5′-Diphosphate Induces Chemokine Expression in Microglia and Astrocytes through Activation of the P2Y6

This information is current as Beomsue Kim, Hey-kyeong Jeong, Jong-hyeon Kim, Sang of September 27, 2021. Yoon Lee, Ilo Jou and Eun-hye Joe J Immunol published online 11 February 2011 http://www.jimmunol.org/content/early/2011/02/11/jimmun ol.1000212 Downloaded from

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 11, 2011, doi:10.4049/jimmunol.1000212 The Journal of Immunology

Uridine 59-Diphosphate Induces Chemokine Expression in Microglia and Astrocytes through Activation of the P2Y6 Receptor

Beomsue Kim,*,† Hey-kyeong Jeong,*,† Jong-hyeon Kim,*,† Sang Yoon Lee,†,‡ Ilo Jou,*,‡ and Eun-hye Joe*,†,‡,x

Chemokines play critical roles in inflammation by recruiting inflammatory cells to injury sites. In this study, we found that UDP induced expression of chemokines CCL2 (MCP-1) and CCL3 (MIP-1a) in microglia, astrocytes, and slice cultures by activation of

P2Y6. Interestingly, CCL2 was more highly expressed than CCL3. However, CCL2 synthesis kinetics in response to UDP differed in microglia and astrocytes; microglia rapidly produced small amounts of CCL2, whereas astrocytes continuously synthesized

large amounts of CCL2, resulting in a high ultimate level of the chemokine. UDP-induced chemokine expression was reduced in Downloaded from

the presence of a specific antagonist of P2Y6 (MRS2578) or small interfering RNA directed against the P2Y6 gene. Inhibition of phospholipase C and increase, downstream signaling pathways of Gq-coupled P2Y6, reduced UDP-induced chemokine expression. UDP activated two calcium-activated transcription factors, NFATc1 and c2. Furthermore, inhibitors of calcineurin (a phosphatase activating NFAT) and NFAT reduced UDP-induced chemokine synthesis. We also found, using a transmigration assay, that UDP-treated astrocytes recruited monocytes. These results suggest that UDP induces chemokine expression in microglia and

astrocytes of the injured brain by activation of P2Y6 receptors. The Journal of Immunology, 2011, 186: 000–000. http://www.jimmunol.org/ icroglia and astrocytes play important roles in the brain have been explored as inflammatory inducers of inflammation that accompanies brain injury. In re- brain inflammation (13). Although the roles of nucleotides M sponse to such injury, microglia and astrocytes release such as ATP in inflammatory processes were earlier intensively inflammatory mediators such as cytokines and chemokines stim- studied, research focus shifted to uridine nucleotides (UTP and ulating subsequent brain inflammation (1–3). Chemokines are UDP) after discovery of specific membrane receptors for these implicated in the pathogenesis of a number of neurologic diseases, factors (14, 15). In the injured brain, uridine nucleotides are including Alzheimer’s disease, cerebral , and multiple rapidly secreted or leaked to the extracellular space. For example,

sclerosis (4). Chemokines are major effector molecules recruiting kainic acid increased the concentration of extracellular UTP (16). by guest on September 27, 2021 blood inflammatory cells to the injury site (5), and recently, sev- In astrocytes, increases in intracellular calcium induced UTP re- eral studies have reported that blood inflammatory cells also enter lease to extracellular levels .20 times normal concentrations (17, the injured brain and participate in brain inflammation (6–8). In 18). Such increases in extracellular UTP also resulted in increases particular, CCL2 (MCP-1) and CCL3 (MIP-1a) are important in extracellular UDP, because secreted UTP is converted to UDP chemokines associated with monocyte infiltration into the injured by ectonucleoside diphosphokinase (19). brain (9–12). Extracellular nucleotides including uridine nucleotides exert their effects through the activation of P2 purinergic receptors. To date, seven ionotropic (P2X1–7) and eight metabotropic receptors *Department of Pharmacology, Ajou University School of Medicine, Suwon, Korea † (P2Y ) have been discovered. It has been reported that 442-721; Neuroscience Graduate Program, Ajou University School of Medicine, 1, 2, 4, 6, 11–14 ‡ Suwon, Korea 442-721; Chronic Inflammatory Disease Research Center, Ajou Uni- both P2Y purinergic receptors and P2X7 receptors play roles in in- x versity School of Medicine, Suwon, Korea 442-721; and Brain Disease Research flammatory processes such as phagocytosis and cytokine/chemokine Center, Ajou University School of Medicine, Suwon, Korea 442-721 production (16, 20–24). However, involvement details of specific Received for publication January 28, 2010. Accepted for publication January 10, P2Y receptors and the exact roles of these receptors in brain in- 2011. flammation are largely unknown. Microglia and astrocytes express This work was supported by Korea Science and Engineering Foundation National Research Laboratory Program Grant 2-2008025-0 funded by the Korean government P2Y1, 2, 6, 12–14 and P2Y1, 2, 4, 6, 13, 14, respectively (14, 16, 18, 25– (Ministry of Education, Science and Technology) and Grant M103KV010011- 28). Each has different affinities for specific nucleo- 06K2201-01110 from the Brain Research Center of the Twenty-First Century Fron- tides; ATP has a relatively high affinity for P2Y and ; ADP tier Research Program funded by the Ministry of Science and Technology, Republic 1, 2, 4, 12, 13 of Korea, and the Korea Science and Engineering Foundation through the Chronic for P2Y1, 12, and 13; UTP for P2Y2, 4, and 6; and UDP for P2Y6 (14). Inflammatory Disease Research Center at Ajou University (Grant R13-2003-019 to The results of the current study showed that UDP significantly E.J.). induces chemokine synthesis, particularly that of CCL2 and CCL3, Address correspondence and reprint requests to Dr. Eunhye Joe, Ajou University in primary cultured microglia, astrocytes, and brain slices by ac- School of Medicine, San-5 Woncheon-dong Youngtong-gu, Kyunggi-do, Korea 442-721. E-mail address: [email protected] tivation of P2Y6 receptors. Furthermore, UDP-treated astrocytes Abbreviations used in this article: BFA, brefeldin A; CFDA, carboxyfluorescein were major recruiters of blood monocytes. diacetate; CsA, cyclosporin A; EthD-1, ethidium homodimer-1; INCA-6, inhibitor of NFAT-calcineurin association-6; PLC, phospholipase C; Probe-1, fluorescence probe based on a perylene-dpa-Zn platform; qPCR, quantitative real-time PCR; Materials and Methods SD, Sprague Dawley; siCont, control siRNA; siP2Y6,P2Y6 siRNA; siRNA, small Reagents interfering RNA. FBS was purchased from Hyclone (Logan, UT). RNAzol B was from Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 iNtRON Biotechnology (Sungnam, Korea) and reverse transcriptase from

