Extracellular ATP Augments Antigen-Induced Murine Degranulation and Allergic Responses via P2X4 Activation This information is current as of September 23, 2021. Kazuki Yoshida, Masa-aki Ito, Naoko Sato, Kosuke Obayashi, Kimiko Yamamoto, Schuichi Koizumi, Satoshi Tanaka, Kazuyuki Furuta and Isao Matsuoka J Immunol published online 1 May 2020 http://www.jimmunol.org/content/early/2020/04/30/jimmun Downloaded from ol.1900954

Supplementary http://www.jimmunol.org/content/suppl/2020/04/30/jimmunol.190095 http://www.jimmunol.org/ Material 4.DCSupplemental

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 1, 2020, doi:10.4049/jimmunol.1900954 The Journal of Immunology

Extracellular ATP Augments Antigen-Induced Murine Mast Cell Degranulation and Allergic Responses via P2X4 Receptor Activation

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 2/2 P2X4 receptor–deficient (P2rx4 ) mice. ATP significantly increased Ag-induced degranulation in BMMCs prepared from wild Downloaded from 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 2+ 2+ external Ca , suggesting that a mechanisms other than Ca influx through activity may be involved. In vivo http://www.jimmunol.org/ 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.

ast cells (MCs) are widely distributed in tissues mainly surface of MCs. Binding of IgE to multivalent Ag induces ag- in contact with the outside, such as skin, airway, and gregation of FcεRI, resulting in phosphorylation of the receptor- intestinal mucous, and are responsible for the immune associated Src family tyrosine kinase Lyn, which initiates a series

M by guest on September 23, 2021 response to parasites and bacterial infections (1). However, once of signal transduction cascades, eventually causing MC activation MCs acquire reactivity to originally innocuous Ag, the resulting (4). Therefore, factors that regulate Ag-induced signaling path- release of multiple chemical mediators leads to allergic inflam- ways have been extensively investigated as a potential therapeutic mation (2). The most important pathway to activate MC functions target for allergic disease. is stimulation via IgE and its high affinity receptor FcεRI (3). IgE A number of studies have suggested that Ag-induced MC ac- confers Ag recognition ability to MCs by binding to FcεRI on the tivation is regulated by various extracellular humoral factors, such as PGE2 (5), sphingosine-1-phosphate (6), and (7) via stimulation of Gi –coupled receptors. Stimulation of these *Laboratory of Pharmacology, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki-shi, Gunma 370-0033, Japan; †Department of Biomedical receptors is known to activate PI3Kg via bg subunit of trimeric Engineering, Graduate School of Medicine, The University of Tokyo, Tokyo 113- G protein, leading to a phosphatidylinositol-3,4,5-trisphosphate- 0033, Japan; ‡Department of Neuropharmacology, Interdisciplinary Graduate School 2+ x dependent sustained Ca influx from external spaces (8, 9). In of Medicine, University of Yamanashi, Chuo, Yamanashi 409-3898, Japan; Depart- ment of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical addition to these factors, we have previously reported that extra- University, Kyoto 607-8414, Japan; and {Department of Immunobiology, Graduate cellular ATP augmented MC functions through distinct systems School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, other than G protein–coupled receptors (10). Namely, bone Okayama 700-8530, Japan marrow–derived MCs (BMMCs) expressed various functional P2 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.). receptors, including ionotropic P2X1, 4, and 7, and G protein– Received for publication August 8, 2019. Accepted for publication April 6, 2020. coupled P2Y1, 2, 13 and 14 receptors, but only two ionotropic re- ceptors were found to regulate the MC degranulation response. 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 Science One was a direct degranulation induced by high concentrations Society Sasakawa Scientific Research Grant (2018-4017) (to K.Y.). of ATP (.1 mM) via the P2X7 receptor, and the other was a Address correspondence and reprint requests to Prof. Isao Matsuoka, Faculty of synergistic enhancement of IgE-induced degranulation induced by Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki, low concentrations of ATP (,100 mM) via P2X4 receptors. Al- Gunma 370-0033, Japan. E-mail address: [email protected] though P2X7 receptor–mediated MC activation has been exten- The online version of this article contains supplemental material. sively investigated in both in vivo and in vitro experiments and has Abbreviations used in this article: BMMC, bone marrow–derived MC; [Ca2+]i, intracellular Ca2+ concentration; CTMC, connective tissue-type MC; DNP-HSA, 2,4- been implicated in MC-mediated bowel inflammation (11–14), DNP human serum albumin; HEK, human embryonic kidney; b-Hex, b-hexosaminidase; little is known about P2X4 receptor involvement in MC function. KRH, Krebs Ringer HEPES buffer; MC, mast cell; PLC, phospholipase C; SCF, stem cell MCs are derived from hematopoietic stem cells as premature MC factor; WT, wild type. progenitors, and differentiate to distinct phenotypes dependent Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 on their tissue localization. They are classified into two main

