RESEARCH Open Access Adenosine A 2B receptor-mediated … · 2017. 8. 25. · RESEARCH Open Access Adenosine A 2B receptor-mediated leukemia inhibitory factor release from astrocytes

RESEARCH Open Access

Adenosine A2B receptor-mediated leukemiainhibitory factor release from astrocytes protectscortical neurons against excitotoxicityShamsudheen Moidunny1, Jonathan Vinet1, Evelyn Wesseling1, Johan Bijzet2, Chu-Hsin Shieh3,Sven CD van Ijzendoorn4, Paola Bezzi5, Hendrikus WGM Boddeke1 and Knut Biber1,3*

Abstract

Background: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widelyreported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which isenhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however,has remained an unanswered question. Since neuronal stress is associated with production of extracellularadenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptorsignaling.

Methods: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-outanimals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to studyLIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed byBonferroni post-hoc test was used for statistical analysis.

Results: We show here that glutamate-stressed cortical neurons induce LIF expression through activation ofadenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC),mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover,LIF concentration in the supernatant in response to 5′-N-ethylcarboxamide (NECA) stimulation was directlycorrelated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated releasepathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11,but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recyclingendosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survivalof cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with ananti-LIF neutralizing antibody.

Conclusions: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid releaseof LIF promotes the survival of cortical neurons against excitotoxicity.

Keywords: 5′-N-Ethylcarboxamide (NECA), Leukemia inhibitory factor, Neuroprotection, Glutamate

* Correspondence: [email protected] of Neuroscience, Section Medical Physiology, University MedicalCenter Groningen, University of Groningen, A. Deusinglaan 1, 9713 AV,Groningen, The Netherlands3Department of Psychiatry and Psychotherapy, Section Molecular Psychiatry,University of Freiburg, Hauptstrasse 5, 79104, Freiburg, GermanyFull list of author information is available at the end of the article

JOURNAL OF NEUROINFLAMMATION

© 2012 Moidunny et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundLeukemia inhibitory factor (LIF) is a soluble glycopro-tein that belongs to the family of interleukin (IL)-6-typecytokines. Other members of this family include IL-6,IL-11, ciliary neurotrophic factor (CNTF), oncostatin M(OSM), cardiotrophin-1 (CT-1) and novel neurotrophin-1 (NNT-1) [1], which display pronounced trophic aswell as protective properties during pathophysiology ofthe central nervous system (CNS) and are hence re-ferred to as neuropoietic cytokines or neurokines [2].Specific functions of LIF in the nervous system includeinduction of cholinergic differentiation of sympatheticneurons, induction of neuropeptide and choline acetyl-transferase (ChAT) gene expression [3], regulation ofpolyneuronal innervation of neuromuscular junction[4,5] and regulation of the HPA axis [6,7]. Furthermore,LIF signaling is crucial for development of the nervoussystem, including development of sensory and motorneurons [8,9] and glial cells [10]. Consistently, reducednumbers of astrocytes and oligodendrocytes are foundin LIF knock-out mice [11]. During inflammation, LIFhas been suggested to be both pro- and anti-inflammatory and appears to play a key role in neuralinjury and regeneration. We and others have previouslydemonstrated the neuroprotective properties of LIFagainst damages caused by excitotoxicity, light, et cetera[12-14]. Moreover, promotion of axonal regenerationand oligodendrocyte growth and survival by LIF suggestsits potential for reducing damage associated with centralinflammatory demyelinating diseases such as multiplesclerosis [15-17].In the CNS, astrocytes are considered to be the major

source for LIF [18,19], and its expression in the brain issignificant during pathological conditions including is-chemia [20,21], multiple sclerosis [22], Alzheimer’s andParkinson’s diseases [23] and brain injury [24]. The fac-tors responsible for elevated LIF induction during CNSpathology are largely unknown. One of the candidatesidentified recently to induce LIF expression in astrocytesis ATP [18,25], levels of which also rise during condi-tions like high-frequency neuronal activity, seizure, is-chemia and hypoxia [26,27]. However, extracellular ATPis rapidly hydrolyzed by a cascade of ectonucleotidasesresulting in an enhanced level of adenosine [26,28]. Cor-respondingly, excitotoxic conditions such as ischemia,hypoxia, seizure and head injury are known to induce arapid increase in extracellular adenosine concentrations,up to 100 times that of the resting concentration [29-33]. There is abundant evidence for immune regulationby adenosine [34] including expression and release ofgrowth factors and cytokines such as nerve growth factor(NGF), S100beta, IL-6 and CCL2 in glial cells [35-39].However, it is not known whether adenosine can induceLIF expression in astrocytes.

