Adenine Nucleotides Inhibit Cytokine
Generation by Human Mast Cells through a
Gs-Coupled Receptor
This information is current as
of June 18, 2017.
Chunli Feng, Amin G. Mery, Elizabeth M. Beller, Christa
Favot and Joshua A. Boyce
J Immunol 2004; 173:7539-7547; ;
doi: 10.4049/jimmunol.173.12.7539
http://www.jimmunol.org/content/173/12/7539
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Copyright © 2004 by The American Association of
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References
The Journal of Immunology
Adenine Nucleotides Inhibit Cytokine Generation by Human
Mast Cells through a Gs-Coupled Receptor1
Chunli Feng,2‡ Amin G. Mery,2*‡ Elizabeth M. Beller,‡ Christa Favot,‡ and
Joshua A. Boyce3*†‡§
N
ucleotides, the substrates for nucleic acid synthesis, are
also ubiquitous extracellular mediators. ATP is stored in
high (micromolar) concentrations in the secretory granules of platelets, neurons, and some leukocytes (reviewed in Ref.
1) and is released in a regulated fashion upon activation of these
cells. In addition, high concentrations of cytosolic nucleotides are
released into the extracellular space in response to cell death, hypoxia, trauma, and infection. Extracellular nucleotides initiate or
modulate cellular responses in multiple organ systems by binding
and activating specific cell surface receptors, collectively termed
purinergic (P2)4 receptors (reviewed in Ref. 2). Nucleotides are
released by endothelial cells, and P2 receptors are essential for
Departments of *Medicine and †Pediatrics, Harvard Medical School; ‡Division of
Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital; and
§
Partners Asthma Center, Boston, MA 02115
Received for publication June 17, 2004. Accepted for publication October 4, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI48802, AI52353,
AI31599, and HL36110 and by grants from the Charles Dana Foundation, the Vinik
Family Fund for Research in Allergic Diseases, and the Hyde and Watson
Foundation.
2
C.F. and A.G.M. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Joshua A. Boyce, One Jimmy
Fund Way, Smith Building Room 626, Boston, MA 02115. E-mail address:
[email protected]
4
Abbreviations used in this paper: P2, purinergic; A3P5P, adenosine 3⬘-phosphate
5⬘-phosphate; ARL67156, 6-N,N-diethyl-D-␤,␥-dibromomethylene 5⬘-triphosphate;
ATP␥S, adenosine 5⬘-O-(3-thiotriphosphate); CREM, cAMP response element modulator protein; cys-LT, cysteinyl leukotriene; GPCR, G protein-coupled receptor;
␤-hex, ␤-hexosaminidase; hMC, human MC; ICER, inducible cAMP early repressor;
LT, leukotriene; LTC4S, leukotriene C4 synthase; MC, mast cell; 2-MesAMP,
2-methylthioadenosine 5⬘-monophosphate; PGN, peptidoglycan; PKA, protein kinase
A; PTX, pertussis toxin; SCF, stem cell factor; TTBS, 1⫻ TBS with 0.5% Tween 20.
Copyright © 2004 by The American Association of Immunologists, Inc.
platelet aggregation and play a role in vascular disease. Two distinct families of P2 receptors exist: P2X receptors are subunits of
multimeric, ligand-gated ion channels that bind ATP (3). P2Y receptors are seven-transmembrane-spanning, G protein-coupled receptors (GPCRs). Both P2 receptor families are expressed on
smooth muscle, neuronal tissues, and hemopoietic cells (4). P2Y
receptors exhibit clear-cut differential specificity for different nucleotides. The P2Y2 receptor binds both ATP and UTP, and the
human P2Y4 receptor prefers UTP, whereas the P2Y11 receptor
preferentially binds ATP. P2Y1, P2Y12, and P2Y13 (5) receptors
all preferentially bind ADP, whereas the P2Y6 receptor exhibits a
binding preference for UDP (reviewed in Refs. 1 and 2). The
P2Y14 receptor is specific for UDP-glucose (6). Not only is the
number of known P2 receptors large, but single-cell types commonly express more than one receptor specific for the same nucleotide (7, 8), suggesting nonredundant, complementary functions
for these receptors. For example, coexpression of ADP-specific
P2Y1 and P2Y12 receptors by platelets is essential for normal
aggregation responses to both exogenous and endogenous ADP in
vitro and in vivo (9 –13). The expression of several P2Y receptors
by leukocytes (4) suggests likely functions in immunity and
inflammation.
Among immune effector cells, mast cells (MCs) are uniquely
situated around blood vessels and nerves throughout the body, especially at interfaces with the external environment. Whereas their
effector role in IgE-dependent allergic and immune responses is
well understood, accumulating evidence supports their involvement in a broad range of nonallergic inflammatory diseases and
indicates a key role for these cells in innate antimicrobial responses (14, 15). MCs abound in atherosclerotic plaques and show
morphological evidence of activation in such lesions (16). Moreover,
cardiovascular events such as myocardial ischemia are associated
0022-1767/04/$02.00
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ATP and ADP activate functionally distinct G protein-coupled purinergic (P2Y) receptors. We determined the expression and
function of adenine nucleotide-specific P2Y receptors on cord blood-derived human mast cells (hMCs). Human MCs expressed
mRNA encoding the ADP-specific P2Y1, P2Y12, and P2Y13 receptors; the ATP/UTP-specific P2Y2 receptor; and the ATPselective P2Y11 receptor. ADP (0.05–50 ␮M) induced calcium flux that was completely blocked by a P2Y1 receptor-selective
antagonist and was not cross-desensitized by ATP. Low doses of ADP induced strong phosphorylation of ERK and p38 MAPKs;
higher doses stimulated eicosanoid production and exocytosis. Although MAPK phosphorylation was blocked by a combination
of P2Y1- and P2Y12-selective antagonists, neither interfered with secretion responses. Unexpectedly, both ADP and ATP inhibited
the generation of TNF-␣ in response to the TLR2 ligand, peptidoglycan, and blocked the production of TNF-␣, IL-8, and MIP-1␤
in response to leukotriene D4. These effects were mimicked by two ATP analogues, adenosine 5ⴕ-O-(3-thiotriphosphate) and
2ⴕ,3ⴕ-O-(4-benzoyl-benzoyl) adenosine 5ⴕ-triphosphate (BzATP), but not by adenosine. ADP, ATP, adenosine 5ⴕ-O-(3-thiotriphosphate), and 2ⴕ,3ⴕ-O-(4-benzoyl-benzoyl) adenosine 5ⴕ-triphosphate each induced cAMP accumulation, stimulated the phosphorylation of CREB, and up-regulated the expression of inducible cAMP early repressor, a CREB-dependent inhibitor of cytokine
transcription. Human MCs thus express several ADP-selective P2Y receptors and at least one Gs-coupled ADP/ATP receptor.
