Differential Regulation of P2X7Receptor Activation by
Extracellular Nicotinamide Adenine Dinucleotide and
Ecto-ADP-Ribosyltransferases in Murine Macrophages
and T Cells
Shiyuan Hong,* Nicole Schwarz,†Anette Brass,†Michel Seman,‡Friedrich Haag,†
Friedrich Koch-Nolte,†William P. Schilling,* and George R. Dubyak1*
Extracellular NAD induces the ATP-independent activation of the ionotropic P2X7purinergic receptor (P2X7R) in murine T
lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X7R ectodomain. This
modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T
cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that con-
stitutively express P2X7R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X7R function in
murine macrophages. Coexpression of the cloned murine P2X7R with ART2.1 or ART2.2 in HEK293 cells verified that P2X7R is
an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of
macrophages or HEK293 cells with NAD alone did not activate the P2X7R. Rather, NAD potentiated ATP-dependent P2X7R
activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X7R in both
macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X7R channel opening
in T cells, this P2X7R modification in macrophages does not gate the channel but decreases the threshold for gating in response
to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X7R signaling in
murine macrophages and also suggest that the cellular context in which P2X7R signaling occurs differs between myeloid and
lymphoid leukocytes. The Journal of Immunology, 2009, 183: 578–592.
rophages and T lymphocytes (1). The subunit structure of this
595-aa receptor includes two transmembrane segments, an intra-
cellular N terminus, an intracellular C terminus, and a large ex-
tracellular loop (47–329 aa) that contain a presumed site or sites
for ATP binding (2). Three of these P2X7subunits assemble to
form the trimeric P2X7R channel (3). Stimulation of the P2X7R
with extracellular ATP rapidly triggers increased Na?, K?, and
Ca2?fluxes across the plasma membrane (2), followed by the
delayed induction of a nonselective pore that facilitates the per-
meation of molecules up to 900 Da in mass (4). Given its expres-
sion in myeloid and lymphoid leukocytes, many studies have
he P2X7purinergic receptor (P2X7R)2is an ATP-gated,
nonselective cation channel that is predominantly ex-
pressed in cells of hematopoietic origin, including mac-
identified roles for the P2X7R in the regulation of various proin-
flammatory and immune responses (reviewed in Refs. 5–7).
An unusual and defining feature of the P2X7R is its high thresh-
old for activation by extracellular ATP (EC50? 500 ?M); this
contrasts with much lower activation thresholds for the other six
members of the P2X family (EC50? 10 ?M) (1, 8, 9). Intracellular
ATP concentration is only 3–5 mM, and most cells express sig-
nificant ecto-ATPase activities. Thus, it is unlikely that submilli-
molar levels of extracellular ATP can be sustained for significant
durations within interstitial tissue compartments except perhaps
during a massive lysis of host cells or the killing of invading patho-
gens. Clearly, the P2X7R is activated within in situ inflammatory
loci or during normal development as indicated by the reduced
levels of cytokines that accumulate within the inflamed footpads of
P2X7R knockout mice (10), as well as the marked changes in bone
density during the aging of these mice (11). Autocrine activation of
P2X7R via the release of endogenous ATP has been recently re-
ported in human monocytes in response to LPS stimulation of
TLR4 signaling (12, 13) and in T cells in response to Ag stimu-
lation of TCR signaling cascades (14). This released ATP may
accumulate within diffusion-restricted microdomains of the cell
surface, such as caveolar or nascent endosomal invaginations not
readily accessible to the bulk extracellular medium.
However, physiological P2X7R activation may involve modes
of regulation in addition to autocrine stimulation. One of these
modes is allosteric modulation of ATP affinity via conformational
changes in P2X7trimeric channels produced by local biophysical
conditions such as pH, ionic composition (15–17), or membrane
lipid composition (18). For example, lysophosphatidylcholine and
other lysolipids reduce the threshold level of ATP required for
*Department of Physiology and Biophysics, Case Western Reserve University School
of Medicine, Cleveland, OH 44120;†Institute of Immunology, University Hospital,
Hamburg, Germany; and‡University of Rouen, Rouen, France
Received for publication January 14, 2009. Accepted for publication May 5, 2009.
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.
