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
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Volume 85, April 2009
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Bulanova et al.
Cytokine expression via P2X1and P2X3receptors in BMMCs 11