ATP induces P2X7receptor-independent cytokine and
chemokine expression through P2X1and P2X3receptors in
murine mast cells
Elena Bulanova,*,1,2Vadim Budagian,*,1Zane Orinska,* Friedrich Koch-Nolte,†Friedrich Haag,†
and Silvia Bulfone-Paus*
*Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany; and†Institute of
Immunology, Hamburg University Medical Center Eppendorf, Hamburg, Germany
array of biological responses in many cell types and
tissues, including immune cells. We have demon-
strated that ATP induces purinergic receptor P2X,
ligand-gated ion channel, 7 (P2X7) receptor-medi-
ated membrane permeabilization, apoptosis, and
cytokine expression in murine mast cells (MCs).
Here, we report that MCs deficient in the expres-
sion of the P2X7receptor are resistant to the ATP-
induced membrane permeabilization and apopto-
sis. However, ATP affects the tyrosine phosphory-
lation pattern of P2X7knockout cells, leading to
the activation of ERK1/2. Furthermore, ATP in-
duces expression of several cytokines and chemo-
kines in these cells, including IL-4, IL-6, IFN-?,
TNF-?, RANTES, and MIP-2, at the mRNA level.
In addition, the release of IL-6 and IL-13 to cell-
conditioned medium was confirmed by ELISA. The
ligand selectivity and pharmacological profile indi-
cate the involvement of two P2X family receptors,
P2X1and P2X3. Thus, depending on genetic back-
ground, particular tissue microenvironment, and
ATP concentration, MCs can presumably engage
different P2X receptor subtypes, which may result
in functionally distinct, biological responses to ex-
tracellular nucleotides. This finding highlights a
novel level of complexity in the sophisticated biol-
ogy of MCs and may facilitate the development of
new, therapeutic approaches to modulate MC
activities. J. Leukoc. Biol. 85: 000–000; 2009.
Extracellul ar ATP mediates a diverse
Key Words: apoptosis ? cell permeabilization ? degranulation
Mast cells (MCs) are major effector cells of IgE-mediated
allergic inflammation, playing a pivotal role in immediate
hypersensitivity and chronic allergic reactions that can con-
tribute to asthma, atopic dermatitis, rheumatoid arthritis, and
other allergic diseases . However, the involvement of MCs in
a surprisingly diverse and complex range of immune functions
goes far beyond allergies. This includes the development of
autoimmune disorders and peripheral tolerance and the initi-
ation and maintenance of innate and adaptive immune re-
sponses [2, 3]. They are also implicated in the pathogenesis of
a number of chronic inflammatory diseases in wound healing
and fibrosis .
MCs are located strategically at host/environment interface
sites such as the skin, airways, and gastrointestinal and uro-
genital tracts and are equipped with a large variety of surface
receptors, which may be activated by diverse inflammatory
stimuli. Following activation, MCs produce a plethora of proin-
flammatory mediators and participate in inflammatory reactions
in many organs. Preformed mediators, such as TNF-?, IL-4,
histamin, heparin, serotonin, kinins, and proteases, are re-
leased immediately from cytoplasmic granules upon MC acti-
vation . Newly synthesized mediators include IL-1–8,
TNF-?, IL-12, IL-13, IL-15, and IL-16, chemokines, PGs,
leukotrienes (LTs), and growth and angiogenesis factors, such
as vascular endothelial factor and platelet-derived growth fac-
Despite the well-characterized role of FcεRI in MC activa-
tion , a variety of other factors can activate these cells. These
include complement fragments, lipid mediators, proteases, hor-
mones, neuropeptides, cytokines, chemokines, microbial prod-
ucts, and extracellular nucleotides [1–3, 5, 6]. Extracellular
ATP and other nucleotides are widely recognized as a ubiqui-
tous family of extracellular signaling molecules, triggering
diverse cellular responses in many cell types and tissues,
including the immune system. These effects include platelet
aggregation, smooth muscle contractility, neurotransmission,
vascular tone, mucociliary clearance, mitogenic stimulation, or
induction of cell death (reviewed in refs. [7, 8]). The biological
effects of extracellular nucleotides are mediated by two pri-
mary classes of specific purinoceptors, P1 and P2. The selec-
tivity of each purinoceptor is defined by its sensitivity to
different purinergic ligands. P1 receptors bind adenosine, and
P2 receptors respond to a variety of nucleotides, including
1These authors contributed equally to the work.
2Correspondence: Department of Immunology and Cell Biology, Research
Center Borstel, Parkallee22, D-23845
Received July 31, 2008; revised December 8, 2008; accepted December 24,
Borstel, Germany. E-mail:
0741-5400/09/0085-0001 © Society for Leukocyte Biology
Journal of Leukocyte Biology
Volume 85, April 2009
Uncorrected Version. Published on January 21, 2009 as DOI:10.1189/jlb.0808470
Copyright 2009 by The Society for Leukocyte Biology.
ATP, and are subdivided further according to agonist selectiv-
ity and mechanisms of signal transduction in two subclasses,
the metabotropic G protein-coupled P2Y receptors and the
ionotropic ligand-gated channel P2X receptors [9, 10].
The P2X receptors represent a family of ligand-gated cation
channels and currently include seven subunits [purinergic
receptor P2X, ligand-gated ion channels, 1–7 (P2X1–P2X7)],
which share 36–48% sequence homology. Signal transduction
by ionotrophic P2X receptors occurs through regulation of
intracellular Ca2?levels via the ligand-stimulated increase in
membrane permeability and is dependent on extracellular
Ca2?ions [11, 12]. P2X receptors exhibit abundant distribu-
tion, and functional responses are observed in neurons, glia,
epithelia, endothelia, bone, muscle, and cells of immune and
hemopoietic origin [7, 8]. In particular, the P2X7receptor has
prominent expression in many immune cells, including lym-
phocytes, monocytes, macrophages, bone marrow (BM), MCs,
dendritic cells (DCs), and mesangial and microglial cells .
