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of March 23, 2018. This information is current as Monosodium Urate Crystals Inflammatory Responses Induced by Receptor Signaling Pathway Mediates 6 P2Y Shinichi Sato and Kunihiko Tamaki Makoto Sugaya, Takafumi Kadono, Akihiko Asahina, Masahiro Kamata, Carren Sy Hau, Yoshihide Asano, Hideya Uratsuji, Yayoi Tada, Tomohiko Kawashima, ol.1003746 http://www.jimmunol.org/content/early/2011/11/18/jimmun published online 18 November 2011 J Immunol Material Supplementary 6.DC1 http://www.jimmunol.org/content/suppl/2011/11/18/jimmunol.100374 average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved. Copyright © 2011 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on March 23, 2018 http://www.jimmunol.org/ Downloaded from by guest on March 23, 2018 http://www.jimmunol.org/ Downloaded from

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Page 1: Monosodium Urate Crystals Inflammatory Responses Induced by

of March 23, 2018.This information is current as

Monosodium Urate CrystalsInflammatory Responses Induced by

Receptor Signaling Pathway Mediates6P2Y

Shinichi Sato and Kunihiko TamakiMakoto Sugaya, Takafumi Kadono, Akihiko Asahina,Masahiro Kamata, Carren Sy Hau, Yoshihide Asano, Hideya Uratsuji, Yayoi Tada, Tomohiko Kawashima,

ol.1003746http://www.jimmunol.org/content/early/2011/11/18/jimmun

published online 18 November 2011J Immunol 

MaterialSupplementary

6.DC1http://www.jimmunol.org/content/suppl/2011/11/18/jimmunol.100374

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2011 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

P2Y6 Receptor Signaling Pathway Mediates InflammatoryResponses Induced by Monosodium Urate Crystals

Hideya Uratsuji,* Yayoi Tada,* Tomohiko Kawashima,* Masahiro Kamata,* Carren Sy Hau,*

Yoshihide Asano,* Makoto Sugaya,* Takafumi Kadono,* Akihiko Asahina,† Shinichi Sato,*

and Kunihiko Tamaki*

Gout occurs in individuals with hyperuricemia when monosodium urate (MSU) crystals precipitate in tissues and induce acute

inflammation via phagocytic cells such as monocytes. MSU crystals have been demonstrated in skin diseases such as tophaceous

gout or psoriasis; however, the importance of MSU crystals in the skin is totally unknown. In this study, we found thatMSU crystals,

through P2Y6 receptors, stimulated normal human keratinocytes (NHK) to produce IL-1a, IL-8/CXCL8, and IL-6. P2Y6 receptor

expression increased in MSU-stimulated NHK. Both P2Y6-specific antagonist and P2Y6 antisense oligonucleotides significantly

inhibited the production of IL-1a, IL-8/CXCL8, and IL-6 by NHK. Similarly, the P2Y6-specific antagonist completely inhibited

the MSU-induced production of IL-1b by THP-1 cells, a human monocytic cell line. Remarkably, the P2Y6-specific antagonist

significantly reduced neutrophil influx in both mouse air pouch and peritonitis models. Thus, these results indicate that the P2Y6

receptor signaling pathway may be a potential therapeutic target for MSU-associated inflammatory diseases, such as tophaceous

gout. The Journal of Immunology, 2012, 188: 000–000.

Uric acid is the natural end product of purine nucleotidecatabolism and is normally present in plasma in solu-ble form. However, beyond maximal solubility, uric acid

can crystallize. These monosodium urate (MSU) crystals are wellknown to be the cause of gout. Gout is generally associated withhyperuricemia and is characterized by deposition of MSU crystalswithin joints or skin, stimulating acute inflammation. In gout, MSUcrystals stimulate the production of inflammatory cytokines such asIL-1, TNF-a, and IL-6 and chemotactic factors for neutrophilssuch as IL-8/CXCL8 and S100A8/A9 (1–6). Because the MSUcrystal-mediated pathways in monocytes/macrophages are beingintensely investigated, the mechanisms are becoming clear. Recentobservations point out that ingestion of MSU crystals by phag-ocytes stimulates the production of inflammatory cytokines andchemokines through activation of the inflammasome (7, 8).Uric acid is released from injured or infected cells and pre-

cipitates and forms MSU crystals. These MSU crystals are knownto act as danger signals that activate the immune system (9). Be-cause epidermis is located in the most external part of the bodycompared with other organs, it is most frequently exposed to var-ious stimuli and Ags such as UV irradiation, physical obstruc-tion, pathogens, allergens, and so on. In addition, apoptosis and

necrosis of keratinocytes (KC) is observed in many inflammatoryskin diseases including atopic eczema, contact dermatitis, lichenplanus, and toxic epidermal necrosis. Therefore, cell injury ornecrosis frequently occurs in KC, and, as a result, MSU wouldeasily precipitate within epidermis. Because uric acid is the lastmetabolic product of nucleic acids, MSU could also appear in skindiseases showing epidermal hyperproliferation. In fact, MSU crys-tals have been found in the epidermis in psoriatic lesions, in whichepidermal hyperproliferation is known (10, 11).However, precisely how MSU crystals trigger inflammation in

the epidermis remains unknown. Therefore, we were prompted toexamine how the MSU crystals might stimulate KC to producecytokines and chemokines. In this study, we showed that MSUcrystals could induce the production of inflammatory cytokines andchemokines and that their production was mainly mediated by theP2Y6 receptor pathway. Furthermore, we showed that P2Y6 re-ceptors were essential in MSU-induced inflammation not only inKC but also in THP-1 cells, a human monocytic cell line, andin vivo in an MSU-induced inflammation model.