1Address correspondence and reprint requests to Dr. George R. Dubyak, Department
of Physiology and Biophysics, Case Western Reserve University School of Medicine,
10900 Euclid Avenue, Cleveland OH, 44120. E-mail address: george.dubyak@
2Abbreviations used in this paper: P2X7R, P2X7receptor; ADP-R, ADP-ribose;
ART, ADP-ribosyltransferase; BMDM, bone marrow-derived macrophage; BSS, ba-
sic salt solution; ?NAD, etheno-NAD; GSH, glutathione; iNOS, inducible NO syn-
thase; mP2X7, murine P2X7; NZW, New Zealand White; PS, phosphatidylserine;
WT, wild type.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
P2X7R-dependent Ca2?influx and pore formation in murine mi-
croglia (19). Another type of regulation involves the gating of
P2X7R channels by mechanisms independent of reversible ATP
binding. Notably, extracellular NAD induces the ATP-independent
activation of P2X7R in murine T lymphocytes via a novel covalent
pathway involving ADP-ribosylation of arginine residues on the
P2X7R ectodomain (20–22).
The ADP-ribosylation of P2X7R is catalyzed by ART2, a GPI-
anchored ADP-ribosyltransferase (ART) constitutively expressed
on the cell surface of murine T cells (23–25). ART2 belongs to a
family of ectoenzymes that use extracellular NAD to transfer the
ADP-ribose (ADP-R) moiety to substrate proteins (26). ART2 me-
diates P2X7R transactivation via ADP-ribosylation of the R125
residue within the extracellular loop of the receptor (21). This co-
valent modification apparently mimics the conformational changes
in P2X7R induced by noncovalent ATP binding and triggers both
Ca2?influx and the secondary nonselective pore permeable to flu-
orescent dyes. The NAD- and ART2-dependent activation of
P2X7R consequently induces phosphatidylserine (PS) exposure on
T cell surfaces, increased shedding of CD62L, and acceleration of
T cell death. Other studies have shown that extracellular NAD
induces P2X7R activation in in vivo models of immune and in-
flammatory responses (21, 27, 28).
ART2 includes two isoforms, ART2.1 and ART2.2, which are
encoded by tandem genes (Art2a and Art2b) located on murine
chromosome 7 (26). Although ART2.1 is functionally and struc-
turally similar to ART2.2, it contains two additional cysteine res-
idues (Cys80and Cys201) that readily form a disulfide bond that
allosterically suppresses catalytic activity (29). This inhibited state
of ART2.1 is reversed by extracellular thiol reductants, such as
exogenous DTT or the endogenous cysteine and glutathione re-
leased by inflamed or damaged tissues (30, 31). T cells from most
inbred mouse strains (e.g., BALB/c) natively express both ART2.1
and ART2.2 (25, 32–34). However, the latter isoform is sufficient
for NAD-induced P2X7R activation and cell death, because these
responses occur in the absence of extracellular thiol reductants and
in T cells from C57BL/6 mice that express a mutated Art2a gene
and no functional ART2.1 protein (20, 35).
Although the NAD-induced, ART2-dependent mechanism is
clearly a major pathway for P2X7R activation in mouse T lym-
phocytes, it is unclear whether this mechanism is operative in mac-
rophages, another class of leukocytes that natively express P2X7R
at high levels. Relevant to this issue, we have reported the induc-
ible expression of ART2.1, but not ART2.2, in murine bone mar-
row-derived macrophages (BMDM) stimulated by multiple in-
flammatory factors (36). This prompted us to examine the role of
ART2.1 as a regulator of P2X7receptors natively expressed in
murine macrophages or heterologously expressed in HEK293
cells. We demonstrate that coexpression of the murine P2X7R with
ART2.1 or ART2.2 in HEK293 cells facilitates similar NAD-
driven ADP-ribosylation of the receptor. However, NAD stimula-
tion of P2X7R in macrophages or HEK293 cells is not sufficient to
activate the receptor. Rather, the NAD/ART2-dependent modifi-
cation of the P2X7R potentiates the ability of ATP to activate the
receptor as indicated by a left shift in the ATP dose-response re-
lationship. Thus, extracellular NAD acts to regulate the ionotropic
P2X7R in both macrophages and T cells but via distinct mecha-
nisms on the gating of channel activity. These observations support
a role for extracellular NAD in the regulation of P2X7R-dependent
inflammatory responses in macrophages and additionally suggest
that the cellular context (i.e., myeloid vs lymphoid cells) dictates
the outcome of signaling through the P2X7R.
Materials and Methods
Recombinant murine IFN-? was from Boehringer Mannheim Biochemica
and recombinant murine IFN-? was from US Biologicals. LPS (Esche-
richia coli serotype 01101:B4) was from List Biological Laboratories.