The P2X7receptor requires millimolar levels of ATP in the
presence of divalent cations to achieve activation, resulting in
the formation of a nonselective, cationic channel with low
affinity for ATP and increased permeability to Ca2?, intracel-
lular depolarization, and equilibration of sodium and potas-
sium gradients [11, 12]. The hallmark of P2X7receptor acti-
vation is the opening of a low selective pore permeable to large,
organic molecules up to 900 Da [11, 12], which may result in
perturbations in ion homeostasis, complete depolarization of
the membrane potential, and ultimately, cell death [8, 13]. The
P2X7receptor mediates a number of biological activities, in-
cluding activation and maturation of T cells , formation of
multinucleated giant cells , killing of invading microorgan-
isms in macrophages [16, 17], activation of various signaling
cascades [18–20], and induction of apoptosis [21, 22].
A number of studies have implicated P2X7in mediating
ATP-induced apoptosis in macrophages, DCs, and mesangial
and microglial cells [18, 21, 23, 24]. We have recently shown
that ATP induces the P2X7-mediated apoptosis in BM-derived
MCs (BMMCs), P815 plasmacytoma, and MC/9 MC lines .
Importantly, in the time lag between the commitment to apo-
ptosis and actual cell death, extracellular ATP stimulated the
phosphorylation of ERK1/2, Jak2, and STAT6 in MCs and
induced expression and release of several proinflammatory
cytokines, such as IL-4, IL-6, IL-13, and TNF-? . In the
present study, we demonstrate that ATP fails to induce apo-
ptosis but preserves the ability to stimulate phosphorylation of
ERK1/2 and induce production of cytokines and chemokines
in BMMCs derived from P2X7knockout (P2X7?/?) mice. The
nucleotide selectivity and pharmacological profile support the
involvement of P2X1and P2X3receptors in mediating the
functional effects of ATP, indicating the functional heteroge-
neity of MC responses to extracellular nucleotides.
MATERIALS AND METHODS
Reagents and antibodies
ATP, ?,?-methyleneadenosine 5?-triphosphate (?,?meATP) agonist of P2X1
and P2X3receptors, 3-O-(4?-benzoyl)-benzoyl-benzoyl-ATP (Bz-ATP) agonist
of the P2X7receptor, and anti-?-actin antibodies were purchased from Sigma-
Aldrich (St. Louis, MO, USA). TNP-ATP antagonist of P2X1and P2X3recep-
tors was from Invitrogen (Groningen, Netherlands). Concentration of IL-6 and
IL-13 in cell supernatants was detected by a standard ELISA procedure using
DuoSet kits from R&D Systems (Wiesbaden, Germany). mAb against murine
CD117 (c-Kit, 2B8) and CD16/32 (Fc? III/II, 2.4G2; all from BD PharMingen,
San Diego, CA, USA) and T1/ST2 (DJ8; Morwell Diagnostics, Zu ¨rich, Swit-
zerland) were used for surface staining and FACS analysis. Anti-P2X3(H-60),
anti-ERK (C-16), and phospho-ERK (pERK; E-4) antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-P2X1, -P2X4, and -P2X7antibodies were obtained by genetic immu-
nization of rats and fusion of spleen cells with Sp2/0 myeloma cells as
described previously . Antibodies were purified from hybridoma superna-
tants and conjugated to Alexa Fluor 488 (Invitrogen) according to the manu-
Cell culture and stimulation
C57BL/6 wild-type (WT) and P2X7?/? mice were provided by C. Gabel
(Pfizer Inc., Ann Arbor, MI, USA) and were backcrossed for ?12 generations
onto C57BL/6 mice, which were bred in a specific pathogen-free facility at the
University Hospital Eppendorf (Hamburg, Germany).
BMMCs were obtained from femoral BM of 6-week-old mice as described
previously , and cultured in IMDM supplemented with 10% FCS (PAA
Laboratories, Coelbe, Germany), 50 ?M ?-ME, 2 mM L-glutamine (Sigma-
Aldrich), 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 ?g/ml
streptomycin, 1 mM sodium pyruvate (all from Invitrogen), and 5 ng/ml each
IL-3 and stem cell factor (R&D Systems). Cells were collected after 4 weeks of
culture. The purity of MCs was assessed by morphological (Toluidine blue and
Giemsa staining) and FACS analysis.
Before stimulation, cells were washed twice with Dulbecco’s PBS. For each
assay, 2 ? 106cells/ml were stimulated with ATP (1–3 mM), Bz-ATP (100–
300 ?M), ?,?meATP (10 ?M), or a combination of ?,?meATP ? TNP-ATP
(30 ?M) for 3 h (for RT-PCR analysis), 18 h (apoptosis), or 48 h (ELISA)
RNA was extracted from cells using Trizol reagent (Invitrogen). cDNA was
synthesized from 5 ?g total RNA using random oligonucleotides and Super-
ScriptIITMkit (Invitrogen). cDNA was amplified by standard PCR procedure as
described previously . For semiquantitative analysis, in addition to 35
cycles, 15 ?l aliquots of the PCR product from 25 and 30 cycles were also
evaluated. Sequences of the primers used are shown in Table 1. All primers
were purchased from Metabion (Planegg-Martinsried, Germany). ?-Actin mes-
sage was used to normalize the cDNA amount to be used. A mock PCR (without
cDNA) was included to exclude contamination in all experiments.
Cell pellets were lysed for 15 min on ice in 1% Nonidet P-40 cell extraction
buffer: 20 mM Tris-HCl, pH 8.0, 15 mM NaCl, 2 mM EDTA, 10 mM sodium
fluoride, 1 ?g/ml pepstatin A, 1 ?g/ml leupeptin, 10 mM PMSF, and 100 ?M
sodium vanadate (all reagents from Sigma-Aldrich). The detergent-insoluble
material was removed by centrifugation at 13,000 rpm for 15 min at 4°C.
Samples were resuspended in SDS-PAGE loading buffer, boiled for 5 min, and
analyzed in 10% SDS-PAGE. The resolved proteins were transferred onto
nitrocellulose (Bio-Rad, Munich, Germany). Blots were blocked for 1 h in PBS
with 0.05% Tween-20 (PBS-T) and 3% BSA (Sigma-Aldrich). After incuba-
tions with first and second antibodies and washing with PBS-T, visualization of
specific proteins was carried out by an ECL method using ECL Western
blotting detection reagents (Amersham Pharmacia, Buckinghamshire, UK)
according to the manufacturer’s instructions.