Materials and MethodsCells

Normal human keratinocytes (NHK) were purchased from Kurabo (Osaka,Japan). The cells were cultured in serum-free medium, Humedia-KG2(Kurabo), supplemented with insulin (10 mg/ml), human recombinantepidermal growth factor (0.1 ng/ml), hydrocortisone (0.5 mg/ml), genta-micin (50 mg/ml), amphotericin B (50 ng/ml), and bovine pituitary extract(0.4%, v/v). THP-1 cells were purchased from American Type CultureCollection (Manassas, VA). THP-1 cells were cultured in RPMI 1640medium supplemented with 10% heat-inactivated FBS and penicillin–streptomycin. THP-1 cells were treated for 3 h with 0.5 mM PMA (Sigma,St. Louis, MO) the day before stimulation. After PMA treatment, THP-1cells were washed with PBS. This treatment increases the phagocyticproperties of the cells and induces a constitutive production of pro–IL-1b.The next day, THP-1 cells were washed with PBS, and OptiMEM (Invi-trogen, Carlsbad, CA) was added for stimulation.

Reagents

MSU crystals were purchased from Alexis (Lausen, Switzerland). Caspase-1inhibitor (z-YVAD-FMK), U-73122, and U-73433 were purchased from

*Department of Dermatology, Graduate School of Medicine, University of Tokyo,Tokyo 113-8655, Japan; and †Department of Dermatology, Sagamihara National Hos-pital, Kanagawa 252-0392, Japan

Received for publication November 11, 2010. Accepted for publication October 20,2011.

This work was supported by grants from the Ministry of Education, Culture, Sports,Science, and Technology of Japan (to K.T. and Y.T.).

Address correspondence and reprint requests to Dr. Yayoi Tada, Department ofDermatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo,Bunkyo-ku, Tokyo 113-8655, Japan. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: IL-1RA, IL-1 receptor antagonist; KC, keratino-cytes; MSU, monosodium urate; NHK, normal human keratinocytes; PI, propidiumiodide; PLC, phospholipase C; RB2, Reactive Blue 2; siRNA, small interfering RNA.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003746

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Merck KGaA (Darmstadt, Germany). Recombinant human TNF-a, recom-binant human IL-1 receptor antagonist (IL-1RA), monoclonal anti-humanTNF-a Ab, monoclonal anti-TNF receptor I Ab, anti-human IL-1a Ab, andanti-human IL-1b Ab were purchased from R&D Systems (Minneapolis,MN). Suramin, KN-62, Reactive Blue 2 (RB2), uricase from Arthrobacterglobiformis, and MRS2578 were purchased from Sigma. Soluble uric acidwas purchased from Wako Pure Chemical (Osaka, Japan).

Cytokine detection by ELISA

NHK were plated at 2.0 3 104 cells/well in a 24-well plate. When theyreached subconfluency, the medium was removed, and fresh medium wasapplied along with MSU (50, 100, 200, 500 mg/ml). After 24 h, the super-natants were collected. NHK were pretreated with IL-1RA, anti-TNF re-ceptor, anti–TNF-a, suramin, RB2, MRS2578, U-73122, and U-73343 for 1h before adding MSU (200 or 500 mg/ml). The supernatants were collectedafter stimulation with MSU for 24 h. IL-1a, IL-1b, TNF-a, IL-8/CXCL8,and IL-6 concentrations in the supernatants were measured by ELISA (R&DSystems) according to the manufacturer’s protocol. IL-18 concentration wasmeasured by ELISA purchased from MBL (Nagoya, Japan). The effect ofreagents on NHK viability was examined by trypan blue staining or MTTassay using MTT cell count kit (Nacalai Tesque, Kyoto, Japan) according tothe manufacturer’s protocol. THP-1 cells were plated at 4.03 105 cells/wellin a 24-well plate after PMA stimulation. The cells were pretreated withz-YVAD, MRS2578, and U-73122 for 1 h before applying MSU (100 mg/ml).The supernatants were collected after stimulation with MSU for 6 h. IL-1bconcentration in the supernatants was measured by ELISA (R&D Systems)according to the manufacturer’s protocol.

