Uridine 5′-Diphosphate Induces Chemokine Expression in Microglia and Astrocytes through Activation of the P2Y6 Receptor 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on September 27, 2021 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 © 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 calcium 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 Nucleotides have been explored as inflammatory inducers of inflammation that accompanies brain injury. In re- brain inflammation (13). Although the roles of adenine 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 ischemia, 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 P2Y receptor 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, suramin, 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 Cell culture 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.