ATP, NAD, etheno-NAD (?NAD), ADP-R, reduced glutathione (GSH),
and TRIzol were from Sigma-Aldrich. Oligo(dT) primer was from Pro-
mega. 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 pro-
vided 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 Ham-
burg 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) supple-
mented with 25% L cell-conditioned medium, 15% calf serum (HyClone
Laboratories), 100 U/ml penicillin, and 100 ?g/ml streptomycin (Invitro-
gen) in the presence of 10% CO2. RAW264.7 macrophages were cultured
in DMEM supplemented with 10% calf serum, 100 U/ml penicillin, and
100 ?g/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-?, (100 U/ml), or IFN-?
(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-strep-
tomycin 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-mP2X7cells) or the mutant
R276K P2X7R (HEK-mP2X7-R276K cells) were selected and maintained
in DMEM supplemented with 400 ?g/ml G418 or 10 ?g/ml blasticidin.
The ART2 plasmids were transiently transfected into HEK P2X7R cells
using cells seeded at 6 ? 105per 35-mm dish 24 h before transient trans-
fection. The cells were transfected using PolyFect reagent (Qiagen) with
2.5 ?g of plasmid DNA per dish followed by incubation at 37°C for 36 h
Total RNA was extracted using TRIzol; all primers and PCR conditions for
ecto-ARTs, inducible NO synthase (iNOS), IFN-?, and GAPDH were pre-
pared 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 ?M ?-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 ?M32P-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)-benzenesul-
fonyl 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
579The Journal of Immunology
at 3 ?g/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 ?l
beads/lysates from 106cells) for 60 min at 4°C. Immunoprecipitates were
washed three times using Triton X-100 buffer. The final product was
eluted into 30 ?l 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. P2X7was detected with a rabbit
anti-P2X7C-terminal peptide Ab (1/1000) (Alomone Labs) and perox-
idase-conjugated anti-rabbit IgG (1/5000) using the ECL system (Am-
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 Bio-
sciences). Gating was performed on living cells on the basis of propidium
Measurement of P2X7R-mediated Ca2?influx or
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 pel-
lets 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 ?M
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 calibra-
tion 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 ?M ADP and 50 ?M 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 ?M 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 ?M NAD.
Measurement of P2X7R-mediated YO-PRO-1 or ethidium dye
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 ?M 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-allophyco-
cyanin (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-?-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 ?M) 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 in-
creases were corrected for background dye fluorescence. The P2X7R-me-
diated 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 (106cells/well in 6-well dishes)
were incubated in 1 ml of BSS at 37°C supplemented with 100 ?M NAD
in the presence or absence of 1 mM ADP-R. At selected times (0–60 min),
100-?l 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 re-
verse-phase HPLC protocol. Briefly, 50-?l 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 KH2PO4and 5% methanol (pH 6).
NAD (elution time 8.2 min) and ADP-R (elution time 4 min) were detected
by absorbance at 254 nm.
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
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 ex-
press 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 en-
zymes, 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).
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 in-
creases in Ca2?in both WT BMDM populations, but not in
BMDMs from P2X7R-deficient mice (Fig. 1F). 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. 1H) or IFNs (not
shown); this is consistent with the general lack of ART2.2 expres-
sion in murine myeloid leukocytes and the specific lack of ART2.1
expression in C57BL/6 myeloid leukocytes (35). As expected, nei-
ther NAD nor ATP elicited a Ca2?influx response in P2X7-knock-
out BMDM (Fig. 1I). Surprisingly, however, inflammatory
BALB/c BMDM primed with IFN-? (Fig. 1G) 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-?ADP- mAbs in FACS anal-
yses to confirm that LPS and IFN-? induced the specific up-reg-
ulation 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 inclu-
sion 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. 1G).
?/?mice (Fig. 1C). (Although
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 mac-
rophages 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
580 NAD, ART2, AND P2X7RECEPTORS IN MACROPHAGES AND LYMPHOCYTES
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. 2A 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. 2B). 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 equiv-
alent [32P]ADP-ribosylation of P2X7R in the absence or presence
of DTT; mutants of P2X7in 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-P2X7Ab and FACS analyses
to confirm similar cell surface expression levels of all WT and
mutant P2X7R constructs (Fig. 2).
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. 2C). 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 P2X7activation. Fig. 2D shows that T lymphocytes
from BALB/c mice, which express high levels of ART2.2, in-
corporate YO-PRO-1 in response to micromolar NAD regard-
less 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
(3 mM) or NAD (100 ?M) 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 ?M ADP and 50 ?M 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-? 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 ?M 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 ?M U0126 for 24 h to induce proinflam-
matory gene expression before the Ca2?assay.
