⁺ was assayed in fura-2-loaded suspensions of BW5147 T cells (A) or IFN-γ-primed Bac1.2F5 macrophages (B) challenged with the indicated concentrations of ADP, UTP, UDP, NAD, or ATP. The results are representative of observations from two experiments. Dig, Digitonin. C and D, Monolayers of Bac1.2F5 macrophages (10⁶ cells/well in 6-well dishes) were incubated in 1 ml of BSS at 37°C supplemented with 100 μM NAD in the presence or absence of 1 mM ADP-R. At selected times (0–60 min), 100-μl aliquots of the extracellular medium were removed and processed for HPLC analysis. C, Stacked HPLC chromatograms of extracellular samples taken at the 0-, 10-, 30-, and 60-min time points. D, NAD concentrations at the indicated times were calculated from the HPLC chromatograms.

Other ectoenzymes that modulate the efficiency of NAD-dependent ADP-ribosylation of cell surface proteins can also be differentially expressed in leukocyte subsets. These include the CD38 NAD-glycohydrolases and CD203 nucleotide pyrophosphatases that metabolize extracellular NAD and thereby reduce substrate drive to the ARTs (26). We used HPLC analyses to test whether the inability of NAD per se to trigger Ca²⁺ influx or mobilization in murine macrophages was due to very rapid metabolism of the added extracellular NAD. Although Bac1 macrophages metabolized 100 μM NAD to ADP-R, the t_1/2 for this reaction was ∼20 min, such that >50 μM NAD was present throughout the 1–10 min test periods used to assay Ca²⁺ influx/mobilization responses (Fig. 6⇑C). Inclusion of exogenous ADP-R (routinely added to maintain ADP-ribosylation of target proteins) further slowed the rate of NAD clearance. Thus, excessive NAD catabolism is an unlikely reason for the differential responses of macrophages vs T cells to extracellular NAD.

NAD potentiates ATP-induced P2X₇R activation in murine macrophages and T lymphocytes

IFN-γ-primed Bac1.2F5 macrophages were briefly pretreated with NAD in the presence of DTT to allow ADP-ribosylation of cell surface proteins and then challenged with various doses of ATP to trigger P2X₇R-dependent Ca²⁺ influx. Similar to its effects in ART2/P2X₇-transfected HEK293 cells, NAD pretreatment increased the sensitivity of the IFN-primed Bac1 macrophages to submillimolar ATP (Figs. 7⇓, A–C). In contrast, naive Bac1.2F5 cells, which were not primed with IFN-γ and thus lacked expression of ART2.1, did not exhibit the NAD-dependent increase in ATP sensitivity (Figs. 7⇓, D and E). Additional experiments demonstrated that NAD pretreatment produced similar increases in ATP sensitivity in other murine macrophage models, including IFN-β-primed RAW264.7 macrophages (Fig. 8⇓A) and IFN-γ-primed BALB/c primary BMDM (Fig. 8⇓B). Similarly as Bac1.2F cells (Fig. 5⇑) and primary BMDM (36), the RAW264.7 macrophage line exhibited up-regulation of ART2.1 mRNA and thiol-dependent ecto-ART activity in response to IFN-β stimulation (data not shown).

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FIGURE 7.