Cells (1?106) were exposed to 3 mM ATP or 100 ?M Bz-ATP for 18 h at 37°C.
After washing cells with cold PBS, the percentage of apoptotic cells was
evaluated by Annexin V-FITC apoptosis kit (Bender MedSystems, Vienna,
Austria) according to the manufacturer’s protocol. Cell viability was deter-
mined by propidium iodide (PI) exclusion. Cells were analyzed by flow cytom-
etry using FACSCalibur (Becton Dickinson, San Jose, CA, USA) and
2Journal of Leukocyte Biology
Volume 85, April 2009
Flow cytometer analysis
Cells were washed twice in FACS buffer (2% newborn calf serum, 0.1% NaN3,
2 mM EDTA in PBS) and stained with anti-CD117, T1/ST2, and CD16/32
antibodies labeled with PE or FITC. Surface detection of FcεRI was done as
described . In brief, cells were preincubated first with purified mouse IgE
(C38-2), then washed twice, and incubated with biotinylated anti-IgE (R35-
118; both from BD PharMingen). This was followed by incubation with strepta-
vidin labeled with PE (Dianova, Hamburg, Germany). Isotype-matched, un-
specific antibodies (BD PharMingen) were used as controls. Samples were
analyzed using a FACSCalibur. For surface staining, gates on viable cells were
set according to exclusion of PI staining.
Changes in plasma membrane permeability
ATP-dependent increase in the plasma membrane permeability was measured
using the extracellular fluorescent tracer Lucifer yellow (Molecular Probes,
Leiden, Netherlands) as described earlier . For Lucifer yellow uptake, cells
were incubated for 15 min at 37°C in PBS containing 250 ?M sulfinpyrazone
(Sigma-Aldrich) and 1 mg/ml Lucifer yellow and stimulated with 3 mM ATP.
After several washing with PBS to remove the extracellular dye, cells were
analyzed with an inverted fluorescence microscope (Diaphot 300, Nikon,
Tokyo, Japan) using a 40? objective and a fluorescein filter.
Cells (2?106) were preincubated with 2 ?M membrane-permeable Fura-2/
acetoxymethyl ester (Molecular Probes) for 30 min at 37°C, washed twice with
PBS, and Ca2?influx was measured in a polarized total internal reflection
fluorescence (PTI-RF-M2001) spectrofluorimeter (Photon Technology Interna-
tional, Wedel, Germany) using 340/380 excitation filters and emission at 510
nm. After recording of background for 20 s, cells were stimulated with 3 mM
ATP. Maximal and minimal fluorescence ratios were determined by lysing the
cells in 0.05% reduced Triton X-100 and subsequent addition of EGTA at a
final concentration of 10 mM (both reagents are from Sigma-Aldrich). Concen-
tration of Ca2?was calculated using the equation of Grynkiewicz et al. .
Protease activity assays
Activity of proteases was measured using the EnzCheck protease assay kit
(Invitrogen). Briefly, 2 ? 106cells were lysed in 200 ?l lysis buffer (150 mM
NaCl, 50 mM Tris-HCl, 1 mM MgCl2, 1 mM CaCl2, 2 ?M ZnCl2, pH 7.4) on
ice for 30 min, centrifuged, and incubated with 10 ?g/ml BODIPY FL casein
solution at 37°C for 1 h. Fluorescence was measured at 485 nm (excitation)/
530 nm (emission) using the Infinite M200 microplate reader (Tecan, Salzburg,
Austria). Trypsin (Sigma-Aldrich; 5 ?g/ml) was used as a positive control, and
lysis buffer was used as a negative control.
MC degranulation was quantified by measuring ?-hexosaminidase in superna-
tants of BMMCs as described . Briefly, BMMCs were loaded with 5 ?g/ml
DNP-specific IgE (SPE-1; Sigma-Aldrich) for 30 min, washed, and resus-
pended in 10 mM HEPES, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2,
1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA (Tyrode’s buffer). Degranulation
was induced by stimulation with DNP-human serum albumin (HSA). After 20
min, samples were centrifuged, supernatants were collected, and cell pellets
were solubilized with 0.5% Triton X-100 in Tyrode’s buffer. Enzymatic activ-
ities of ?-hexosaminidase were assessed after incubation for 40 min at 37°C
with p-nitrophenyl-N-acetyl-?-D-glucosaminide (Sigma-Aldrich) in 0.1 M so-
dium citrate (pH 4.5). Production of p-nitrophenol was stopped by addition of
0.2 M glycine (pH 10.7) and quantified by absorbance measurement at 405 nm.
The extent of degranulation was calculated by dividing p-nitrophenol absor-
bance in the supernatants by the sum of absorbance in the supernatant and cell
All experiments were performed in at least four independent assays, which
yielded highly comparable results. Data are summarized as mean ? SD.
Statistical analysis of the results was performed by Student’s t-test for unpaired
samples. A P value of ?0.05 was considered statistically significant.
Phenotypic and functional characterization
Expression of c-Kit (CD117), FcεRI, CD16/32, and T1/ST2
(MC-specific markers) was analyzed in WT and P2X7?/?
TABLE 1. Sequences of Primers
Name of primerSequenceSize, bp
Bulanova et al.