NAD induces the activation of P2X7R in murine lymphocytes but not murine macrophages. Changes in cytosolic Ca2?in response to ATP
581 The Journal of Immunology
with ART2.1 in the HEK293 cell background. However, we have
reported that this covalent modification of the WT P2X7R ex-
pressed in HEK293 cells is not sufficient for functional activa-
tion of the receptor (21). Fig. 3A shows this by comparing NAD
with ATP as stimuli for Ca2?influx in a HEK293 line (HEK-
mP2X7cells) stably transfected with murine P2X7R cDNA be-
fore 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. 3C). This assay involves incubation of intact cells
with ?NAD (an NAD analog) to covalently ?-ADP-ribosylate
cell surface proteins followed by cell extraction, SDS-PAGE,
and probing with the anti-?-ADP-R 1G4 mAb. Fig. 3C also
shows that inclusion of extracellular ADP-R potentiated the ac-
cumulation of ADP-ribosylated proteins by attenuating the me-
tabolism of ?NAD (or NAD) by ectonucleotidases. Despite the
robust ART activity in the cotransfected HEK-mP2X7cells, ex-
tracellular NAD did not mimic the ability of ATP to trigger
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. 3B shows that the HEK293 line (HEK-
mP2X7-R276K), stably transfected with the R276K gain of func-
tion mutant of P2X7R, exhibited a maximal Ca2?influx response
to 50 ?M ATP, which is a subthreshold concentration in cells
expressing WT P2X7R (see Fig. 4A). Notably, this R276K muta-
tion also facilitated the gating of P2X7R channel activity in re-
sponse to NAD in HEK293 cells cotransfected with ART2.2
Fig. 3D demonstrates that ART2.1 also mediates the NAD-de-
pendent 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 an-
nexin 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 an-
nexin 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 in-
flux responses to NAD were absent when the R276K mutation was
combined with the double R125K/R133K substitutions that
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-P2X7K1G 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 ?M [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.
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,
582NAD, ART2, AND P2X7RECEPTORS IN MACROPHAGES AND LYMPHOCYTES
eliminate the P2X7ADP-ribosylation sites targeted by ART2.1. In
contrast, and in accord with our previous report (21), the triple
R276K/R125K/R133K mutant of P2X7retained robust responses
The differential ability of NAD/ART2 to activate the R276K-
mutated P2X7R (Fig. 3, B and D), but not the WT P2X7R (Fig.
3A), 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 macro-
phages. These differences indicate that the consequences of
ADP-ribosylation on P2X7R function are cell type specific, per-
haps 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 coex-
pressing ART2.2 and WT P2X7R were prestimulated with or
without 100 ?M 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-
mP2X7cells. The NAD-induced shift in ATP sensitivity was
most apparent with [ATP] in the 200–600 ?M range. NAD did
not further increase the Ca2?influx response to [ATP] ? 1 mM,
which maximally activates P2X7R function. Notably, NAD pre-
treatment did not shift the ATP dose-response relationship in
were stably transfected with either WT murine P2X7R (HEK-mP2X7cells) 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 ?M ADP and 50 ?M 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 ?M ?-NAD with or without 1 mM ADP-R
before extraction, SDS-PAGE, and Western blotting with the 1G4 mAb that detects ?-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 ?M NAD, or 500 ?M ATP separately in the presence of 1 mM DTT. The cells were
then treated with 1 ?M 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.
Differential effects of NAD/ART2 on activation of the murine P2X7R vs R276K-mutated P2X7R in HEK293 cells. A and B, HEK293 cells
583The Journal of Immunology
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-EC50range 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 extracel-
lular Mg2?and Ca2?on P2X7R activity; and 3) the strong
chelation of divalent cations by ATP (a divalent anion at phys-
iological pH and ionic strength), such that the extracellular con-
centrations of free Mg2?and free Ca2?are decreased as the
concentration of added ATP is increased to supramillimolar lev-
els. 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]. Be-
cause 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
EC50as the simplest index of increased ATP potency at the
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 ex-
periments for E and F, the HEK-
mP2X7cells that lack ART2.2 expres-
sion were directly assayed. Cytosolic
Ca2?was measured in fura-2-loaded
HEK cell suspensions as described in
Fig. 3. The cell suspensions were pre-
treated with a mixture of 50 ?M ADP
and 50 ?M UTP to activate and de-
sensitize P2Y receptors 5 min before
P2X7R stimulation by the indicated
concentrations of ATP; where indi-
cated (as in the traces shown in B),
100 ?M NAD (plus ADP-R) was also
added 5 min prior to the addition of
ATP. In each assay, the cells were per-
meabilized with digitonin (Digi) for
the calibration of Ca2?-dependent
fura-2 fluorescence. A and B, ATP-in-
duced Ca2?influx without (A) or with
(B) NAD pretreatment for 5 min.