In the present study, we investigated the potential in-fluence of adenosine receptor activity on LIF releasefrom cultured astrocytes.

MethodsChemicals and reagentsNeurobasal media, Hank’s balanced salt solution (HBSS),phosphate-buffered saline (PBS), sodium pyruvate, L-glutamine, penicillin-streptomycin, hydroxyethyl pipera-zineethanesulfonic acid (HEPES), glutaMAX-1 and B27supplement were obtained from Gibco (Breda, TheNetherlands). Dulbecco’s modified Eagle’s medium(DMEM) and fetal calf serum (FCS) were obtained fromPAA Laboratories (Cölbe, Germany). Trypsin was ob-tained from Life Technologies (Breda, The Netherlands).L-leucine methyl ester (LME) and the remaining cellmedium components were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Recombinantmouse LIF (rmLIF: LIF2005) was obtained from Milli-pore (Amsterdam, The Netherlands). Brefeldin A (BFA),caffeine, L-glutamate, adenosine A2B receptor antagonist(MRS 1754), protein kinase A (PKA) inhibitor (KT5720), protein kinase C (PKC) inhibitor (Ro 31–8220),p38 mitogen-activated protein kinases (MAPK) inhibitor(SB 203580), and adenosine analog (5′-N-Ethylcarboxa-mide or NECA) were obtained from Sigma-Aldrich(Zwijndrecht, The Netherlands). Non-hydrolysable ATP(2MeSATP), adenosine A2A receptor antagonist (ZM241385), adenosine A2A receptor agonist (CGS 21680)and MEK1/2 inhibitor (U 0126) were obtained fromTocris Bioscience (Bristol, UK). NF-kB inhibitor (BAY11–7082) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125) were obtained from Calbiochem (Darmstadt,Germany). Reagents used in immunoblotting experimentswere purchased from Bio-Rad Laboratories (Veenendaal,The Netherlands) with the exception of the polyvinylidenefluoride (PVDF) membranes that were obtained fromMillipore (Bedford, MA).

AnimalsWild-type C57BL/6 J (1 to 2 days postnatal) mice wereobtained from Central Laboratory Animal Facility (Uni-versity of Groningen, The Netherlands). Adenosine A2B

receptor knock-out (A2B KO) mice (1 to 2 days postna-tal) with the same genetic background were kindly pro-vided by Professor Marco Idzko (University of Freiburg,Germany). Wild-type C57BL/6 J (14 to 15 days em-bryonic) mice were obtained from Harlan (Horst, TheNetherlands). All procedures were in accordance withthe regulation of the Ethical Committee for the use ofexperimental animals of the University of Groningen,The Netherlands (License number DEC 4623A and DEC5913A). Animals were housed in standard MakrolonTM

(Bayer AG, Leverkusen, Germany) cages and maintained

Moidunny et al. Journal of Neuroinflammation 2012, 9:198 Page 2 of 18http://www.jneuroinflammation.com/content/9/1/198

on a 12 hour light/dark cycle. They received food andwater ad libitum.

Primary neuronal culturePrimary culture of cortical neurons from mouse embryo(~E15) was established as described previously [13].Briefly, cortices from embryonic brains were dissected inice-cold HBSS supplemented with 30% glucose. Menin-ges were removed, and the tissues were treated withtrypsin before they were gently dissociated by triturationin neuronal culture media (neurobasal medium supple-mented with 2% B27, 1 mM sodium pyruvate, 2 mM L-glutamine and 50 U/mL penicillin-streptomycin). Thecell suspension was filtered using cell strainer (70 μm)(BD Falcon, Franklin Lakes, NJ, USA) before centrifuga-tion (800 rpm for 10 minutes). Cells were then seededon poly-D-lysine-coated six-well plates (1.5 x 106 cells/well) and maintained in neuronal culture media in a hu-midified atmosphere with 5% CO2 at 37°C. The culturemedium was refreshed the next day to get rid of debris.The neuronal purity as determined by Microtubule-associated protein 2 (MAP2)-staining was around 98%(data not shown) [13]. Cultures were used after 5 daysin vitro.

