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P2X4 Receptor ActivationDegranulation and Allergic Responses viaAntigen-Induced Murine Mast Cell Extracellular ATP Augments

Tanaka, Kazuyuki Furuta and Isao MatsuokaObayashi, Kimiko Yamamoto, Schuichi Koizumi, Satoshi Kazuki Yoshida, Masa-aki Ito, Naoko Sato, Kosuke

ol.1900954http://www.jimmunol.org/content/early/2020/04/30/jimmun

published online 1 May 2020J Immunol 

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The Journal of Immunology

Extracellular ATP Augments Antigen-Induced Murine MastCell Degranulation and Allergic Responses via P2X4 ReceptorActivation

Kazuki Yoshida,* Masa-aki Ito,* Naoko Sato,* Kosuke Obayashi,* Kimiko Yamamoto,†

Schuichi Koizumi,‡ Satoshi Tanaka,x Kazuyuki Furuta,{ and Isao Matsuoka*

Extracellular ATP released from stimulated and/or damaged cells modulates physiological responses via stimulation of various

purinoceptors. We previously showed that ATP potentiated the Ag-induced mast cell (MC) degranulation via purinoceptors phar-

macologically similar to the ionotropic P2X4 receptor. In this study, we investigated the role of P2X4 receptor in MC degranulation

induced by stimulation of IgE-Fc«RI complex with Ag, using bone marrow–derived MCs (BMMCs) prepared from wild type and

P2X4 receptor–deficient (P2rx42/2) mice. ATP significantly increased Ag-induced degranulation in BMMCs prepared from wild

type mice. This effect of ATP was reduced in BMMCs prepared from P2rx42/2 mice. The potentiating effect of ATP was restored

by expressing P2X4 receptor in P2rx42/2 BMMCs. The P2X4 receptor–mediated effects were maintained even after differenti-

ating into the connective tissue-type MCs. P2X4 receptor stimulation did not affect the Ag-induced Ca2+ response but enhanced

Ag-induced early signals, such as tyrosine phosphorylation of Syk and phospholipase C-g. Interestingly, these effects of ATP on

Syk phosphorylation were not impaired by pretreatment with Cu2+, an inhibitor of the P2X4 receptor channel, or removal of

external Ca2+, suggesting that a mechanisms other than Ca2+ influx through ion channel activity may be involved. In vivo

experiments revealed that systemic and intradermal passive anaphylaxis responses were significantly alleviated in P2rx42/2 mice.

Taken together, the present data suggest that the P2X4 receptor plays an essential role in ATP-induced upregulation of MC

degranulation in response to Ag, and also contributes to the Ag-induced allergic response in vivo. The Journal of Immunology,

2020, 204: 000–000.

Mast cells (MCs) are widely distributed in tissues mainlyin contact with the outside, such as skin, airway, andintestinal mucous, and are responsible for the immune

response to parasites and bacterial infections (1). However, onceMCs acquire reactivity to originally innocuous Ag, the resultingrelease of multiple chemical mediators leads to allergic inflam-mation (2). The most important pathway to activate MC functionsis stimulation via IgE and its high affinity receptor FcεRI (3). IgEconfers Ag recognition ability to MCs by binding to FcεRI on the

surface of MCs. Binding of IgE to multivalent Ag induces ag-gregation of FcεRI, resulting in phosphorylation of the receptor-associated Src family tyrosine kinase Lyn, which initiates a seriesof signal transduction cascades, eventually causing MC activation(4). Therefore, factors that regulate Ag-induced signaling path-ways have been extensively investigated as a potential therapeutictarget for allergic disease.A number of studies have suggested that Ag-induced MC ac-

tivation is regulated by various extracellular humoral factors, suchas PGE2 (5), sphingosine-1-phosphate (6), and adenosine (7) viastimulation of Gi protein–coupled receptors. Stimulation of thesereceptors is known to activate PI3Kg via bg subunit of trimericG protein, leading to a phosphatidylinositol-3,4,5-trisphosphate-dependent sustained Ca2+ influx from external spaces (8, 9). Inaddition to these factors, we have previously reported that extra-cellular ATP augmented MC functions through distinct systemsother than G protein–coupled receptors (10). Namely, bonemarrow–derived MCs (BMMCs) expressed various functional P2receptors, including ionotropic P2X1, 4, and 7, and G protein–coupled P2Y1, 2, 13 and 14 receptors, but only two ionotropic re-ceptors were found to regulate the MC degranulation response.One was a direct degranulation induced by high concentrationsof ATP (.1 mM) via the P2X7 receptor, and the other was asynergistic enhancement of IgE-induced degranulation induced bylow concentrations of ATP (,100 mM) via P2X4 receptors. Al-though P2X7 receptor–mediated MC activation has been exten-sively investigated in both in vivo and in vitro experiments and hasbeen implicated in MC-mediated bowel inflammation (11–14),little is known about P2X4 receptor involvement in MC function.MCs are derived from hematopoietic stem cells as prematureMC

progenitors, and differentiate to distinct phenotypes dependenton their tissue localization. They are classified into two main

*Laboratory of Pharmacology, Faculty of Pharmacy, Takasaki University of Healthand Welfare, Takasaki-shi, Gunma 370-0033, Japan; †Department of BiomedicalEngineering, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan; ‡Department of Neuropharmacology, Interdisciplinary Graduate Schoolof Medicine, University of Yamanashi, Chuo, Yamanashi 409-3898, Japan; xDepart-ment of Pharmacology, Division of Pathological Sciences, Kyoto PharmaceuticalUniversity, Kyoto 607-8414, Japan; and {Department of Immunobiology, GraduateSchool of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University,Okayama 700-8530, Japan

ORCIDs: 0000-0001-7785-5021 (K.Y.); 0000-0003-0154-9516 (M.-a.I.); 0000-0001-6184-3106 (S.K.); 0000-0002-3468-7694 (S.T.); 0000-0002-7170-3630 (I.M.).