These traces are representative of ob-
servations from four experiments. C
and D, Changes in cytosolic Ca2?in
ART2.2-transfected cells were quanti-
fied at 30 s (C) or 3 min (D) after the
addition of the indicated concentra-
tions 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-mP2X7cells not
transfected with ART2.2. Data bars
represent the mean ? SE from three
NAD potentiates ATP-
584 NAD, ART2, AND P2X7RECEPTORS IN MACROPHAGES AND LYMPHOCYTES
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 ex-
pressed 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 dif-
ferential effects of NAD/ART2 on P2X7R function in primary mu-
rine 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. 5A) 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. 5C), and this up-regulation of ART2.1 correlated with in-
creased expression of iNOS and IFN-?, 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. 5D). Multiple cell surface proteins were
ADP-ribosylated in a strictly DTT-dependent manner in Bac1.2F5
macrophages primed by LPS, IFN-?, or IFN-? to up-regulate
ART2.1 expression (Fig. 5D). Extracellular GSH, a physiological
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-1?, or GAPDH content. D,
Bac1.2F5 macrophages were transferred to M-CSF-free medium and stimulated with or without IFN-? (100 U/ml), IFN-? (100 U/ml), or LPS (100 ng/ml)
for 24 h (priming incubation). The primed cells were then stimulated with 50 ?M ?-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, ?ADP-ribosylated proteins. IB, Immu-
noblotting. E, Bac1.2F5 macrophages were stimulated with or without IFN-? (100 U/ml) for 24 h. The primed cells were then stimulated with 50 ?M
?-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-?
(G). For Bac1 cells, the 100 ?M NAD was added together 1 mM ADP-R and 1 mM DTT. These results are representative of observations from three or
Differential effects of NAD/ART2 on P2X7R function in established murine macrophage and T lymphocyte cell lines. A, Expression of
585The Journal of Immunology
thiol reductant, also supported the ADP-ribosylation of multiple
surface proteins in the IFN-primed macrophages (Fig. 5E). Simi-
larly 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.
5F) as observed in primary splenic lymphocytes (Fig. 1, A and B).
In contrast, IFN-?-primed Bac1.2F5 macrophages responded to
exogenous ATP, but not to NAD alone (Fig. 5G), similarly as IFN-
or LPS-primed primary BMDM (Fig. 1D).
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 ex-
pressed 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-nucle-
otide agonists with no prior desensitization incubation. Al-
though murine T cells have been reported to express mRNA for
various P2Y receptor subtypes (14), we observed no Ca2?-mo-
bilizing responses to micromolar concentrations of ADP (P2Y1
agonist), UTP (P2Y2/P2Y4 agonist), or UDP (P2Y6 agonist) in
the BW5147 T cells (Fig. 6A). 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 func-
tionally 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. 6B). Thus, the differential re-
sponses of T cells vs macrophages to NAD do not involve ob-
vious roles for NAD-sensitive P2Y receptor subtypes.
Other ectoenzymes that modulate the efficiency of NAD-de-
pendent ADP-ribosylation of cell surface proteins can also be
differentially expressed in leukocyte subsets. These include the
CD38 NAD-glycohydrolases and CD203 nucleotide pyrophos-
phatases that metabolize extracellular NAD and thereby reduce
Cytosolic Ca2?was assayed in fura-2-loaded suspensions of BW5147 T cells (A) or IFN-?-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 (106cells/well in 6-well dishes) were incubated in 1 ml of BSS at 37°C supplemented with 100 ?M NAD in the
presence or absence of 1 mM ADP-R. At selected times (0–60 min), 100-?l 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.
P2Y receptor-based Ca2?signaling and extracellular NAD metabolism in murine macrophage and T lymphocyte cell lines. A and B,
586NAD, ART2, AND P2X7RECEPTORS IN MACROPHAGES AND LYMPHOCYTES
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 ?M NAD to ADP-R, the t1/2
for this reaction was ?20 min, such that ?50 ?M NAD was
present throughout the 1–10 min test periods used to assay
Ca2?influx/mobilization responses (Fig. 6C). Inclusion of
exogenous ADP-R (routinely added to maintain ADP-ribosyla-
tion of target proteins) further slowed the rate of NAD clear-
ance. Thus, excessive NAD catabolism is an unlikely reason for
the differential responses of macrophages vs T cells to extra-
NAD potentiates ATP-induced P2X7R activation in murine
macrophages and T lymphocytes
IFN-?-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 in-
creased 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-? and thus lacked expres-
sion of ART2.1, did not exhibit the NAD-dependent increase in
ATP sensitivity (Figs. 7, D and E). Additional experiments dem-
onstrated that NAD pretreatment produced similar increases in
ATP sensitivity in other murine macrophage models, including
IFN-?-primed RAW264.7 macrophages (Fig. 8A) and IFN-?-
primed BALB/c primary BMDM (Fig. 8B). Similarly as Bac1.2F
cells (Fig. 5) and primary BMDM (36), the RAW264.7 macro-
phage line exhibited up-regulation of ART2.1 mRNA and thiol-
dependent ecto-ART activity in response to IFN-? stimulation
(data not shown).