Nucleotides could therefore contribute to MC-dependent microvascular leakage in atherosclerosis, tissue injury, and innate immunity while simultaneously limiting the extent of subsequent inflammation by attenuating the generation of inducible cytokines
by MCs. The Journal of Immunology, 2004, 173: 7539 –7547.
7540
Materials and Methods
Nucleotides, analogues, and antagonists
The following reagents were purchased from Sigma-Aldrich (St. Louis,
MO): ATP, ADP, UTP, and UDP; and the ATP analogues adenosine 5⬘O-(3-thiotriphosphate) (ATP␥S), 2⬘,3⬘-O-(4-benzoyl-benzoyl) adenosine 5⬘triphosphate (BzATP), and dATP; the P2Y1-selective antagonist adenosine
3⬘-phosphate 5⬘-phosphate (A3P5P); the P2Y12-selective antagonist 2-methylthioadenosine 5⬘-monophosphate (2-MesAMP); and the nonselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid.
Derivation of hMCs
Human MCs were derived from cord blood mononuclear cells cultured in
the presence of stem cell factor (SCF; 100 ng/ml), IL-6 (50 ng/ml), and
IL-10 (10 ng/ml; all from R&D Systems, Minneapolis, MN), as previously
described (31). Cells were harvested for studies when ⬎95% stained metachromatically with toluidine blue dye (typically 6 –9 wk). To optimize
conditions for Fc⑀RI-dependent exocytosis, cytokine generation, and eicosanoid production, hMCs were transferred to new medium containing SCF
and IL-4 (10 ng/ml; R&D Systems) for 5 days at 37°C and 5% CO2 before
activation (32, 33).
RT-PCR
To determine the steady state expression by cultured hMCs of mRNAs
encoding P2Y receptors, total RNA was extracted from hMCs at 6 – 8 wk
of culture with Tri-Reagent (Molecular Research, Cincinnati, OH). For
RT-PCR, RNA samples were primed with oligo(dT) and reverse transcribed according to the manufacturer’s protocol in an RT kit (BD Clontech, Palo Alto, CA). The following primers were designed to amplify
coding sequences of the indicated receptors: P2Y1 sense strand, 5⬘-AT
GACCGAGGTGCTGTGGCC-3⬘; P2Y1 antisense strand, 5⬘-AGAATG
GAGATACAAGCCTGTGA-3⬘; P2Y12 sense strand, 5⬘-CCGTCGAC
AATCTCACC-3⬘; P2Y12 antisense strand, 5⬘-GCCCAGATGACAA
CAGAG-3⬘; P2Y13 sense strand, 5⬘-GGTGTTTGTTCACATCCCCAG3⬘; P2Y13 antisense strand, 5⬘-CTTTAAGGAAGCACACTTTTTCAC-3⬘;
P2Y2 sense strand, 5⬘-ATGGCAGCAGACCTGGGCCCCTGG-3⬘; P2Y2
antisense strand, 5⬘-CTACAGCCGAATGTCCTTAGTGTTC-3⬘; P2Y11
sense strand, 5⬘-CTCGGGTGCCAAGTCCTGCCC-3⬘; P2Y11 antisense
strand, 5⬘-CAGTCATGCCTGGGCTGCGTAG-3⬘; P2X1 sense strand, 5⬘CAAGAACAGCATCAGCTTTCCACG-3⬘; P2X1 antisense strand, 5⬘CCTGTTGACCTTGAAGCGTGGAAA-3⬘; P2X4 sense strand, 5⬘-GGT
TAAGAACAACATCTGGTATCC-3⬘; P2X4 antisense strand, 5⬘-GGGA
TACCAGATGTTGTTCTTAACC-3⬘; P2X5 sense strand, 5⬘-GTTACCAA
GACGTCGACACCTC-3⬘; P2X5 antisense strand, 5⬘-CCCCTGGCCAAG
TTCTCTC-3⬘; P2X7 sense strand, 5⬘-AGGGGAACTCTTTCTTCGT
GAT-3⬘; and P2X7 antisense strand, 5⬘-CAATGCCCATTATTCCGCCCTG3⬘. All these primers readily amplified bands from genomic DNA that were
consistent with the sizes of the respective target sequences. PCR was performed with 0.4 U of Taq polymerase (PerkinElmer, Foster City, CA) for
35 cycles in a PerkinElmer Thermal Cycler with the following parameters:
94°C for 1 min, 94°C for 30 s, 50°C (or, in the case of the P2X primers,
55°C) for 2 min, and 72°C for 4 min. Specific primers for human GAPDH
(BD Clontech) were run as positive controls. The PCR products were resolved on ethidium bromide-stained gels containing 0.5% agarose/0.5%
Synergel (Diversified Biotech, Boston, MA).
Calcium mobilization
To measure changes in the cytosolic concentration of free calcium, hMCs
(0.5–1 ⫻ 106 cells/sample) were washed and resuspended in HBSS containing 1 mM CaCl2, 1 mM MgCl2, and 0.1% BSA. The cells were labeled
with fura 2-AM (Molecular Probes, Eugene, OR) for 30 min at 37°C,
washed, and resuspended in the same HBSS buffer. Some cell samples
were pretreated with A3P5P (0.5–100 ␮M), 2-MesAMP (0.5–100 ␮M), or
6-N,N-diethyl-D-␤,␥-dibromomethylene 5⬘-triphosphate (ARL67156; 50
␮M; Sigma-Aldrich), an inhibitor of ectonucleotidase activity (34), before
the agonists were added. Other cell samples were stimulated after incubation for 4 h with pertussis toxin (PTX; 100 ng/ml; Sigma-Aldrich) to interfere with Gi/Go protein signaling. The hMCs were stimulated with various concentrations of ATP, ADP, UTP, adenosine, or the analogues of
ATP (35). The changes in intracellular calcium were measured using excitation at 340 and 380 nm in a fluorescence spectrophotometer (F-4500;
Hitachi, Hialeah, FL). The relative ratios of fluorescence emitted at 510 nm
were recorded and displayed as a reflection of intracellular concentrations
of calcium.