Received for publication August 8, 2019. Accepted for publication April 6, 2020.

This work was supported by Japan Society for the Promotion of Science (JSPS)KAKENHI Grants 18K14925 (to K.Y.) and 19K07328 (to I.M.) and Japan ScienceSociety Sasakawa Scientific Research Grant (2018-4017) (to K.Y.).

Address correspondence and reprint requests to Prof. Isao Matsuoka, Faculty ofPharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki,Gunma 370-0033, Japan. E-mail address: isao@takasaki-u.ac.jp

The online version of this article contains supplemental material.

Abbreviations used in this article: BMMC, bone marrow–derived MC; [Ca2+]i,intracellular Ca2+ concentration; CTMC, connective tissue-type MC; DNP-HSA, 2,4-DNP human serum albumin; HEK, human embryonic kidney; b-Hex, b-hexosaminidase;KRH, Krebs Ringer HEPES buffer; MC, mast cell; PLC, phospholipase C; SCF, stem cellfactor; WT, wild type.

Copyright� 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900954

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subpopulations: connective tissue-type MCs (CTMCs) and mucosal-type MCs (15). These two MC populations are known to differ inhistamine content and response to poly cationic secretagogue, suchas compound 48/80 (16). Skin MCs, representative of CTMCs,contain low amounts of P2X7 receptors, whereas colonic MCs,representative of mucosal-type MCs, highly express P2X7 receptors(12). However, P2X4 receptor expression in CTMCs and their effecton MC function is unknown. In this study, using P2rx42/2 mice, weinvestigated the role of P2X4 receptors in MC function in bothBMMCs and CTMC-like MCs, and in allergic responses in vivo.

Materials and MethodsMaterials

ATP, adenosine, UTP, GenElute Mammalian Total RNA Miniprep Kit, hista-mine dichloride, ionomycin, 2,4-DNP human serum albumin (DNP-HSA), anti-DNP IgE (clone SPE-7), and p-nitrophenyl N-acetyl-b-D-glucosaminidewere from Sigma-Aldrich Japan (Tokyo, Japan). Allophycocyanin-conjugated rat anti-mouse CD117 (c-Kit) Ab (clone 2B8) was from BDPharmingen (Tokyo, Japan). PE-conjugated mouse anti-mouse FcεRIa Ab(clone MAR-1) was from eBioscience (San Diego, CA). Recombinantmouse IL-3 and recombinant mouse stem cell factor (SCF) were fromPeproTech (London, U.K.). Fura-2-AM was from Wako (Osaka, Japan).Anti-P2X4 receptor Ab (APR-002) was from Almone Labs (Jerusalem,Israel). Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) rabbit mAb (no.2717), phospho-Syk (Tyr525/526) (C87C1) rabbit mAb (no. 2710), phos-pho-PLCg1 (Tyr783) Ab (no. 2821), phospho-Akt (Thr308) Ab (no. 9275),and anti-rabbit IgG, HRP-linked Ab (no. 7074) were from Cell SignalingTechnology (Danvers, MA). Anti-actin Ab was from Santa Cruz Bio-technology (Dallas, TX). All other chemicals used were of reagent gradeor of the highest quality available.

Animals

C57BL/6 mice, 8–10 wk old, were obtained from SLC Japan (Hamamatsu,Japan). P2X4 receptor–deficient (P2rx42/2) (17) and P2X7 receptor–deficient (P2rx72/2) mice (18) were of C57BL/6 background. Mice weremaintained under specific pathogen-free conditions at the animal facility ofTakasaki University of Health and Welfare. All experiments were per-formed in accordance with the regulations of the Animal Research Com-mittee of Takasaki University of Health and Welfare.

Cell culture

BMMCs were established using bone marrow obtained from C57BL/6 wildtype (WT), P2rx42/2, and P2rx72/2 mice (designated as WT, P2rx42/2,and P2rx72/2 BMMCs, respectively). Briefly, bone marrow cells werecultured with RPMI 1640 growth medium containing 10% FBS, 100 U/mlpenicillin, 100 mg/ml streptomycin, and 10 ng/ml rIL-3. After 2 wk, cellswere cultured with 10 ng/ml recombinant SCF for 4–6 wk. Subsequently,almost all (.95%) cells displayed a MC phenotype, as assessed by theexpression of CD117 (c-Kit) and FcεRI determined with FACSCanto IIflow cytometer (BD Biosciences, Tokyo, Japan). Cytospin preparations ofBMMCs were analyzed after toluidine blue staining.

CTMC-like MCs were prepared as previously described (16). Briefly,bone marrow cells obtained from C57BL/6 WT and P2rx42/2 mice werecultured in the presence of 10 ng/ml IL-3 for 21 d. At this period,.95% ofcells became CD117 (c-Kit) and FcεRI double positive. These cells werethen cocultured with mitomycin C–treated Swiss 3T3 fibroblasts in thepresence of 100 ng/ml recombinant murine SCF for 16 d. After thesetreatments,.90% of cells were confirmed as mature MCs with Safranin-Ostaining (16).

Peritoneal MCs were prepared from peritoneal cells obtained from WTand P2rx42/2 mice. Peritoneal cells were collected by washing the peri-toneal cavity with 5 ml of RPMI 1640 medium and cultured in the pres-ence of 10 ng/ml IL-3 and 10 ng/ml SCF for 14 d. At this period, more than95% of cells were double positive for c-Kit and FcεRI.

Human embryonic kidney (HEK) 293 cells (American Type CultureCollection, Manassas, VA) were cultured in DMEM containing 5% FBS,100 U/ml penicillin, and 100 mg/ml streptomycin and were used for heterolo-gous expression of mouse P2X4 receptor and electrophysiological experiments.