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 ?M) and/or ATP (300 ?M). Notably, when combined, 10 ?M
NAD and 300 ?M ATP acted synergistically to increase P2X7R-
mediated Ca2?influx (Fig. 8C).
ART2 can use dinucleotide substrates other than NAD to co-
valently ribosylate arginine residues in target proteins; these alter-
native substrates include ?-NAD and nicotinamide guanine dinu-
cleotide (NGD). We previously reported that ribosylation of the
P2X7R by ?-NAD or NGD in primary murine T cells does not
stimulate the receptor but rather antagonizes NAD-induced recep-
tor activation (20). We observed similar effects of ?-NAD on
P2X7R function in the BW5147 T cell line (Figs. 9, A and B).
Notably, P2X7receptors ribosylated by ?NAD also have decreased
sensitivity to activation by submaximal ATP as shown in primary
transferred to M-CSF-free medium and then stimulated with IFN-? (100 U/ml) for 24 h (A–C) or were incubated in M-CSF-free medium for 24 h in the
absence of IFN-? (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 ?M ADP and 50 ?M 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 ?M 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-?-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-?. Data bars represent the mean ? SE from four experiments.
NAD potentiates ATP-induced P2X7R activation in IFN-primed murine macrophages that express ART2.1. Bac1.2F5 macrophages were
587 The Journal of Immunology
murine T cells (20), BW5147 T lymphoma cells (Figs. 9, A and B),
IFN-?-primed Bac1.2F5 macrophages (Fig. 9C), and IFN-?-
primed RAW264.7 macrophages (Fig. 9D).
NAD potentiates ATP-induced P2X7R-dependent pore formation
in murine macrophages
The previous data showing that ADP-ribosylation of P2X7R potenti-
ates ATP activation of these receptors in murine macrophages and
HEK293 cells used increases in cytosolic Ca2?as a sensitive readout
prestimulation protocol (used to desensitize Ca2?-mobilizing P2Y re-
ceptors) triggered G protein-coupled signaling pathways that modu-
late the functional interactions between ART2 and P2X7R in macro-
phages 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 desensiti-
zation of P2Y receptors. Pore formation was assayed by ethidium
accumulation in IFN-?-primed (Fig. 10A) vs control (Fig. 10B)
previously described for HEK293 cells (Fig. 3D). NAD (plus DTT)
by itself did not stimulate ethidium accumulation but did increase the
rate of accumulation triggered by submillimolar ATP in the IFN-?
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
(49) that involves gating of pannexin-1 hemichannels (4). Previous
studies have indicated that the ATP concentration-response relation-
ship describing this secondary response is right shifted relative to the
concentration-response relationship describing the primary gating of
P2X7R channels (2, 50).
From the initial functional characterization of the permeabilizing
“P2Z” ATP receptor 30 years ago (51–53), through the molecular
identification of the P2Z receptor phenotype as the product of the
P2X7R gene (2), and up to the most recent analyses of in vivo
functional deficits in P2X7-knockout mice (10, 11, 54), two fun-
damental and perplexing questions regarding the P2X7R have been
repeatedly considered. First, why does this particular ATP recep-
tor, in sharp contrast to the six other P2X receptor subtypes, re-
quire millimolar levels of extracellular ATP for activation when
studied in isolated cells? This unusual characteristic suggests that
low affinity variants of an ancestral P2X7R were favored by pos-
itive selection as the receptor acquired its physiological roles as a
regulator of proinflammatory signaling and cell death. Low ATP
affinity prevents inadvertent activation of these highly consequen-
tial but poorly reversible responses until leukocytes accumulate at
sites of tissue damage or microbial invasion. However, this raises
a second and corollary question: how does the P2X7R become
activated in leukocytes within these latter tissue compartments
given the receptor’s low affinity of ATP? Recent studies support
three possible mechanisms that are not mutually exclusive: 1)
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-? (100 U/ml) for 24 h. The primed macrophages
were stimulated with 1 mM ATP alone, 100 ?M NAD (plus DTT) alone, or with 300 ?M ATP in the absence or presence of a pretreatment with 100 ?M
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 ?M ATP with and without NAD pretreatment. B, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with IFN-?