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900954 2 ROLE OF P2X4R IN MAST CELL ACTIVATION AND ALLERGIC RESPONSE subpopulations: connective tissue-type MCs (CTMCs) and mucosal- full-length cDNA encoding the P2X4 receptor was cloned into the mammalian type MCs (15). These two MC populations are known to differ in expression vector using pcDNA3.3-TOPO TA Cloning Kit (Thermo Fisher histamine content and response to poly cationic secretagogue, such Scientific, Tokyo, Japan). The clone was verified by DNA sequencing and then used for transfection. BMMCs (2.5 3 106) were suspended in Opti-MEM I as compound 48/80 (16). Skin MCs, representative of CTMCs, (Thermo Fisher Scientific) and transfected with 5 mgofplasmidDNAby contain low amounts of P2X7 receptors, whereas colonic MCs, electroporation, using Amaxa Nucleofector 2b system (Lonza, Tokyo, Japan) representative of mucosal-type MCs, highly express P2X7 receptors with the Y-001 program. HEK293 cells (5 3 105) were cotransfected with 0.8 (12). However, P2X4 receptor expression in CTMCs and their effect mgofmouseP2X4plasmidDNAand0.4mgofpEGFP-C1plasmidDNA 2/2 using FuGENE HD transfection reagent (Promega, Tokyo, Japan). After 24–48 on MC function is unknown. In this study, using P2rx4 mice, we h, GFP-positive cells were subjected to patch clamp analysis. investigated the role of P2X4 receptors in MC function in both BMMCs and CTMC-like MCs, and in allergic responses in vivo. Degranulation assay Degranulation was evaluated by measuring b-hexosaminidase (b-Hex) Materials and Methods release. BMMCs were sensitized with 50 ng/ml anti-DNP IgE overnight in RPMI 1640 growth medium. Cells were washed twice and suspended in Materials Krebs Ringer HEPES buffer (KRH; 130 mM NaCl, 4.7 mM KCl, 4.0 mM ATP, adenosine, UTP, GenElute Mammalian Total RNA Miniprep Kit, hista- NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.8 mM CaCl2, 11.5 mM glucose, and 10 mM HEPES [pH 7.4]) containing 0.1% BSA. Some ex- mine dichloride, ionomycin, 2,4-DNP human serum albumin (DNP-HSA), anti- + DNP IgE (clone SPE-7), and p-nitrophenyl N-acetyl-b-D-glucosaminide periments were performed in high K buffer, in which the amounts of NaCl were from Sigma-Aldrich Japan (Tokyo, Japan). Allophycocyanin- and KCl were exchanged (130 mM KCl and 4.7 mM NaCl). Cells were conjugated rat anti-mouse CD117 (c-Kit) Ab (clone 2B8) was from BD stimulated under various conditions at 37˚C for 5 min, at which time the ε initial degranulation response reached a steady-state level (10). Reactions Pharmingen (Tokyo, Japan). PE-conjugated mouse anti-mouse Fc RIa Ab Downloaded from 3 (clone MAR-1) was from eBioscience (San Diego, CA). Recombinant were terminated by rapid chilling on ice and centrifuging at 300 g for mouse IL-3 and recombinant mouse stem cell factor (SCF) were from 5 min. Supernatants were collected and the cell pellets were then lysed in PeproTech (London, U.K.). Fura-2-AM was from Wako (Osaka, Japan). 0.1% Triton X-100. Supernatant and cell lysates were incubated with an Anti-P2X4 receptor Ab (APR-002) was from Almone Labs (Jerusalem, equal volume of 1 mM p-nitrophenyl N-acetyl-b-D-glucosaminide dis- Israel). Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (65E4) rabbit mAb (no. solved in citrate buffer (pH 4.5) in a 96-well plate at 37˚C for 30 min. 2717), phospho-Syk (Tyr525/526) (C87C1) rabbit mAb (no. 2710), phos- Reactions were stopped by adding 0.1 M sodium carbonate buffer (pH 10.4), pho-PLCg1 (Tyr783) Ab (no. 2821), phospho-Akt (Thr308) Ab (no. 9275), and the absorbance was measured at 405/655 nm. Percentage degranulation and anti-rabbit IgG, HRP-linked Ab (no. 7074) were from Cell Signaling was calculated using the following formula: b-Hex release (%) = supernatant http://www.jimmunol.org/ 3 Technology (Danvers, MA). Anti-actin Ab was from Santa Cruz Bio- absorbance/(supernatant absorbance + lysate absorbance) 100. technology (Dallas, TX). All other chemicals used were of reagent grade Measurement of intracellular Ca2+ concentration or of the highest quality available. Cells were collected and washed twice with KRH containing 0.1% BSA. Animals Cells were loaded with 1 mM fura 2-AM at 37˚C for 20 min and were then 3 5 C57BL/6 mice, 8–10 wk old, were obtained from SLC Japan (Hamamatsu, washed twice with KRH-BSA buffer and adjusted to 1–2 10 cells/ml. Japan). P2X4 receptor–deficient (P2rx42/2) (17) and P2X7 receptor– Changes in the fura 2 fluorescence at 510 nm with dual excitation wave- 2/2 lengths at 340 and 380 nm were monitored as previously described (19), deficient (P2rx7 ) mice (18) were of C57BL/6 background. Mice were 2+ 2+ maintained under specific pathogen-free conditions at the animal facility of and intracellular Ca concentration ([Ca ]i) was calculated using the