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000212 2 UDP INDUCES CHEMOKINE EXPRESSION IN GLIA

Promega (Madison, WI). Real-time master mix was from Kapa Biosystems as relative fold induction. The following primers were used for amplifi- (Cape Town, South Africa). Oligonucleotide primers were purchased from cation of rat CCL2, CCL3, P2Y6, P2X7, Iba-1, GFAP, and GAPDH: CCL2, IDT (Coralville, IA). Small interfering RNAs (siRNAs) including control 59-ATG CAG GTC TCT GTC ACG CT-39 (sense) and 59-CTA GTT CTC siRNA and siGLO were purchased from Dharmacon (Lafayette, CO). TGT CAT ACT GG-39 (antisense); CCL3, 59-ATG AAG GTC TCC ACC Opti-MEM and RNAiMAX were from Invitrogen (Basel, Switzerland). ACT-39 (sense) and 59-TCA GGC ATT CAG TTC CAG-39 (antisense); Transwell plates (pore size 5.0 mm) were from Corning (Lowell, MA). P2Y6,59-GTG GTATGT GGA GTC GTT TG-39 (sense) and 59-CTG TAG BAPTA-AM was purchased from Biomol International (Plymouth Meet- GAG ATC GTG TG GTT-39; P2X7,59-GTG CCA TTC TGA CCA GGG ing, PA). Cyclosporin A (CsA) and inhibitor of NFAT-calcineurin asso- TTG TAT AAA-39 (sense) and 59-GCC ACC TCT GTA AAG TTC TCT ciation-6 (INCA-6) were from Calbiochem (San Diego, CA). ATP, ADP, CCG ATT-39 (antisense); Iba-1, 59-TTG ATC TGA ATG GCA ATG GA-39 UDP, UMP, uridine, LPS, apyrase, , MRS2578, U73122, MEM, (sense) and 59-CCT CCA ATT AGG GCA ACT CA-39 (antisense); GFAP, and other reagents were from Sigma-Aldrich (St. Louis, MO). 59-TTC CTG TAC AGA CTT TCT CC-39 (sense) and 59-CCC TTC AGG ACT GCC TTA GT-39 (antisense); and GAPDH, 59-TCC CTC AAG ATT GTC AGC AA-39 (sense) and 59-AGA TCC ACA ACG GAT ACA TT-39 Primary microglia were cultured from the cerebral cortices of 1–3-d-old (antisense). Sprague Dawley (SD) rats (Samtako, Osan, Korea) as described previously (29, 30). All animal procedures were approved by the Ajou University ELISA Institutional Animal Experimentation Committee. Briefly, cortices were Levels of chemokines CCL2 and CCL3 secreted to the media were mea- triturated into single cells using Pasteur pipettes in MEM containing 10 sured using ELISA kits according to the manufacturer’s (Antigenix Ameri- mM HEPES (pH 7.4), 2 mM glutamine, and 10% (v/v) FBS and plated into 2 ca) instructions. We also analyzed CXCL2/3 (MIP-2) secretion. But be- 75 cm T-flasks (half a hemisphere in each flask) using the same culture cause UDP increased CXCL2/3 secretion in microglia but not in astrocytes medium. After 2 wk of culture, microglia were detached from flasks by (data not shown), we focused on CCL2 and CCL3. gentle shaking and filtered through nylon mesh to remove astrocytes and 3 4 2 Downloaded from cell clumps. Microglia were counted and plated (5 10 cells/cm ). siRNA transfection of primary microglia and astrocytes Isolated microglia were allowed to attach to plates for 3 to 4 h, and un- attached cells were removed by changing the medium. Microglial purity For knockdown of P2Y6 receptor expression, we used ON-TARGET plus was confirmed by morphology analysis and/or by staining using a micro- siRNAs targeting the rat P2Y6 gene (Dharmacon, Lafayette, CO). Primary 4 2 4 2 glial marker isolectin B4 (from Griffonia simplicifolia) (31). More than astrocytes (2 3 10 cells/cm ) and microglia (5 3 10 cells/cm ) were 95% of cells were isolectin B4 positive (data not shown) and subsequently seeded into 12-well plates and used at 50–70% or 90% confluence, re- used in experiments. spectively. One day before transfection, medium was replaced by Opti-