(100 U/ml) for 24 h. The cells were stimulated and assayed as described in A, but with 200 ?M 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 ?M ATP with and without NAD pretreatment.
C, BW5147 T lymphocytes were stimulated with 1 mM ATP alone, 10 ?M NAD alone, or 300 ?M ATP in the absence or presence of a pretreatment with
10 ?M NAD for 5 min. These results are representative of observations from three experiments.
Comparative effects of NAD on the potentiation of ATP-induced P2X7R activation in primary macrophages, a macrophage cell line, and
588 NAD, ART2, AND P2X7RECEPTORS IN MACROPHAGES AND LYMPHOCYTES
highly localized accumulation of ATP for autocrine activation of
P2X7R within diffusion-restricted cell surface compartments (12–
14); 2) allosteric modulation of ATP affinity via conformational
changes in P2X7trimeric channels produced by local biophysical
conditions or covalent modification of the P2X7R protein itself
(15, 16, 18, 19, 55, 56); and 3) the ATP-independent activation of
P2X7R via conformational changes produced by ADP-ribosylation
of key arginines within the extracellular loop of the P2X7R (20, 21,
57). The experiments described in this report provide new insights
into the latter two regulatory mechanisms and additionally suggest
that a fourth mechanism, one involving tissue/cell-selective ex-
pression of accessory molecules and/or of P2X7R splice variants,
contributes to the regulation of P2X7R function.
The ability of NAD to drive the covalent modification of extra-
cellular residues of the P2X7R comprises a novel mechanism to
produce relatively long-lasting changes in the conformational state
of these ligand-gated ion channels during transient increases in
extracellular NAD, ATP, and other normally intracellular metab-
olites, such as lysolipids, that can regulate P2X7R function (22, 57,
58). Previous studies demonstrated that NAD can trigger P2X7R
activation in murine T lymphocytes even when these cells were
incubated in the presence of apyrase to scavenge any released ATP
(20). Moreover, P2X7R activation was sustained in T cells briefly
treated with NAD and then washed free of this nucleotide. In con-
trast, ATP-stimulated P2X7R rapidly deactivated when T cells
were transferred to ATP-free medium. These findings indicate that
ADP-ribosylation of P2X7R subunits in murine T cells induces a
conformational change sufficient to gate the opening of these tri-
meric channels even in the absence of ATP binding. However, our
studies of P2X7R function in murine macrophages and HEK293
cells indicate that this ATP-independent activation of P2X7R by
ADP-ribosylation is not a general mode of P2X7R regulation but
rather reflects the specialized conditions present in murine T
Notably, although NAD by itself was able to gate P2X7R in
NZW T lymphocytes expressing solely ART2.1, it failed to acti-
vate P2X7R either in murine macrophages that coexpress native
P2X7R and ART2.1 or in HEK293 cells engineered to coexpress
7 were performed but used the NAD analog, ?NAD, 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 ?M ATP alone (added as two sequential 100 ?M pulses), or 100 ?M 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
?M ?NAD for 5 min before stimulation with 0.5 mM ATP alone, 200 ?M ATP alone (added as two sequential 100 ?M pulses), or 100 ?M 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-? (100 U/ml) for 24 h. The primed macrophages were acutely stimulated with 1 mM ATP alone, 100 ?M ?NAD (plus DTT) alone,
or 300 ?M ATP in the absence or presence of a pretreatment with 100 ?M ?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 ?M ATP with and without ?NAD pretreatment. D, RAW264.7
macrophages were transferred to M-CSF-free medium and stimulated with IFN-? (100 U/ml) for 24 h. The primed macrophages were acutely stimulated
with 1 mM ATP alone, 100 ?M ?NAD (plus DTT) alone, or 400 ?M ATP in the absence or presence of a pretreatment with 100 ?M ?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 400 ?M
ATP with and without ?NAD pretreatment.
?-NAD, an NAD analog, desensitizes P2X7R to ATP in macrophages and T lymphocytes. Experiments similar to those described in Fig.
589The Journal of Immunology
murine P2X7R and murine ART2 ectoenzymes. However, NAD
acted synergistically with ATP to regulate P2X7R in both the mac-
rophages and the engineered HEK cells, and this effect of NAD
was strictly dependent on the expression of ART2.1 or ART2.2 in
both cell models. How can these regulatory effects of NAD/ART2
on ATP-dependent P2X7R activation observed in murine macro-
phages and HEK293 cells be reconciled with the robust ATP-in-
dependent activation by NAD/ART2 in murine T cells? Possible
explanations for the difference in P2X7R signaling observed
between myeloid and lymphoid cells are that T cells, but not mac-
rophages or HEK cells, express other regulatory proteins that fa-
cilitate the ATP-independent gating of P2X7R in response to ADP-
ribosylation, or that the local membrane microenvironments
containing P2X7R and ART2 are different in the two cell types.