Cell activation for secretion
Human MCs primed with IL-4 were passively sensitized with human myeloma IgE (Chemicon International, Temecula, CA; 2 ␮g/ml) overnight on
the fourth day of priming to saturate Fc⑀RI. On the following day, the cells
were washed and resuspended in fresh medium at a concentration of 1 ⫻
106/ml, except in the case of samples used for analysis of exocytosis, which
were suspended at a 10-fold higher cell density. For exocytosis, samples of
2 ⫻ 106 hMCs were challenged with ADP or ATP at the concentrations
eliciting maximal calcium fluxes (5 ␮M each). Replicate cell samples were
stimulated with a rabbit anti-human IgE polyclonal Ab (Calbiochem, San
Diego, CA; 1 ␮g/ml) or with staphylococcal PGN (10 ␮g/ml; SigmaAldrich) with or without the addition of ADP, ATP, or ATP analogues.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
with elevations in plasma levels of histamine (17) and tryptase
(18), two products of MC secretory granules that are released on
cell activation. Activated MCs are also abundant sources of leukotrienes (LTs), which are products of the 5-lipoxygenase pathway. These smooth muscle-active mediators have an accepted role
in asthma and have recently been implicated in the pathophysiology of atherosclerosis (19). Although the mechanisms responsible
for MC activation in atherosclerosis are not known, MCs express
a range of activating receptors for small soluble mediators as well
as Ig and microbial constituents (20 –24). Human MCs (hMCs)
from a variety of sources express several mRNAs that encode P2X
receptor subtypes (25). Rat peritoneal MCs release histamine and
generate PGD2 when stimulated with ATP (26); this response is
blocked by antagonists of both P2X and P2Y receptors. ATP and
UTP (1–100 ␮M) each enhance exocytosis of histamine by human
lung MCs stimulated by cross-linkage of Fc⑀RI (27), an effect
attributed to the P2Y2 receptor. ATP is also stored in MC granules,
and its release by a rat MC line activated by Fc⑀RI cross-linkage
resulted in propagation and radial spread of calcium fluxes in adjacent cells, which were blocked by a global P2 receptor antagonist, suramin (28). Taken together, these studies support the concepts that MCs express more than one P2 receptor type and that
ATP and/or its metabolites modulate MC activation through both
autocrine and paracrine pathways. These possibilities have potential relevance for multiple disease processes.
Extracellular ATP in vivo is probably maintained in equilibrium
with ADP through ectonucleotidases and nucleoside diphosphokinases (29). The capacity for ATP to modulate MC activation, and
the existence of P2Y receptor subtypes with distinct preferences for
ATP or ADP, prompted us to study whether cultured cord bloodderived hMCs express ADP-selective P2Y receptors and whether
these receptors modulate the activation of hMCs. The results of our
studies indicate that hMCs express mRNAs for the ADP-preferring
P2Y1, P2Y12, and P2Y13 receptors as well as the P2X1 and P2X4
receptors and demonstrate functionality of the corresponding proteins
by characteristic biochemical signatures. The P2Y1 receptor mediates
calcium flux by hMCs in response to ADP (but not ATP) and synergizes with P2Y12 receptors to mediate phosphorylation of ERK and
p38 MAPKs. Despite these signatures, ADP and ATP both fail to
induce cytokine generation de novo, even at high (100 ␮M) doses.
Instead, ADP, ATP, and a panel of ATP analogues (but not adenosine) interfere with TNF-␣ production by hMCs activated by peptidoglycan (PGN), a component of Staphylococcus aureus cell walls
that is an agonist of TLR2, and block LTD4-induced production of a
panel of cytokines. ADP, ATP, and its analogues all induce accumulation of cAMP in hMCs and up-regulate the expression of inducible
cAMP early repressor (ICER), a repressor of NF-AT and AP-1-mediated transcription (30). The presence on MCs of excitatory receptors
for adenine nucleotides may facilitate the acute tissue swelling response to nucleotides in atherosclerosis, tissue injury, and innate immunity, whereas the presence of at least one Gs-coupled inhibitory
receptor for ATP and ADP may limit consequent inflammation in
such circumstances by interfering with inducible cytokine gene
expression.
ADENINE NUCLEOTIDE RECEPTORS AND MAST CELLS
The Journal of Immunology
cAMP measurements
Primed hMCs were stimulated with various nucleotides for 10 min in triplicate samples of 2 ⫻ 105 cells. The stimulations were performed in medium, except for experiments in which the effect of extracellular calcium
was determined. In these experiments, cells were stimulated in HBSS either with or without calcium and magnesium. In some experiments cells
were pretreated with indomethacin (1 ␮M), a dose that completely inhibited PGD2 production, to rule out a contribution from endogenous prostanoids to cAMP accumulation. The reactions were stopped by incubating
the samples on ice. Cells were lysed with a lysis buffer provided in a
commercial Biotrack cAMP ELISA kit (Amersham Biosciences), which
was used to measure cAMP according to the manufacturer’s protocol. In
some experiments the cells were pretreated for 4 –24 h with PTX, or A3P5P
was added immediately before stimulation. The cAMP values for each
condition were normalized to the values for the unstimulated cells and are
expressed as a percentage of the control value.