mRNA expression analysis

Total RNAwas extracted with RNeasy kit (Qiagen, Valencia, CA). DNAse I(Invitrogen) was used to avoid genomic DNA contamination. We synthesizedcDNA from total RNA (1mg) using Superscript First-Strand Synthesis Systemfor RT-PCR (Invitrogen). We performed PCR analysis as templates of thesynthesized cDNA. Nucleotide sequences for RT-PCR are shown in Table I.The mixture was pre-denatured for 5 min at 94˚C and then subjected to 35cycles of 94˚C for 30 s, 55˚C for 30 s, 72˚C for 1 min, then 72˚C for 10 min.Amplified DNA fragments were judged from 2% agarose gel electrophore-sis. For real-time RT-PCR, we used the ABI Prism 7000 sequence detectionsystem (Applied Biosystems, Carlsbad, CA). A 50-ml reaction mixture con-taining cDNA diluted in RNase-free water and 2.5 ml TaqMan Gene Ex-pression Assays Inventoried was mixed with 25 ml TaqMan Universal PCRMaster Mix (23; Applied Biosystems). Gene Expression Assays Inventoriedconsists of a 203 mix of unlabeled PCR primers and TaqMan MGB probes(FAM dye labeled). Assay IDs Hs99999028_m1, Hs00602548_m1, andHs99999905_m1 were used for IL-1a, P2Y6, and GAPDH, respectively. Thereaction conditions were designed as followed: 50˚C for 2 min, 95˚C for 10min, followed by 40 cycles of 15 s at 95˚C for denaturation and 1 min at 60˚Cfor annealing and extension. The threshold cycle, the cycle number at whichthe amount of amplified gene of interest reached a fixed threshold, wassubsequently determined. Expression was normalized to mRNA for GAPDH.

Immunoblot analysis

Cells were disrupted in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophos-phate, and 1 mM b-glycerophosphate) with 1 mM PMSF (Nacalai Tesque),1 mg/ml leupeptin (Peptide Institute, Louisville, KY), and 1 mM sodiumorthovanadate (Sigma). The concentrations of the extracted proteins weremeasured using a BCA Protein Assay Kit (Thermo Fisher Scientific,Waltham, MA). The samples were mixed with equal volume of 23 samplebuffer (100 mM Tris HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue,20% glycerol, 1% 2-mercaptoethanol, v/v), boiled for 3 min, and separatedby 12.5% SDS-PAGE (3 mg of protein per lane). After transfer to anImmobilon-P transfer membrane (Millipore, Billerica, MA), the membranewas incubated in blocking buffer (5% skim milk in 25 mM Tris/0.02%KCl/0.8% NaCl, pH 7.4; Tris-buffered saline) for 1 h at room temperature,followed by an appropriate primary Ab overnight at 4˚C. The membranewas washed and incubated with a secondary Ab for 30 min, and the bandswere visualized using a chemiluminescence method (LumiGLO Reagentand Peroxide; Cell Signaling). We used rabbit polyclonal anti-human P2Y6

Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonalanti-human b-actin Ab (Cell Signaling, Danvers, MA) as primary Ab andanti-rabbit IgG HRP-linked Ab (Cell Signaling) as secondary Ab.

Treatment with antisense oligonucleotides

Antisense or sense oligonucleotides against P2Y6 protein were synthesizedas phosphorothioate-modified oligonucleotides and were purified by high-

pressure liquid chromatography. The oligonucleotides were P2Y6 antisense(59-CATTGTCCCATTCCATGGC-39), and P2Y6 sense (59-GCCATGGA-ATGGGACAATG-39). NHK were transfected with a final volume of 0.5mM of the indicated oligonucleotides premixed with FuGENE 6 (RocheDiagnostics, Basel, Switzerland) in KG2 for 24 h. The medium wasaspirated, and the cells were cultured with KG2 for 24 h and then treatedwith MSU (200 mg/ml) for 24 h. THP-1 cells were washed twice in PBSand cultured in serum-free RPMI 1640 medium. Cells were supplementedwith 2.5% oligofectamine (Invitrogen) and various concentrations of oli-gonucleotides and were incubated for 4 h. Then, 4 volumes of completeculture medium was added and cultured overnight. The next day, the cellmedium was replaced with OptiMEM, and the cells were treated withMSU (100 mg/ml) for 6 h.

Transfection with small interfering RNA

All transfections were done using Lipofectamine 2000 following themanufacturer’s instructions (Invitrogen). P2Y6 small interfering RNA(siRNA; M-004579-02-0005; Thermo Scientific Dharmacon) or negativecontrol siRNA (D-001210-05-05; Thermo Scientific Dharmacon) wastransfected into 30–50% subconfluent NHK in 6-well plates by Lipofect-amine 2000. Medium was changed after 6 h. After another 24 h, freshmedium with MSU 200 mg/ml was infused. After 24 h of incubation withMSU, P2Y6 mRNA and protein expressions were assayed by RT-PCR andimmunoblot, respectively. Cell supernatants were collected, and IL-1a andIL-8/CXCL8 levels were assessed by ELISA.