Cytokine expression via P2X1and P2X3receptors in BMMCs3
BMMCs after 4 weeks of culture (Fig. 1A). T1/ST2/IL-1R4,
known previously as an orphaned IL-1 family receptor, is now
recognized as a receptor for IL-33, highly expressed on MCs
. As shown in Figure 1A, WT and P2X7?/? BMMCs
develop normally, yielding more than 98% of c-Kit?, FceRI?,
and T1/ST2?cells. Both cell types also highly express the
CD16/32 receptor, and P2X7?/? cells lack expression of the
P2X7receptor. Furthermore, P2X7?/? cells were morpholog-
ically indistinguishable from WT BMMCs according to Giemsa
staining (data not shown). In addition, the histochemical hall-
marks of WT and P2X7?/? BMMCs were comparable after
staining of dense MC metachromatic granules by Toluidine
blue or intracellular proteoglycans by alcian blue/safranin
(data not shown). Moreover, quantification of cell numbers by
FACS failed to reveal any significant differences between
peritoneal MC numbers from WT and P2X7?/? mice (data not
Degranulation of WT and P2X7?/? BMMCs in response to
IgE and antigen (DNP) stimulation exhibited no difference, as
assessed by ?-hexosaminidase release (Fig. 1B, upper panel),
indicating that the absence of P2X7receptor expression does
not affect IgE-mediated MC degranulation. However, extracel-
lular ATP-induced degranulation  was dramatically lower
(P?0.05) in P2X7?/? BMMCs, even in response to high ATP
concentrations (Fig. 1B, lower panel). Finally, we examined
whether deletion of the P2X7?/? receptor affects the ability of
BMMCs to produce, store, and release MC-specific proteases.
As shown in Figure 1C, WT and P2X7?/? BMMCs exhibited
similar, total protease activity, as assessed by measuring flu-
orescent casein degradation by MC lysates. Taken together,
these findings indicate that the absence of the P2X7?/?
receptor leaves key MC features unaltered, not affecting the
morphology, distribution, numbers, key surface marker expres-
sion, and selected activities of BMMCs.
ATP does not induce membrane
permeabilization, Ca2?influx, and apoptosis in
Recent study from our laboratory demonstrated that ATP in
millimolar range is capable of inducing the P2X7-dependent
Ca2?influx, permeabilization of the cell membrane, and apo-
ptosis in murine MCs, and these effects are inhibited by a
selective P2X7receptor antagonist KN-62 or oATP . To
investigate whether these effects are mediated exclusively by
the P2X7receptor, WT and P2X7?/? BMMCs were treated
with various concentrations of ATP (1–3 mM) and Ca2?influx;
membrane permeabilization and apoptosis were measured. In
agreement with earlier findings, concentrations of ATP in the
range of 1–3 mM rendered WT BMMCs permeable for Lucifer
yellow (Fig. 2A). This fluorescent dye exhibited diffuse dis-
tribution in the cytoplasm of ATP-treated BMMCs. A molecu-
lar weight limitation for the permeabilization was confirmed by
phase-contrast microscopy analysis, which demonstrated that
MCs did not become permeable to trypan blue (MW 961 Da)
after ATP stimulation (data not shown). Furthermore, ATP also
triggered an increase in intracellular Ca2?levels (Fig. 2B).
Fig. 1. Characterization of P2X7receptor-
deficient BMMCs, which (A) were stained
for P2X7, CD117 (c-Kit), FcεRI, T1/ST2,
and CD16/32. Percentage of FcεRI?/
positive cells is shown. Staining with iso-
type-matched antibodies was used as a neg-
ative control (open histograms). (B) IgE
(upper panel)- and ATP (lower panel)-de-
pendent degranulation of BMMCs. Degran-
ulation was analyzed as percentage of
?-hexosaminidase release from BMMCs.
Cells were preloaded with DNP-specific IgE
(5 ?g/ml) for 30 min and stimulated with
specific antigen DNP-HSA (20 ng/ml; 20
min). Unstimulated (medium) or preloaded
but not DNP-HSA-stimulated cells served
as negative controls. For ATP-induced de-
granulation, cells were incubated for 1 h
with 1 or 5 mM ATP. One representative
experiment of three is shown. (C) Protease
activity of BMMCs was measured as fluo-
rescence intensity 24 h after degradation of
fluorescent casein by cell lysates. Results
represent mean ? SD of four independent
experiments (*, P?0.05, vs. control).
4Journal of Leukocyte Biology
Volume 85, April 2009
Conversely, membrane permeabilization and Ca2?influx were
undetectable in BMMCs from P2X7?/? animals (Fig. 2, A and
B), thereby indicating an absolute requirement of the P2X7
receptor for mediating these effects. It should, however, be
noted that ATP was able to induce a brief spike in intracellular
Ca2?concentration in P2X7?/? BMMCs within the first sec-
onds of action, which returned rapidly to near basal levels (Fig.
2B). Given that KN-62 was not able to abrogate this effect (data
not shown), this fact supports the involvement of other purino-
A major effect of extracellular ATP is the ability to induce
apoptosis in different cell types [20, 21, 32]. Recent data from
our laboratory demonstrated that millimolar concentrations of
ATP induced P2X7-mediated apoptosis in murine BMMCs as
well as MC/9 and P815 MC lines . Therefore, we proposed
that P2X7?/? BMMCs should be resistant to the cytotoxic
action of ATP, as compared with WT cells. To prove this
hypothesis, WT and P2X7?/? BMMCs were treated with
extracellular ATP. After 18 h, cells were analyzed by the
presence of apoptotic cells. These experiments showed that
ATP at millimolar concentrations induced apoptosis in ?40%
of WT BMMCs, whereas P2X7?/? BMMCs remained viable
Given that Bz-ATP is a more potent agonist for the P2X7
receptor than ATP and can induce the P2X7-mediated re-
sponses at lower concentrations [8, 18], we also stimulated
cells with this chemical compound. Indeed, Bz-ATP (100 ?M)
was almost as equipotent as ATP in inducing apoptosis of WT
but not P2X7?/? BMMCs (Fig. 2C). Notably, this agent failed
to trigger apoptosis at concentrations below 10 ?M (data not
Fig. 2. ATP induces plasma membrane
permeabilization, Ca2?influx, and apopto-
sis in WT but not in P2X7?/? BMMCs. (A)
Cells were incubated in PBS containing 1
mg/ml Lucifer yellow and 250 ?M sulfin-
pyrazone in the absence of stimuli or in the
presence of 3 mM ATP. After 15 min, the
cells were washed twice with serum-con-
taining culture medium and photographed
with ?40 objective. (B) Ca2?influx in MCs
stimulated with ATP (3 mM) was measured
as described in Materials and Methods. (C)
BMMCs were incubated with ATP (3 mM) or
Bz-ATP (100 ?M) for 18 h at 37°C. Per-
centage of apoptotic cells was analyzed by
Annexin V-FITC/PI staining and FACS.