software FL Solution 4.2 (Hitachi, Tokyo, Japan), with Kd value of by guest on September 23, 2021 Takasaki University of Health and Welfare. All experiments were per- 2+ formed in accordance with the regulations of the Animal Research Com- 224 nM for the fura-2/Ca equilibrium. mittee of Takasaki University of Health and Welfare. Western blot Cell culture Cells were collected and washed with PBS and then resuspended in KRH 3 5 BMMCs were established using bone marrow obtained from C57BL/6 wild (1–5 10 /100 ml). The reaction was performed in KRH buffer and ter- 2 2 2 2 2 2 3 type (WT), P2rx4 / , and P2rx7 / mice (designated as WT, P2rx4 / , minated by adding 4 Laemmli sample buffer (Bio Rad, Tokyo, Japan). and P2rx72/2 BMMCs, respectively). Briefly, bone marrow cells were were separated by 10% SDS-PAGE and transferred to Immobilon- cultured with RPMI 1640 growth medium containing 10% FBS, 100 U/ml P polyvinylidene fluoride membranes (Merck Millipore, Tokyo, Japan). penicillin, 100 mg/ml streptomycin, and 10 ng/ml rIL-3. After 2 wk, cells The membranes were incubated with primary Abs overnight at 4˚C and were cultured with 10 ng/ml recombinant SCF for 4–6 wk. Subsequently, with secondary Abs for 2 h at 25˚C. The primary Abs were used at 1000- almost all (.95%) cells displayed a MC phenotype, as assessed by the fold dilutions, and the HRP-conjugated secondary Ab was used at a expression of CD117 (c-Kit) and FcεRI determined with FACSCanto II 10,000-fold dilution. The immunoreactive proteins were detected by ECL flow cytometer (BD Biosciences, Tokyo, Japan). Cytospin preparations of (GE Healthcare Bio-sciences, Tokyo, Japan) using Image Reader LAS- BMMCs were analyzed after toluidine blue staining. 3000 (FUJIFILM, Tokyo, Japan). The density of bands was quantified CTMC-like MCs were prepared as previously described (16). Briefly, using Multi Gauge Version 3.0 software (FUJIFILM), and fold change in bone marrow cells obtained from C57BL/6 WT and P2rx42/2 mice were phosphorylation was calculated from the amount of phosphoprotein rela- cultured in the presence of 10 ng/ml IL-3 for 21 d. At this period, .95% of tive to actin as a loading index. ε cells became CD117 (c-Kit) and Fc RI double positive. These cells were Quantitative RT-PCR then cocultured with mitomycin C–treated Swiss 3T3 fibroblasts in the presence of 100 ng/ml recombinant murine SCF for 16 d. After these Total RNA was isolated using the acid guanidine thiocyanate–phenol– treatments, .90% of cells were confirmed as mature MCs with Safranin-O chloroform extraction method (20). First-strand cDNA was synthesized staining (16). using Moloney–murine leukemia virus reverse transcriptase with a 6-mer Peritoneal MCs were prepared from peritoneal cells obtained from WT random primers (Takara Bio, Tokyo, Japan), and quantitative RT-PCR 2/2 and P2rx4 mice. Peritoneal cells were collected by washing the peri- was performed using a SYBR green kit (Takara Bio) as previously de- toneal cavity with 5 ml of RPMI 1640 medium and cultured in the pres- scribed (19). ence of 10 ng/ml IL-3 and 10 ng/ml SCF for 14 d. At this period, more than 95% of cells were double positive for c-Kit and FcεRI. Passive cutaneous anaphylaxis Human embryonic kidney (HEK) 293 cells (American Type Culture The left ear of WT and P2rx42/2 mice was sensitized by intradermal Collection, Manassas, VA) were cultured in DMEM containing 5% FBS, injection of 100 ng anti-DNP IgE diluted in 20 ml saline, whereas a sham m 100 U/ml penicillin, and 100 g/ml streptomycin and were used for heterolo- saline injection was given in the other ear. After 24 h, mice were injected gous expression of mouse P2X4 receptor and electrophysiological experiments. i.v. with 100 mgofDNP-HSAin200ml 0.5% in saline. After Cloning and transfection 30 min, mice were sacrificed, ears were collected and weighed, and extravasated Evans blue dye was extracted by incubation of biopsies in Full-length mouse P2X4 receptor cDNA was amplified from BMMC cDNA by 1 ml formamide at 55˚C for 24 h before absorbance was measured at PCR using sense primer 59-ATATGCTAGCGGGCGGCGGAGCCATGGCAG- 620 nm. Data were expressed as microgram of Evans blue per milligram 3 and anti-sense primer 59-GTCTCTAGAGTGACAGACGCAGCAG-3. The of ear. The Journal of Immunology 3