Induction of excitotoxicityCortical neuron cultures were subjected to an excito-toxic challenge with glutamate (50 μM, for 1 hour), afterwhich cultures were refreshed with fresh media andwere incubated at 37°C. Supernatants from neuron cul-tures (untreated and glutamate-treated) were collected18 hours after glutamate challenge and were applied tothe primary astrocyte cultures.

Primary astrocyte culturesPrimary astrocyte cultures were established from cere-bral cortices of postnatal (1 to 2 days) C57BL/6 J andA2B KO mice according to a previously described pro-cedure [40], which was modified to reduce microglialcontamination [41]. Microglial cells were separated fromthe astrocytic monolayer by 1-hour shake-off at150 rpm. This procedure was repeated two times withan interval of 4 days in vitro between each shake off, fol-lowed by an overnight shake-off at 240 rpm to removeoligodendrocyte precursor cells. Purified astrocytes werewashed with HBSS buffer containing 1 mM ethylenedia-minetetraacetic acid (EDTA) and further detached usingHBSS with 0.1% trypsin. Cells were reseeded with freshastrocyte culture medium (DMEM supplemented with5% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate and50 U/mL penicillin-streptomycin) in multi-well plates(5 x 104 cells/cm2) and maintained in culture to con-fluency. To further reduce microglial contamination,confluent astrocyte cultures were treated with 5 mM

LME, a lysosomotropic agent [42], for 4 to 5 hours.Astrocytes were ready for experiments after 1 to 2 days.Our cell preparations had a high percentage of astro-cytes (≥95%), which was confirmed by immunostainingagainst GFAP (astrocyte specific marker) and CD11b(microglial specific marker) (data not shown).

Real-time polymerase chain reactionTotal RNA of primary astrocytes was extracted, puri-fied and transcribed into cDNA as described previ-ously [13]. Quality of the cDNA was examined usingthe following housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer pairs: Fw5′-CATCCTGCACCACCAACTGCTTAG-3′ and Rev 5′-GCCTGCTTCACCACCTTCTTGATG-3′[Accessionnum-ber: NM-008084]. The effect of neuronal supernatantsand NECA on LIF mRNA expression in cultured astro-cytes was analyzed by real-time PCR (qPCR) using theiCycler and iQ™ SYBR™ Green supermix (Bio-Rad, Vee-nendaal, The Netherlands ). Mouse Hypoxanthine phos-phoribosyltransferase 1 (HPRT1) and GAPDH primerswere used for normalization to housekeeping genes(data normalized to HPRT1 are not shown), and thesegenes showed no variations in response to the experimen-tal treatments. The primer pairs used for qPCR were: LIF(Fw 5′-ATGTGCGCCTAACATGACAG-3′ and Rev 5′-TATGCGACCATCCGATACAG-3′) [Accession number:NM-008501]; GAPDH (Fw 5′-ATGGCCTTCCGTGTTCCTAC-3′ and Rev 5′-GCCTGCTTCACCACCTTCTT-3′) [Accession number: AF106860] and HPRT1 (Fw 5′-GACTTGCTCGAGATGTCA-3′ and Rev 5′-TGTAATCCAGCAGGTCAG-3′) [Accession number: NM-013556].The comparative Ct method (amount of target ampliconX in Sample S, normalized to a reference R and relatedto a control sample C), was calculated by:

2� CtX; S� CtR; Sð Þ � CtX;C� CtR;Cð Þð Þ

and was used to determine the relative gene expressionlevels [43].

Western blotWestern blotting on cultured cortical astrocytes was per-formed as previously described [13]. Equal amounts ofprotein (30 μg) were loaded to 12.5 or 15% sodiumdodecyl sulfate-polyacrylamide gels and subsequentlytransferred to PVDF membranes. The membranes wereblocked using Odyssey™ Blocking Buffer (OBB; LI-CORBiosciences, Cambridge, UK; diluted 1:1 in PBS) for1 hour and incubated overnight at 4°C with differentcombinations of primary antibodies (diluted in 1:1 OBBand PBS + 0.1% Tween 20 (PBS-T)): mouse monoclonalanti-β-actin (1:8000, Abcam, Cambridge, UK); rabbitmonoclonal anti-phospho-NF-κB p65 (Ser536) (1:1000,