Primary astrocytes remaining in the flask were harvested with 0.1% (w/v) MEM (Invitrogen). Transfection was performed using 20–40 nM siRNA http://www.jimmunol.org/ trypsin, plated (3 3 104 cells/cm2), and cultured with 5% (v/v) FBS- and the RNAiMAX transfection reagent according to the manufacturer’s containing MEM for 1 to 2 d and serum-free MEM for 2 to 3 d. Then, instructions (Invitrogen). Transfection efficiency was measured by fluo- astrocytes were detached with trypsin once again and plated (2 to 3 3 104 rescence microscopy using siGLO Red (Dharmacon). At least 90% of cells 2 cells/cm ) with 5% (v/v) FBS-containing MEM. Astrocytes were in- were transfected (data not shown). Four to 5 d post-transfection, cells were cubated at least overnight to minimize any effects induced by changing used in knockdown experiments. medium before they were used in experiments. More than 95% of purified astrocytes were GFAP positive (data not shown). EMSA and supershift assay Adult astrocytes were cultured as described previously (32) with some modifications. Briefly, cortices obtained from 6–8-wk-old SD rats were NFAT binding activities were measured using an EMSA, as described chopped with a razor blade, incubated with 0.1% (w/v) trypsin (Hyclone) previously (34). Primary microglia (1.5 3 106 cells) were harvested and for 10 min, and washed with MEM containing 10% (v/v) FBS. Single cells suspended in 900 ml hypotonic solution (10 mM HEPES, 10 mM KCl, 0.1 by guest on September 27, 2021 were obtained by titration, layered over a 0.4 M sucrose solution, and mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF [pH 7.9]) for centrifuged at 400 3 g for 10 min. The pellet was washed and resuspended 15 min, followed by incubation in hypotonic solution containing 0.5% in MEM containing 10 mM HEPES (pH 7.4), 2 mM glutamine, and 10% (v/v) Nonidet P-40 for 5 min. Cells were centrifuged at 500 3 g for 10 min (v/v) FBS and cultured in 75-cm2 culture flasks (one half hemisphere per at 4˚C and the pellet containing the nuclear fraction resuspended in a so- two flasks). Three days later, floating cells were removed by changing the lution containing 20 mM HEPES, 20% (v/v) glycerol, 0.4 M NaCl, 1 mM medium. When cells reached confluence 3 to 4 wk later, they were sub- EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF (pH 7.9). Samples cultured and incubated at least overnight before experiments. were centrifuged at 10,000 3 g for 10 min, and crude nuclear fractions 2 For experiments employing inhibitors of P2Y6, calcium, phospholipase (supernatants) were collected and stored at 70˚C until use. C (PLC), and/or mNFAT, cultures were pretreated for 5 min with the ap- The NFATc consensus oligonucleotide (59-CGC CCA AAG AGG AAA propriate inhibitors before UDP treatment. ATT TGT TTC ATA-39) and mutant oligonucleotide (59-CGC CCA AAG CTT AAA ATT TGT TTC ATA-39) (Santa Cruz Biotechnology, Santa Organotypic cortical culture Cruz, CA) were end-labeled using T4 polynucleotide kinase (Promega) and [g-32P] ATP (PerkinElmer, Waltham, MA). The labeled DNA probe Cortical slices were prepared using a modified Stoppini method (33). ∼ Briefly, 1-d-old SD rats were anesthetized and then decapitated. Brains ( 0.5 ng) was incubated for 30 min with 2 mg nuclear proteins in a re- were rapidly removed and cortices separated by thin forceps in culture action mixture containing 21.4 mM EDTA, 21.4 mM EGTA, 20% (v/v) medium. Coronal cortical slices (400-mm thick) were prepared using glycerol, 0.29 mM ZnSO4, 10 ng/ml poly(deoxyinosinic-deoxycytidylic) a McIlwain tissue chopper (Mickle Laboratory Engineering, Goose Green, acid, 1 mM DTT, 0.4 mg/ml BSA, and 8 mM MgCl2. The reaction mixture U.K.). Slices were placed into 24-well plates in which each well was filled was resolved on a 6% (w/v) polyacrylamide gel. For supershift assays, nu- with 500 ml culture medium (MEM containing 25% [v/v] HBSS, 25% clear extracts were preincubated with a 1-mg combination of anti-NFATc1, anti-NFATc2, and control IgG Abs (Affinity Bioreagents, Golden, CO) for [v/v] heat-inactivated horse serum [Hyclone], 6.5 mg/ml glucose, 1 mM L- glutamine, 10 U/ml penicillin-G, and 10 mg/ml streptomycin). Using ethid- 10 min at 4˚C before probe addition. The reaction mixture was subjected to ium homodimer-1 (EthD-1; Molecular Probes, Eugene, OR), cell viability in electrophoresis on a 6% (w/v) polyacrylamide gel. slices was assayed. Immunofluorescence staining PCR Primary microglia were seeded onto coverslips (Fisher Scientific, Pitts- mRNA expression was detected with quantitative real-time PCR (qPCR). burgh, PA) and incubated overnight. After incubation with UDP (100 mM), Total RNA was isolated using RNAzol B (iNtRON Biotechnology) and cells were fixed in 4% (w/v) paraformaldehyde for 15 min, permeabilized in cDNA prepared using reverse transcriptase from avian myeloblastosis virus, 0.1% (v/v) Triton X-100, and blocked with 1% (v/v) bovine serum. Next, according to the manufacturer’s (Promega) instructions. For qPCR, cDNA microglia were incubated with Abs to NFATc1 (1:2000; Affinity Biore- and forward/reverse primers (200 nM) were added to 23 KAPA SYBR agents), NFATc2 (1:200; Affinity Bioreagents), or anti-ionized calcium- Fast Master Mix (Kapa Biosystems). qPCR were performed on RG-6000 binding adaptor molecule (Iba-1; 1:1000, Wako Pure Chemicals) over- real-time amplification instrument (Corbett Research, Sydney, Australia). night at 4˚C. Alexa 555-conjugated anti-mouse donkey IgG and Alexa 488- qPCR reaction protocol was denaturation at 95˚C for 30 s, followed by 40 conjugated anti-rabbit donkey IgG (Invitrogen) were used to detect NFATcs cycles of 95˚C for 3 s, 55˚C for 20 s, and 72˚C for 3 s. Threshold cycle and Iba-1, respectively. Immunostained cells were mounted using VEC- number of each gene was calculated, and GAPDH was used as a reference TASHIELD containing DAPI (Vector Laboratories, Burlingame, CA). gene. The D-d threshold cycle values of CCL2 and CCL3 were presented Confocal microscopy images were obtained (LSM510; Carl Zeiss). The Journal of Immunology 3