Cloning and transfection

Full-length mouse P2X4 receptor cDNAwas amplified from BMMC cDNA byPCR using sense primer 59-ATATGCTAGCGGGCGGCGGAGCCATGGCAG-3 and anti-sense primer 59-GTCTCTAGAGTGACAGACGCAGCAG-3. The

full-length cDNA encoding the P2X4 receptor was cloned into the mammalianexpression vector using pcDNA3.3-TOPO TA Cloning Kit (Thermo FisherScientific, Tokyo, Japan). The clone was verified by DNA sequencing and thenused for transfection. BMMCs (2.5 3 106) were suspended in Opti-MEM I(Thermo Fisher Scientific) and transfected with 5 mg of plasmid DNA byelectroporation, using Amaxa Nucleofector 2b system (Lonza, Tokyo, Japan)with the Y-001 program. HEK293 cells (53 105) were cotransfected with 0.8mg of mouse P2X4 plasmid DNA and 0.4 mg of pEGFP-C1 plasmid DNAusing FuGENE HD transfection reagent (Promega, Tokyo, Japan). After 24–48h, GFP-positive cells were subjected to patch clamp analysis.

Degranulation assay

Degranulation was evaluated by measuring b-hexosaminidase (b-Hex)release. BMMCs were sensitized with 50 ng/ml anti-DNP IgE overnight inRPMI 1640 growth medium. Cells were washed twice and suspended inKrebs Ringer HEPES buffer (KRH; 130 mM NaCl, 4.7 mM KCl, 4.0 mMNaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.8 mM CaCl2, 11.5 mMglucose, and 10 mM HEPES [pH 7.4]) containing 0.1% BSA. Some ex-periments were performed in high K+ buffer, in which the amounts of NaCland KCl were exchanged (130 mM KCl and 4.7 mM NaCl). Cells werestimulated under various conditions at 37˚C for 5 min, at which time theinitial degranulation response reached a steady-state level (10). Reactionswere terminated by rapid chilling on ice and centrifuging at 300 3 g for5 min. Supernatants were collected and the cell pellets were then lysed in0.1% Triton X-100. Supernatant and cell lysates were incubated with anequal volume of 1 mM p-nitrophenyl N-acetyl-b-D-glucosaminide dis-solved in citrate buffer (pH 4.5) in a 96-well plate at 37˚C for 30 min.Reactions were stopped by adding 0.1 M sodium carbonate buffer (pH 10.4),and the absorbance was measured at 405/655 nm. Percentage degranulationwas calculated using the following formula: b-Hex release (%) = supernatantabsorbance/(supernatant absorbance + lysate absorbance) 3 100.

Measurement of intracellular Ca2+ concentration

Cells were collected and washed twice with KRH containing 0.1% BSA.Cells were loaded with 1 mM fura 2-AM at 37˚C for 20 min and were thenwashed twice with KRH-BSA buffer and adjusted to 1–2 3 105 cells/ml.Changes in the fura 2 fluorescence at 510 nm with dual excitation wave-lengths at 340 and 380 nm were monitored as previously described (19),and intracellular Ca2+ concentration ([Ca2+]i) was calculated using thesoftware FL Solution 4.2 (Hitachi, Tokyo, Japan), with Kd value of224 nM for the fura-2/Ca2+ equilibrium.

Western blot

Cells were collected and washed with PBS and then resuspended in KRH(1–5 3 105/100 ml). The reaction was performed in KRH buffer and ter-minated by adding 43 Laemmli sample buffer (Bio Rad, Tokyo, Japan).Proteins were separated by 10% SDS-PAGE and transferred to Immobilon-P polyvinylidene fluoride membranes (Merck Millipore, Tokyo, Japan).The membranes were incubated with primary Abs overnight at 4˚C andwith secondary Abs for 2 h at 25˚C. The primary Abs were used at 1000-fold dilutions, and the HRP-conjugated secondary Ab was used at a10,000-fold dilution. The immunoreactive proteins were detected by ECL(GE Healthcare Bio-sciences, Tokyo, Japan) using Image Reader LAS-3000 (FUJIFILM, Tokyo, Japan). The density of bands was quantifiedusing Multi Gauge Version 3.0 software (FUJIFILM), and fold change inphosphorylation was calculated from the amount of phosphoprotein rela-tive to actin as a loading index.

Quantitative RT-PCR

Total RNA was isolated using the acid guanidine thiocyanate–phenol–chloroform extraction method (20). First-strand cDNA was synthesizedusing Moloney–murine leukemia virus reverse transcriptase with a 6-merrandom primers (Takara Bio, Tokyo, Japan), and quantitative RT-PCRwas performed using a SYBR green kit (Takara Bio) as previously de-scribed (19).

Passive cutaneous anaphylaxis

The left ear of WT and P2rx42/2 mice was sensitized by intradermalinjection of 100 ng anti-DNP IgE diluted in 20 ml saline, whereas a shamsaline injection was given in the other ear. After 24 h, mice were injectedi.v. with 100 mg of DNP-HSA in 200 ml 0.5% Evans blue in saline. After30 min, mice were sacrificed, ears were collected and weighed, andextravasated Evans blue dye was extracted by incubation of biopsies in1 ml formamide at 55˚C for 24 h before absorbance was measured at620 nm. Data were expressed as microgram of Evans blue per milligramof ear.

2 ROLE OF P2X4R IN MAST CELL ACTIVATION AND ALLERGIC RESPONSE

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Passive systemic anaphylaxis

Mice were sensitized with 10 mg anti-DNP IgE diluted in 200 ml saline byi.v. injection in the tail vain. After 24 h, mice were injected i.v. with100 mg of DNP-HSA in 200 ml in saline. After Ag challenge, rectaltemperature was measured every 5 min for 70 min with a digital ther-mometer (Physitemp Instruments, Clifton, NJ). Blood was then collectedby cardiac puncture with the anticoagulant EDTA and was centrifuged at3300 3 g for 1 min. The supernatant was collected to evaluate plasmahistamine levels using the Histamine ELISA kit (Enzo Life Sciences,Farmingdale, NY).

Histamine-induced hypothermia

To observe the direct effect of histamine on body temperature, WT andP2rx42/2 mice were administrated with 5 mg histamine dichloride in200 ml PBS by i.p. injection. After the administration, body temperaturewas monitored as described above.