In vivo MSU-induced mouse inflammation model

Air pouches were created on the backs of 8-wk-old female BALB/c mice(Japan SLC, Hamamatsu, Japan). Three milliliters of filtered air was injecteds.c., followed by a second injection of 3ml of filtered air after 3 d. Seven daysafter the first injection, 2 mg MSU crystals in 1 ml PBS or 1 ml PBS alonewas injected into the air pouches. MRS2578 or PBS was injected into the airpouch 1 h before MSU injection. After 9 h, the pouch fluid was harvested byinjecting 1ml PBS. Peritonitis was induced by injection of 1mgMSU in 1mlPBS. Equal volumes of PBS were injected into mice and served as negativecontrols. Six hours after injection, peritoneal cavities were harvested with 8ml PBS. To analyze the involvement of P2Y6 receptors, mice were injectedwith MRS2578 (5 mg/body) in 1 ml PBS 1 h before MSU. The harvestedcells were incubated with anti-mouse CD16/CD32 mAb 2.4G2 (BD Bio-sciences, Franklin Lakes, NJ) to block FcgII/III receptors and stainedwith FITC-conjugated Ly-6G (BD Biosciences). After propidium iodide (PI)was added to exclude dead cells, the cells were analyzed using a FACScanflow cytometer equipped with CellQuest software (FACSCaliber; BD Bio-sciences). The number of neutrophils was determined by measuring thenumber of Ly-6G(+)/PI(2) cells. All animal experiments were approved bythe Animal Research Committee of the University of Tokyo.

Data analysis and statistics

Data represent mean 6 SEM. Statistical differences between two groupswere determined using two-tailed Student and Aspin–Welch t tests fol-lowing F-test. Differences were considered to be significant at p , 0.05.

ResultsIL-1a plays a central role in MSU-induced inflammation inhuman KC

In monocytes, MSU is reported to induce the production of vari-ous inflammatory cytokines and chemokines including IL-1b andIL-18 (2–4). In NHK, MSU also induced the production of smallbut significant amounts of TNF-a and IL-1b and large amountsof IL-8/CXCL8 together with IL-18 and IL-6 (Fig. 1A). Thesecytokine productions were induced in a time-dependent manner.When we used soluble uric acid or uricase-digested MSU for stim-ulation, IL-8/CXCL8 production from NHK was impaired (Sup-plemental Fig. 1). These findings indicated that MSU crystals caninduce the production of inflammatory cytokines and chemokinesfrom KC, as from monocytes.Because the activation of IL-1R is known to be essential for

propagation of the inflammatory cascade triggered by MSUcrystals (1), we next studied the importance of IL-1R in the MSU-induced production of cytokines by NHK. IL-1RA significantlyinhibited the production of both IL-8/CXCL8 and IL-6 from

2 P2Y6 RECEPTOR MEDIATES RESPONSES BY MSU

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MSU-stimulated NHK (Fig. 1B). TNF-a is also known to be aninflammation-inducing cytokine; however, neither anti–TNF-a Abnor anti-TNF receptor Ab inhibited the production of these cyto-

kines, although these Abs inhibited TNF-a–induced IL-8/CXCL8production (data not shown). These results showed that IL-1 playsa central role in inducing IL-8/CXCL8 and IL-6 production from

FIGURE 1. IL-1a and P2Y receptors regulate MSU-induced IL-8/CXCL8 and IL-6 production from NHK. A, IL-1b, IL-18, TNF-a, IL-8/CXCL8, and

IL-6 in supernatants of NHK stimulated by indicated dose of MSU for 24 h. B, IL-8/CXCL8 and IL-6 concentration in supernatants of NHK stimulated by

MSU (200 mg/ml) for 24 h in presence of IL-1RA. C, IL-1a concentration in supernatants of NHK stimulated by indicated dose of MSU for 24 h. D, IL-8/

CXCL8 and IL-6 concentration in supernatants of NHK stimulated by MSU (200 mg/ml) for 24 h in presence of anti–IL-1a or anti–IL-1b Ab. E, IL-1a, IL-8/

CXCL8, and IL-6 concentration in supernatants of NHK stimulated by MSU (200 mg/ml) for 24 h in the presence of suramin or RB2. Data show mean 6SEM of three or four independent experiments. *p , 0.05, **p , 0.01 (significant differences from the response to MSU alone).

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MSU-stimulated NHK. Although IL-1b appears to be the domi-nant form of IL-1 produced by monocytes, macrophages, and den-dritic cells, IL-1a predominates in the epithelial cells, including

KC. Thus, we investigated the production of IL-1a by MSU-stimulated NHK. We observed large amounts of IL-1a secretedfrom the MSU-stimulated NHK compared with the amounts of

Table I. List of primers for P2Y receptors and GAPDH

Gene SequencesAccession Number

in GenBankProductSize (bp)

P2Y1 Forward 59-GGTCTAGCAAGTCTCAACAG-39 NM002563 359Reverse 59-AAGCTAAGTGTGGATGTGGG-39

P2Y2 Forward 59-TTGCCGTCATCCTTGTCTGT-39 NM002564 433Reverse 59-CTGCCCAACACATCTTCTAT-39

P2Y4 Forward 59-TGTCCTTTTCCTCACCTGCA-39 NM002565 440Reverse 59-AGTAAATGGTGCGGGTGATG-39

P2Y6 Forward 59-CACATCACCAAGACAGCCTA-39 NM004154 340Reverse 59-TCTTAACTCCATGCCCAGCT-39

P2Y11 Forward 59-AGAAGCTGCGTGTGGCAGCGTTGTT-39 NM002566 369Reverse 59-ACGGTTTAGGGGCGGCTGTGGCATT-39

P2Y12 Forward 59-GTGTCAAGTTACCTCCGTCA-39 NM022788 274Reverse 59-ATGCCAGACTAGACCGAACT-39