Measurement of UDP secreted from cortical slices were either untreated or preactivated with UDP (100 mM) 1 h prior to addition of monocytes. Freshly purified CFDA-labeled monocytes (4 3 Cortical slices were placed in a 96-well plate (one slice per well) filled with 105 cells/100 ml) were added to the top of each transwell insert of 5-mm 80 ml HEPES-buffered solution (130 mM NaCl, 5 mM KCl, 1.5 mM pore size (Corning) and allowed to migrate 8–10 h. The number of CFDA- CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES [pH 7.4]) positive monocytes transmigrating into the bottom chamber were counted immediately after slicing and incubated with or without in three randomly selected fields (320 objective) under a fluorescent mi- inhibitor (10 mM ) for 10 min to 1 h. The levels of UDP croscope (Zeiss Axiovert 200M; Carl Zeiss). secreted into the solution were measured using a fluorescence probe based on a perylene-dpa-Zn platform (Probe-1) provided by Dr. Juyoung Yoon Statistical analysis (Department of Chemistry and Nano Science, Ewha Woman’s University, Suwon, Korea) (35). HEPES-buffered solution (80 ml) was mixed with Data were analyzed by Student t test, one-way ANOVA, or two-way Probe-1 (10 mM), and fluorescence levels were measured at 485/535 nm ANOVA followed by post hoc comparisons (Student-Newman-Keuls ap- (excitation/emission) with a Victor3 fluorescence reader (PerkinElmer). proach) using the Statistical Package for Social Sciences version 8.0 Probe-1 has greater specificity for UDP than UTP and has little specificity (SPSS, Chicago, IL). for ATP, ADP, AMP, UMP, or GTP (35). Results In vitro transmigration assay UDP induces chemokine expression in cultured microglia Rat monocytes were isolated and labeled as described (6), with some modifications. Briefly, blood was collected from 5-wk-old SD rats by car- In the injured brain, nucleotides such as ATP, ADP, UTP, and UDP diac puncture. Blood was mixed with PBS containing 2.5% (w/v) dextran are released at high levels from neurons and astrocytes (16, 36, 37). (blood/PBS = 1:4) to sediment erythrocytes. The plasma layer was spun As microglial activation accompanies brain injury, we examined

down, and the pellet was suspended in PBS, incubated with 10 mMcar- whether extracellular nucleotides could alter microglial function. Downloaded from boxyfluorescein diacetate (CFDA; Molecular Probes), loaded onto a Ficoll Thus, we treated microglia with 100 mM ATP, ADP, UTP, or UDP gradient (Amersham Pharmacia Biotech, San Francisco, CA), and centri- fuged (400 3 g) for 30 min. The monocyte/lymphocyte layer at the interface and examined the expression levels of chemokines CCL2 and was carefully collected and washed three times with sterile PBS. Cells were CCL3 by ELISA (Fig. 1A). Microglia released CCL2 and CCL3 in suspended in HBSS containing calcium (140 mg/l) and seeded in a Petri the range of 1–20 pg/mg protein. UDP was the most powerful dish for 30 min. Unattached lymphocytes were removed and adherent inducer of CCL2, and ADP, UTP, and UDP induced CCL3 to monocytes collected and suspended in MEM containing 1% (v/v) FBS.

Primary astrocytes were cultured in 24-well plates (3 3 104 cells/cm2), similar extents, whereas ATP barely induced both chemokines http://www.jimmunol.org/ and medium was replaced with 600 ml/well MEM with 1% (v/v) FBS (Fig. 1A). Based on these results, we selected UDP for use in 1 d before the transmigration assay was conducted. Primary astrocytes further experiments. by guest on September 27, 2021