MAPK phosphorylation
Samples of 2 ⫻ 105 hMCs were stimulated for various periods with ADP
(5 ␮M) in the presence or the absence of A3P5P, 2-MesAMP, or both (50
␮M each). The reactions were stopped by incubating the samples on ice at
5 min (the time at which maximal phosphorylation of ERK was observed),
and the cells were lysed in a buffer containing 1% SDS, 1% Triton X-100,
1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 5 ␮g/ml leupeptin, and 1
␮g/ml pepstatin in 10 mM Tris (pH 8.0). The samples were loaded with an
equal volume of Tris-glycine-SDS loading buffer (NOVEX, San Diego,
CA) onto precast 12% Tris-glycine-SDS gels, and the proteins were separated by electrophoresis at 30 mA and 150 mV. After overnight transfer
to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA), the blots
were blocked for 1 h in 1⫻ TBS with 0.5% Tween 20 (Bio-Rad; TTBS)
containing 3% nonfat dry milk and 0.25% normal goat serum and were
incubated in the same buffer containing 1/2000 dilutions of anti-Active
ERK, c-JNK, and p38 MAPK Abs (Promega, Madison, WI) for 2 h at room
temperature. After extensive washing in TTBS, the blots were incubated
again for 1 h in blocking buffer with a 1/5000 dilution of a goat anti-rabbit
HRP-conjugated secondary Ab (Bio-Rad). The blots were washed again in
TTBS, and bands were detected with ECL. The same blots were stripped
and probed again with rabbit polyclonal Abs that detect total ERK, p38,
and JNK at dilutions of 1/2000 (Cell Signaling Technologies, Beverley,
MA). Maximal phosphorylation of ERK was observed at 5 min; thus, this
period was chosen as the harvest time for subsequent experiments.
Immunoblotting for phosphorylation of CREB and induced
expression of ICER
Primed hMCs were stimulated for 30 min to 3 h with various concentrations of nucleotides and were lysed as described above. In some experiments the cells were pretreated with H89 (10 ␮M) for 1 h. Samples of 3 ⫻
105 cells were loaded onto precast gels and subjected to electrophoresis and
transblotting. The blots were incubated with rabbit polyclonal Abs specific
for total CREB and for CREB phosphorylated at serine 133 (both from Cell
Signaling Technologies) at dilutions of 1/500 and with a rabbit polyclonal
antiserum that recognizes all forms of cAMP response element modulator
(CREM), including ICER (provided by Dr. C. Molina, University of New
Jersey Medical School, Newark, NJ) at a concentration of 1/1000 (30). The
20-kDa band corresponding to ICER was visualized with ECL.
Real-time PCR
Expression of TNF-␣ and MIP-1␤ mRNA was determined using real-time
PCR performed on ABI PRISM 7700 Sequence Detection System (Applied
Biosystems, Foster City, CA). Samples of 5–10 ⫻ 106 primed, sensitized
hMCs were stimulated for 2 h with anti-IgE, PGN, or medium alone in the
presence of SCF at a constant concentration of 100 ng/ml. RNA was prepared using an RNeasy Mini kit (Qiagen, Valencia, CA) and was treated
with RNase-free DNase (Qiagen, Valencia, CA) according to the manufacturer’s protocols. RT was performed on samples of 2 ␮g of DNasetreated RNA using TaqMan RT reagents (Applied Biosystems). Primers
and minor groove binder-labeled probes for the amplification of TNF-␣
and human ␤2-microglobulin and the corresponding VIC dye were purchased from Applied Biosystems.
Results
Identification and pharmacologic characterization of ADP and
ATP-binding P2Y receptors on hMCs
To determine whether hMCs expressed functional P2Y receptors
for ADP and ATP, fura 2-loaded hMCs were stimulated with various concentrations of these nucleotides and monitored for changes
in absorbance at 380 and 510 nm. ADP strongly induced dosedependent increases in intracellular calcium, with maximal responses at concentrations between 0.5 and 5 ␮M in most experiments (n ⫽ 4; as shown for a single donor; Fig. 1A), nearing a
plateau at 5 ␮M. ATP (5 ␮M; Fig. 1B) and UTP (not shown) also
induced a calcium flux of a consistently smaller magnitude than
that observed in response to the same dose of ADP. BzATP, dATP,
ATP␥S, adenosine, and AMP failed to cause calcium fluxes at
concentrations up to 100 ␮M (n ⫽ 2; not shown). Priming of the
hMCs for 5 days with IL-4 (10 ng/ml) in the continued presence of
SCF did not alter the magnitude of the ADP- or ATP-induced
calcium fluxes and did not change the dose-response characteristics (not shown). Neither the magnitude nor the duration of the
ADP-induced calcium fluxes were altered by the addition of
ARL67156 or by PTX pretreatment (n ⫽ 2 for each; as shown for
individual experiments; Fig. 1B). ATP and ADP (5 ␮M each) did
not cross-desensitize each other’s calcium fluxes (Fig. 1C). ADPinduced (but not ATP-induced) calcium flux was blocked completely by the P2Y1-selective antagonist, A3P5P, at 50 ␮M and
was unaltered by 2-MesAMP (Fig. 1B). RT-PCR revealed the
presence of transcripts encoding the ADP-selective P2Y1, P2Y12,
and P2Y13 receptors as well as the ATP-preferring P2Y2 and
P2Y11 receptors (n ⫽ 3; as shown for one experiment; Fig. 1D).
Transcripts encoding the P2X1 and P2X4 receptors were also
readily detected (n ⫽ 3; as shown for one experiment; Fig. 1D),
whereas those encoding P2X5 and P2X7 receptors were either
only weakly detectable or undetectable in the same samples. The
profile of detected transcripts was not altered by DNase treatment
of the RNA before reverse transcription (n ⫽ 2; data not shown).
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Throughout the activation process, SCF was maintained at a constant concentration of 100 ng/ml to ensure optimal cell viability. Activation was
stopped by incubating the samples on ice, and the supernatants were separated from the cell pellets by centrifugation at 200 ⫻ g in an Eppendorf
microcentrifuge at 4°C. After lysis of the cell pellet by resuspension in
medium and sonication, the content of ␤-hexosaminidase (␤-hex) was measured by spectrophotometric analysis of the hydrolysis of p-nitrophenyl␤-D-2-acetamido-2-deoxyglucopyranoside as described previously (36).
The percent release values were calculated according to the formula [S/S ⫹
P] ⫻ 100, where S and P refer to the absorbance detected in equal volumes
of supernatant and pellet, respectively, after subtraction of the background
absorbance of medium alone. Nonspecific release from replicate samples
treated with medium alone was subtracted, and the data were expressed as
the mean net release from duplicate readings.
For eicosanoid production, samples of 1 ⫻ 105 hMCs were stimulated
in triplicate with various doses of ADP and ATP (0.05–50 ␮M), with and
without anti-IgE (1 ␮g/ml), in the wells of 96-well, flat-bottom plates.