Percentage of living cells is shown in the
bottom left quadrant, early apoptotic cells in
bottom right, and late apoptotic in the top
right quadrant. The data shown are repre-
sentative of three independent experiments
with similar results.
Bulanova et al.
Cytokine expression via P2X1and P2X3receptors in BMMCs5
shown). The inability of ATP and Bz-ATP to induce cell death
in BMMCs from P2X7?/? animals indicates that deletion of
the P2X7receptor renders these MCs resistant to the cytotoxic
action of ATP.
ATP induces the phosphorylation of ERK in
BMMCs from P2X7?/? mice
Recent data from our laboratory indicate that ATP induces
phopshorylation of several intracellular proteins including
ERK1/2 in murine BMMCs and the MC/9 MC line  There-
fore, our next goal was to determine whether ATP is still able
to induce activation of intracellular signaling molecules in
P2X7-deficient BMMCs. To this end, WT and P2X7?/? BM-
MCs were pulse-stimulated with extracellular ATP for short
time-periods (5 and 15 min). Given that extracellular ATP is
rapidly degraded by ubiquitous ecto-ATPases/ectonucleoti-
dases, such pulses presumably represent a more “physiologic”
stimuli, limiting the exposure of MCs to high ATP concentra-
tion. Analysis of tyrosine phosphorylation by Western blotting
showed that ATP preserved the ability to mediate weak phos-
phorylation of several intracellular substrates in P2X7?/?
BMMCs, whereas KN-62 was not able to abrogate this effect
(data not shown). Probing the membranes with phosphospecific
antibodies allowed us to identify among the phosphorylated
proteins ERK1 (p44) and ERK2 (p42; Fig. 3A). However, the
phosphorylation of ERK1/2 in the P2X7-deficient cells was
weaker as compared with WT cells and was most prominent
after 5 min of ATP stimulation, returning to near-basal level
after 15 min. Taken together, these results indicate that extra-
cellular ATP can induce P2X7receptor-independent activation
Extracellular ATP induces expression of
cytokines and chemokines in P2X7?/? BMMCs
Many key inflammatory mediators, including cytokines and
chemokines, are synthesized de novo by MCs in response to
stimulation with a variety of stimuli [1–5]. Given the ability of
ATP to induce ERK1/2 phosphorylation in P2X7?/? BMMCs,
we tested whether the expression of selected cytokines and
chemokines is dependent on the P2X7receptor expression in
MCs. To this end, semiquantitative RT-PCR analysis of various
cytokine and chemokine expressions (IL-4, IL-6, IL-13,
TNF-?, IFN-?, RANTES, and MIP-2) in response to ATP
stimulation was performed. Figure 3B shows that ATP in
millimolar range was a potent stimulus for the expression of
IL-4, IL-6, IFN-?, and TNF-?, as well as RANTES and MIP-2.
ATP was more potent in inducing expression of these mediators
in WT BMMCs, whereas P2X7?/? cells exhibited weaker
up-regulation of all tested cytokines and chemokines (Fig. 3B).
Notwithstanding, message for IL-13 was up-regulated only in
response to 3 mM ATP in both cell types, whereas TNF-?
expression was also prominent in control cells (Fig. 3B). Re-
markably, low ATP concentrations (10–500 ?M) were ineffec-
tive in inducing cytokine and chemokine expressions in WT
and P2X7?/? BMMCs (Fig. 3C). As a next step, we investi-
gated whether WT or P2X7-deficient BMMCs release selected
cytokines to the culture medium in response to the stimulation
with ATP. To this end, WT and P2X7?/? BMMCs were
Fig. 3. ATP induces ERK1/2 phosphoryla-
tion, cytokine, and chemokine expression in
MCs. (A) Cells were stimulated with 3 mM ATP
for 5 or 15 min and lysed, and total protein
anti-phosphotyrosine or anti-pERK antibodies.
To prove that the equal amount of proteins was
loaded in each sample, the blots were stripped
MCs were incubated with indicated concentra-
tions of ATP for 3 h. Total RNA was extracted
from cells, reverse-transcribed, and subjected to
semiquantitative PCR amplification using spe-
cific primers for IL-4, IL-6, IL-13, RANTES,
IFN-?, TNF-?, and MIP-2, as described in Ma-
terials and Methods. The picture shows the am-
plified bands after 35 cycles. The amount of
cDNA was equalized by PCR amplification of
?-actin. A mock PCR (no cDNA) was included
as a negative control. The data represent three
separate experiments with comparable results.
trations of ATP for 3 h, and RNA was extracted
ers for IL-6, IL-13, MIP-2, and ?-actin (for nor-
malization of DNA amount). The picture shows
were treated with indicated concentrations of
ATP. Supernatants were collected after 48 h
and analyzed for IL-6 and IL-13 release by
ELISA. Results represent mean ? SD of five
independent experiments (*, P?0.05, vs.
control and **, P?0.01, vs. control).
6 Journal of Leukocyte Biology
Volume 85, April 2009
stimulated, and the cell supernatants were tested by ELISA
after 48 h, as our previous experiments demonstrated that
cytokine release from WT BMMCs in response to ATP was
rather low after 24 h . These experiments showed the
ability of ATP to up-regulate the secretion of IL-6 and IL-13 in
both cell types. As shown in Figure 3C, ATP in concentration
of 1 and 3 mM induced the secretion of both cytokines from
WT BMMCs (400–650 pg/ml and 250–440 pg/ml IL-6 and
IL-13, respectively). Importantly, these two cytokines were also
secreted, although at lower levels, from P2X7?/? BMMCs
(240–250 and 200–250 pg/ml IL-6 and IL-13, respectively;
P?0.05) upon stimulation with extracellular ATP. Conversely,
no changes in the release of IL-4, TNF-?, IFN-?, RANTES, or
MIP-2 were observed (data not shown). Taken together, these
data indicate that BMMCs, which are deficient in the expres-
sion of the P2X7receptor, express and release a number of
mediators in response to ATP stimulation, thereby suggesting
that other receptors of the P2X family can mediate these
BMMCs express P2X1and P2X3receptors
Given that other P2X receptors may mediate ATP-induced
effects in P2X7?/?BMMCs, we tested by RT-PCR a panel of
mouse P2X subunits using specific primers. These experiments
revealed that P2X7?/? and WT BMMCs express transcripts
coding for several members of the P2X family, including P2X1,
P2X3, P2X4, and P2X6(Fig. 4A), whereas WT BMMCs also
expressed the P2X7subunit but not P2X5receptors .