Passive systemic anaphylaxis adenosine remained unchanged in P2rx42/2 BMMCs (Fig. 1A). Mice were sensitized with 10 mg anti-DNP IgE diluted in 200 ml saline by To further confirm the role of P2X4 receptor in ATP-induced i.v. injection in the tail vain. After 24 h, mice were injected i.v. with enhancement of MC degranulation, P2X4 receptors were recon- 2 2 100 mg of DNP-HSA in 200 ml in saline. After Ag challenge, rectal stituted in P2rx4 / BMMCs by transfecting P2X4 receptor temperature was measured every 5 min for 70 min with a digital ther- expressing plasmid (Fig. 1B). Although transfection experiments mometer (Physitemp Instruments, Clifton, NJ). Blood was then collected using the electroporation technique increased the basal release of by cardiac puncture with the anticoagulant EDTA and was centrifuged at 3300 3 g for 1 min. The supernatant was collected to evaluate plasma granule contents, expression of P2X4 receptor restored the ATP- 2/2 histamine levels using the Histamine ELISA kit (Enzo Life Sciences, mediated enhancement of Ag-induced degranulation in P2rx4 Farmingdale, NY). BMMCs (Fig. 1C). Histamine-induced hypothermia P2X4 receptor functions in CTMC-like MCs To observe the direct effect of histamine on body temperature, WT and MCs in vivo are classified into two major subsets: mucosal-type P2rx42/2 mice were administrated with 5 mg histamine dichloride in 200 ml PBS by i.p. injection. After the administration, body temperature MCs that are induced in the gastrointestinal tract upon parasitic was monitored as described above. infection and CTMCs that are distributed in the skin and peritoneal cavity (15). BMMCs are thought to be relevant to mucosal-type Frozen section and staining MCs. Therefore, we investigated whether P2X4 receptor–medi- Tissues were wrapped in Tissue-Tec Optimal Cutting Temperature Com- ated effects were retained in CTMCs. CTMC-like MCs were pound (Sakura Finetek, Japan, Tokyo) and frozen. Sections (10 mm) were generated by a method established by Takano et al. (16). As shown cut on cryostat, thaw-mounted on gelatin-coated slides, and fixed with 4% in Fig. 2A, differentiation was confirmed by Alcian blue/safranin Downloaded from formaldehyde for 10 min and subsequently stained with toluidine blue. staining (16). The acquisition of response to compound 48/80 is a Patch clamp analysis typical feature of CTMCs (16). As shown in Fig. 2B, compound 2/2 P2X4 receptor–mediated membrane current was recorded using the whole- 48/80 induced only minor degranulation in WT- and P2rx4 cell patch clamp technique as described previously (21). Patch pipettes had BMMCs but stimulated a significant degranulation in CTMC-like a tip resistance of 4–6 MV when filled with an intracellular solution MCs obtained from both WT and P2rx42/2 mice. Quantitative (30 mM CsCl, 110 mM CsOH, 5 mM MgATP, 3 mM MgCl･6H O, 10 mM 2 RT-PCR analysis showed that although differentiation to http://www.jimmunol.org/ EGTA, 20 mM HEPES, 50 mM aspartic acid [pH 7.2]). Cells dispersed on CTMC-like MCs altered mRNA expression of some P2 re- a poly-L-lysine–coated chamber were perfused with Tyrode solution (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 0.33 mM NaH2PO4,5mM ceptors, such as upregulation of P2X3 and downregulation of HEPES, 1.8 mM CaCl2, 5.5 mM glucose [pH 7.4]) at 37˚C. Membrane P2X7, P2Y1,andP2Y14, there was little difference in P2X4 potentials were controlled by a model Axopatch 200B amplifier (Axon receptor expression (Supplemental Fig. 3A–D). In those prep- Instruments, Foster City, CA). Membrane currents were acquired online arations, ATP clearly enhanced Ag-induced degranulation in and analyzed using pCLAMP10.4 software (Axon). The current–voltage relationship was obtained using ramp pulses from a holding potential of WT-CTMCs, whereas the enhancing effect of ATP was markedly 2/2 210 mV, initially depolarized to 60 mV, then hyperpolarized to 2120 mV, reduced in P2rx4 -CTMCs (Fig. 2C). Similar results were ob- before depolarization back to the holding potential with a speed of 1.0 V/s. tained in peritoneal MCs, a representative CTMCs. Thus, ATP The ramp pulse was applied every 3 s. The P2X4 receptor current was markedly enhanced Ag-induced degranulation in peritoneal MCs by guest on September 23, 2021 calculated at 260 mV in the presence or absence of CuSO4 and was normalized to cell membrane capacitance. obtained from WT mice. This enhancement was significantly re- duced in peritoneal MCs obtained from P2rx42/2-mice, but a Statistics P2X4-independent enhancing component was also retained in All experiments were repeated at least three times. Each value indicates a these cells (Fig. 2D). mean 6 SEM. Statistical analyses of the data were performed using a Student t test for two sample comparison, and one-way ANOVA with Mechanism underlying P2X4 receptor–mediated upregulation Dunnett two-tailed test. The p values ,0.05 were statistically significant. of Ag-induced degranulation Because the P2X4 receptor is a -gated nonselective cation Results channel with high Ca2+ permeability, various physiological Effects of ATP on Ag-induced degranulation in 2/2 responses mediated by P2X4 receptors have been reported as P2rx4 BMMCs Ca2+-dependent. An Ag-induced increase in [Ca2+]i did not differ Bone marrow cells obtained from P2rx42/2 mice were normally between BMMCs from WT and P2rx42/2 mice (Fig. 3A, 3C). differentiated to MCs, and .95% of cells were c-kit and FcεRI Although the ATP-induced Ca2+ response was significantly double positive after 5 wk, with few morphological differences smaller in P2rx42/2 BMMCs, the Ag-induced Ca2+ response in compared with WT-BMMCs (Supplemental Fig. 1A, 1B). In ad- the presence of ATP did not differ between WT- and P2rx42/2 dition, the degranulation responses induced by different concen- BMMCs (Fig. 3B, 3D). trations of Ag or by 1 mM ATP, which were mediated by IgE-bound To evaluate the role of increased [Ca2+]i, the effect of ATP was FcεRI or P2X7 receptors, respectively, did not differ between WT- compared with UTP (100 mM) and ionomycin (0.3 mM), which and P2rx42/2 BMMCs (Supplemental Fig. 1C, 1D). Quantita- increased [Ca2+]i to a similar degree as ATP (Fig. 4A). As shown tive RT-PCR analysis showed that overall mRNA expression of in Fig. 4B, unlike ATP, neither UTP nor ionomycin enhanced purinergic signaling molecules, including P2X and P2Y recep- Ag-induced degranulation. In , K+ efflux via P2X7 tors, P1 adenosine receptors, and , did not receptor channels was shown to trigger NLRP3 inflammasome differbetweenWT-andP2rx42/2 BMMCs except for the P2X4 activation (22). However, ATP enhancement of degranulation by receptor (Supplemental Fig. 2A–D). Ag did not change with high K+ buffer, a condition that impairs K+ As we previously showed (10), ATP enhanced the degranulation efflux (Fig. 4C). We observed that the Ag- and ATP-induced Ca2+ response induced by the threshold concentrations of Ag DNP- response was not affected in high K+ buffer, (Supplemental Fig. HSA in WT-BMMCs. This response to ATP was significantly 4A, 4B). We next tested the effects of Cu2+, a divalent cation that reduced in P2rx42/2 BMMCs (Fig. 1A). Adenosine also increased blocks P2X4 receptor current (23). Because it was difficult to the degranulation response induced by threshold concentrations of demonstrate the P2X4 current in BMMCs, we confirmed the in- 2+ Ag in WT-BMMCs via adenosine A3 receptors (10). This effect of hibitory effects of Cu on ATP-induced current in HEK293 cells 4 ROLE OF P2X4R IN MAST CELL ACTIVATION AND ALLERGIC RESPONSE

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 Downloaded from three independent experiments (n = 3 per group). *p , 0.05, **p , 0.01. ##p , 0.01 compared with the response to Ag alone (-). expressing mouse P2X4 receptor (Supplemental Fig. 4C, 4D). in Fig. 5, ATP itself had no effect but significantly increased Ag- Under similar conditions, ATP-induced enhancement of the de- induced Syk phosphorylation at tyrosine residues 346 and 519/520 granulation response to Ag was not inhibited by Cu2+. These re- in WT-BMMCs. This effect of ATP was reduced in P2rx42/2 sults suggest that ATP enhancement of the degranulation by Ag BMMCs (Fig. 5A–C). Similar effects of ATP were observed with http://www.jimmunol.org/ may involve a novel P2X4 receptor–signal transduction pathway PLCg1 phosphorylation at tyrosine residue 783 in a manner re- that is independent of ion channel activity. quiring P2X4 receptor expression (Fig. 5A, 5D). Although ATP increases Akt phosphorylation, synergistic effects of ATP and Ag Effects of ATP on Ag-induced tyrosine-kinase on Akt phosphorylation were not evident (Fig. 5A, 5E). signaling cascade We observed ATP-induced enhancement of Ag-triggered phos- Cross-linking of IgE-bound FcεRI by Ag triggers activation of a phorylation of Syk at tyrosine residue 346 even in the absence of subset of protein-tyrosine kinases, including the nonreceptor extracellular Ca2+ (Fig. 6A, 6D). Although ATP also increased Ag- protein-tyrosine kinase Syk, which further propagates the signal to induced Syk phosphorylation in P2rx42/2 BMMCs, the extent of various effector proteins, such as PLCg1, and Akt (4). As shown ATP-induced enhancement in P2rx42/2 BMMCs was significantly by guest on September 23, 2021

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 (-). The Journal of Immunology 5

2+ FIGURE 3. Effects of ATP on Ag-induced increase in [Ca ]i in WT- Downloaded from and P2rx42/2 BMMCs. (A and B)WT-orP2rx42/2 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 FIGURE 4. Relationship between P2X4 receptor–mediated enhance- obtained in (A) and (B). Data are presented as the mean 6 SEM (n = 3–4). ment of Ag-induced degranulation and receptor ion channel activity.