Supernatants were collected at 30 min and stored at ⫺20°C until further
analysis. Specific ELISAs were used to detect LTC4, LTD4, LTE4 (Cayman Chemical, Ann Arbor, MI), and PGD2 (Amersham Biosciences, Arlington Heights, IL), respectively. For measurements of cytokine generation, triplicate samples of 1 ⫻ 105 passively sensitized, primed hMCs were
challenged with PGN (10 ␮g/ml; Sigma-Aldrich), anti-IgE (1 ␮g/ml),
LTD4 (100 nM; Cayman), or medium alone in the presence or the absence
of ADP, ATP, ATP␥S, BzATP, or adenosine at various concentrations in
the wells of 96-well, flat-bottom plates. The agonist doses were optimal for
cytokine generation by IL-4-primed hMCs and were chosen on the basis of
preliminary dose-response experiments as well as previously published
data (32, 37). In some experiments the cells were pretreated for 30 min
with H89 (10 ␮M; Sigma-Aldrich), a selective inhibitor of protein kinase
A (PKA). At 6 h, supernatants were harvested and frozen at ⫺70°C until
further analysis. Specific ELISAs were used to measure the concentrations
of TNF-␣, IL-8, and IL-5 (all from eBiosciences, San Diego, CA) and
MIP-1␤ (Endogen, Woburn, MA), according to the directions provided by
the manufacturers. The data for each experiment were tabulated as the
mean values from triplicate samples. The cytokine quantities in the samples
stimulated with medium without agonists were subtracted from the corresponding quantities in the samples stimulated with agonists to obtain the
net quantity produced for each cytokine.
7541
7542
ADENINE NUCLEOTIDE RECEPTORS AND MAST CELLS
Stimulation of hMCs with ADP (5 ␮M) for 5 min strongly induced the phosphorylation of ERK MAPK and more weakly elicited p38 MAPK phosphorylation (Fig. 2). A3P5P and 2-MesAMP
(100 ␮M each) each attenuated ADP-induced ERK phosphorylation when used alone, and the combination of the two inhibitors
was additive, while also decreasing p38 phosphorylation (n ⫽ 2; as
shown for one experiment; Fig. 2). JNK phosphorylation was not
detected under these conditions (n ⫽ 2; not shown).
Secretion responses to stimulation with ADP and ATP
IL-4-primed hMCs were stimulated with various concentrations of
ADP or ATP, both with and without Fc⑀RI cross-linkage. The
content of ␤-hex was measured in both the supernatants and pellets, and these values were used to calculate the percent release.
Supernatants were also assayed for the presence of cysteinyl LTs
(cys-LTs) and PGD2 as indices of arachidonic acid metabolism.
Neither ADP nor ATP directly induced exocytosis at 0.05–5 ␮M,
whereas both weakly induced exocytosis at 50 ␮M (10 ⫾ 4 and
7 ⫾ 4% net ␤-hex release (n ⫽ 4) vs 19 ⫾ 2% net release in
FIGURE 2. Phosphorylation of ERK and p38 MAPKs. IL-4-primed
hMCs were stimulated for 5 min with ADP (5 ␮M) in the presence of the
indicated inhibitors or with a buffer control. Whole-cell lysates were resolved by SDS-PAGE, and Western blotting was performed with rabbit
polyclonal Abs that recognize the phosphorylated forms of ERK (P-ERK)
and p38 (P-p38). The blot was then stripped and reprobed with Abs that
recognize total ERK and total p38. The data presented are from a single
experiment that is representative of two performed.
response to Fc⑀RI cross-linkage; not shown). Spontaneous release
was low (generally ⬍5%). Both ADP and ATP amplified Fc⑀RIinduced net ␤-hex release by an additional ⬃5% (⬃25% increase
over anti-IgE alone) when added as a second stimulus (n ⫽ 3; not
shown). At 50 ␮M ADP or ATP, primed hMCs generated small
quantities of PGD2 (4.6 ⫾ 4 and 9 ⫾ 2 ng/106 hMCs in response
to ADP and ATP vs 59 ⫾ 41 ng/106 hMCs in response to Fc⑀RI
cross-linkage; n ⫽ 3; data not shown) and cys-LTs (466 ⫾ 172 and
334 ⫾ 150 pg/106 hMCs in response to ADP and ATP vs 18 ⫾ 4
ng/106 hMCs in response to Fc⑀RI cross-linkage; n ⫽ 3; data not
shown). Stimulation with ATP␥S, BzATP, and dATP (50 ␮M
each) also elicited small amounts of PGD2 and cys-LT generation,
which approached 25–70% of that induced by ADP (n ⫽ 2; data
not shown). PPADs (100 ␮M) decreased cys-LT generation in
response to both ADP (98 ⫾ 1% inhibition) and ATP (70 ⫾ 11%),
whereas A3P5P and 2-MeSAMP had no effect (data not shown).
Effect of nucleotides on cytokine generation
To determine whether adenine nucleotides would induce the generation of cytokines, primed hMCs were stimulated with various
concentrations of ADP or ATP in the presence of SCF to maintain
optimal viability. As positive control stimuli, other cell samples
were activated with Fc⑀RI cross-linkage, staphylococcal PGN (10
␮g/ml), or LTD4 (100 nM) in some experiments. Some of these
samples were treated concomitantly with various concentrations of
ADP or ATP. Supernatants were harvested at 6 h for measurement
of cytokines. Neither ATP nor ADP used alone elicited TNF-␣,
IL-8, or MIP-1␤ generation at doses up to 100 ␮M (n ⫽ 6; data not
shown). Fc⑀RI cross-linkage, PGN, and LTD4 each induced the
generation of these cytokines by IL-4-primed hMCs. When added
as a concomitant stimulus, neither ADP nor ATP significantly altered Fc⑀RI-induced generation of TNF-␣ (Fig. 3B), IL-8, or
MIP-1␤ (not shown), although there was a trend toward inhibition
at higher nucleotide doses. However, both nucleotides significantly
decreased the production of TNF-␣ in response to PGN (Fig. 3A)
and LTD4 (B) in a dose-dependent fashion, decreased IL-5 production in a similar dose-dependent fashion (n ⫽ 2; not shown),
and at 5 ␮M also inhibited the LTD4-mediated generation of IL-8
and MIP-1␤ (Fig. 3D). The inhibitory effects of ADP and ATP on
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FIGURE 1. Functional characterization
of receptors for adenine nucleotides on
hMCs. A, Calcium flux induced by stimulation of fura 2-loaded hMCs with various
doses of ADP. B, Effect of inhibitors of
ectonucleotidase (ARL67156), Gi/Go signaling (PTX), P2Y12 receptors (2MesAMP), and P2Y1 receptors (A3P5P)
on calcium fluxes induced by stimulation
of fura 2-loaded hMCs with ADP (1 ␮M).