Cell Signaling Technology, Leiden, The Netherlands);and rat monoclonal anti-LIF (MAB449; 1 μg/mL, R&DSystems, Oxford, UK). The next day, membraneswere washed in PBS-T (four times for 5 minuteseach time) and incubated for 1 hour at roomtemperature with appropriate fluorescence conjugatedsecondary antibodies (diluted in PBS-T): donkey anti-mouse IR Dye 680 (1:10000, LI-COR Biosciences,Cambridge, UK); goat anti-rat IR Dye 680 (1:10000,LI-COR); and donkey anti-rabbit IR Dye 800CW(1:10000, LI-COR). Membranes were washed again inPBS-T (four times for 5 minutes each time) and thefluorescent bands were detected using LI-COR’sOdyssey™ infrared imaging system.

Leukemia inhibitory factor ELISAA total of 1 mL of supernatant was collected from eachwell of the six-well plates of primary mouse astrocyte cul-tures, and these samples were stored at −20°C. ELISAplates (96-well, Costar, Corning Life Sciences, Amsterdam,The Netherlands) were coated overnight at roomtemperature with 100 μl/well of primary antibody goatanti-LIF (AF449; 0.5 μg/mL, R&D Systems, Oxford, UK)diluted in 0.01 M PBS (pH 7.4). The following day, theplates were washed six times with wash buffer (0.25 MTris–HCl pH 8, 0.15 M NaCl, 0.05% Tween-20) using anautomated microplate washer and air dried (this step isrepeated after each incubation step). Plates were subse-quently incubated for 1 hour at room temperature with200 μl/well of blocking buffer (0.01 M PBS, 2% BSA).After blocking, the plates were incubated with superna-tants from astrocyte cultures (100 μl/well) for 2 hours atroom temperature. Two dilutions (1:2 and 1:4) of eachsample, diluted in incubation buffer (0.01 M PBS, 0.2%gelatin, 0.05% Tween-20), were made in triplicates. Theplates were then incubated for 1 hour at roomtemperature with 100 μl/well of the detection antibody,biotinylated goat anti-LIF (BAF449; 0.05 μg/mL; R&D Sys-tems, Oxford, UK) diluted in incubation buffer, followedby an incubation for 30 minutes at room temperature with100 μl/well of Streptavidin-horseradish peroxidase (HRP)conjugate (1:8000, Sanquin Reagents, Amsterdam, TheNetherlands). The plates were then incubated for 15to 20 minutes at room temperature with 100 μl/wellof TMB detection buffer (0.1 M acetate buffer, 0.1 Msodium-acetate, pH adjusted with 1 M citric acid(0.21 g/mL; dissolve 2 tablets of 3, 3′, 5, 5′-tetramethyl benzidinedihydrochloride in 11 mL ofTMB buffer and add 2 μl of 30% H2O2)). Upon stablecolor formation the reactions were stopped by adding100 μl/well of 1 M H2SO4. Absorbance of the sampleswas measured using VersaMax, a spectrophotometricELISA plate reader, and SoftMax Pro software (Molecu-lar Devices, CA, USA) at 450 nm, with a background

correction at 575 nm. Recombinant mouse LIF (15 to2000 pg/mL) was used to plot the standard curve.

MTT assaySurvival of cultured embryonic cortical neurons orcultured neonatal astrocytes against various experi-mental treatments was measured by the colorimetricMTT (3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetra-zolium bromide) assay, as described previously [44].MTT solution (0.5 mg/mL final concentration) wasadded to cultured cells and incubated for 4 hours,after which, cells were lysed and MTT-formazan solu-bilized in dimethyl sulfoxide (DMSO) on an orbitalshaker for 15 minutes. Optical density measure of eachsample was determined using an automated ELISAreader - the Varioskan Flash spectral scanning multimodereader (Thermo Scientific, FL, USA) at 570 nm, with abackground correction at 630 nm.