P2Y13 Forward 59-CCTTTCAAAATCCTCTCTGACTC-39 NM023914 266Reverse 59-TCCTTGTTGCTCAAGATCGT-39

P2Y14 Forward 59-CTCTGCCGTGCTCTTCTACGTCAA-39 NM014879 275Reverse 59-TTGATGCTTTGTGCCACTTCCGT-39

GAPDH Forward 59-TGAAGGTCGGAGTCAACGGATTTGGT-39 NM002046 983Reverse 59-CATGTGGGCCATGAGGTCCACCAC-39

FIGURE 2. MSU increases P2Y6 receptor expression in NHK. A, The mRNA expression of P2Y receptors was compared between NHK stimulated with

MSU for 24 h and no stimulation. Human reference cDNAwas used as positive control for expression of P2Y receptors. RT-PCR analysis was performed

using primer sets in Table I. B, Quantification of P2Y6 receptor mRNA expression in MSU-stimulated NHK normalized by GAPDH using real-time RT-

PCR. Data show mean 6 SEM of three independent experiments. Student t test was used to calculate p values. C, Comparison of P2Y6 receptor protein

expression in NHK between no stimulation and stimulated by MSU (200 mg/ml) using immunoblot analysis.

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IL-1b (Fig. 1A, 1C). MSU not only increased the IL-1a secretionbut also increased IL-1a mRNA expression in a dose- and time-dependent manner (data not shown). Furthermore, anti–IL-1a neu-tralizing Ab, but not anti–IL-1b Ab, significantly suppressed theproduction of IL-8/CXCL8 and IL-6 (Fig. 1D). Additionally,when we treated NHK with caspase-1 inhibitor z-YVAD, theproduction of IL-1b was inhibited, however, IL-1a, IL-8/CXCL8,and IL-6 production was not (Supplemental Fig. 2). Thus, these

results indicate that IL-1a–mediated signaling through IL-1R ispivotal for subsequent cytokine and chemokine secretion, such asIL-8/CXCL8 and IL-6, from NHK after MSU stimulation.

MSU induces inflammation via P2Y receptors in human KC

Our next interest was how MSU crystals stimulate KC. AlthoughKC are capable of phagocytosing melanosomes (12), the resultusing electron microscopy did not suggest any phagocytosis of

FIGURE 3. MSU crystals induce inflammation via P2Y6 receptors in NHK. A, IL-1a, IL-8/CXCL8, and IL-6 concentration in supernatants of NHK

stimulated by MSU (200 mg/ml) for 24 h in the presence of MRS2578. B, P2Y6 receptor protein expression in NHK treated with the indicated oligo-

nucleotides. C, IL-1a, IL-8/CXCL8, and IL-6 concentration in supernatants of NHK stimulated by MSU (200 mg/ml) for 24 h after treatment with the

indicated oligonucleotides. D, IL-1a, IL-8/CXCL8, and IL-6 concentration in supernatants of NHK stimulated by MSU (200 mg/ml) for 24 h in presence of

U-73122 or U-73343. Data show mean 6 SEM of three or four independent experiments. A and D, *p , 0.05, **p , 0.01 (significant differences from the

response to MSU alone). C, Student or Aspin–Welch t test was used to calculate p values.

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MSU crystals by NHK (Supplemental Fig. 3), and cytochalasin D,a phagocytosis inhibitor (8), did not suppress the production ofIL-1a, IL-8/CXCL8, and IL-6 (data not shown). We speculated thatNHK might be stimulated by MSU in a different manner fromphagocytes; therefore, we attempted to identify the cell surfacereceptors for MSU on KC. IL-1RA, which inhibited IL-8/CXCL8and IL-6 production from NHK, did not inhibit IL-1a productionby MSU-stimulated NHK (data not shown), indicating that IL-1Ris not the cell surface receptor for MSU on NHK. We next ex-amined the role of P2 receptors in the MSU-induced cytokineproduction by NHK, as uric acid is a breakdown product of pu-rines (ATP, GTP, and nucleic acids), and P2 receptors senseextracellular nucleotides such as ATP, ADP, UTP, and UDP (13).We first focused on the P2X7 receptors, as P2X7 receptor an-tagonist is known to inhibit MSU-induced IL-1b production inmonocytes by inhibiting autocrine ATP stimulation (14). However,KN-62, a specific P2X7 receptor antagonist, did not inhibit theMSU-induced production of IL-1a, IL-8/CXCL8, or IL-6 withNHK (data not shown). We next examined the effects of suramin,a P2 receptor antagonist, and RB2, a P2Y receptor antagonist, onNHK. It should be noted that suramin is not a P2X7 receptorantagonist (15). We first confirmed that none of these antagonistswas toxic to NHK by MTT assay and trypan blue staining (datanot shown). Surprisingly, both suramin and RB2 almost completelyinhibited the MSU-induced production of IL-1a, IL-8/CXCL8,and IL-6 (Fig. 1E). These results revealed that the stimulation ofNHK by MSU is mediated by P2Y receptors.