The responses to ATP (5 ␮M) were not
inhibited by either 2-MesAMP or A3P5P.
C, Cross-desensitization experiment between ATP and ADP, added in both forward and reverse orders. D, Ethidium bromide-stained agarose gel of DNA
fragments amplified by 35 cycles of PCR
from DNase-treated, reverse transcribed
RNA from hMCs showing bands that correspond to the steady state mRNA encoding the indicated P2Y and P2X receptors.
The m.w. markers are indicated on the left.
Data depicted in A–D are from single experiments representative of at least two
performed.
The Journal of Immunology
7543
PGN-induced TNF-␣ generation were mimicked by both ATP␥S
and BzATP at the same dosing ranges (64 ⫾ 25 and 56 ⫾ 6%
inhibition, respectively, at 50 ␮M; mean ⫾ half-range; n ⫽ 2).
Real-time PCR analysis revealed that both ADP and ATP␥S at 50
␮M significantly attenuated the steady state expression of TNF-␣
mRNA induced by PGN (n ⫽ 3; Fig. 4A) and also decreased
LTD4-induced TNF-␣ mRNA expression by ⬎50% (n ⫽ 2; Fig.
4B). In the cases of all agonists used, the inhibitory effects of ADP
and ATP were not reversed by the presence of H89 and were not
blocked by PPADs, A3P5P, or 2-MesAMP, alone or in combina-
FIGURE 4. Real-time PCR analysis showing the effects of ADP and
ATP␥S on the steady state levels of TNF-␣ mRNA expressed by hMCs
stimulated with PGN (A; 10 ␮g/ml) and LTD4 (B; 100 nM). IL-4-primed
hMCs were stimulated for 2 h in the absence or the presence of each
nucleotide at the indicated concentrations. The level of TNF-␣ mRNA is
normalized to the internal control gene, ␤2-microglobulin, and is expressed
as a percentage of the expression by the cells not treated with nucleotides.
The results for PGN-stimulated samples are the mean ⫾ SEM from three
experiments, whereas those for LTD4 are the mean ⫾ half-range from two
experiments. ⴱ, Statistical significance (p ⬍ 0.05).
tion. Adenosine did not affect cytokine generation at concentrations up to 100 ␮M (n ⫽ 2; data not shown).
Effect of adenine nucleotides on adenylyl cyclase-dependent
signaling pathways
To determine whether adenine nucleotides exerted their inhibitory
effects through receptors that stimulate adenylyl cyclase, cAMP
was measured in hMCs stimulated for 10 min with various concentrations of these nucleotides. ADP and ATP (0.5–100 ␮M)
were equipotent for increasing intracellular levels of cAMP in a
dose-dependent fashion (n ⫽ 4; Fig. 5A). This effect was slightly
potentiated by overnight pretreatment of the cells with PTX (n ⫽
2; data not shown). A3P5P did not affect ADP-induced cAMP
accumulation, whereas 2-MesAMP cross-reacted with AMP in the
detection assay and could not be studied (not shown). BzATP,
ATP␥S, and dATP also strongly stimulated cAMP accumulation,
each being more potent than ADP and ATP at equivalent concentrations (n ⫽ 2; Fig. 5A). UDP did not affect cAMP accumulation
(n ⫽ 1; data not shown). Neither pretreatment with indomethacin
nor depletion of extracellular calcium affected cAMP accumulation in response to ADP, ATP, ATP␥S, and BzATP (50 ␮M each;
n ⫽ 2; data not shown). Stimulation with ATP␥S transiently induced the phosphorylation of CREB and up-regulated the expression of ICER in a time- and dose-dependent fashion (n ⫽ 3; as
shown for one experiment; Fig. 5B). These effects were mimicked
by ADP (n ⫽ 3; as shown for one experiment; Fig. 5B) and by
ATP and BzATP at a concentration of 50 ␮M each (n ⫽ 1 for
each; data not shown). Pretreatment of the hMCs with H89 attenuated CREB phosphorylation at 30 min and the up-regulation of
ICER at 30 min and 2 h, although it did not prevent substantial
up-regulation of ICER at 3 h (n ⫽ 3; shown for a single experiment
in Fig. 5B). Preincubation of hMCs with ADP or ATP␥S (50 ␮M)
for various time periods to up-regulate ICER expression before
activation did not further inhibit PGN-induced TNF-␣ generation,
but tended to enhance inhibition of Fc⑀RI-dependent TNF-␣ generation relative to the addition of nucleotides concomitantly with
anti-IgE (n ⫽ 2; Fig. 5C). Preincubation with nucleotides did not
inhibit Fc⑀RI-dependent exocytosis (n ⫽ 2; not shown).
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FIGURE 3. Effect of adenine nucleotides on cytokine generation. IL-4-primed,
sensitized hMCs were stimulated with PGN
(10 ␮g/ml; A), anti-IgE (1 ␮g/ml; B), or a
buffer control in the presence of the indicated nucleotide concentrations. Supernatants were harvested 6 h after stimulation,
and the presence of TNF-␣ was assessed by
ELISA. Data for PGN are the mean ⫾ SEM
from four experiments, whereas the data for
anti-IgE are the mean ⫾ SEM from three
experiments. ⴱ, Significant inhibition (p ⱕ
0.05). Adenosine at 0.05–50 ␮M had no effect (n ⫽ 2; not shown). C, Effects of various concentrations of ADP on TNF-␣ production by hMCs in response to LTD4 (100
nM). Data are the mean ⫾ half-range from
two experiments. D, Effect of ADP (5 ␮M)
on the production of TNF-␣, IL-8, and
MIP-1␤ by primed hMCs in response to
LTD4. Data displayed are the mean ⫾ SEM
from three experiments.