NAD potentiates ATP-induced P2X₇R activation in IFN-primed murine macrophages that express ART2.1. Bac1.2F5 macrophages were transferred to M-CSF-free medium and then stimulated with IFN-γ (100 U/ml) for 24 h (A–C) or were incubated in M-CSF-free medium for 24 h in the absence of IFN-γ (D and E). Cytosolic Ca²⁺ was measured in fura-2-loaded Bac1 cell suspensions as previously described but with the inclusion of 1 mM DTT and 1 mM ADP-R in all test media. The cell suspensions were pretreated with a mixture of 50 μM ADP and 50 μM UTP to activate and desensitize P2Y receptors 5 min before P2X₇R stimulation by the indicated concentrations of ATP; where indicated (as in the traces shown in A), 100 μM NAD was also added 5 min prior to the addition of ATP. In each assay, the cells were permeabilized with digitonin (Digi) for calibration of Ca²⁺-dependent fura-2 fluorescence. A, ATP-induced Ca²⁺ influx without or with NAD pretreatment for 5 min. These traces are representative of observations from four experiments. B and C, Changes in cytosolic Ca²⁺ in IFN-γ-primed Bac1 macrophages were quantified at 30 s (B) or 3 min (C) after the addition of the indicated concentrations of ATP with or without NAD pretreatment. Data bars represent the mean ± SE from four experiments. D and E, Identical experiments to those in B and C, but with Bac1 macrophages that were not primed with IFN-γ. Data bars represent the mean ± SE from four experiments.

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FIGURE 8.

Comparative effects of NAD on the potentiation of ATP-induced P2X₇R activation in primary macrophages, a macrophage cell line, and a T cell line. Experiments similar to those described in Fig. 6 with Bac1.2F5 macrophages were performed using other murine leukocytes that express P2X₇R. A, RAW264.7 macrophages were transferred to M-CSF-free medium and stimulated with IFN-β (100 U/ml) for 24 h. The primed macrophages were stimulated with 1 mM ATP alone, 100 μM NAD (plus DTT) alone, or with 300 μM ATP in the absence or presence of a pretreatment with 100 μM NAD (plus DTT) for 5 min. The accompanying histograms (mean ± SE from three experiments) show the quantified changes in Ca²⁺ at 3 min after the addition of 300 μM ATP with and without NAD pretreatment. B, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with IFN-γ (100 U/ml) for 24 h. The cells were stimulated and assayed as described in A, but with 200 μM ATP as the test pulse. The accompanying histograms (mean ± SE from three experiments) show the quantified changes in Ca²⁺ 3 min after the addition of 200 μM ATP with and without NAD pretreatment. C, BW5147 T lymphocytes were stimulated with 1 mM ATP alone, 10 μM NAD alone, or 300 μM ATP in the absence or presence of a pretreatment with 10 μM NAD for 5 min. These results are representative of observations from three experiments.

NAD pretreatment also increased the sensitivity to ATP in the BW5147 T lymphocytes. These experiments involved stimulation of these cells with submaximally active concentrations of NAD (10 μM) and/or ATP (300 μM). Notably, when combined, 10 μM NAD and 300 μM ATP acted synergistically to increase P2X₇R-mediated Ca²⁺ influx (Fig. 8⇑C).

ART2 can use dinucleotide substrates other than NAD to covalently ribosylate arginine residues in target proteins; these alternative substrates include ε-NAD and nicotinamide guanine dinucleotide (NGD). We previously reported that ribosylation of the P2X₇R by ε-NAD or NGD in primary murine T cells does not stimulate the receptor but rather antagonizes NAD-induced receptor activation (20). We observed similar effects of ε-NAD on P2X₇R function in the BW5147 T cell line (Figs. 9⇓, A and B). Notably, P2X₇ receptors ribosylated by εNAD also have decreased sensitivity to activation by submaximal ATP as shown in primary murine T cells (20), BW5147 T lymphoma cells (Figs. 9⇓, A and B), IFN-γ-primed Bac1.2F5 macrophages (Fig. 9⇓C), and IFN-β-primed RAW264.7 macrophages (Fig. 9⇓D).

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FIGURE 9.