P2Y6 receptors mediate MSU-induced inflammation in humanKC

To identify which of the P2Y receptors might be responsible forthe stimulation of NHK by MSU, we first attempted to identifyP2Y receptors expressed in NHK (Table I). As shown in Fig. 2A,the results of RT-PCR analysis revealed that NHK expressed themRNA for P2Y1,2,6,11. MSU stimulation of NHK enhanced orinduced the expression of P2Y6,12,14 but not other P2Y receptorsubtypes. Among these P2Y receptors, we focused on the P2Y6

receptors, as their mRNA expression was enhanced after MSUstimulation, and both suramin and RB2 are known to be P2Y6

antagonists but not P2Y14 antagonists (13). The P2Y6 receptorswere expressed constitutively in NHK, and we confirmed thatMSU stimulation enhanced their expression at both mRNA andprotein levels by real-time RT-PCR and immunoblot, respectively(Fig. 2B, 2C). To examine the role of P2Y6 receptors in MSU-induced production of cytokines by NHK, we used MRS2578,a specific P2Y6 antagonist (16), and P2Y6 antisense oligonucleo-tides. MRS2578 significantly inhibited the MSU-induced produc-tion of IL-1a, IL-8/CXCL8, and IL-6 by NHK (Fig. 3A). TheP2Y6 antisense oligonucleotide decreased the P2Y6 protein ex-pression (Fig. 3B). Furthermore, treatment of NHK with P2Y6

antisense significantly inhibited the MSU-induced cytokine pro-duction (Fig. 3C). To complement the experiments with antisenseoligonucleotide, we also examined the effect of P2Y6 siRNA. TheP2Y6 siRNA decreased both the P2Y6 mRNA and protein ex-pression (Fig. 4A, 4B). As shown in Fig. 4C, P2Y6 siRNA in-hibited IL-1a and IL-8/CXCL8 production. The P2Y6 receptorsare known to be coupled to the activation of phospholipase C(PLC) (13). Consistent with this, U-73122, a PLC inhibitor, sig-nificantly suppressed the MSU-induced production of IL-1a, IL-8/CXCL8, and IL-6 by NHK, whereas U-73343, the inactiveanalogue of U-73122, did not (Fig. 3D). These results indicatedthat a functional P2Y6–PLC pathway plays a pivotal role in theMSU-induced inflammatory cytokine and chemokine productionby KC.

P2Y6 receptors mediate MSU-induced inflammation in THP-1cells

In acute gouty inflammation, it is reported that MSU crystals in-ternalized by monocytes activate the caspase-1–activating NALP3inflammasome, subsequently resulting in the production of IL-1b.In fact, MSU induced a large amount of IL-1b production fromTHP-1 cells (Fig. 5B) compared with that induced from NHK(Fig. 1A). We investigated whether P2Y6 receptors are also im-portant in this MSU-induced IL-1b release by monocytes. P2Y6

protein expression in THP-1 cells was increased by MSU stimu-lation (Fig. 5A). A caspase-1–specific inhibitor (z-YVAD) com-pletely suppressed MSU-induced IL-1b production in THP-1 cells(Fig. 5B), indicating that MSU-induced IL-1b production fromTHP-1 cells is dependent on caspase-1 activation, which is con-sistent with previous reports (17). The specific P2Y6 antagonist,MRS2578, almost completely inhibited the MSU-induced pro-duction of IL-1b by THP-1 cells (Fig. 5B). Furthermore, thetreatment of THP-1 cells with P2Y6 antisense oligonucleotidessignificantly inhibited MSU-induced IL-1b production (Fig. 5C).Moreover, U-73122 significantly suppressed the MSU-inducedIL-1b production by THP-1 cells (Fig. 5D). Thus, these resultsshowed that the P2Y6–PLC signaling pathway mediates MSU-induced inflammatory responses not only in KC but also in mono-cytes.

P2Y6 receptors mediate MSU-induced inflammation in vivo

Finally, we examined whether the P2Y6 receptors may also beresponsible for MSU-induced inflammation in vivo. In the mouseair pouch model, MSU induced neutrophil infiltration into the airpouch (17). The number of neutrophils infiltrating into the airpouch was assayed by staining the cells with neutrophils markerLy-6G. As shown in Fig. 6A, MSU enhanced the infiltration ofneutrophils into the air pouch, which was significantly inhibitedby MSR2578 (Fig. 6B). We also investigated the effect ofMRS2578 using MSU-induced peritonitis model, as there weresome previous reports showing the different effect of reagents

FIGURE 4. P2Y6 siRNA transfection inhibits IL-1a and IL-8/CXCL8

production from MSU-treated NHK. A and B, RT-PCR (A) and immuno-

blot (B) of NHK transfected with P2Y6 or negative control siRNA and

treated with MSU (200 mg/ml) for 24 h. C, IL-1a and IL-8/CXCL8 pro-

duced by NHK treated with or without P2Y6 or negative control siRNA

and stimulated with MSU 200 mg/ml for 24 h. *p , 0.05 (significant differ-

ences from negative control siRNA-transfected NHK stimulated with MSU).

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between these two models (1, 17). MSU induced neutrophil in-filtration into the peritoneum, which was significantly suppressedby MRS2578 as expected (Fig. 6C). Thus, these findings led to theconclusion that P2Y6 receptors are pivotal for MSU-induced in-flammation in vivo.