7544
ADENINE NUCLEOTIDE RECEPTORS AND MAST CELLS
Discussion
Nucleotides are probably among the first and most abundant soluble mediators to be released in tissue injury, infection, hypoxia,
and shear stress (38 – 40). Their release by cell lysis, exocytosis of
nucleotide-containing granules, and efflux through membrane
transport proteins causes their concentrations in extracellular fluids
to increase into micromolar ranges, and higher concentrations are
probably achieved at cell-cell synapses (1). The key role for ADP
and its receptors in vascular biology and platelet aggregation in
vivo is well established by studies with P2Y1 and P2Y12 receptornull mice (12, 13) and is validated in humans by the clinical efficacy of P2Y1 receptor antagonists as antiplatelet agents (41, 42).
The constitutive perivascular location of MCs throughout the body
and their abundance in atherosclerotic plaques probably permit
their exposure to endothelial- and platelet-derived nucleotides in
vivo (43). Reports that ATP activates rodent MCs or hMCs suggest
that these cells express functionally significant P2X and/or P2Y
receptors (26, 27). The fact that other hemopoietic cells express
multiple members of this family (44, 45) prompted the current
study, in which we sought to define the profile of P2Y receptors for
adenine nucleotides expressed by hMCs and to explore their potential complementary or opposing functions in cell activation.
After binding of their cognate ligands, most P2Y receptors initiate signaling through Gq and/or Gi family proteins, activating
phospholipase C and inducing calcium flux and inositol phosphate
accumulation (for review, see Ref. 1). The hMCs in our study
responded to low concentrations of ADP (0.05–50 ␮M) with a
PTX-resistant calcium flux (Fig. 1, A and B), consistent with a
functional response through a Gq-coupled receptor. The fact that
this calcium flux was unaffected by an ectonucleotidase inhibitor
(Fig. 1B) and was not induced by adenosine or AMP indicates that
it was not due to a response from the A3, A2A, or A2B adenosine
receptors that are expressed by rodent MCs (23) and an hMC line,
HMC-1 (24). The complete blockade of ADP-mediated calcium
flux by A3P5P (Fig. 1B) indicates that this response requires the
P2Y1 receptor, the mRNA for which was readily detected by RTPCR (Fig. 1C). Although heterologously expressed P2Y1 receptors can also bind and respond to ATP (with 30-fold lower affinity
than to ADP) (46), the lack of cross-desensitization between ADP
and ATP for calcium flux on hMCs (Fig. 1C) and the lack of
blockade of the ATP-induced calcium flux by A3P5P are consistent
with the additional presence of a response through one or more of the
ATP-preferring receptors. Although we detected transcripts encoding
the ATP-preferring P2Y11 receptor in the cells of two of four donors
tested (as shown for one donor; Fig. 1), the lack of calcium fluxes in
response to BzATP, ATP␥S, and dATP (each of which is a potent
agonist for P2Y11 receptors) (47, 48) mitigates against a substantial
P2Y11 contribution to the ATP-induced calcium response. The P2Y2
receptor (also detected by RT-PCR at low levels) prefers ATP and
UTP over ADP (49). Thus, the fact that UTP and ATP both caused
weak calcium fluxes is consistent with the function of the P2Y2
receptor in hMCs and with the results of earlier studies in lung hMCs
(27), as is the modest enhancement of exocytosis by ATP (and, in our
study, ADP). Transcripts encoding the P2X1 and P2X4 receptors
were also detected, a profile consistent with that previously reported
by others using cord blood-derived hMCs or lung MCs (25). Although additional contributions from these P2X receptors to the
ATP-dependent calcium flux is possible, their ability to serve as
sodium channels may ablate the ability to detect their calcium fluxes
in sodium-containing buffer solutions (50) such as that used in our
study.
Unlike the Gq-coupled P2Y1 receptor, the ADP-selective
P2Y12 and P2Y13 receptors both use Gi proteins to inhibit adenylate cyclase (51–53). Stimulation of platelets with ADP through
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FIGURE 5. Adenylyl cyclase-related signaling by adenine nucleotides and temporal effects on inhibition of TNF-␣ production. A, Accumulation of
cAMP in primed hMCs stimulated for 10 min with the indicated doses of ADP, ATP, BzATP, and ATP␥S. The data are expressed as a percentage of the
control value (taken as the average cAMP content of unstimulated hMCs in each experiment) and are the mean ⫾ SEM for three experiments for ADP and
ATP. Data for BzATP and ATP␥S are the mean ⫾ half-range from two experiments. B, Up-regulation of ICER expression and phosphorylation of CREB
in hMCs stimulated for various periods with ATP␥ S (50 ␮M) in the presence or the absence of H89 (10 ␮M). Dose-dependent effects of ADP and ATP␥S
on ICER expression are depicted below. Data are from single experiments, representative of three performed. Similar data were obtained with BzATP and
ATP as stimuli (n ⫽ 2; not shown). C, Time-dependent effects of nucleotides on suppression of TNF-␣ generation by hMCs. Cells were preincubated for
the indicated times with ADP or ATP␥S (50 ␮M) to up-regulate ICER expression before subsequent stimulation for 6 h with Fc⑀RI cross-linkage or PGN
(10 ␮M). Data are the mean ⫾ half-range for two experiments and are expressed as a percentage of the control value (amount of TNF-␣ generated by
samples not treated with nucleotides).
The Journal of Immunology
lation (Fig. 5A), being equipotent and effective at the same dose
range at which they interfered with TNF-␣ generation (Fig. 3).
Moreover, ATP␥S, BzATP, and dATP more potently stimulated
cAMP accumulation despite their inability to elicit calcium fluxes.
The accumulation of cAMP did not require extracellular calcium,
implying a lack of involvement P2X receptor-mediated ion flux in
this function, whereas the lack of effect of indomethacin indicates
that endogenous generation of PGs (which may also stimulate Gs)
is not required. This pharmacologic profile is thus similar to that
reported by Duhant and suggests the existence of a dominantly
Gs-coupled receptor for adenine nucleotides that is expressed by
both hMCs and lymphocytes. Although it is possible that this activity reflects the P2Y11 receptor, the lack of calcium flux in response to ATP and its analogues argues against this idea. Alternately, the responsible receptor may be among several orphan
GPCRs that have sequence homology to the P2Y family (62).
ICER is a product of an alternate promoter in the CREM gene
that autoregulates CREM-dependent transcription. Stimuli that elevate cAMP levels transiently induce ICER expression in human
thymocytes. In these cells, ICER blocks binding of NF-AT and
AP-1 transcription factors to their target sites in the promoters of
TNF-␣ and other cytokines and suppresses their expression (63).