*p , 0.05. N.S. no significant difference. (A) Fura 2–loaded BMMCs were stimulated with ATP (100 mM), UTP http://www.jimmunol.org/ (100 mM), or ionomycin (0.3 mM), and changes in [Ca2+]i were monitored. B lesser than in WT-BMMCs (Fig. 6D). Consistent with results Results shown are representative of three independent experiments. ( ) BMMCs were stimulated with vehicle (-), ATP (100 mM), UTP (100 mM), obtained for the degranulation response, ATP enhancement of or ionomycin (0.3 mM) in the presence or absence of Ag (DNP-HSA, Ag-triggered phosphorylation of Syk at tyrosine residue 346 was + 10 ng/ml) for 5 min, and b-Hex released into the reaction medium was observed in the high K -buffer (Fig. 6B, 6E) and in the presence of measured. (C) BMMCs were suspended in normal KRH or high K+ buffer 2+ 2/2 Cu (Fig. 6C, 6F) in WT-BMMCs but not in P2rx4 BMMCs. and stimulated with vehicle (-) or ATP (100 mM) in the presence or ab- P2X7 receptor assembly does not contribute to increased P2X4 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 receptor–mediated degranulation by guest on September 23, 2021 presence or absence of 10 mM CuSO4 for 10 min, and then stimulated with Our results above suggest that P2X4 receptor transduces the vehicle (-) or Ag (DNP-HSA, 10 ng/ml) in the presence or absence of ATP ATP binding signal to intracellular in an ion channel- (100 mM) for 5 min, and b-Hex released into the reaction medium was independent manner. Such a signaling system has been proposed measured. Data are presented as the mean 6 SEM of three independent , for the P2X7 receptor, which has a long cytoplasmic C terminus experiments (n = 3 per group). **p 0.01. (24). In addition, the P2X4 receptor is thought to form a functional 2/2 heterotrimeric structure with the P2X7 receptor (25). We therefore P2rx4 mice (Fig. 8D). Additionally, we further confirmed the examined the effects of ATP on Ag-induced degranulation in absence of any difference between the histamine-induced hypo- 2/2 P2rX72/2 BMMCs. Although P2X7 receptor–mediated degranu- thermia response in WT and P2rx4 mice (Supplemental Fig. 5). lation induced by 1 mM ATP was completely absent in P2rX72/2 BMMCs (Fig. 7A), ATP-induced enhancement of the degranula- Discussion tion response to Ag was observed in P2rX72/2 BMMCs (Fig. 7B), We previously reported that Ag-induced degranulation in BMMCs 2/2 suggesting that the heterotrimeric structure formed with the P2X7 was enhanced by extracellular ATP. Using P2rx4 mice, the receptor is not necessary for P2X4 receptor–mediated enhance- current study demonstrates that the P2X4 receptor was essential ment of degranulation. for ATP-induced enhancement of the degranulation response in BMMCs. ATP-induced enhancement of the degranulation re- Comparison of passive anaphylaxis responses in WT and 2/2 2/2 sponse was absent in BMMCs prepared from P2rx4 mice, P2rx4 mice which showed only minor differences in cell morphology, de- We finally examined whether the absence of P2X4 receptors affects granulation response to Ag, and overall expression of P2X and MC-mediated allergic responses in vivo using two passive ana- P2Y receptors (other than P2X4 receptor) than the WT mice. phylaxis models. For FcεRI-mediated passive cutaneous anaphy- Furthermore, ATP-induced enhancement of degranulation was laxis, mice received intradermal injection of anti-DNP IgE or restored in a rescue experiment, in which P2rx42/2 BMMCs were saline in their ears. After 24 h, mice were challenged by i.v. in- transfected with P2X4 receptor expression plasmid. Based on jection of DNP-HSA and Evans blue. Although the s.c. MC dis- these results, we confirmed that the P2X4 receptor plays an es- tribution in the ear was similar in both WT and P2rx42/2 mice sential role in ATP enhancement of Ag-induced degranulation in (Fig. 8A), dye extravasation for the IgE-injected side in P2rx42/2 BMMCs. mice was significantly alleviated compared with WT mice BMMCs used in the current study, which are dependent on IL-3 (Fig. 8B). In the FcεRI-mediated passive systemic anaphylaxis for proliferation, are relatively immature MCs. Therefore, we also model, Ag-induced body temperature decrease was also signifi- evaluated the effects of ATP on Ag-induced degranulation in cantly alleviated in P2rx42/2 mice (Fig. 8C). Plasma histamine CTMC-like MCs, which were positive for Safranin-O staining and levels after passive systemic anaphylaxis were significantly low in acquired reactivity to the cationic secretagogue compound 48/80. 6 ROLE OF P2X4R IN MAST CELL ACTIVATION AND ALLERGIC RESPONSE

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 (-). Downloaded from

Although this CTMC-like MC preparation showed altered ex- presence of ATP. This result is in clear contrast to the response pression of some P2 receptors, such as upregulation of P2X3 re- after costimulation of MCs with Ag and G protein (Gi)-coupled ceptor and downregulation of P2X7, P2Y1, and P2Y14 receptors, receptor , such as PGE2 or adenosine (7). Specifically, expression of P2X4 receptor was not significantly different from stimulation of Gi-coupled receptor with PGE or adenosine has