FIGURE 1. UDP induces chemokine expression in microglia. A, Pri- mary microglia were treated with 100 mM ATP, ADP, UTP, or UDP for 3 h. The amounts of CCL2 and CCL3 secreted to media were analyzed by ELISA. Media chemokine levels were normalized to microglial total FIGURE 2. Time- and dose-dependent chemokine expression in UDP- protein concentrations. Cells were treated with 100 mM UDP, UMP, or treated microglia. Primary microglia were incubated with 100 mM (unless uridine (B) or 100 mM UDP preincubated with apyrase (0.1–1 U) for 15 indicated) UDP for the indicated times (A, B) or incubated with the in- min (C). After 1 h, mRNA levels encoding CCL2 and CCL3 were analyzed dicated amount of UDP for 1 (C)or3h(D). A and C, Expression of by qPCR. Relative fold induction compared with control was represented. mRNAs encoding CCL2 and CCL3 was analyzed by qPCR. B and D, GAPDH mRNA levels were analyzed as internal controls. Values are Chemokine levels in media were analyzed by ELISA. Closed and open means 6 SEMs of three independent experiments (A, C) or three samples symbols in B indicate the presence or absence of UDP, respectively. Values of representative of at least three independent experiments unless indicated are means 6 SEMs of three independent experiments (A, C) or three (B, D). *p , 0.05 versus values in the absence of any activator (A, B)orin samples (B, D). *p , 0.05, **p , 0.01 versus values in the presence and the presence of UDP (C). absence of UDP. 4 UDP INDUCES CHEMOKINE EXPRESSION IN GLIA

We next examined the effect of UDP metabolites on chemokine mokine expression in microglia. As expected, U73122 (1–5 mM) expression, because UDP can be metabolized into UMP and uridine and BAPTA-AM (10–50 mM) significantly reduced UDP-induced in culture media. In qPCR, UDP significantly induced microglial chemokine mRNA expression (Fig. 3C,3D). CCL2 and CCL3 mRNA expression. However, both UMP and P2Y6 siRNA (siP2Y6) also significantly inhibited UDP-induced uridine had little effect on chemokine expression (Fig. 1B). Ac- chemokine secretion in primary microglia, whereas nontargeting cordingly, apyrase, an degrading UDP into UMP or uri- control siRNA (siCont) had virtually no effect (Fig. 3E,3F). dine, also inhibited UDP-induced CCL2 and CCL3 mRNA ex- Taken together, these results indicate that UDP induces chemokine pression (Fig. 1C). These results suggest that UDP per se is the expression in microglia by activation of P2Y6 receptors. active component inducing chemokine expression in microglia. NFATc1 and NFATc2 mediate UDP-induced chemokine In the following experiments, UDP (100 mM) induced expres- expression sion of mRNA encoding CCL2 and CCL3 within 30 min, and such expression reached maximal levels at 1 h, returning to basal levels Next, we investigated the involvement of transcription factors at 3–12 h (Fig. 2A). Secretion of CCL2 and CCL3 increased mediating UDP-induced chemokine expression. NF-kB is widely within 1 h, peaked at 3–6 h, and was maintained for up to 24 h known as a transcription factor involved in chemokine expression (Fig. 2B). UDP induced a concentration-dependent increase in the by various cell types including microglia (40–43). However, UDP expression of chemokines at both the mRNA and protein level barely activated NF-kB in primary microglia compared with LPS over a concentration range of 10 mM to 1 mM (Fig. 2C,2D). (data not shown). As P2Y6 is a Gq-coupled receptor, and as in- tracellular calcium increase is required for UDP-induced chemo- UDP induces chemokines through the activation and signaling kine expression, as shown above (Fig. 3), we hypothesized that Downloaded from of P2Y6 NFAT would be involved in this process. In EMSA, microglial UDP is known to be a specific agonist of the P2Y6 purinergic NFAT activation was detected at 15–30 min post-treatment with receptor (14), and P2Y6 is known to be expressed in microglia (25, either UDP or ionomycin (positive control) (Fig. 4A). The NFAT– 26). Thus, we investigated whether P2Y6 mediated UDP-induced probe complex was not formed with NFAT mutant (mut) probe production of chemokines in cultured microglia. MRS2578, a changing AGG to CTT in the binding motif (Fig. 4B, lane 3).

specific antagonist of the P2Y6 receptor (38), significantly inhibi- Moreover, the UDP-induced NFAT–probe complex was effec- http://www.jimmunol.org/ ted UDP-induced mRNA and protein expression of both chemo- tively competed by excessive cold wild-type probe (Fig. 4B, lane kines at 2–5 mM (Fig. 3A,3B). No cytotoxic effects of MRS2578 4) but not by the mutant cold probe (Fig. 4B, lane 5). The NFAT– were evident at 5 mM (data not shown). probe complex was disappeared or supershifted in the presence of As the P2Y6 receptor is a Gq/G11-coupled receptor activating Abs against NFATc1 and c2 but not in the presence of control IgG PLC and subsequently increasing intracellular calcium (39), we Abs (Fig. 4C). We also found that both NFATc1 and c2 were examined the effects of a PLC inhibitor (U73122) and an in- translocated from the cytosol to the nucleus within 15 min after tracellular calcium chelator (BAPTA-AM) on UDP-induced che- UDP treatment, and MRS2578 inhibited translocation of NFATs by guest on September 27, 2021

FIGURE 3. Inhibition of P2Y6 signaling pathways and knockdown of P2Y6 reduce UDP-induced chemokine expression in microglia. Microglia were treated with UDP in the presence of the indicated amounts of MRS2578 (a P2Y6 receptor antagonist) (A, B), U73122 (a PLC inhibitor) (C), or BAPTA-AM (an intracellular calcium chelator) (D). mRNA levels (A, C, D) and secretion levels (B) of CCL2 and CCL3 were analyzed with qPCR and ELISA 1 and 3 h after UDP treatment, respectively. E and F, P2Y6 expression was knocked down by transfecting microglia with 40 nM siP2Y6 for 4 d; a nontargeted siRNA (siCont) was used as a control. Specific knockdown of P2Y6 was confirmed by analyzing P2Y6 and P2X7 mRNA levels using qPCR (E). Secretion levels of CCL2 and CCL3 were analyzed with ELISA 3 h after UDP treatment (F). Values are means 6 SEMs of three independent experiments (A, C–E) or three samples (B, F). *p , 0.05, **p , 0.01 versus values in the presence of UDP. The Journal of Immunology 5