Consistent with these same actions in hMCs, ADP, ATP, ATP␥S,
and BzATP all strongly up-regulated the expression of ICER (Fig.
5B). Pretreatment of hMCs with ADP or ATP␥S before activation
tended to inhibit Fc⑀RI-dependent TNF-␣ generation to a greater
extent than did concomitant nucleotide treatment. Phosphorylation
of CREB at serine 133, a prerequisite for induced ICER expression, was observed at 30 min in ATP␥S-stimulated hMCs and was
decreased by pretreatment with the PKA inhibitor H89. Nonetheless, there was residual CREB phosphorylation, and ICER expression was still strongly up-regulated at 3 h even in cells treated with
H89. Moreover, H89 failed to reverse the inhibitory effect of ADP
or ATP on cytokine generation, a finding reminiscent of previous
studies examining the inhibitory effect of exogenous PGE2 and
cAMP on cytokine generation by human T lymphocytes (64) and
macrophages (58). Our data (Fig. 5) thus suggest a component of
ICER induction that is H89 resistant and that may recapitulate
inhibitory pathways initiated by other Gs-coupled receptors. This
component may reflect the capacity of such receptor systems to
activate kinases other than PKA that can target CREB in immune
cells (65). Based on our previous studies (37), the particular sensitivity of cys-LT-mediated cytokine generation to inhibition by
nucleotides may reflect an absolute requirement for NF-AT, a target of ICER.
This study reveals that hMCs express complementary receptors
for ADP in addition to ATP receptors. Moreover, the results implicate at least one Gs-coupled ADP/ATP receptor in a nucleotideinitiated signaling pathway that is inhibitory for cytokine production, and adenine may prevent nucleotides themselves from
initiating this process while attenuating cytokine generation initiated by other receptor systems. Adenine nucleotide-mediated stimulation of MCs through activating P2 receptors could relate to MC
activation during coronary events, tissue injury, and infections.
Although such activation could facilitate vascular permeability responses by permitting the rapid release of smooth muscle-active
mediators, the simultaneous stimulation of inhibitory pathways
could prevent overexuberant secretion of potentially harmful cytokines into the tissue microenvironment, thus limiting the extent
of tissue pathology. Identification of the responsible receptor(s)
could present a novel therapeutic target for asthma, arthritis, and
other disorders with a prominent contribution from MCs and their
products.
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the P2Y12 receptor provides a calcium-independent signal that
activates the small GTPase RAP-1B (51, 52) and is complementary with the calcium-dependent signal from P2Y1 for MAPK activation and aggregation in response to exogenous or endogenous
ADP (9, 11). Although ADP-mediated calcium fluxes in hMCs
were P2Y1-dependent in our study, the combined inhibitory effects
of A3P5P and 2-MesAMP on ADP-induced phosphorylation of
ERK and p38 MAPKs suggests contributions from both P2Y1 and
P2Y12 receptors to this response (Fig. 2), similar to their behavior
in platelets. Although calcium flux and ERK phosphorylation are
prerequisites to the activation of cytosolic phospholipase A2, the
initial step in the synthesis of both cys-LTs and PGD2 by MCs and
other cell types (54, 55), only small amounts of PGD2 and cys-LTs
were secreted in response to ADP or ATP, and their secretion was
only evident at higher doses of nucleotides. Furthermore, P2Y1
and P2Y12 blockade did not prevent this response, possibly reflecting the inability of these antagonists to inhibit at higher agonist concentrations. Alternatively, 50 ␮M doses of nucleotides
may elicit contributions from P2Y13, P2X, or even adenosine receptors. Indeed, the capacity of PPADs to block cys-LT generation
supports the involvement of P2X receptors, consistent with prior
observations made using rat peritoneal MCs (22).
Calcium flux and MAPK phosphorylation are both early and
essential events in de novo generation of cytokines by MCs activated through other GPCRs (56). In our previous studies, exogenous LTC4, LTD4, and the nucleotide UDP each induced cytokine
generation by IL-4-primed hMCs through a pathway involving the
complementary functions of cys LT1 and cys LT2 receptors, which
are structural homologues of the P2Y receptors (21, 37). Given the
evident signaling parallels between the cys-LT receptors and the
P2Y receptors, it was initially surprising that adenine nucleotides
failed to elicit cytokine generation by primed hMCs, even at high
(100 ␮M) doses. Indeed, ADP, ATP (Fig. 3), and ATP analogues
significantly suppressed TNF-␣ production by cells stimulated
with PGN, a ligand for the TLR2/TLR6 heterodimer than mediates
innate immune responses to Gram-positive organisms (15). The
inhibitory effect was probably mediated, at least in part, at the level
of transcription or transcript stability, based on decrements in
steady state TNF-␣ mRNA (Fig. 4). ADP also strikingly suppressed the generation of TNF-␣, IL-8, and MIP-1␤ by cells stimulated with LTD4 (Fig. 3). The fact that adenosine did not interfere
(or augment) cytokine generation implies a lack of involvement of
P1 receptors. Moreover, the inability of A3P5P, 2-MesAMP, and
pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid to reverse
the suppressive effect of nucleotides on cytokine production suggests that this action may be independent of a known member of
the P2 receptor family.
GPCRs that interfere with activation of immune cells commonly
couple to Gs proteins, stimulating adenylyl cyclase and initiating
cAMP-dependent inhibitory signaling pathways. Examples include the receptors for PGE2 (EP2 and EP4 receptors) (57, 58) and
the ␤2-adrenergic receptor (59). To date, the P2Y11 receptor is the
only cloned ATP-binding P2Y family member known to stimulate
Gs proteins. When expressed heterologously in transfected cells,
P2Y11 receptors also mediate strong calcium fluxes in response to
ATP and its analogues (60). Recently, Duhant et al. (61) reported
that ATP, ATP␥S, and BzATP all suppress cytokine secretion by
polyclonally stimulated human T lymphocytes through a putative,
unidentified P2 receptor that stimulates the accumulation of
cAMP; ADP was not examined. Because the ability of both ADP
and ATP to interfere with cytokine production in our study suggested the potential presence of a Gs-coupled P2Y family member,
we measured cAMP accumulation in hMCs stimulated by these
nucleotides. ADP and ATP both induced modest cAMP accumu-
7545
7546
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