2 http://www.jimmunol.org/ expression levels seen in BMMCs. These results indicate that ATP been reported to increase the Ag-induced Ca2+ response markedly, augments Ag-mediated degranulation in CTMC-like cells pre- leading to an augmented Ag-induced degranulation in BMMCs pared from WT mice, whereas ATP effects on degranulation are (7). In addition, the P2X4 receptor–mediated effect of ATP on MC 2/2 largely decreased in cells from P2rx4 mice. We observed an degranulation was not mimicked by the P2Y2 receptor effect of the P2X4 receptor on ATP-mediated enhancement in Ag- UTP or lower concentrations of Ca2+ ionophore ionomycin, which induced degranulation in primary cultured peritoneal MCs, which increased [Ca2+]i to a similar extent as ATP. These results sug- are representative of CTMCs. This result suggests that upregula- gested that although [Ca2+]i measurement in this study could not tion of the MC degranulation response to Ag by P2X4 recep- evaluate a local change in [Ca2+]i, which is effectively coupled to 2+ tor activation is maintained even after differentiation of cells to the degranulation process, P2X4 receptor–mediated Ca influx by guest on September 23, 2021 CTMCs. may not be sufficient to induce augmentation of Ag-induced de- The P2X4 receptor is a ligand-gated nonselective cation channel granulation. In macrophages, intracellular K+ efflux has been with high Ca2+ permeability. Many physiological responses trig- proposed as a mechanism by which inflammasomes are activated gered by activation of this receptor are mediated by the rise via the ionotropic P2X7 receptor (22). However, this mechanism in [Ca2+]i due to Ca2+influx through receptor channels (26). is unlikely to be involved in the augmentation of Ag-induced However, the Ag-induced Ca2+-response was not altered in the degranulation in BMMCs, because even when all extracellular

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 (-). The Journal of Immunology 7

the presence of Cu2+, a condition which inhibits the P2X4 receptor current (23). These results suggest that enhancement of Ag- induced degranulation by ATP may not depend on ion channel activity of the P2X4 receptor. The P2X7 receptor in macrophages is an example of an ionotropic receptor that transmits a signal into cells by an action other than through ion channel activity (24). The P2X7 receptor has a long intracellular C-terminal domain, which interacts with various intracellular signaling proteins. Because the P2X4 receptor is thought to form a functional heterotrimer with the P2X7 receptor (25), and MCs express the P2X7 receptor, as we demonstrate in this study, it is possible that the P2X4 receptor subunit forms a functional heterotrimer with the P2X7 receptor FIGURE 7. P2X7 receptor does not function in ATP-induced augmen- subunit, resulting in transmission of a degranulation-promoting A tation of degranulation in response to Ag. ( ) BMMCs prepared from WT signal through the P2X7 receptor C-terminal domain. However, and P2rx72/2 mice were stimulated with vehicle (-) or ATP (1 mM) for this possibility is unlikely because ATP enhancement of Ag- 5 min, and b-Hex released into the reaction medium was measured. (B) 2/2 induced degranulation was observed in BMMCs prepared from WT- and P2rx7 BMMCs were stimulated with vehicle (-) or Ag (DNP- 2/2 HSA, 10 ng/ml) in the presence or absence of ATP (100 mM) for 5 min, P2rX7 mice. Taken together, our results suggest that the P2X4 and b-Hex released into the reaction medium was measured. Data are receptor may enhance Ag-induced degranulation through an ad- presented as mean 6 SEM from three independent experiments (n = 3 per ditional signal other than that resulting from ion channel activity. Downloaded from group). **p , 0.01. ##p , 0.01 compared with Ag alone (-). 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 activates Na + was replaced with K+ to abrogate K+ movement, augmen- downstream PLCg to transmit signal to degranulation machineries tation of Ag-induced degranulation by ATP was not affected. In (4, 8). Although stimulation of BMMCs with ATP alone failed to 2+ + Ag-mediated MC degranulation, the Ca -activated K channel activate these signaling pathways, ATP augmented Ag-induced http://www.jimmunol.org/ KCa3.1 is known to play a critical role by regulating membrane tyrosine-phosphorylation of Syk and PLCg. This effect of ATP potential (23). This channel is activated through an initial increase was diminished in P2rx42/2 BMMCs, suggesting involvement of in [Ca2+]i, thereby eliciting membrane hyperpolarization, which the P2X4 receptor. An increase in Ag-induced Syk phosphoryla- enhances the driving force for Ca2+ influx through nonselective tion by ATP was also observed in the absence of external Ca2+.In cation channels, such as the P2X4 receptor (24). This mechanism addition, the increase in Ag-induced Syk phosphorylation by ATP should not occur in the K+ buffer, because the opening of the K+ was observed even in the high K+ buffer, and in the presence of channel depolarizes the membrane potential. However, our results Cu2+, in a P2X4 receptor–dependent manner. Therefore, ATP showed that the high K+ buffer did not affect P2X4 receptor– modulation of phosphorylation is most likely mediated by the mediated enhancement of degranulation by Ag. Furthermore, the P2X4 receptor in a manner independent of Ca2+ influx. The P2X4 by guest on September 23, 2021 effect of ATP on Ag-induced degranulation was observed even in receptor contains a short C-terminal domain composed of 26 aa,

FIGURE 8. Role of P2X4 receptor in passive cuta- neous and systemic anaphylaxis. (A) Ear sections from WT or 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 6 SEM (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)WTorP2rx42/2 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. 8 ROLE OF P2X4R IN MAST CELL ACTIVATION AND ALLERGIC RESPONSE including a polar amino acid rich region with six tyrosine resides expression of P2X4 receptor as a functional ATP receptor has been (26). This region has been demonstrated to contain a putative reported in human MCs (38), the role of P2X4 receptor signal in noncanonical tyrosine-based endocytic motif (27). It is therefore human MC function is largely unknown. Therefore, experiments possible that the P2X4 receptor signal may cross-talk with FcεRI- using human MCs are important to translate the results of this induced tyrosine kinase signals due to the short C-terminal do- study to human diseases. main of the P2X4 receptor. Further study is needed to clarify the In conclusion, our results demonstrated that P2X4 receptor detailed mechanism. activation augments Ag-induced MC degranulation in BMMCs and In this study, in vivo experiments using WT and P2rx42/2 mice CTMCs through a novel mechanism in which FcεRI-induced ty- showed that P2X4 receptor–mediated MC hyperactivity was also rosine kinase signaling is increased. We further demonstrated involved in the in vivo allergic reactions. We observed that the using P2rx42/2 mice that the allergic response was up-regulated passive cutaneous anaphylactic reaction was significantly attenu- in vivo in a P2X4 receptor–dependent manner. Although further ated in P2rx42/2 mice. In addition, the decrease in rectal tem- studies are needed in human MCs, our results provide the indi- perature due to the passive whole-body anaphylactic reaction was cation that the P2X4 receptor may be a potential therapeutic target also attenuated in P2rx42/2 mice. We confirmed that the rectal for allergic diseases. temperature decrease induced by histamine did not differ between WT and P2rx42/2 mice, suggesting that Ag-induced MC de- Disclosures 2 2 granulation was abrogated in P2rx4 / mice. Consistently, plasma The authors have no financial conflicts of interest. histamine levels after the passive whole-body anaphylactic reac- 2 2