Frozen section and staining

Tissues were wrapped in Tissue-Tec Optimal Cutting Temperature Com-pound (Sakura Finetek, Japan, Tokyo) and frozen. Sections (10 mm) werecut on cryostat, thaw-mounted on gelatin-coated slides, and fixed with 4%formaldehyde for 10 min and subsequently stained with toluidine blue.

Patch clamp analysis

P2X4 receptor–mediated membrane current was recorded using the whole-cell patch clamp technique as described previously (21). Patch pipettes hada tip resistance of 4–6 MV when filled with an intracellular solution(30 mM CsCl, 110 mM CsOH, 5 mM MgATP, 3 mMMgCl･6H2O, 10 mMEGTA, 20 mM HEPES, 50 mM aspartic acid [pH 7.2]). Cells dispersed ona poly-L-lysine–coated chamber were perfused with Tyrode solution(140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 0.33 mM NaH2PO4, 5 mMHEPES, 1.8 mM CaCl2, 5.5 mM glucose [pH 7.4]) at 37˚C. Membranepotentials were controlled by a model Axopatch 200B amplifier (AxonInstruments, Foster City, CA). Membrane currents were acquired onlineand analyzed using pCLAMP10.4 software (Axon). The current–voltagerelationship was obtained using ramp pulses from a holding potential of210 mV, initially depolarized to 60 mV, then hyperpolarized to 2120 mV,before depolarization back to the holding potential with a speed of 1.0 V/s.The ramp pulse was applied every 3 s. The P2X4 receptor current wascalculated at 260 mV in the presence or absence of CuSO4 and wasnormalized to cell membrane capacitance.

Statistics

All experiments were repeated at least three times. Each value indicates amean 6 SEM. Statistical analyses of the data were performed using aStudent t test for two sample comparison, and one-way ANOVA withDunnett two-tailed test. The p values ,0.05 were statistically significant.

ResultsEffects of ATP on Ag-induced degranulation inP2rx42/2 BMMCs

Bone marrow cells obtained from P2rx42/2 mice were normallydifferentiated to MCs, and .95% of cells were c-kit and FcεRIdouble positive after 5 wk, with few morphological differencescompared with WT-BMMCs (Supplemental Fig. 1A, 1B). In ad-dition, the degranulation responses induced by different concen-trations of Ag or by 1 mMATP, which were mediated by IgE-boundFcεRI or P2X7 receptors, respectively, did not differ between WT-and P2rx42/2 BMMCs (Supplemental Fig. 1C, 1D). Quantita-tive RT-PCR analysis showed that overall mRNA expression ofpurinergic signaling molecules, including P2X and P2Y recep-tors, P1 adenosine receptors, and ectonucleotidases, did notdiffer between WT- and P2rx42/2 BMMCs except for the P2X4receptor (Supplemental Fig. 2A–D).As we previously showed (10), ATP enhanced the degranulation

response induced by the threshold concentrations of Ag DNP-HSA in WT-BMMCs. This response to ATP was significantlyreduced in P2rx42/2 BMMCs (Fig. 1A). Adenosine also increasedthe degranulation response induced by threshold concentrations ofAg in WT-BMMCs via adenosine A3 receptors (10). This effect of

adenosine remained unchanged in P2rx42/2 BMMCs (Fig. 1A).To further confirm the role of P2X4 receptor in ATP-inducedenhancement of MC degranulation, P2X4 receptors were recon-stituted in P2rx42/2 BMMCs by transfecting P2X4 receptorexpressing plasmid (Fig. 1B). Although transfection experimentsusing the electroporation technique increased the basal release ofgranule contents, expression of P2X4 receptor restored the ATP-mediated enhancement of Ag-induced degranulation in P2rx42/2

BMMCs (Fig. 1C).

P2X4 receptor functions in CTMC-like MCs

MCs in vivo are classified into two major subsets: mucosal-typeMCs that are induced in the gastrointestinal tract upon parasiticinfection and CTMCs that are distributed in the skin and peritonealcavity (15). BMMCs are thought to be relevant to mucosal-typeMCs. Therefore, we investigated whether P2X4 receptor–medi-ated effects were retained in CTMCs. CTMC-like MCs weregenerated by a method established by Takano et al. (16). As shownin Fig. 2A, differentiation was confirmed by Alcian blue/safraninstaining (16). The acquisition of response to compound 48/80 is atypical feature of CTMCs (16). As shown in Fig. 2B, compound48/80 induced only minor degranulation in WT- and P2rx42/2

BMMCs but stimulated a significant degranulation in CTMC-likeMCs obtained from both WT and P2rx42/2 mice. QuantitativeRT-PCR analysis showed that although differentiation toCTMC-like MCs altered mRNA expression of some P2 re-ceptors, such as upregulation of P2X3 and downregulation ofP2X7, P2Y1, and P2Y14, there was little difference in P2X4receptor expression (Supplemental Fig. 3A–D). In those prep-arations, ATP clearly enhanced Ag-induced degranulation inWT-CTMCs, whereas the enhancing effect of ATP was markedlyreduced in P2rx42/2-CTMCs (Fig. 2C). Similar results were ob-tained in peritoneal MCs, a representative CTMCs. Thus, ATPmarkedly enhanced Ag-induced degranulation in peritoneal MCsobtained from WT mice. This enhancement was significantly re-duced in peritoneal MCs obtained from P2rx42/2-mice, but aP2X4-independent enhancing component was also retained inthese cells (Fig. 2D).