Another possible explanation might be the expression of different,
recently identified splice variants of rodent P2X7R in T cells vs
macrophages. Indeed, Taylor et al. have recently reported that
P2X7R function is preserved in the T lymphocytes, but not in
macrophages, from one strain of P2X7-null mice that was gener-
ated by lacZ insertion into exon 1 of the p2rx7 gene (59). Deter-
mining whether different murine tissues and cells, particularly he-
matopoietic cell types, express splice variants of the P2X7R with
altered functional responses to ART2-mediated modification is an
important goal for future experiments.
It is important to consider how ADP-ribosylation of key Arg
residues may affect the conformation of these trimeric channels.
Electrophysiological analyses of P2X-family channels at the whole
cell and single-channel levels indicate that at least two, and prob-
ably three, molecules of ATP need to be bound per channel for
optimal gating (9, 60). Moreover, the critical ATP binding sites
appear to be formed at the interfaces between the extracellular
loops of individual subunits, rather than within each subunit loop
as initially hypothesized (61, 62). In this regard, the covalently
associated ADP-R at Arg125of a P2X7subunit may interact with
the key interfacial amino residues that form the ATP-binding site.
However, ADP-R is larger than ATP per se, and it is unclear
whether the P2X7R channel complex can accommodate ADP-ri-
bosylation of all three subunits or whether only one or two sub-
units per channel can be efficiently modified. Differences in the
number of covalently modified subunits per channel, due possibly
to steric hindrance, may underlie the distinctive consequences of
NAD/ART2 action on P2X7R function in macrophages vs T cells.
ADP-ribosylation of these receptors in macrophages may be lim-
ited to only one or two subunits per channel, which is insufficient
for gating but sufficient for positive allosteric action at the remain-
ing interfacial ATP-binding sites. This would be consistent with
the observed increase in potency of ATP at P2X7R in ART2.1-
expressing macrophages (or HEK293 cells) pretreated with NAD.
In contrast, the predominant P2X7R channels in T cells or the
mutant P2X7R-R276K channels in HEK cells may have confor-
mations that accommodate and permit ADP-ribosylation of all
It is currently unclear whether ADP-ribosylation is a common
mechanism for the activation or sensitization of P2X7R signaling
in other tissues or organisms. Notably, the Arg125and Arg133res-
idues are conserved in the human P2X7R (63), but the human
ART2A and ART2B loci are transcriptionally silent pseudogenes
(26, 64). Thus, human T cells and macrophages lack the capacity
for cis-regulation of P2X7R by a coexpressed ecto-ART. However,
human ART1 is constitutively expressed in neutrophils, and this
GPI-anchored enzyme is rapidly shed during neutrophil activation
in response to bacterial infection (65). ART1 is also expressed by
human airway epithelial cells basally and at increased levels in
response to bacterial mediators (66–68). Thus, the P2X7R in hu-
man macrophages and T cells might be trans-regulated by shed
ART1 that accumulates at sites of bacterial infection and neutro-
phil recruitment. Such a mechanism may also be operative in mice
that also express ART1 in other tissues such as cardiac and skeletal
NAD is released to extracellular environments during the early
stage of inflammatory response (57). Besides its ability to trigger
P2X7R-dependent T cell death (20), extracellular NAD has been
reported as an agonist for P2Y11 receptors in human granulocytes
(48). Our study now shows that NAD also increases the sensitivity
of the P2X7R to ATP gating in macrophages. This action of NAD
requires expression of the thiol-sensitive ART2.1 enzymes and re-
duced thiols, such as glutathione and cysteine, that can accumulate
at inflammatory loci due to release from activated macrophages
and the hypoxia that often characterizes such loci (31). We have
found that ART2.1 is widely expressed in other leukocytes, such as
dendritic cells and B lymphocytes (70). Thus, the sensitization of
ATP-dependent P2X7R activation by NAD/ART2.1 may provide
an additional layer of regulatory control in multiple phases of in-
nate and adaptive immunity.
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-? (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 ?M 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
NAD potentiates ATP-induced pore formation in IFN-?-primed murine macrophages. Bac1.2F5 macrophages were suspended in BSS, and
590NAD, ART2, AND P2X7RECEPTORS IN MACROPHAGES AND LYMPHOCYTES
The authors have no financial conflict of interest.
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