DiscussionOne of the central observations in this report is that MSU-inducedinflammatory cytokine and chemokine production is predominantlyregulated by the G protein-coupled P2Y6 receptors–PLC signalingpathway in both KC and monocytes. Some cell surface receptorshave been proposed to be involved in MSU-induced inflammation.They are TLR2/4 and CD14 on macrophages (17, 18), CD16 andcomplement receptors on neutrophils (19), and membrane integrins(GPIIb/IIIa) on platelets (20). In contrast, Ng et al. (21) recentlyreported that in the case of dendritic cells, direct binding of MSU tothe cell membrane, not onto the dendritic cell receptor, is important.In the current study, we investigated the role of P2 receptors inMSU-induced inflammation. P2 receptors are classified into P2Xand P2Y receptors based on their molecular structure and signaltransduction pathways. P2X receptors are ligand-gated ion chan-nels, and seven receptor subtypes exist (P2X1–7). P2Y receptors,which are G protein-coupled metabotropic receptors, have eightreceptor subtypes (P2Y1,2,4,6,11–14) in humans. P2Y1,2,4,6,11 arecoupled to PLC via Gq/11 protein, whereas P2Y12,13,14 are coupledto adenylate cyclase via Gi protein (13). The activation of P2Yreceptors, which are distributed widely among various tissues,

leads to cell proliferation, differentiation, and inflammation inmany kinds of cells. Among the P2Y receptors on KC, the P2Y2

receptors are reported to be involved in KC proliferation (22) andinhibition of KC migration in wounds (23). In addition, ATP andUTP induce the production of IL-6 from KC through P2Yreceptors (24), and ATP enhances the production of IL-8/CXCL8from IFN-g–stimulated KC (25). Although expression of P2Y6

receptors on KC has been controversial (26), we demonstrated thatthe expression of P2Y6 receptors was increased at both mRNA andprotein levels in MSU-stimulated KC. Furthermore, MSU-inducedIL-8/CXCL8 and IL-6 production was inhibited by P2Y6 antag-onist and P2Y6 antisense oligonucleotides. These results stronglysuggest that KC express functional P2Y6 receptors under MSUstimulation. Consistent with this, epithelial cells in lung and in-testine are demonstrated to produce IL-8/CXCL8 via P2Y6 recep-tors (27, 28).Regarding monocytes, THP-1 cells also express functional P2Y6

receptors. THP-1 cell activation through the endogenous ligand,UDP, leads to IL-8/CXCL8 production (29). Furthermore, UDPupregulates the mRNA expression of TNF-a, IL-8/CXCL8, andIL-1b in another monocytic cell line, U937, when U937 is stablytransfected with P2Y6 receptors (30). UDP is shown to facilitatethe phagocytosis in a P2Y6 receptor-dependent manner in micro-glia (31); therefore, phagocytosis of MSU by monocytes could befacilitated via P2Y6 receptor signaling. It is known that IL-1breleased from MSU-stimulated monocytes triggers the inductionof acute gouty inflammation. Our current study showed that P2Y6

FIGURE 5. The role of P2Y6 receptors in MSU-induced inflammation by THP-1 cells. A, Comparison of P2Y6 receptor protein expression in THP-1

between no stimulation and stimulated by MSU (100 mg/ml) using immunoblot analysis. B, IL-1b concentration in supernatants of THP-1 stimulated by

MSU (100 mg/ml) for 6 h in presence of z-YVAD or MRS2578. C, P2Y6 receptor protein expression in THP-1 treated with the indicated oligonucleotides,

and IL-1b concentration in supernatants of THP-1 stimulated by MSU (100 mg/ml) for 6 h after treatment with the indicated oligonucleotides. D, IL-1b

concentration in supernatants of THP-1 stimulated by MSU (100 mg/ml) for 6 h in presence of U-73122. Data show mean 6 SEM of three or four in-

dependent experiments. B, C, and D, *p , 0.05, *p , 0.01 (significant differences from the response to MSU alone).

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receptors play a pivotal role in IL-1b release from MSU-stim-ulated monocytes in vitro.Moreover, we showed that P2Y6 antagonist significantly in-

hibited the infiltration of neutrophils in both air pouch and peri-tonitis models. Our results clearly demonstrate that P2Y6 aremajor receptors mediating the neutrophilic gouty inflammationin vivo. P2Y6 receptors are reported to be involved in inflamma-tory bowel diseases, as their expression is upregulated in humanT cells infiltrating the bowel of affected patients (32). There havebeen no reports suggesting the role of P2Y6 receptors in gout;however, recent reports have suggested that the activation ofprotein kinase C is important for neutrophil activation by MSUcrystals in gout (33). P2Y6 receptors are coupled to the Gq/11