Immunocytochemistry and confocal microscopyAstrocytes cultured on glass cover slips were fixed for15 minutes in 4% paraformaldehyde. After severalwashes in PBS, the cells were blocked for 45 minuteswith 5% normal goat serum (Vector Laboratories,Burlingame, CA, USA) in PBS containing 0.1% TritonX(Sigma, Zwijndrecht, The Netherlands). The cover slipswere then incubated overnight at 4°C with rat anti-LIFprimary antibody (5 μg/mL, R&D Systems, Oxford, UK)in combination with one of the following primary anti-bodies: rabbit anti-Rab11 (1:400, Zymed, San Francisco,CA, USA); rabbit anti-chromogranin A&B (1:100,Novus Biologicals, Cambridge, UK); mouse anti-clathrin(1:1000, Abcam, Cambridge, UK); rabbit anti-pHogrinC-terminal (1:100, kind gift of Professor J.C. Hutton(Denver, USA)) and rabbit anti-giantin (1:1000, Covance,Princeton, NJ, USA). The following day, cells wererinsed three times with PBS and incubated for 1 hourwith the appropriate secondary antibodies: donkey anti-rat CY3 (1:500, Jackson ImmunoResearch Laboratories,Uden, The Netherlands); donkey anti-rabbit Alexa Fluor488 (1:500, Molecular Probes, Leiden, The Netherlands)and donkey anti-mouse Alexa Fluor 488 (1:500, Molecu-lar Probes). The cover slips were then rinsed with PBSand mounted on microscopic slide with Mowiol(Sigma, Zwijndrecht, The Netherlands) and analyzedwith a Leica SP2 AOBS system (Leica Microsystems,Rijswijk, The Netherlands). Pictures were deconvolutedusing the software Huygens Pro (SVI, Hilversum, TheNetherlands). Primary antibody omission served as thecontrol.

Statistical data analysisThe absolute data values were normalized to the control inorder to allow multiple comparisons. Statistical analyses


were performed by one-way analysis of variance (ANOVA)followed by Bonferroni post-hoc test, using the StatisticalPackage for the Social Sciences (SPSS, Chicago, IL, USA).In all cases, P values < 0.05 were considered statisticallysignificant.

ResultsGlutamate-challenged cortical neurons induce LIFexpression in cultured astrocytes through adenosinereceptor activationWe have previously shown that treatment of culturedcortical neurons with glutamate (50 μM, for 1 hour)reduces cell survival by 60%, when compared to un-treated controls [13]. In order to investigate whetherneuronal death induces LIF synthesis in astrocytes,supernatants from cultured cortical neurons were col-lected 18 hours after glutamate treatment (50 μM for1 hour) and applied to cultured cortical astrocytes. It isshown here that treatment of cultured astrocytes for2 hours with supernatant from untreated neurons didnot change LIF expression (Figure 1). On the otherhand, supernatant from glutamate-challenged neuronsinduced approximately three times greater expression ofastrocytic LIF mRNA (Figure 1). The induction of LIFmRNA expression by glutamate-challenged neuronal

supernatants was absent in the presence of the non-specific adenosine receptor antagonist (caffeine, 50 μM)(Figure 1) and by cocktail of the specific adenosine A2

receptors antagonists (A2A antagonist: ZM 241385, 250nM; A2B antagonist: MRS 1754, 250 nM) (Figure 1), sug-gesting that enhanced LIF expression in astrocytesinduced by glutamate-challenged neuronal supernatantsis mediated through adenosine A2A and/or A2B receptorsubtypes.

NECA-induced LIF expression and secretion levels incultured mouse astrocytes is concentration- andtime-dependentIn order to further investigate adenosine receptor-mediated LIF expression in astrocytes, we used NECA, anon-selective adenosine receptor agonist. As shown inFigure 2A, NECA-induced LIF mRNA expression in cul-tured astrocytes was concentration- and time-dependent,with maximum induction after 2 hours of incubationwith 1 and 10 μM NECA. Subsequently, the effect ofNECA (1 μM) on LIF protein expression was analyzedby Western blot. Elevated LIF protein expression wasdetected after 1 hour of NECA treatment, with a max-imum induction after 2 to 4 hours (Figure 2B). Consist-ently, ELISA analysis revealed LIF protein content insupernatants from untreated astrocyte cultures, whichincreased in time upon treatment with NECA (1 μM)(Figure 2C).