P2X7?/? BMMCs lack expression of P2X7and P2X5recep-
tors at the mRNA level (data not shown). To prove the expres-
sion of respective proteins, we performed flow cytometry anal-
ysis, which confirmed the presence of the P2X1receptor on the
cell membrane of P2X7?/? and WT BMMCs (Fig. 4B). Con-
versely, flow cytometry failed to detect the membrane-associ-
ated P2X4receptor despite the presence of mRNA for this
protein. Anti-P2X4antibodies detected the expression of the
P2X4receptor on the surface of mouse DCs, which were used
as a positive control (data not shown). The lack of the P2X4
receptor was also confirmed by Western blotting analysis (data
not shown). These data essentially rule out the involvement of
the P2X4receptor in the mediating, ATP-induced effects in
BMMCs from P2X7-deficient animals. The expression of the
P2X3receptor was corroborated further by Western blotting
analysis using antibodies directed against this receptor, which
confirmed the presence of respective protein in the cell lysates
from P2X7?/? and WT BMMCs (Fig. 4C). Thus, BMMCs
generated from WT and P2X7-deficient mice express several
P2X receptor subtypes, which might be responsible for the
expression of cytokines and chemokines in response to stimu-
lation with extracellular ATP.
WT and P2X7?/? BMMCs express cytokines
and chemokines in response to P2X1and P2X3
Given the absence of mRNA for the P2X5receptor and the
inability of antibodies targeting the P2X4receptor to detect this
subunit on the plasma membrane, we focused on P2X1and
P2X3receptors as most appropriate candidates for mediating
the ATP-induced effects in P2X7?/? BMMCs. Homomeric
P2X1and P2X3receptors are readily distinguishable by their
rapid desensitization, the agonistic action of ?,?meATP, and
the antagonistic activity of TNP-ATP . First, we assessed
the ability of ?,?meATP to induce Ca2?influx in WT and
P2X7-deficient BMMCs. Figure 5A illustrates that this chem-
ical agent induces rapid Ca2?influx, which is most prominent
Fig. 4. Expression of purinoceptors of P2X subtypes in murine MCs. (A)
RT-PCR analysis of P2X1–P2X4and P2X6receptors expression in BMMCs.
Total RNA was extracted from cells, and RT-PCR was performed as described
in Materials and Methods. The amplified products were electrophoresed on
1.5% agarose gel. RNAs derived from BMMCs of at least six mice were tested
separately: #1 or #2 represents the highest or the lowest level of expression for
each purinoceptor, respectively. The amount of cDNA analyzed was similar in
different samples, as shown by PCR amplification of ?-actin cDNA. A mock
PCR (without cDNA) was included to exclude contamination. (B) BMMCs were
stained for P2X1and P2X4receptors. Staining with isotype-matched antibodies
was used as a negative control (white histograms). (C) Expression of P2X3
protein in murine MCs was analyzed by Western blotting in cell lysates using
specific anti-P2X3antibodies. Expression of ?-actin on the same blot was used
as a control for loading.
Bulanova et al.
Cytokine expression via P2X1and P2X3receptors in BMMCs7
within seconds of ?,?meATP action but shows quick desensi-
tization. Given the rapidly desensitization properties of P2X1
and P2X3channels, a brief increase in Ca2?influx in response
to ?,?meATP is presumably not enough to affect intracellular
Ca2?levels substantially. Blockade of ?,?meATP by pretreat-
ment of BMMCs with TNP-ATP essentially abrogated Ca2?
influx, indicating that this effect is sensitive to its inhibitory
Next, the influence of ?,?meATP and TNP-ATP treatment
on expression and secretion of selected cytokines and chemo-
kines by WT and P2X7-deficient BMMCs was analyzed. Cells
were assayed for the expression of mRNA for IL-6, IL-13, and
RANTES by semiquantitative RT-PCR. In fact, stimulation
with ?,?meATP induced a considerable increase in the level of
IL-6 and IL-13 mRNA, and this effect was inhibited signifi-
cantly by the pretreatment of cells with TNP-ATP (Fig. 5B).
?,?meATP was less potent in inducing up-regulation of RAN-
TES, presumably as a result of the fact that the cells already
expressed the message for this chemokine at a rather high
level. Finally, we evaluated the release of IL-6 and IL-13 from
WT and P2X7?/? BMMCs in response to ?,?meATP stimu-
lation. As shown in Figure 5C, stimulation with ?,?meATP was
able to enhance secretion of IL-6 and IL-13 (320–350 pg/ml
and 290–320 pg/ml; P?0.05), whereas pretreatment with
TNP-ATP almost fully abrogated this effect. The effect of
TNP-ATP was not caused by cellular damage or cytotoxicity, as
PI or trypan blue staining (indicators of cell death) was not
different from that of untreated cells (not shown). In summary,
these experiments show that in the absence of the P2X7re-
ceptor, ATP-induced release of selected mediators from MCs
occurs through other P2X receptor subunits. The nucleotide
selectivity, pharmacological profile, and desensitization prop-
erties strongly suggest that the ATP-induced effects are medi-
ated by P2X1and P2X3receptors.