Mechanism underlying P2X4 receptor–mediated upregulationof Ag-induced degranulation

Because the P2X4 receptor is a ligand-gated nonselective cationchannel with high Ca2+ permeability, various physiologicalresponses mediated by P2X4 receptors have been reported asCa2+-dependent. An Ag-induced increase in [Ca2+]i did not differbetween BMMCs from WT and P2rx42/2 mice (Fig. 3A, 3C).Although the ATP-induced Ca2+ response was significantlysmaller in P2rx42/2 BMMCs, the Ag-induced Ca2+ response inthe presence of ATP did not differ between WT- and P2rx42/2

BMMCs (Fig. 3B, 3D).To evaluate the role of increased [Ca2+]i, the effect of ATP was

compared with UTP (100 mM) and ionomycin (0.3 mM), whichincreased [Ca2+]i to a similar degree as ATP (Fig. 4A). As shownin Fig. 4B, unlike ATP, neither UTP nor ionomycin enhancedAg-induced degranulation. In macrophages, K+ efflux via P2X7receptor channels was shown to trigger NLRP3 inflammasomeactivation (22). However, ATP enhancement of degranulation byAg did not change with high K+ buffer, a condition that impairs K+

efflux (Fig. 4C). We observed that the Ag- and ATP-induced Ca2+

response was not affected in high K+ buffer, (Supplemental Fig.4A, 4B). We next tested the effects of Cu2+, a divalent cation thatblocks P2X4 receptor current (23). Because it was difficult todemonstrate the P2X4 current in BMMCs, we confirmed the in-hibitory effects of Cu2+ on ATP-induced current in HEK293 cells

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expressing mouse P2X4 receptor (Supplemental Fig. 4C, 4D).Under similar conditions, ATP-induced enhancement of the de-granulation response to Ag was not inhibited by Cu2+. These re-sults suggest that ATP enhancement of the degranulation by Agmay involve a novel P2X4 receptor–signal transduction pathwaythat is independent of ion channel activity.

Effects of ATP on Ag-induced tyrosine-kinasesignaling cascade

Cross-linking of IgE-bound FcεRI by Ag triggers activation of asubset of protein-tyrosine kinases, including the nonreceptorprotein-tyrosine kinase Syk, which further propagates the signal tovarious effector proteins, such as PLCg1, and Akt (4). As shown

in Fig. 5, ATP itself had no effect but significantly increased Ag-induced Syk phosphorylation at tyrosine residues 346 and 519/520in WT-BMMCs. This effect of ATP was reduced in P2rx42/2

BMMCs (Fig. 5A–C). Similar effects of ATP were observed withPLCg1 phosphorylation at tyrosine residue 783 in a manner re-quiring P2X4 receptor expression (Fig. 5A, 5D). Although ATPincreases Akt phosphorylation, synergistic effects of ATP and Agon Akt phosphorylation were not evident (Fig. 5A, 5E).We observed ATP-induced enhancement of Ag-triggered phos-

phorylation of Syk at tyrosine residue 346 even in the absence ofextracellular Ca2+ (Fig. 6A, 6D). Although ATP also increased Ag-induced Syk phosphorylation in P2rx42/2 BMMCs, the extent ofATP-induced enhancement in P2rx42/2 BMMCs was significantly

FIGURE 1. Role of P2X4 receptors in the potentiating effects of ATP on Ag-induced BMMC degranulation. (A) WT- and P2rx42/2 BMMCs were

stimulated with ATP (100 mM) and adenosine (Ado, 100 mM) in the presence or absence of Ag (DNP-HSA, 10 ng/ml) for 5 min, and b-Hex released into

the reaction medium was measured. (B) Reconstitution of P2X4 receptor in P2rx42/2 BMMCs. P2rx42/2 BMMCs were transfected with control vector or

P2X4 receptor expression plasmid. The expression of P2X4 receptor protein was confirmed by Western blot. Arrow indicates recombinant P2X4 receptor.

(C) P2rx42/2 BMMCs transfected with control vector or P2X4 receptor expression plasmid were stimulated with vehicle (-) or ATP (100 mM) in the

presence of Ag (DNP-HSA, 10 ng/ml) for 5 min, and b-Hex released into the reaction medium was measured. Data are presented as the mean 6 SEM of

three independent experiments (n = 3 per group). *p , 0.05, **p , 0.01. ##p , 0.01 compared with the response to Ag alone (-).

FIGURE 2. P2X4 receptor–mediated augmentation of Ag-induced degranulation was maintained in CTMC-like MCs. (A) BMMCs and CTMC-like MCs

were stained with Alcian blue and safranin. Scale bar, 10 mm. (B) BMMCs or CTMC-like MCs were prepared from WT and P2rx42/2 mice and stimulated

with compound 48/80 (100 mM) for 5 min, and b-Hex released into the reaction medium was measured. (C) CTMC-like MCs prepared from WT and

P2rx42/2 mice were stimulated with vehicle (-) or ATP (100 mM) in the presence of Ag (DNP-HSA, 10 ng/ml) for 5 min, and b-Hex released into the

reaction medium was measured. (D) Peritoneal MCs prepared from WT and P2rx42/2 mice were stimulated with vehicle (-) or ATP (100 mM) in the

presence of Ag (DNP-HSA, 10 ng/ml) for 5 min, and b-Hex released into the reaction medium was measured. All data shown are from a representative

experiment of at least three individually performed experiments containing three to four mice per group. Data are presented as the mean 6 SEM of three

independent experiments (n = 3 per group). *p , 0.05, **p , 0.01. ##p , 0.01 compared with the response to Ag alone (-).

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lesser than in WT-BMMCs (Fig. 6D). Consistent with resultsobtained for the degranulation response, ATP enhancement ofAg-triggered phosphorylation of Syk at tyrosine residue 346 wasobserved in the high K+-buffer (Fig. 6B, 6E) and in the presence ofCu2+ (Fig. 6C, 6F) in WT-BMMCs but not in P2rx42/2 BMMCs.

