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    by IFN-Astrocytes and Microglial Cells: Modulation ATP Mediates Calcium Signaling Between

    Claudia Verderio and Michela Matteoli 10.4049/jimmunol.166.10.6383

    2001; 166:6383-6391; ;J Immunol


    , 22 of which you can access for free at: cites 54 articlesThis article


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    Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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  • ATP Mediates Calcium Signaling Between Astrocytes andMicroglial Cells: Modulation by IFN-g1

    Claudia Verderio2 and Michela Matteoli

    Calcium-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulatethe activity of adjacent cells, including neurons and oligodendrocytes. We have investigated whether microglia, the immuneeffector cells involved in several diseases of the CNS, are actively involved in this communication network. To address this issue,we analyzed calcium dynamics in fura-2-loaded cocultures of astrocytes and microglia under physiological conditions and in thepresence of the inflammatory cytokine IFN-g. The intracellular calcium increases in astrocytes, occurring spontaneously or as aresult of mechanical or bradykinin stimulation, induced the release of ATP, which, in turn, was responsible for triggering adelayed calcium response in microglial cells. Repeated stimulations of microglial cells by astrocyte-released ATP activated P2X7purinergic receptor on microglial cells and greatly increased membrane permeability, eventually leading to microglial apoptosis.IFN-g increased ATP release and potentiated the P2X7-mediated cytolytic effect. This is the first study showing that ATP mediatesa form of calcium signaling between astrocytes and microglia. This mechanism of intercellular communication may be involvedin controlling the number and function of microglial cells under pathophysiologic CNS conditions. The Journal of Immunology,2001, 166: 63836391.

    R esident microglia are CNS immune effector cells that par-ticipate in many different diseases (1, 2). Under patho-logical conditions they change their morphology, up-reg-ulate a number of surface molecules, and acquire the features ofcytotoxic, phagocytic cells. Although the phenomenology of theiractivation has been well documented, the signals by which theyinteract with their environment are still elusive (2). Microenviron-ment plays a key role in CNS immunopathogenetic events, and theinteractions between microglia and lymphocytes or other immu-nocompetent cells probably play a pivotal role in the developmentof CNS inflammation (3). Identifying the key signals governing theinteractions between microglial cells and their surroundings is ofparamount importance for understanding both normal and patho-logic functioning of the nervous system.

    ATP is a major factor mediating intercellular communication inthe immune and nervous systems and triggers a variety of strik-ingly different biological effects (47). In the brain it is consideredto be the dominant extracellular messenger for astrocyte-to-astro-cyte calcium-mediated communication. Astrocytes release ATPupon mechanical stimulation (8) or glutamatergic receptor activa-tion (9) and respond to ATP with a propagating wave of intracel-lular calcium ([Ca21]i)

    3 increases (8), a process that is thought toserve as a long-range signaling system in the CNS (10, 11).

    Functionally active purinergic receptors have been detected incultured and in situ microglial cells (1217), thus suggesting thepossibility that ATP may also act in astrocyte-to-microglia com-munication. In this study we demonstrate the existence of an ATP-mediated calcium signaling mechanism between astrocytes andmicroglial cells that is increased in the presence of the inflamma-tory cytokine IFN-g and eventually leads to microglial apoptosis.

    Materials and MethodsCell cultures

    Hippocampal mixed glia cultures from embryonic rat pups (embryonic day18) were obtained using previously described methods (18). The cultureswere grown in MEM supplemented with 20% FCS and 5.5 g/L glucose.The astrocytic and microglial components of the cultures were determinedby means of immunostaining for glial fibrillar acidic protein (GFAP) andCSF-1R. The purified microglia were harvested by shaking 3-wk-old cul-tures, seeded on glass coverslips, and cultured in the same medium.

    Fura-2 videomicroscopy

    The cultures were loaded with 5 mM fura-2 pentacetoxy-methylester inKrebs-Ringer solution buffered with HEPES (KRH; 150 mM NaCl, 5 mMKCl, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES/NaOH, pH 7.4) for 1 h at 37C, washed in the same solution to allowde-esterification of the dye, and transferred to the recording chamber of aninverted microscope (Axiovert 100; Zeiss, New York, NY) equipped witha calcium-imaging unit. A modified CAM-230 dual wavelength microflu-orometer (Jasco, Tokyo, Japan) was used as a light source for the assays.The experiments were performed at room temperature (2425C) using anAxon Imaging Workbench 2.2 equipped with a PCO Super VGA Sensi-Cam (Axon Instruments, Foster City, CA). A single astrocyte in the fieldwas gently mechanical stimulated with a glass microelectrode to evokeintercellular calcium waves.

    ATP measurements

    Bioluminescence assay. ATP levels in the extracellular saline incubated30 min with dishes containing a pure hippocampal astrocytic monolayerwere measured using a luciferin/luciferase assay (Molecular Probes, Lei-den, The Netherlands) and a luminometer (Lumat LB9501; Berthold,Nashua, NH). The experimental samples were compared with a standardATP curve created on the basis of saline samples containing known con-centrations of ATP. Each sample was run in duplicate. Most of the sampleswere assayed within 510 min of collection; the others were frozen forsubsequent ATP determination.

    Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology and B.Ceccarelli Centers, Department of Medical Pharmacology, Milan, Italy

    Received for publication October 16, 2000. Accepted for publication March 7, 2001.

    The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from Istituto Superiore di Sanita (Project Mul-tiple Sclerosis 61), Cofin 2000, and Azienda Spaziale Italiana (I/R/149/00).2 Address correspondence and reprint requests to Dr. Claudia Verderio, ConsiglioNazionale delle Ricerche, Cellular and Molecular Pharmacology and B. CeccarelliCenters, Department of Medical Pharmacology, University of Milan, Via Vanvitelli32, 20129 Milan, Italy. E-mail address: claudiav@farma.csfic.mi.cnr.it3 Abbreviations used in this paper: [Ca21]i, intracellular calcium; KRH, Krebs-Ringersolution with HEPES; CSF-1R, CSF receptor; PPADS, pyridoxalphosphate-6-azo-phenyl-2,4-disulfonic acid, tetrasodium; oATP, oxidized ATP; MS, multiple sclero-sis; GFAP, glial fibrillar acidic protein.

    Copyright 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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  • Extracellular ATP bioassay. One milliliter of extracellular saline incu-bated for 30 min with dishes containing a pure hippocampal astrocyticmonolayer in the presence or in the absence of IFN-g was split into twoaliquots before testing on fura-2-loaded astrocytes. One aliquot was pre-treated with apyrase (30 U/ml) for 15 min before testing. Five hundredmicroliters of test solution or saline containing known concentrations ofATP were applied to astrocytes as ATP sensor cells during image acqui-sition, and the amplitude of the [Ca21]i response was measured.


    At the end of the recording session, cultures were fixed at room tempera-ture with 4% paraformaldehyde in 0.1 M phosphate buffer containing 0.12M sucrose for 25 min. Fixed cells were permeabilized with detergent andlabeled with anti-GFAP monoclonal Abs (Sigma, Milan, Italy), and anti-CSF-R1 po