In this study, we demonstrate a functional dichotomy of ATP-
mediated responses in normal MCs versus cells deficient in the
expression of a biologically active P2X7receptor. Despite the
fact that the ATP-induced cell membrane permeabilization and
apoptosis were inhibited by a specific antagonist of the P2X7
receptor KN-62 , decisive, experimental evidence for the
major role of P2X7in mediating ATP cytotoxicity was provided
by studies using BMMCs generated from animals bearing a
homozygous disruption of the P2X7-encoding gene. However,
although the P2X7receptor is critical for conferring sensitivity
to apoptosis, other P2X receptor subtypes mediate the release
of several proinflamatory mediators in response to ATP stim-
ulation. These facts highlight the functional heterogeneity of
MC responses to extracellular nucleotides, supporting the idea
that a diverse array of P2X purinoceptors expressed by MCs—
key protagonists of innate immunity—can be selectively acti-
vated according to particular requirements in a given tissue
In a previous report, we provided several lines of experi-
mental evidence supporting the role for the P2X7receptor as
the mediator of the ATP-induced responses in murine BMMCs
and two MC lines, MC/9 and P815 . The current study
Fig. 5. Specific stimulation of P2X1and P2X3receptors
leads to Ca2?influx and cytokine expression in WT and
P2X7?/? BMMCs. (A) Ca2?influx in MCs stimulated with
?,?meATP (10 ?M) or pretreated with 30 ?M TNP-ATP for
30 min prior to ?,?meATP stimulation was measured as
described in Materials and Methods. The data shown are
representative of three independent experiments with simi-
lar results. (B) Cells were stimulated with ?,?meATP or with
?,?meATP in the presence of TNP-ATP for 3 h. Total RNA
was extracted from cells, reverse-transcribed, and subjected to PCR amplification using primers for IL-6, IL-13, and RANTES as described in Materials and
Methods. The picture of semiquantitative analysis shows the amplified bands after 35 cycles. The amount of cDNA was equalized by PCR amplification of ?-actin.
A mock PCR (no cDNA) was included as a negative control. (C) BMMCs were treated with ?,?meATP or TNP-ATP ? ?,?meATP, and the supernatants were
collected after 48 h and analyzed for IL-6 and IL-13 by ELISA. Results represent mean ? SD of four independent experiments (*, P?0.05).
8 Journal of Leukocyte Biology
Volume 85, April 2009
extends our initial findings by showing that BMMCs generated
from the P2X7-deficient mice are resistant to the ATP-induced
membrane permeabilization and apoptosis yet respond by sig-
nificant increase in the expression of several cytokines and
chemokines to ATP stimulation. ATP also affected the pattern
of tyrosine phosphorylation in these cells, leading, e.g., to the
activation of the ERK pathway, as demonstrated by the phos-
phorylation of ERK1/2. The activation of ERKs might explain,
at least partially, the observed increase in the expression of
proinflammatory mediators. The nucleotide selectivity, phar-
macological profile, and desensitization data suggest that the
ATP-mediated cytokine and chemokine expression involves
the activity of P2X1and P2X3receptors. The assessment of
functionality and discrimination between P2X receptors criti-
cally depends on the use of receptor agonists and antagonists
and the desensitization properties, which define a particular
pharmacological profile for a distinct receptor subtype. This
approach allowed identification of rapidly desensitizing and
?,?meATP-sensitive receptors, P2X1and P2X3. Homomeric
P2X1and P2X3receptors are highly sensitive to stimulating
action of ?,?meATP (EC50?1 ?M), and agonist application
induces rapid desensitization of the current through these
receptors [11, 12], which is in accord with our findings.
Conversely, P2X2, P2X4, P2X5, P2X6, and P2X7receptors
are much less sensitive to ?,?meATP. In particular, P2X2
receptors are not activated by ?,?meATP, at least at concen-
trations up to 300 ?M, and P2X4–7also respond to rather high
(?100 ?M) doses of this chemical compound . Further-
more, these receptors are sensitive to inhibitory action of
suramin and pyridoxalphosphate-6-azophenyl-2?,4?-disulfonic
acid but not TNP-ATP . In addition, P2X4and P2X6
exhibit a moderate speed of desensitization, whereas P2X2,
P2X5, and P2X7are nondesensitizing . Of note, there are
no agonists or antagonists that selectively recognize the homo-
meric P2X2receptor, which renders linking of this receptor to
a particular biological response a difficult task. On the other
hand, TNP-ATP is ?1000-fold more effective by blocking
ATP-induced currents at P2X1receptors (50% IC50?1 nM)
than at P2X2, P2X4, and P2X7. Although Bz-ATP is active
at P2X1and P2X3receptors, exhibiting 60% activity when
compared with ATP, it was also reported to have high potency
at all P2X receptors , which invalidated its use for dis-
crimination between different receptor subtypes. The fact that
ATP and ?,?meATP induce cytokine and chemokine expres-
sion and brief Ca2?influx in the P2X7-deficient BMMCs,
whereas TNP-ATP abrogates these effects, taken together with
the receptor desensitization properties, strongly supports the
involvement of P2X1and P2X3receptors in mediating func-
tional effects of ATP in the P2X7?/? MCs.
Another important feature is the ability of P2X1–P2X6re-
ceptors to associate with each other, assembling into the ATP-
activated ion channels as homomers or heteromers (reviewed in
ref. ). Heteromeric receptors include P2X2/3, P2X1/5, and
P2X4/6. For example, a distinct class of receptors is formed by
the coexpression of P2X2and P2X3subunits; these hetero-
meric channels are activated by ?,?meATP, blocked by TNP-
ATP, but exhibit little or no desensitization. Given the rapid
desensitization channel properties in response to ?,?meATP,
the involvement of heteromeric P2X2/3receptors in the ATP-
mediated cytokine and chemokine expression in BMMCs ap-
pears unlikely, although cannot be ruled out completely.