P2X7 receptor assembly does not contribute to increased P2X4receptor–mediated degranulation

Our results above suggest that P2X4 receptor transduces theATP binding signal to intracellular enzymes in an ion channel-independent manner. Such a signaling system has been proposedfor the P2X7 receptor, which has a long cytoplasmic C terminus(24). In addition, the P2X4 receptor is thought to form a functionalheterotrimeric structure with the P2X7 receptor (25). We thereforeexamined the effects of ATP on Ag-induced degranulation inP2rX72/2 BMMCs. Although P2X7 receptor–mediated degranu-lation induced by 1 mM ATP was completely absent in P2rX72/2

BMMCs (Fig. 7A), ATP-induced enhancement of the degranula-tion response to Ag was observed in P2rX72/2 BMMCs (Fig. 7B),suggesting that the heterotrimeric structure formed with the P2X7receptor is not necessary for P2X4 receptor–mediated enhance-ment of degranulation.

Comparison of passive anaphylaxis responses in WT andP2rx42/2 mice

We finally examined whether the absence of P2X4 receptors affectsMC-mediated allergic responses in vivo using two passive ana-phylaxis models. For FcεRI-mediated passive cutaneous anaphy-laxis, mice received intradermal injection of anti-DNP IgE orsaline in their ears. After 24 h, mice were challenged by i.v. in-jection of DNP-HSA and Evans blue. Although the s.c. MC dis-tribution in the ear was similar in both WT and P2rx42/2 mice(Fig. 8A), dye extravasation for the IgE-injected side in P2rx42/2

mice was significantly alleviated compared with WT mice(Fig. 8B). In the FcεRI-mediated passive systemic anaphylaxismodel, Ag-induced body temperature decrease was also signifi-cantly alleviated in P2rx42/2 mice (Fig. 8C). Plasma histaminelevels after passive systemic anaphylaxis were significantly low in

P2rx42/2 mice (Fig. 8D). Additionally, we further confirmed theabsence of any difference between the histamine-induced hypo-thermia response in WT and P2rx42/2 mice (Supplemental Fig. 5).

DiscussionWe previously reported that Ag-induced degranulation in BMMCswas enhanced by extracellular ATP. Using P2rx42/2 mice, thecurrent study demonstrates that the P2X4 receptor was essentialfor ATP-induced enhancement of the degranulation response inBMMCs. ATP-induced enhancement of the degranulation re-sponse was absent in BMMCs prepared from P2rx42/2 mice,which showed only minor differences in cell morphology, de-granulation response to Ag, and overall expression of P2X andP2Y receptors (other than P2X4 receptor) than the WT mice.Furthermore, ATP-induced enhancement of degranulation wasrestored in a rescue experiment, in which P2rx42/2 BMMCs weretransfected with P2X4 receptor expression plasmid. Based onthese results, we confirmed that the P2X4 receptor plays an es-sential role in ATP enhancement of Ag-induced degranulation inBMMCs.BMMCs used in the current study, which are dependent on IL-3

for proliferation, are relatively immature MCs. Therefore, we alsoevaluated the effects of ATP on Ag-induced degranulation inCTMC-like MCs, which were positive for Safranin-O staining andacquired reactivity to the cationic secretagogue compound 48/80.

FIGURE 3. Effects of ATP on Ag-induced increase in [Ca2+]i in WT-

and P2rx42/2 BMMCs. (A and B) WT- or P2rx42/2 BMMCs preloaded

with fura 2-AM were stimulated with Ag (10 ng/ml) in the absence (A) or

presence (B) of ATP (100 mM). Typical responses obtained from WT- and

P2rx42/2 BMMCs were superimposed. The Ca2+ data are representative of

three to four independent experiments. (C and D) Summary of the data

obtained in (A) and (B). Data are presented as the mean 6 SEM (n = 3–4).

*p , 0.05. N.S. no significant difference.

FIGURE 4. Relationship between P2X4 receptor–mediated enhance-

ment of Ag-induced degranulation and receptor ion channel activity.

(A) Fura 2–loaded BMMCs were stimulated with ATP (100 mM), UTP

(100 mM), or ionomycin (0.3 mM), and changes in [Ca2+]i were monitored.

Results shown are representative of three independent experiments. (B)

BMMCs were stimulated with vehicle (-), ATP (100 mM), UTP (100 mM),

or ionomycin (0.3 mM) in the presence or absence of Ag (DNP-HSA,

10 ng/ml) for 5 min, and b-Hex released into the reaction medium was

measured. (C) BMMCs were suspended in normal KRH or high K+ buffer

and stimulated with vehicle (-) or ATP (100 mM) in the presence or ab-

sence of Ag (DNP-HSA, 10 ng/ml) for 5 min, and b-Hex released into the

reaction medium was measured. (D) BMMCs were preincubated in the

presence or absence of 10 mM CuSO4 for 10 min, and then stimulated with

vehicle (-) or Ag (DNP-HSA, 10 ng/ml) in the presence or absence of ATP

(100 mM) for 5 min, and b-Hex released into the reaction medium was

measured. Data are presented as the mean 6 SEM of three independent

experiments (n = 3 per group). **p , 0.01.

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Although this CTMC-like MC preparation showed altered ex-pression of some P2 receptors, such as upregulation of P2X3 re-ceptor and downregulation of P2X7, P2Y1, and P2Y14 receptors,expression of P2X4 receptor was not significantly different fromexpression levels seen in BMMCs. These results indicate that ATPaugments Ag-mediated degranulation in CTMC-like cells pre-pared from WT mice, whereas ATP effects on degranulation arelargely decreased in cells from P2rx42/2 mice. We observed aneffect of the P2X4 receptor on ATP-mediated enhancement in Ag-induced degranulation in primary cultured peritoneal MCs, whichare representative of CTMCs. This result suggests that upregula-tion of the MC degranulation response to Ag by P2X4 recep-tor activation is maintained even after differentiation of cells toCTMCs.The P2X4 receptor is a ligand-gated nonselective cation channel

with high Ca2+ permeability. Many physiological responses trig-gered by activation of this receptor are mediated by the risein [Ca2+]i due to Ca2+influx through receptor channels (26).However, the Ag-induced Ca2+-response was not altered in the