FIGURE 5. NFAT mediates UDP-induced chemokine expression in microglia. Microglia were treated with UDP for 1 h in the absence or presence of the indicated amounts of INCA-6 (A) or CsA (B). mRNA levels of chemokines were examined using qPCR. Values are means 6

SEMs of three independent experiments. *p , 0.05, **p , 0.01 versus Downloaded from values in the presence of UDP.

functioning as a downstream transcription factor of the P2Y6 re- ceptor. http://www.jimmunol.org/

P2Y6 activation induces chemokine expression in cultured astrocytes from neonate and adult rat brain

FIGURE 4. UDP induces activation of NFATc1 and NFATc2 in Several lines of evidence indicate that astrocytes are the main source microglia. A, Nuclear extracts were prepared from microglia treated with of chemokines in many neurologic diseases, although microglia play UDP or ionomycin (4 mM) for the indicated times. NFAT activation (ar- central roles in initiation of inflammatory processes (1, 46). Thus, we row) was assayed by EMSA as described in Materials and Methods. B, examined whether UDP induced chemokine expression in astro- Nuclear extracts obtained 15 min after UDP treatment, and NFAT activa- cytes by activation of P2Y6, as in microglia. Primary astrocytes by guest on September 27, 2021 tion was confirmed using consensus NFAT probe (wt) and mutant NFAT express P2Y6 (25) and, in response to UDP (100 mM), astrocytes probe (mut, lane 3). Competitive assay was performed with excessive increased expression of mRNAs encoding CCL2 and CCL3 within 3 amount (20 ) of consensus NFAT sequence (c-wt, lane 4), or mutant 30 min (Fig. 6A). The elevated mRNA levels were maintained for up sequence oligonucleotide (c-mut, lane 5). C, A supershift assay performed to 3 h and then rapidly decreased to the basal level by 12 h (Fig. 6A). as described in Materials and Methods using anti-NFATc1 (c1), anti- CCL2 secretion increased continuously from 3 h (∼10 pg/mgpro- NFATc2 (c2), and control IgG Abs (IgG). NFAT–probe complex (arrow) ∼ and Ab-bounded complex (arrowhead) was indicated. Nuclear extracts tein) to 24 h ( 120 pg/mg protein) post-treatment. However, CCL3 , obtained 15 min after UDP treatment. D, Nuclear translocation of NFAT was barely produced ( 0.5 pg/mg protein), even 24 h after UDP was determined by immunofluorescence staining. Microglia were treated treatment (Fig. 6B). We further tested whether adult astrocytes also with 100 mM UDP in the absence or presence of MRS2578 (5 mM) for 15 express chemokines in response to UDP. In adult cultures, most cells min. Cells were stained with anti-NFATc1/NFATc2 and anti–Iba-1 Abs and (∼90%) were GFAP positive, but Iba-1–positive cells were also visualized with Alexa 555- (red) and Alexa 488-conjugated (green) sec- detected (data not shown). As was the case in neonatal astrocyte ondary Abs, respectively. Nuclei were stained with DAPI (blue). Control cultures, UDP (100 mM) also enhanced CCL2 levels in adult cells stained with Iba-1 and a combination of secondary Abs, but without astrocytes, increasing production from 90 to 260 pg/mg protein be- anti-NFATc1/NFATc2 Abs, confirmed the specificity of NFATc1/NFATc2 tween 3 and 24 h. CCL3 production was also increased by UDP staining. Images were obtained using a Zeiss LSM510 microscope (Carl (from 0.4 to 2.6 pg/mg protein between 3 and 24 h) (Fig. 6C). Zeiss). Scale bar, 20 mm. Data are representative of three independent experiments. Involvement of P2Y6 in UDP-induced chemokine expression was assayed in two ways. First, MRS2578 (2–10 mM) inhibited che- mokine expression (Fig. 7A). Furthermore, use of siP2Y6 supported the involvement of P2Y6 in UDP-induced chemokine expression in (Fig. 4D). The specificity of NFATc1 and NFATc2 immunoreac- astrocytes as in microglia. In astrocytes transfected with siP2Y6, tivity was confirmed by staining with control IgG Abs in the ab- P2Y6 expression was dramatically reduced to levels ,10% of those sence of NFAT Abs (Fig. 4D). in siCont-treated cells, without any effect on expression of P2X7, The involvement of NFAT in UDP-induced chemokine ex- another (Fig. 7B). P2Y6 siRNA significantly pression was further confirmed using inhibitors of NFAT (INCA-6) decreased UDP-induced mRNA expression encoding CCL2 and (44) and calcineurin (CsA). Calcineurin is a calcium-dependent CCL3, whereas control siRNA had little effect on mRNA levels phosphatase activating NFAT (45). qPCR showed that 10–20 mM encoding either P2Y6 or the chemokines (Fig. 7C). Accordingly, INCA-6 significantly reduced UDP-induced CCL2 and CCL3 siP2Y6 reduced CCL2 secretion in response to extracellular UDP mRNA expression (Fig. 5A). As expected, CsA in the range of (Fig. 7D). In additional experiments, we found that UDP decreased 10–50 mM also attenuated UDP-induced mRNA expression of P2Y6 mRNA levels within 1 h, and this was sustained for up to 3 h CCL2 and CCL3 in qPCR (Fig. 5B). These results indicated that (data not shown). Taken together, these results indicate that P2Y6 NFAT mediates UDP-induced chemokine production in microglia, mediates UDP-induced chemokine expression in astrocytes. 6 UDP INDUCES CHEMOKINE EXPRESSION IN GLIA