NECA-induced LIF expression and secretion levels isdependent on adenosine A2B receptor activationIn subsequent experiments, specific antagonists of ad-enosine A2A and A2B receptors (ZM 241385 and MRS1754, respectively) were used to identify the receptorsubtype involved in NECA-induced LIF expression andrelease in cultured astrocytes. Pre-treatment of astro-cytes with ZM 241385 (250 nM, added 30 minutes be-fore NECA) did not abolish NECA-induced LIF mRNAand protein expression (Figure 3A and 3C). In contrast,NECA-induced LIF mRNA (Figure 3A) and protein ex-pression (Figure 3C) and release (Figure 3D) were com-pletely inhibited by MRS 1754 pre-incubation (250 nM,added 30 minutes before NECA). In addition, specificadenosine A2A receptor agonist (CGS 21680; 1 μM)failed to induce LIF mRNA or protein expression andrelease (Figure 3A, C, D). The involvement of A2B recep-tors was further confirmed in A2B KO astrocytes whereNECA stimulation for up to 24 hours did not induceLIF mRNA expression (Figure 3B). Instead, astrocyteswithout A2B receptors responded to NECA stimulationwith a down-regulation of LIF mRNA at 8 and 24 hours(Figure 3B). Taken together, these results clearly showthat NECA-induced LIF expression and release from

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Figure 1 Glutamate-stressed cortical neurons induce leukemiainhibitory factor (LIF) gene expression in primary corticalastrocytes, by a mechanism dependent on astrocytic adenosinereceptor activation. Supernatants of primary mouse corticalneurons (NS) collected 18 hours following treatment without (untr.)or with glutamate (glut.; 50 μM, for 1 hour) were applied (1:1dilution by volume) to the primary cultured cortical astrocytes for2 hours. Where indicated, astrocytes were pre-treated with caffeine(50 μM) or a cocktail of adenosine A2A and A2B receptor antagonists(A2A antagonist: ZM 241385, 250 nM; A2B antagonist: MRS 1754, 250nM), for 30 minutes before incubation with neuronal supernatantsand were analyzed for LIF mRNA expression (relative to GAPDH)using real-time PCR. Data are normalized to the control andpresented as Mean± SEM of three independent experiments.P< 0.05.


cultured mouse astrocytes is dependent on the activationof adenosine A2B receptors.NECA-induced LIF expression and secretion levels in

primary astrocytes are mediated through the Gq/11-PLC-PKC and MAPKs, but not through Gs-cAMP-PKApathway.In order to analyze the intracellular signaling pathways

that couple A2B receptor activity and LIF expression andrelease in astrocytes, various specific blockers of signal-ing routes were used. To determine the potential toxicity

of these blockers, cultured astrocytes were incubated for24 hours with the used and the doubled concentrationof these blockers, and cellular survival was assessed byMTT assay. None of the used blockers at the appropriateconcentration caused significant toxicity; the onlyblocker that negatively influenced astrocytic survival atthe double concentration was the NF-κB inhibitorBAY 11-7082 (Figure 4).Adenosine A2B receptors are coupled to two types of

G-proteins: Gs and Gq/11 [45]. Activation of Gs proteins

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Figure 2 NECA increases leukemia inhibitory factor (LIF) expression and secretion levels in primary mouse astrocytes. (A) Primarycortical astrocytes were treated without or with NECA (1, 10 or 20 μM) for 0.5, 1, 2, 4, 8, 12 and 24 hours. Cells were then analyzed for LIF mRNAexpression (relative to GAPDH) by real-time PCR. Data are normalized to the control and presented as Mean± SEM of three independentexperiments. P< 0.05. (B) shows Western blot analysis of cultured astrocytes treated without or with NECA (1 μM) for 1, 2, 4, 6, 8 and 12 hours todetermine LIF protein levels. β-actin served as a loading control. (C) shows a representation of sandwich ELISA experiment performed to detectLIF content in supernatants of cultured astrocytes that were treated without or with NECA (1 μM) for 1, 2, 4, 6, 8 and 12 hours. Each barcorresponds to the mean concentration of LIF in triplicate samples; error bars indicate SEM. Observations were confirmed by repeating theexperiments two additional times. P< 0.05. NECA, 5′-N-ethylcarboxamide.


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stimulates cyclic AMP (cAMP) leading to PKA activa-tion or Exchange Protein Activated by cAMP (EPAC)signaling pathways [46], whereas Gq/11 proteins stimu-late PKC via phospholipase C (PLC). In order to deter-mine which pathway downstream of the A2B receptor isresponsible for NECA-induced LIF expression and re-lease, we used specific inhibitors of PKA and PKC (KT5720 and Ro 31–8220, respectively) [47]. Pre-incubationof astrocytes with KT 5720 (250 nM, added 30 minutesbefore NECA) did not affect NECA-induced LIF expres-sion (both mRNA and proteins) (Figure 5A and 5B).However, pre-treatment with Ro 31-8220 (250 nM,added 30 minutes before NECA) reduced NECA-induced LIF mRNA (Figure 5A) as well as protein levels(Figure 5B). Consistently, Ro 31-8220, but not KT 5720,suppressed LIF content in the supernatant from NECA-treated astrocyte cultures (Figure 5C), indicating thatadenosine A2B receptor-mediated LIF expression andrelease from astrocytes require PKC, but not PKA,activation.