presence of ATP. This result is in clear contrast to the responseafter costimulation of MCs with Ag and G protein (Gi)-coupledreceptor agonists, such as PGE2 or adenosine (7). Specifically,stimulation of Gi-coupled receptor with PGE2 or adenosine hasbeen reported to increase the Ag-induced Ca2+ response markedly,leading to an augmented Ag-induced degranulation in BMMCs(7). In addition, the P2X4 receptor–mediated effect of ATP on MCdegranulation was not mimicked by the P2Y2 receptor agonistUTP or lower concentrations of Ca2+ ionophore ionomycin, whichincreased [Ca2+]i to a similar extent as ATP. These results sug-gested that although [Ca2+]i measurement in this study could notevaluate a local change in [Ca2+]i, which is effectively coupled tothe degranulation process, P2X4 receptor–mediated Ca2+influxmay not be sufficient to induce augmentation of Ag-induced de-granulation. In macrophages, intracellular K+ efflux has beenproposed as a mechanism by which inflammasomes are activatedvia the ionotropic P2X7 receptor (22). However, this mechanismis unlikely to be involved in the augmentation of Ag-induceddegranulation in BMMCs, because even when all extracellular

FIGURE 5. Effects of ATP on Ag-induced

phosphorylation of Syk, PLCg, and Akt in WT- and

P2rx42/2 BMMCs. (A) Cells were stimulated with

vehicle (-) or Ag (DNP-HSA, 10 ng/ml) in the

presence or absence of ATP (100 mM) for 1 min,

and phosphorylation of Syk (Y346), Syk (Y519/

520), PLCg1 (Y783), and Akt (T308) and the

loading control actin were analyzed by Western

blotting. (B–E) The densitometry values of protein

bands of (B) p-Syk (Y346), (C) p-Syk (Y519/520),

(D) p-PLCg1, and (E) p-Akt are shown as relative

intensities, with the results obtained with Ag

alone designated as 1. Data are presented as

mean 6 SEM from three independent experi-

ments. **p , 0.01, ##p , 0.01 compared with the

response to Ag alone (-).

FIGURE 6. Effect extracellular Ca2+ removal, replacement of extracellular Na+ with K+, or inhibition of P2X4 receptor current with Cu2+ on ATP

augmentation of Ag-induced phosphorylation of Syk in BMMCs. (A–C) BMMCs suspended in Ca2+-free KRH containing 1 mM EGTA (A), high K+ buffer

(B), or normal KRH containing 10 mM CuSO4 (C) were stimulated with vehicle (-) or Ag (DNP-HSA, 10 ng/ml) in the presence or absence of ATP

(100 mM) for 1 min, and p-Syk (Y346) was analyzed by Western blotting. (D–F) The densitometry values of protein bands of p-Syk (Y346) in (A)–(C) are

shown in (D)–(F), respectively, as relative intensities, with the results obtained using Ag alone designated as 1. Data are presented as mean 6 SEM from

three independent experiments. *p , 0.05, **p , 0.01. ##p , 0.01 compared with the response to Ag alone (-).

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Na + was replaced with K+ to abrogate K+ movement, augmen-tation of Ag-induced degranulation by ATP was not affected. InAg-mediated MC degranulation, the Ca2+-activated K+ channelKCa3.1 is known to play a critical role by regulating membranepotential (23). This channel is activated through an initial increasein [Ca2+]i, thereby eliciting membrane hyperpolarization, whichenhances the driving force for Ca2+ influx through nonselectivecation channels, such as the P2X4 receptor (24). This mechanismshould not occur in the K+ buffer, because the opening of the K+

channel depolarizes the membrane potential. However, our resultsshowed that the high K+ buffer did not affect P2X4 receptor–mediated enhancement of degranulation by Ag. Furthermore, theeffect of ATP on Ag-induced degranulation was observed even in

the presence of Cu2+, a condition which inhibits the P2X4 receptorcurrent (23). These results suggest that enhancement of Ag-induced degranulation by ATP may not depend on ion channelactivity of the P2X4 receptor. The P2X7 receptor in macrophagesis an example of an ionotropic receptor that transmits a signal intocells by an action other than through ion channel activity (24). TheP2X7 receptor has a long intracellular C-terminal domain, whichinteracts with various intracellular signaling proteins. Because theP2X4 receptor is thought to form a functional heterotrimer withthe P2X7 receptor (25), and MCs express the P2X7 receptor, as wedemonstrate in this study, it is possible that the P2X4 receptorsubunit forms a functional heterotrimer with the P2X7 receptorsubunit, resulting in transmission of a degranulation-promotingsignal through the P2X7 receptor C-terminal domain. However,this possibility is unlikely because ATP enhancement of Ag-induced degranulation was observed in BMMCs prepared fromP2rX72/2 mice. Taken together, our results suggest that the P2X4receptor may enhance Ag-induced degranulation through an ad-ditional signal other than that resulting from ion channel activity.In Ag-stimulated degranulation in MCs, tyrosine phosphoryla-

tion of ITAM, an intracellular domain of FcεRI, activates non-receptor tyrosine kinases Lyn and Syk, which in turn activatesdownstream PLCg to transmit signal to degranulation machineries(4, 8). Although stimulation of BMMCs with ATP alone failed toactivate these signaling pathways, ATP augmented Ag-inducedtyrosine-phosphorylation of Syk and PLCg. This effect of ATPwas diminished in P2rx42/2 BMMCs, suggesting involvement ofthe P2X4 receptor. An increase in Ag-induced Syk phosphoryla-tion by ATP was also observed in the absence of external Ca2+. Inaddition, the increase in Ag-induced Syk phosphorylation by ATPwas observed even in the high K+ buffer, and in the presence ofCu2+, in a P2X4 receptor–dependent manner. Therefore, ATPmodulation of phosphorylation is most likely mediated by theP2X4 receptor in a manner independent of Ca2+ influx. The P2X4receptor contains a short C-terminal domain composed of 26 aa,

FIGURE 7. P2X7 receptor does not function in ATP-induced augmen-

tation of degranulation in response to Ag. (A) BMMCs prepared from WT

and P2rx72/2 mice were stimulated with vehicle (-) or ATP (1 mM) for

5 min, and b-Hex released into the reaction medium was measured. (B)

WT- and P2rx72/2 BMMCs were stimulated with vehicle (-) or Ag (DNP-

HSA, 10 ng/ml) in the presence or absence of ATP (100 mM) for 5 min,

and b-Hex released into the reaction medium was measured. Data are

presented as mean 6 SEM from three independent experiments (n = 3 per

group). **p , 0.01. ##p , 0.01 compared with Ag alone (-).