/ Downloaded from tion were significantly lower in P2rx4 mice. ATP is known to References be released from organs in response to various stimuli (28). The 1. Marshall, J. S. 2004. Mast-cell responses to pathogens. Nat. Rev. Immunol. 4: results of this study suggest that MCs in the body may be stim- 787–799. ulated by ATP constantly released from the surrounding micro- 2. Galli, S. J., S. Nakae, and M. Tsai. 2005. Mast cells in the development of adaptive immune responses. Nat. Immunol. 6: 135–142. environment and that their responsiveness to Ag is enhanced by 3. Metzger, H. 1992. The receptor with high affinity for IgE. Immunol. Rev. 125: P2X4 receptor signal. In general, allergic reactions are known 37–48. 4. Gilfillan, A. M., and J. Rivera. 2009. The tyrosine kinase network regulating to be exacerbated when tissues are scraped due to itching (29). http://www.jimmunol.org/ mast cell activation. Immunol. Rev. 228: 149–169. Because there is ample evidence that physical stimuli such as 5. Morimoto, K., N. Shirata, Y. Taketomi, S. Tsuchiya, E. Segi-Nishida, T. Inazumi, scratching trigger the release of ATP from cells (28, 30, 31), the K. Kabashima, S. Tanaka, M. Murakami, S. Narumiya, and Y. Sugimoto. 2014. activation of MCs via the P2X4 receptor shown in this study may Prostaglandin E2-EP3 signaling induces inflammatory swelling by mast cell activation. J. Immunol. 192: 1130–1137. be important for understanding mechanical stimuli-induced ex- 6. Jolly, P. S., M. Bektas, A. Olivera, C. Gonzalez-Espinosa, R. L. Proia, J. Rivera, acerbation of allergic reactions. S. Milstien, and S. Spiegel. 2004. Transactivation of sphingosine-1-phosphate receptors by FcepsilonRI triggering is required for normal mast cell degranu- To date, many critical physiological and pathophysiological lation and chemotaxis. J. Exp. Med. 199: 959–970. responses occurring via the P2X4 receptor have been reported, 7. Laffargue, M., R. Calvez, P. Finan, A. Trifilieff, M. Barbier, F. Altruda, indicating that this receptor acts as a sensor of shear stress-induced E. Hirsch, and M. P. Wymann. 2002. Phosphoinositide 3-kinase g is an essential amplifier of mast cell function. Immunity 16: 441–451. by guest on September 23, 2021 ATP release from vascular endothelial cells and plays a role in 8. Nunomura, S., Y. Gon, T. Yoshimaru, J. Kashiwakura, T. Kawakami, and C. Ra. regulating vascular tension via NO release (17). In the nervous 2010. FcepsilonRI b-chain ITAM amplifies PI3K-signaling to ensure synergistic system, P2X4 receptors are upregulated in spinal during degranulation response via FcepsilonRI and adenosine receptors. Eur. J. Immu- nol. 40: 1205–1217. nerve injury, leading to (32). The P2X4 receptor 9. Kuehn, H. S., and A. M. Gilfillan. 2007. G protein-coupled receptors and the is thought to be critical for activation and migration, which modification of FcepsilonRI-mediated mast cell activation. Immunol. Lett. 113: 59–69. are closely related to rejection of transplanted tissue (33). It has 10. Yoshida, K., M. Ito, and I. Matsuoka. 2017. Divergent regulatory roles of ex- been reported that P2X4 receptor expression is increased in the tracellular ATP in the degranulation response of mouse bone marrow-derived airway of asthmatic model mice, and P2X4 deficiency alleviates mast cells. Int. Immunopharmacol. 43: 99–107. 11. Kurashima, Y., T. Amiya, T. Nochi, K. Fujisawa, T. Haraguchi, H. Iba, allergic inflammation (34). The results presented in this study on H. Tsutsui, S. Sato, S. Nakajima, H. Iijima, et al. 2012. Extracellular ATP me- MC function contribute a novel role for P2X4 receptors in the diates mast cell-dependent intestinal inflammation through P2X7 purinoceptors. regulation of biological functions. Nat. Commun. 3: 1034. 12. Kurashima, Y., T. Amiya, K. Fujisawa, N. Shibata, Y. Suzuki, Y. Kogure, The present study has focused on the role of the P2X4 receptor in E. Hashimoto, A. Otsuka, K. Kabashima, S. Sato, et al. 2014. The regulating MC function. However, other –mediated Cyp26b1 mediates inhibition of mast cell activation by fibroblasts to maintain skin-barrier homeostasis. Immunity 40: 530–541. enhancement of Ag-induced degranulation has already been 13. Wareham, K. J., and E. P. Seward. 2016. P2X7 receptors induce degranulation in reported. For example, Gao et al. (35, 36) demonstrated that human mast cells. Purinergic Signal. 12: 235–246. Ag-induced degranulation was enhanced by the P2Y or P2Y 14. Yoshida, K., M. Ito, Y. Hoshino, and I. Matsuoka. 2017. Effects of dexameth- 13 14 asone on purinergic signaling in murine mast cells: selective suppression of receptors. In this study, we also observed P2X4 receptor– P2X7 receptor expression. Biochem. Biophys. Res. Commun. 493: 1587–1593. independent regulatory action under certain conditions. For ex- 15. Gurish, M. F., and K. F. Austen. 2012. Developmental origin and functional ample, enhancement of Ag-induced degranulation by ATP in specialization of mast cell subsets. Immunity 37: 25–33. 16. Takano, H., S. Nakazawa, Y. Okuno, N. Shirata, S. Tsuchiya, T. Kainoh, CTMC-like cells and peritoneal MCs possessed a P2X4 receptor– S. Takamatsu, K. Furuta, Y. Taketomi, Y. Naito, et al. 2008. Establishment of the independent component in P2rx42/2 MCs and the enhancement culture model system that reflects the process of terminal differentiation of 2+ connective tissue-type mast cells. FEBS Lett. 582: 1444–1450. of Ag-induced phosphorylation of Syk by ATP under Ca -free 17.Yamamoto,K.,T.Sokabe,T.Matsumoto,K.Yoshimura,M.Shibata,N.Ohura, 2/2 conditions remained in P2rx4 MCs. Analysis of such re- T. Fukuda, T. Sato, K. Sekine, S. Kato, et al. 2006. Impaired flow-dependent control sponses may contribute to elucidation of the diversity of puri- of vascular tone and remodeling in P2X4-deficient mice. Nat. Med. 12: 133–137. 18. Hirayama, Y., and S. Koizumi. 2017. Hypoxia-independent mechanisms of HIF- nergic regulation. 1a expression in after ischemic preconditioning. Glia 65: 523–530. Because this study was conducted using a mouse model, it was 19. Ito, M., and I. Matsuoka. 2008. Regulation of purinergic signaling by prosta- necessary to confirm whether the same mechanism operates in glandin E2 in murine macrophages. J. Pharmacol. Sci. 107: 443–450. 20. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by human MCs. In fact, adenosine is well known to enhance Ag- acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159. induced degranulation through the adenosine A3 receptor in mice (7, 8), whereas in human MCs, adenosine is shown to inhibit 21. Watano, T., I. Matsuoka, and J. Kimura. 2002. Characteristics of ATP-induced current through P2X7 receptor in NG108-15 cells: unique antagonist sensitivity Ag-induced degranulation via the A2A receptor (37). Although and lack of pore formation. Jpn. J. Pharmacol. 88: 428–435. The Journal of Immunology 9