Basal and NECA-induced LIF expression and secretionlevels in primary astrocytes are dependent on ERK1/2-and p38- but not on JNK-MAPK activationActivation of the PKC pathway has been associatedwith effects mediated through mitogen-activated proteinkinase (MAPK) signaling [48,49]. In order to determinethe involvement of MAPKs in NECA-induced LIF ex-pression and release, specific inhibitors of the threeMAPK cascades: p38, extracellular signal-regulated kin-ase (ERK) 1/2, and c-Jun N-terminal kinase (JNK) (SB203580, U 0126 and SP 600125, respectively), wereused. It is shown here that pre-treatment (2 hours be-fore NECA) of cultured astrocytes with SB 203580(10 μM) and U 0126 (5 μM) significantly reduced basalas well as NECA-induced LIF mRNA expression(Figure 6A) and protein release (Figure 6B). On theother hand, pre-treatment with SP 600125 (10 μM,added 2 hours before NECA) affected neither basal, norNECA-induced LIF mRNA expression or protein re-lease (Figure 6A and 6B), suggesting that both p38 andERK1/2, but not JNK, are important for basal, as well

as NECA-induced, LIF expression and release in cul-tured astrocytes.

Basal and NECA-induced LIF expression and secretionlevels in primary astrocytes are dependent on NF-κBactivationIL-6 gene expression in cultured astrocytes is enhancedby NECA [36,38], similar to our present findings withLIF. Since NF-κB is a key transcription factor that regu-lates IL-6 gene expression [50,51], we wondered whetherNECA-induced LIF gene expression is similarly regu-lated by NF-κB. Analysis of the mouse LIF promoter re-gion (using Genomatix-MatInspector software (http://www.genomatix.de/)), identified multiple consensus bind-ing sequences for NF-κB at the first 500 bp upstream tothe transcription start site (data not shown). Treatmentof cultured astrocytes with NECA (1 μM) for 0.5, 1, 2and 4 hours, induced activation of NF-κB, which wasdetected by phosphorylation of NF-κB p65 subunit byWestern blot (Figure 7A). In addition, the specific in-hibitor of NF-κB activation, BAY 11-7082 (10 μM;added 2 hours before NECA) reduced NECA-inducedphosphorylation of NF-κB p65 (Figure 7A) and signifi-cantly inhibited basal as well as NECA-induced LIF ex-pression (both mRNA and protein) and release(Figure 7B–D), strongly indicating that LIF gene expres-sion is regulated by NF-κB.

LIF secretion in primary astrocytes is constitutive andindependent of NECA stimulationSupernatants from untreated astrocytes contained basallevels of LIF, suggesting that it could be constitutivelyreleased from these cells. Since we observed increasedLIF concentrations in astrocyte supernatants after NECAstimulation, we investigated whether or not NECAplayed a direct role in LIF secretion. Thus, we blockedthe early secretory pathway with Brefeldin A (BFA), afungal metabolite that causes Golgi-derived proteinsto accumulate in the endoplasmic reticulum [52]. Cul-tured astrocytes were pre-treated for 1 hour with BFA(5 μg/mL), followed by treatment without or with NECA(1 μM) for 1 or 4 hours (for immunocytochemistry and