we analyzed the levels of UDP secreted from cortical slices using Probe-1. This probe specifically detects UDP and UTP (with higher sensitivity for UDP) and not other nucleotides, including ATP, ADP, and UMP/uridine (35). Within 10 min after slicing, ∼6.4 6 3.5 mM UDP was detected, and the levels increased to 21.7 6 4.7 mM after 1 h (Table I). In the presence of dipyr- idamole, an inhibitor of ectonucleotidase that inhibits degradation of UTP/UDP into UMP/uridine, UDP levels further increased to 228.5 6 22.7 mM at 10 min (Table I). In slice cultures, the mRNAs encoding CCL2 and CCL3 in- creased within 30 min of slicing and reached the first peak at 6 h (Fig. 8A). CCL2 mRNA expression further increased up to 72 h, whereas CCL3 mRNA levels rapidly decreased at 12 h (Fig. 8A). Analysis of chemokine secretion showed that CCL2 levels sig- nificantly increased from 3 to 72 h (0.4 to 30 pg/mg protein) compared with those of CCL3 (0.5 to 0.9 pg/mg protein) (Fig. 8B). We further found that exogenously added UDP (100 mM) did not enhance chemokine secretion from the slices (Fig. 8C). These

results suggest that the amount of UDP released from slices was Downloaded from sufficient to induce chemokine expression. We next investigated involvement of P2Y6 in CCL2 expression from slice cultures using the inhibitor MRS2578. When slices were cultured with MRS2578 (10 mM), both mRNA expression encoding CCL2 (Fig. 8D) and chemokine secretion (Fig. 8E) were

FIGURE 6. UDP induces chemokine expression in astrocytes cultured significantly reduced. We confirmed that MRS2578 had little ef- http://www.jimmunol.org/ from neonate and adult rat brain. Primary astrocytes cultured from neonate fect on astrocyte and microglial viability in slices, as shown by rat brain (A, B) or adult brain (C) were incubated with UDP (100 mM) for both EthD-1 staining and analysis of mRNA levels encoding the indicated times. mRNA expression of chemokine genes (A) and che- GFAP and Iba-1, markers of astrocytes and microglia, respectively mokine secretion (B, C) were examined using qPCR and ELISA, re- (Fig. 8F). These results suggest that P2Y6 may mediate chemo- spectively. Closed and open symbols indicate the presence or absence of kine expression in the injured brain. UDP, respectively. Values are means 6 SEMs of three samples. UDP-treated astrocytes recruit monocytes

P2Y6 mediates chemokine production from cortical slice As CCL2 is well known to function in monocyte recruitment, cultures we examined whether UDP-treated astrocytes were able to recruit by guest on September 27, 2021 monocytes. To this end, monocytes labeled with CFDA- Next, we investigated whether P2Y regulated chemokine ex- 6 succinimidyl ester and astrocytes were cultured in the upper pression in slice cultures of rat brain cortex. It is well known that and lower chambers, respectively, of transwells, and the numbers the process of slice preparation caused traumatic injury (47). First, of monocytes migrating to lower chambers were counted (Fig. 9). Monocyte levels detected in lower chambers increased ∼1.6-fold when astrocytes were treated with UDP (Fig. 9A,9B). However, in the presence of brefeldin A (BFA), an inhibitor of protein traf- ficking from the endoplasmic reticulum to the Golgi, migration of monocytes toward UDP-treated astrocytes significantly decreased (Fig. 8A,8B). Astrocytes treated with UDP for only 5 min still induced monocyte migration (Fig. 9B). The extent of monocyte migration correlated with the extent of CCL2 secretion because astrocytes treated with UDP either overnight or for 5 min pro- duced similar levels of CCL2, and astrocytes treated with UDP in the presence of BFA did not produce CCL2 (Fig. 9C). We also examined the effect of UDP-treated microglia on transmigration of monocytes. In accordance with differences in secretion levels of CCL2 from astrocytes and microglia (.100 pg/mg protein from

Table I. Levels of extracellular (secreted) UDP in slice cultures

FIGURE 7. P2Y6 mediates UDP-induced chemokine expression in 10 min 30 min 1 h astrocytes. A, Astrocytes were treated with 100 mM UDP for 1 h in the 6 6 6 absence or presence of the indicated amounts of MRS2578 (MRS). Che- Slices 6.4 3.5 13.5 2.9 21.7 4.7 6 mokine expression was examined using qPCR. B–D, Astrocytes were Plus dipyridamole 228.5 27.7 NA NA transfected with 20 nM siP2Y6 or siCont for 4 d. mRNA levels of P2Y6 Cortical slices from P1 rats were incubated with HEPES-buffered saline for the indicated times. Levels of secreted UDP (mM) were measured using Probe 1, as was analyzed with qPCR (B). Effect of siP2Y6 on UDP-induced expression of CCL2 mRNA (C) and secretion (D) was analyzed at 1 and 6 h after UDP described in Materials and Methods. Dipyridamole (10 mM) was used to inhibit ectonucleotidase activity. 6 treatment, respectively. Values are means SEMs of three independent Data are presented as means 6 SEMs (n = 4). experiments (A–C) or three samples (D). *p , 0.05, **p , 0.01. NA, not assayed. The Journal of Immunology 7