ε-NAD, an NAD analog, desensitizes P2X₇R to ATP in macrophages and T lymphocytes. Experiments similar to those described in Fig. 7 were performed but used the NAD analog, εNAD, as the ART2 substrate for the pretreatment of murine leukocytes that express P2X₇R. A, BW5147 T lymphocytes were stimulated with 0.5 mM ATP alone, 200 μM ATP alone (added as two sequential 100 μM pulses), or 100 μM NAD alone. These results are representative of observations from two experiments. B, Identical experiment as in A but with BW5147 T lymphocytes that were pretreated with 50 μM εNAD for 5 min before stimulation with 0.5 mM ATP alone, 200 μM ATP alone (added as two sequential 100 μM pulses), or 100 μM NAD alone. These results are representative of observations from two experiments. Digi, Digitonin. C, Bac1.2F5 macrophages were transferred to M-CSF-free medium and stimulated with IFN-γ (100 U/ml) for 24 h. The primed macrophages were acutely stimulated with 1 mM ATP alone, 100 μM εNAD (plus DTT) alone, or 300 μM ATP in the absence or presence of a pretreatment with 100 μM εNAD (plus DTT) for 5 min. The accompanying histograms (mean ± SE from three experiments) show the quantified changes in Ca²⁺ at 3 min after the addition of 300 μM ATP with and without εNAD pretreatment. D, RAW264.7 macrophages were transferred to M-CSF-free medium and stimulated with IFN-β (100 U/ml) for 24 h. The primed macrophages were acutely stimulated with 1 mM ATP alone, 100 μM εNAD (plus DTT) alone, or 400 μM ATP in the absence or presence of a pretreatment with 100 μM εNAD (plus DTT) for 5 min. The accompanying histograms (mean ± SE from three experiments) show the quantified changes in Ca²⁺ at 3 min after the addition of 400 μM ATP with and without εNAD pretreatment.

NAD potentiates ATP-induced P2X₇R-dependent pore formation in murine macrophages

The previous data showing that ADP-ribosylation of P2X₇R potentiates ATP activation of these receptors in murine macrophages and HEK293 cells used increases in cytosolic Ca²⁺ as a sensitive readout of P2X₇R channel activity. We considered the possibility that the P2Y prestimulation protocol (used to desensitize Ca²⁺-mobilizing P2Y receptors) triggered G protein-coupled signaling pathways that modulate the functional interactions between ART2 and P2X₇R in macrophages and HEK293 cells. Thus, we measured the effects of NAD/ART2 on nonselective pore formation as an alternative readout of P2X₇R activation that does not require prestimulation and desensitization of P2Y receptors. Pore formation was assayed by ethidium accumulation in IFN-γ-primed (Fig. 10⇓A) vs control (Fig. 10⇓B) Bac1.2F5 macrophages, similar to the YO-PRO-1 accumulation assay previously described for HEK293 cells (Fig. 3⇑D). NAD (plus DTT) by itself did not stimulate ethidium accumulation but did increase the rate of accumulation triggered by submillimolar ATP in the IFN-γ primed, but not control, Bac1 macrophages. Notably, the potentiating effects of NAD on ethidium influx were observed at a higher range of extracellular ATP concentrations (0.5–1 mM) than the effects on Ca²⁺ influx (0.1–0.5 mM ATP; Fig. 7⇑). This is consistent with the fact that ethidium influx is a secondary response to P2X₇R stimulation (49) that involves gating of pannexin-1 hemichannels (4). Previous studies have indicated that the ATP concentration-response relationship describing this secondary response is right shifted relative to the concentration-response relationship describing the primary gating of P2X₇R channels (2, 50).

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FIGURE 10.

NAD potentiates ATP-induced pore formation in IFN-γ-primed murine macrophages. Bac1.2F5 macrophages were suspended in BSS, and ATP-induced ethidium accumulation was assayed as described in Materials and Methods. Bac1.2F5 macrophages were transferred to M-CSF-free medium and stimulated with (A) or without (B) IFN-γ (100 U/ml) for 24 h before an assay of ethidium accumulation. The primed macrophages were stimulated with the indicated concentrations of ATP in the absence or presence of pretreatment with 100 μM NAD (plus 1 mM DTT) for 5 min before the addition of ATP. Accumulation of fluorescent ethidium/DNA/RNA complexes was measured at 350 s after ATP addition. Data bars represent the mean ± SE from four experiments.