22. Katsnelson, M. A., L. G. Rucker, H. M. Russo, and G. R. Dubyak. 2015. K+ 32. Tsuda, M., Y. Shigemoto-Mogami, S. Koizumi, A. Mizokoshi, S. Kohsaka, efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ M. W. Salter, and K. Inoue. 2003. P2X4 receptors induced in spinal microglia signaling. J. Immunol. 194: 3937–3952. gate tactile allodynia after nerve injury. Nature 424: 778–783. 23. Acun˜a-Castillo, C., B. Morales, and J. P. Huidobro-Toro. 2000. and copper 33. Ledderose, C., K. Liu, Y. Kondo, C. J. Slubowski, T. Dertnig, S. Denicolo´, modulate differentially the P2X4 receptor. J. Neurochem. 74: 1529–1537. M. Arbab, J. Hubner, K. Konrad, M. Fakhari, et al. 2018. Purinergic P2X4 re- 24. Wilson, H. L., S. A. Wilson, A. Surprenant, and R. A. North. 2002. Epithelial ceptors and mitochondrial ATP production regulate T cell migration. J. Clin. membrane proteins induce membrane blebbing and interact with the P2X7 re- Invest. 128: 3583–3594. ceptor C terminus. J. Biol. Chem. 277: 34017–34023. 34. Zech, A., B. Wiesler, C. K. Ayata, T. Schlaich, T. Du¨rk, M. Hoßfeld, N. Ehrat, 25. Guo, C., M. Masin, O. S. Qureshi, and R. D. Murrell-Lagnado. 2007. Evidence for S. Cicko, and M. Idzko. 2016. P2rx4 deficiency in mice alleviates allergen- functional P2X4/P2X7 heteromeric receptors. Mol. Pharmacol. 72: 1447–1456. induced airway inflammation. Oncotarget 7: 80288–80297. 26. Suurva¨li, J., P. Boudinot, J. Kanellopoulos, and S. Ru¨u¨tel Boudinot. 2017. P2X4: 35. Gao, Z.-G., Y. Ding, and K. A. Jacobson. 2010. P2Y(13) receptor is responsible a fast and sensitive . Biomed. J. 40: 245–256. for ADP-mediated degranulation in RBL-2H3 rat mast cells. Pharmacol. Res. 27. Royle, S. J., L. K. Bobanovic´, and R. D. Murrell-Lagnado. 2002. Identification 62: 500–505. of a non-canonical tyrosine-based endocytic motif in an ionotropic receptor. 36. Gao, Z.-G., Y. Ding, and K. A. Jacobson. 2010. UDP-glucose acting at P2Y14 J. Biol. Chem. 277: 35378–35385. receptors is a mediator of mast cell degranulation. Biochem. Pharmacol. 79: 28. Lazarowski, E. R. 2012. Vesicular and conductive mechanisms of 873–879. release. Purinergic Signal. 8: 359–373. 37. Matsuo, Y., Y. Yanase, R. Irifuku, K. Ishii, T. Kawaguchi, S. Takahagi, I. Hide, 29. Leslie, T. A., M. W. Greaves, and G. Yosipovitch. 2015. Current topical and and M. Hide. 2018. The role of adenosine for IgE receptor-dependent degran- systemic therapies for itch. Handb. Exp. Pharmacol. 226: 337–356. ulation of human peripheral basophils and skin mast cells. Allergol. Int. 67: 524– 30. Dosch, M., J. Gerber, F. Jebbawi, and G. Beldi. 2018. Mechanisms of ATP re- 526. lease by inflammatory cells. Int. J. Mol. Sci. DOI: 10.3390/ijms19041222. 38. Wareham, K., C. Vial, R. C. Wykes, P. Bradding, and E. P. Seward. 2009. 31. Giuliani, A. L., A. C. Sarti, and F. Di Virgilio. 2019. Extracellular Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in and nucleosides as signalling molecules. Immunol. Lett. 205: 16–24. human lung mast cells. Br. J. Pharmacol. 157: 1215–1224. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021