FIGURE 8. P2Y6 mediate CCL2 expression in neonate slice cultures. mRNA expression of chemokine genes (A, D) and chemokine secretion (B, C, E)in neonatal cortical slices were assayed. Cortical slices were prepared from neonatal rat brains as described in Materials and Methods and cultivated for the indicated times (A–C)or24h(D, E) in the absence or presence of exogenously added UDP (100 mM) (C) or 2–10 mM MRS2578 (D, E). F, Absence of Downloaded from cytotoxic effects of MRS2578 was confirmed by staining slices with EthD-1 (left panel) or analyzing mRNA levels of GFAP and Iba-1 (right panel)24h after 10 mM MRS2578 treatment. Images were obtained using a Zeiss Axiovert 200M microscope (Carl Zeiss). Scale bar, 500 mm. Values are means 6 SEMs of three independent experiments (A, D, F) or three samples (B, C, E). *p , 0.05, **p , 0.01 versus control CCL2. http://www.jimmunol.org/ astrocytes versus ,20 pg/mg protein from microglia), UDP- Discussion activated microglia barely induced monocyte migration (data not In this study, we have demonstrated a novel pathway for induction shown). Taken together, the results suggest that UDP released of chemokine expression in microglia and astrocytes. UDP released from injured brain cells may activate astrocytes and microglia from damaged cells could induce chemokine synthesis in microglia to secrete chemokines. Furthermore, astrocytes could play major and astrocytes through activation of P2Y6. In fact, significant roles in recruiting blood inflammatory cells into the brain. amounts of UDP were rapidly released from cortical slices (Table I), and exogenously added UDP had no additional effect on che- mokine expression. In response to UDP, microglia rapidly (within 3 h) produced by guest on September 27, 2021 small amounts of chemokines (,20 pg/mg protein), whereas as- trocytes cultured from both neonatal and adult rats continuously (over 9–24 h) produced large amounts of chemokines, particularly CCL2. Furthermore, UDP-treated microglia barely enhanced monocyte migration (data not shown) whereas UDP-treated as- trocytes strongly recruited monocytes. These findings led us to speculate that the functions of chemokines produced by microglia and astrocytes may differ. Chemokines not only recruit inflam- matory cells to injury sites but also exert several other functions. Recently, neuroprotection effects of chemokines such as CCL2, CCL5 (RANTES), CXCL8 (IL-8), and CXCL2/3 (MIP-2) have been reported (48–50). Chemokines also modulate ion channel activities: CCL2 activates nonselective cation channels and in- hibits delayed-rectifier potassium channels (51), and CCL3 mod- ulates the activities of transient receptor potential channels (51). Chemokines may sensitize immune cells to inflammatory stimuli. FIGURE 9. UDP treatment of astrocytes enhances transmigration ac- 2/2 tivity of monocytes. A, Cultured astrocytes were treated with or with- Thus, when LPS was injected into the brain of CCL2 mice, out UDP (100 mM) with or without a 30 min-pretreatment with BFA (5 less IL-1b and TNF-a were produced compared with levels seen mg/ml). CFDA-labeled monocytes prepared as described in Materials and in normal mice (52). Therefore, rapidly released small amounts of Methods were placed in transwells and cocultured with astrocytes over- chemokines from UDP-activated microglia may function to pro- night. Phase-contrast and CFDA fluorescence images were obtained using tect neurons or to modulate channel activity in the injured brain a Zeiss Axiovert 200M microscope (Carl Zeiss). Scale bar, 50 mm. B, rather than to recruit blood inflammatory cells. In contrast, large Numbers of transmigrated monocytes were counted under a fluorescence amounts of chemokines produced by astrocytes may play major microscope at a minimum of three different fields. The mean values were roles in recruitment of blood inflammatory cells. In animal models achieved, and three independent experiments were performed. The number of the ischemic and traumatic brain, the duration (9–24 h) of che- of transmigrated cells in control condition considered as 1 U. UDP (59) mokine production from astrocytes was correlated with the time indicates an experiment in which astrocytes were treated with UDP for 5 min, washed, changed to UDP-free medium, and incubated with labeled (12–24 h) required for infiltration of neutrophils and monocytes monocytes overnight. C, Secretion of CCL2 was assayed. Values in B and (53). These findings support the hypothesis that astrocyte-derived C are means 6 SEMs of three samples. Data are representative of at least chemokines play important roles in recruitment of blood inflam- three independent experiments. *p , 0.05 versus control. matory cells. 8 UDP INDUCES CHEMOKINE EXPRESSION IN GLIA

Recently, several lines of evidence have indicated that purinergic of various neurologic injuries associated with brain inflammatory receptors activated by extracellular nucleotides mediate various processes. inflammatory responses, including production of reactive oxygen species and expression of cytokines and chemokines (54, 55). In Acknowledgments the current study, we showed that UDP-activated P2Y6 mediated The fluorescent Probe-1 was kindly provided by Dr. Juyoung Yoon (Ewha chemokine production in both microglia and astrocytes, based on Womans University, Seoul, South Korea). several experimental approaches. First, among ATP, ADP, UTP, and UDP (all at 100 mM), UDP, with the highest affinity for P2Y6 Disclosures (14), was the most potent chemokine inducer (Fig. 1). Second, The authors have no financial conflicts of interest. siRNA directed against P2Y6 abrogated UDP-induced chemokine expression (Figs. 3, 7). 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