Considering that many physiologic or pathologic conditions
may result in ATP release into an extracellular environment,
this chemical agent represents a good candidate to the role of
nonimmune activation stimulus for MCs. Reportedly, extracel-
lular ATP induces pore formation in the plasma membrane of
rat peritoneal MCs , triggers release of hexosaminidase, an
elevation in Ca2?level and protein tyrosine phosphorylation in
the MC/9 MC line , and stimulates release of histamine and
LT from murine BMMCs [31, 35]. However, ATP was only able
to modulate anti-IgE-induced release of histamine in human
lung MCs , which indicates heterogeneity of MCs from
different sources. Recent findings from our laboratory indicate
that ATP induces a number of the P2X7-mediated cell re-
sponses in murine MCs, including the phosphorylation of sig-
naling molecules, expression of several proinflammatory cyto-
kines, membrane permeabilization, and induction of caspase-
dependent apoptosis . Conversely, the absence of a
functional P2X7receptor fully abrogated MC membrane per-
meabilization to low MW hydrophilic solutes, such as Lucifer
yellow, and substantially affected the kinetics and duration of
Ca2?influx across the cellular membrane, leading to quick
desensitization of the channel. However, ATP still induced,
although at lower levels, the phosphorylation of ERK1/2 and
expression of transcripts for several important, proinflamma-
tory mediators, including IL-4, IL-6, IFN-?, TNF-?, RANTES,
and MIP-2. The up-regulation of IL-6 and IL-13 expression
was corroborated further by ELISA analysis, which demon-
strated the presence of IL-6 and IL-13 in the cell-conditioned
medium from ATP-stimulated, P2X7-deficient BMMCs. The
ATP effects were mimicked by low concentrations of P2X1and
P2X3agonist ??meATP and inhibited by pretreatment of the
cells by the antagonist of P2X1and P2X3, TNP-ATP. Never-
theless, the P2X1- and P2X3-induced expression and secretion
of proinflamatory mediators (e.g., IL-6 and IL-13) were lower in
P2X7-deficient BMMCs as compared with WT MCs. Given that
the increase in the cytokine expression might take place pre-
dominantly in the apoptosis-resistant MCs, the release of cy-
tokines normalized on a per-cell basis from the viable WT cells
would be even higher (relative to the receptor-deficient cells).
This fact suggests that the P2X7receptor is particularly im-
portant in MC biology and essential for mediating full-range
cellular effects in response to extracellular ATP. In addition,
this experimental evidence supports the idea that depending on
a particular tissue microenvironment and ATP concentration,
MCs may respond in a restricted way and engage different P2X
receptor subtypes (i.e., P2X7- vs. P2X1- and P2X3-dependent),
which could result in a release of a limited subset of mediators
and affect the amount and/or kinetics of the production of
MC-specific bioactive substances.
Thus, the complexity of the functional responsiveness of
MCs to ATP seems to be determined at least on two distinct
levels. High millimolar ATP concentrations presumably trigger
activation of the low-affinity P2X7receptor and P2X1and P2X3
receptors, whereas in the absence of P2X7, only P2X1and
P2X3receptors are able to respond to ATP stimulation. The
inability of ATP to induce P2X1and P2X3 receptor activation
at low concentrations (below 1 mM) is intriguing and might be
Bulanova et al.
Cytokine expression via P2X1and P2X3receptors in BMMCs9
explained by the fact that BMMCs exhibit high activity of the
ATP-hydrolyzing enzyme(s) (such as CD39) on the cell surface
(E. Bulanova, unpublished observation). In addition, the num-
ber and sensitivity of P2X1and P2X3receptors on MCs derived
from the P2X7?/? animals may adjust accordingly in an
attempt to compensate the absence of the P2X7receptor, which
appears to be the primary mediator of the ATP-induced re-
sponses in these cells. However, the existence of such com-
pensatory mechanism(s) in P2X7?/? cells should be con-
firmed experimentally, and further studies are required to
clarify this issue.
Another essential question is whether such high ATP levels
could be achieved and maintained in the extracellular space.
Experimental evidence supports the idea that concentrations of
ATP in the protected compartments at the level of the cell
membrane could easily reach amounts sufficient to activate the
low-affinity P2X7receptor . Furthermore, many cell types
including MCs release ATP: In fact, ATP released from one
MC was shown to diffuse several tens of micrometers and elicit
rises in intracellular Ca2?in the neighboring cells, thereby
indicating an existence of an autocrine/paracrine mechanism of
ATP-dependent cell activation . As ATP release might be
able to alter function of the same or nearby cells by an
autocrine/paracrine mechanism, proinflammatory cytokine se-
cretion by MCs in response to ATP may thus lead to autocrine
activation of MCs and paracrine activation of neighboring T
and B lymphocytes, monocytes/macrophages, and DCs.
An emerging concept implicates MCs as repair cells, which
provide antithrombotic and/or profibrinolytic mediators to pre-
vent thrombus formation or help to dissolve thrombotic mate-
rial in the course of the vascular repair process [38, 39]. Given
that MCs act as fibrinolytic, their apoptosis at initial stages of
acute vascular injury in response to high extracellular ATP
concentrations, which may be achieved as a result of its release
from damaged blood cells, endothelial cells, and activated
platelets, seems to be physiologically relevant, preventing their
function as antithrombotic and/or profibrinolytic cells. Note-
worthy, only 40–45% of BMMCs were apoptotic in response to
millimolar ATP concentrations, indicating different suscepti-
bility to the ATP-induced cytotoxicity. This fact indicates
heterogeneity of the MC population, which may reflect varia-
tions in the amount and/or kinetics of activation of functional
P2X receptors upon the cell surface of BMMCs, thereby con-
ferring distinct, functional responses to ATP.
In summary, our results indicate that stimulation with ex-
tracellular ATP involves complex responses in murine MCs
through activation of different P2X receptor subtypes. Al-
though P2X7-dependent apoptosis appears to represent a major
functional outcome of ATP action, other P2X receptors are also
activated and may potentially govern fine-tuned MC activities
in a specific tissue microenvironment and/or at different ATP
levels. Given unique properties of MCs as potential linkers
between innate and acquired immunity, the regulation of their
cytokine and chemokine expression by P2X1and P2X3recep-
tors highlights novel approaches for the development of new
therapeutic strategies to modulate MC functions in local im-
We are grateful to Martina Hein and Kathleen Wilke for
excellent technical assistance, to Katrin Westfall for genera-
tion of BMMCs, and to Florian Schiemann for the help with
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Cytokine expression via P2X1and P2X3receptors in BMMCs 11