protein, which activates PLC and results in the activation of pro-tein kinase C. Therefore, this report may implicate the involve-ment of P2Y6 receptors in gout. We investigated several possibili-ties to explain the mechanism. First, P2Y6 receptors may act asa receptor for MSU. To examine whether MSU crystals candirectly bind to P2Y6 receptors, we assessed the calcium mobili-zation by MSU stimulation using 1321N1 astrocytoma transfectedwith P2Y6 receptors; however, we could not detect the calciumflux immediately after MSU stimulation (data not shown), sug-gesting that MSU crystals do not bind directly to P2Y6 receptors.We next investigated the possibility that UDP produced by MSUstimulation contributes to cytokine production via P2Y6 receptors,as UDP stimulation has been reported to induce the production ofIL-8/CXCL8 from lung epithelial cells (27). However, UDP didnot induce IL-1a, IL-8/CXCL8, or IL-6 production from NHK(data not shown), which is consistent with a previous report (24).Furthermore, UDP (1, 10, 100, 1000 mM) did not induce IL-1bproduction from THP-1 cells (data not shown). We also consid-ered the possibility that MSU induces ATP release from NHK,which subsequently activate KC. However, this was unlikely,because ATP did not induce IL-1a or IL-8/CXCL8 productionfrom NHK (data not shown). These results indicate that MSU

crystals act on cells via the P2Y6 receptor-signaling pathwaythrough unknown mechanisms. Further investigation is needed toclarify the exact mechanism by which MSU signals through P2Y6

receptors.Another major finding in this study is that MSU crystals can

induce various inflammatory cytokines and chemokines from hu-man KC, in which the IL-1a and IL-1R pathway plays a pivotalrole. MSU crystals have recently been reported to induce IL-1b byactivating caspase-1 through the formation of NALP3 inflamma-some in monocytes and to induce other inflammatory cytokinesincluding TNF-a, IL-6, and IL-8/CXCL8 through the IL-1R–MyD88–dependent pathway (1, 7). Watanabe et al. (34) haveshown that KC also have all the necessary components to formNALP3 inflammasome and that it can be formed by skin irritantsand UV B, which results in IL-1b release. In this study, MSUinduced the production of IL-1b from KC; however, the amountof IL-1b produced by KC was much smaller than that by mono-cytes. Furthermore, when we treated NHK with caspase-1 inhib-itor z-YVAD, the production of IL-1b was inhibited; however, IL-1a,IL-8/CXCL8, and IL-6 production was not. These data demon-strated that there might be pathways that could be activated byP2Y6 receptors to promote inflammatory signals, without involv-ing inflammasomes in KC. In contrast, a large amount of IL-8/CXCL8 and IL-6 was produced from MSU-stimulated KC in anIL-1R–dependent manner. Therefore, we focused on IL-1a, whichshares the receptor with IL-1b.IL-1a, a major subtype of IL-1 secreted by KC, can be quickly

released in case of epidermal infection or injury (35). IL-1a pro-duced by KC can induce other inflammatory cytokines and chem-okines through the autocrine pathway (35, 36). Overexpressionof IL-1a in murine epidermis produces spontaneous inflammatorylesions, suggesting the potency of IL-1a to drive skin inflamma-tion (37). MSU crystals induced a large amount of IL-1a fromKC. Furthermore, IL-1RA and anti–IL-1a neutralizing Ab sig-nificantly inhibited the production of IL-6 and IL-8/CXCL8 by

FIGURE 6. The effect of P2Y6 antagonist (MRS2578) in MSU-induced neutrophil infiltration in vivo. A, Representative dot plots of Ly-6G expression on

the harvested cells in the air pouch model. The Ly-6G(+)/PI(2) gate represents neutrophils. B and C, The number of neutrophils in air pouch (B) or

peritoneal cavities (C). Data show mean 6 SEM: n = 5 mice per group (B); n = 3 mice per group (C). Student t test was used to calculate p values.

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KC. Thus, these results indicate that IL-1a, but not IL-1b, is es-sential for MSU-induced inflammation in KC, which is differentfrom monocytes. To our knowledge, this is the first report to revealthe relationship between MSU crystals and IL-1a. A previousreport has shown that the ingestion of MSU crystals by phago-cytosis is central to MSU-induced gouty inflammation (8);therefore, it is interesting that MSU crystals can induce inflam-mation in epithelial cells such as KC through the IL-1a/IL-1Rpathway without phagocytosis.In conclusion, we showed that P2Y6 receptors mediated MSU-

induced inflammation in both KC and monocytes and that P2Y6

antagonist suppressed neutrophil infiltration in both mouse airpouch and peritonitis models. We also demonstrated in this studythat MSU-induced inflammatory cytokine and chemokine produc-tion by KC was regulated by the IL-1a/IL-1R pathway. Thus, ourcurrent findings could provide the possibility that P2Y6 receptor-signaling pathway is a potential therapeutic target for MSU-associ-ated inflammatory diseases. Regarding skin diseases other thantophaceous gout, skin disorders showing epidermal proliferationor apoptosis and necrosis of KC, such as psoriasis or atopic ecze-ma, could also be the target.

AcknowledgmentsWe thank T. Shimizu, S. Ishii, and K. Yanagida (University of Tokyo) for

helping to perform the calciummobilization assay and for discussions about

the manuscript. We thank N. Kanda (Teikyo University) and M. Komine

(Jichi Medical University) for technical advice and discussions about the

experiments.

DisclosuresThe authors have no financial conflicts of interest.

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