FIGURE 8. Role of P2X4 receptor in passive cuta-

neous and systemic anaphylaxis. (A) Ear sections from

WTor P2rx42/2 mice were stained with toluidine blue

(scale bar, 50 mm), and toluidine blue–positive MCs

(indicated by arrows) per field were counted, as shown

in the lower panel. Data are presented as the mean 6SEM (n = 4). (B) WT and P2rx42/2 mouse ears were

intradermally injected with saline or anti-DNP IgE.

After 24 h, mice were injected i.v. with Evans blue

containing Ag (DNP-HSA), and extravasated Evans

blue dye was measured. Data are presented as the

mean 6 SEM (n = 5). (C) WT or P2rx42/2 mice were

sensitized with i.v. injection of anti-DNP-IgE. After

24 h, mice were injected i.v with Ag (DNP-HSA), and

rectal temperatures were measured every 5 min for

70 min. (D) Plasma histamine levels were measured

after the experiments performed in (C). Data are

presented as the mean 6 SEM (n = 4). *p , 0.05,

**p , 0.01. N.S. no significant difference.

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including a polar amino acid rich region with six tyrosine resides(26). This region has been demonstrated to contain a putativenoncanonical tyrosine-based endocytic motif (27). It is thereforepossible that the P2X4 receptor signal may cross-talk with FcεRI-induced tyrosine kinase signals due to the short C-terminal do-main of the P2X4 receptor. Further study is needed to clarify thedetailed mechanism.In this study, in vivo experiments using WT and P2rx42/2 mice

showed that P2X4 receptor–mediated MC hyperactivity was alsoinvolved in the in vivo allergic reactions. We observed that thepassive cutaneous anaphylactic reaction was significantly attenu-ated in P2rx42/2 mice. In addition, the decrease in rectal tem-perature due to the passive whole-body anaphylactic reaction wasalso attenuated in P2rx42/2 mice. We confirmed that the rectaltemperature decrease induced by histamine did not differ betweenWT and P2rx42/2 mice, suggesting that Ag-induced MC de-granulation was abrogated in P2rx42/2 mice. Consistently, plasmahistamine levels after the passive whole-body anaphylactic reac-tion were significantly lower in P2rx42/2 mice. ATP is known tobe released from organs in response to various stimuli (28). Theresults of this study suggest that MCs in the body may be stim-ulated by ATP constantly released from the surrounding micro-environment and that their responsiveness to Ag is enhanced byP2X4 receptor signal. In general, allergic reactions are knownto be exacerbated when tissues are scraped due to itching (29).Because there is ample evidence that physical stimuli such asscratching trigger the release of ATP from cells (28, 30, 31), theactivation of MCs via the P2X4 receptor shown in this study maybe important for understanding mechanical stimuli-induced ex-acerbation of allergic reactions.To date, many critical physiological and pathophysiological

responses occurring via the P2X4 receptor have been reported,indicating that this receptor acts as a sensor of shear stress-inducedATP release from vascular endothelial cells and plays a role inregulating vascular tension via NO release (17). In the nervoussystem, P2X4 receptors are upregulated in spinal microglia duringnerve injury, leading to neuropathic pain (32). The P2X4 receptoris thought to be critical for T cell activation and migration, whichare closely related to rejection of transplanted tissue (33). It hasbeen reported that P2X4 receptor expression is increased in theairway of asthmatic model mice, and P2X4 deficiency alleviatesallergic inflammation (34). The results presented in this study onMC function contribute a novel role for P2X4 receptors in theregulation of biological functions.The present study has focused on the role of the P2X4 receptor in

regulating MC function. However, other P2 receptor–mediatedenhancement of Ag-induced degranulation has already beenreported. For example, Gao et al. (35, 36) demonstrated thatAg-induced degranulation was enhanced by the P2Y13 or P2Y14

receptors. In this study, we also observed P2X4 receptor–independent regulatory action under certain conditions. For ex-ample, enhancement of Ag-induced degranulation by ATP inCTMC-like cells and peritoneal MCs possessed a P2X4 receptor–independent component in P2rx42/2 MCs and the enhancementof Ag-induced phosphorylation of Syk by ATP under Ca2+-freeconditions remained in P2rx42/2 MCs. Analysis of such re-sponses may contribute to elucidation of the diversity of puri-nergic regulation.Because this study was conducted using a mouse model, it was

necessary to confirm whether the same mechanism operates inhuman MCs. In fact, adenosine is well known to enhance Ag-induced degranulation through the adenosine A3 receptor inmice (7, 8), whereas in human MCs, adenosine is shown to inhibitAg-induced degranulation via the A2A receptor (37). Although

expression of P2X4 receptor as a functional ATP receptor has beenreported in human MCs (38), the role of P2X4 receptor signal inhuman MC function is largely unknown. Therefore, experimentsusing human MCs are important to translate the results of thisstudy to human diseases.In conclusion, our results demonstrated that P2X4 receptor

activation augments Ag-inducedMC degranulation in BMMCs andCTMCs through a novel mechanism in which FcεRI-induced ty-rosine kinase signaling is increased. We further demonstratedusing P2rx42/2 mice that the allergic response was up-regulatedin vivo in a P2X4 receptor–dependent manner. Although furtherstudies are needed in human MCs, our results provide the indi-cation that the P2X4 receptor may be a potential therapeutic targetfor allergic diseases.

DisclosuresThe authors have no financial conflicts of interest.

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