(See figure on previous page.)Figure 3 NECA-induced leukemia inhibitory factor (LIF) expression and secretion levels in primary astrocytes are dependent onadenosine A2B receptor activation. Primary cortical astrocytes were treated with the adenosine analog, NECA (1 μM) or a selective adenosineA2A receptor agonist (CGS 21680, 1 μM) for 2 hours (for real-time PCR) or 4 hours (for Western blot and ELISA). Where indicated, astrocytes werepre-treated for 30 minutes with selective adenosine A2A receptor antagonist (ZM 241385, 250 nM) or adenosine A2B receptor antagonist (MRS1754, 250 nM), prior to NECA stimulation. (A and B) show real-time PCR analyses of LIF gene expression (relative to GAPDH) in wild-type and A2Bknock-out astrocytes, respectively. Data are normalized to the control and presented as Mean± SEM of three independent experiments. P< 0.05.(C) shows Western blot analyses to detect LIF protein levels in wild-type astrocytes. β-actin served as a loading control. (D) shows arepresentation of sandwich ELISA experiment performed to detect LIF content in supernatants of cultured wild-type astrocytes. Each barcorresponds to the mean concentration of LIF in triplicate samples; error bars indicate SEM. Observations were confirmed by repeating theexperiments two additional times. P< 0.05. NECA, 5′-N-ethylcarboxamide.


http://www.genomatix.de/

http://www.genomatix.de/

LIF-ELISA, respectively). In BFA-treated cells, all LIF-immunoreactivity co-localized with the Golgi markerGiantin (Figure 8B), compared to control conditions,where punctate LIF stainings could be observed all overthe cytoplasm (Figure 8A). LIF levels in the supernatantsfrom BFA-treated (5 μg/mL, for 5 hours) astrocyte cul-tures were significantly lower than that of the control(Figure 8C), implying that LIF is indeed constitutivelysecreted. Moreover, NECA treatment (1 μM, for 4 hours)did not stimulate LIF secretion in BFA-treated astrocytes(Figure 8C), further indicating that increased LIF levelsin astrocytic culture supernatants after NECA stimula-tion require synthesis of new proteins and does not in-volve a ready-releasable post-Golgi reservoir of LIF.

LIF secretion in primary astrocytes is mediated throughrecycling endosomesWe further investigated the type of organelles respon-sible for LIF release. Several reports have shown thatcytokines such as IL-6 and transforming growth factor β(TGFβ) are secreted through specialized secretory gran-ules called large dense-core vesicles (LDCV) [53,54].However, when astrocytes were co-stained for LDCVmarkers such as Chromogranin A&B, or pHogrin andLIF, no co-localization was observed (Figure 9A and 9B).In contrast, co-localization between LIF and clathrinwas observed (Figure 9C). Clathrin is a marker forendosomal vesicles and is sometimes used as a marker

for constitutive release [55,56]. Furthermore, LIF par-tially co-localized with Rab11 (Figure 9D), which is amarker for recycling endosomes [57,58], suggestingthat recycling endosomes, rather than LDCV, mediatesecretion of LIF.

NECA-treated astrocytes induce LIF-mediated protectionof cultured cortical neurons against excitotoxicityWe have previously shown that recombinant mouse LIF(rmLIF) protein protects mouse cortical neurons againstexcitotoxicity [13]. In order to understand whetherNECA stimulation of astrocytes specifically would in-duce accumulation of neuroprotective LIF, astrocyte cul-tures were refreshed with new media shortly beforeNECA stimulation (1 μM for 4 hours) and the super-natant was collected. As shown in Figure 10, pre-treatment (1:1 dilution, for 24 hours) with supernatantfrom NECA-treated astrocytes significantly reducedthe glutamate-induced cell death of cultured corticalneurons. A similar protective effect was observed bypre-treatment with rmLIF (0.1 ng/mL, for 24 hours)(Figure 10). Pre-treatment of the neurons (for 24 hoursbefore application of glutamate) with NECA (1 μM) orsupernatant from untreated astrocytes did not affectglutamate-induced neuronal cell death (Figure 10). Wefurther investigated whether neuroprotection induced byNECA-treated astrocyte supernatant was mediated byLIF, by incubating the supernatants for 1.5 hours at 37°C

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with a LIF-neutralizing antibody (goat polyclonal anti-LIF, AF449, 100 ng/mL, R&D Systems, Oxford, UK) be-fore applying to the neuronal cultures. The optimumconcentration of LIF-neutralizing antibody was standar-dized by an efficiency test, performed according to man-ufacturer’s recommendations (data not shown). Inaddition, the effect of rmLIF protein treated with LIF-neutralizing antibody, on neuronal survival against glu-tamate, served as a control (Figure 10). Interestingly,LIF-neutralized supernatant from NECA-treated astrocyte

cultures failed to protect neurons against glutamate(Figure 10), suggesting a direct neuroprotective mechanismof the endogenous LIF produced by astrocytes in responseto NECA stimulation.

DiscussionWe have previously shown that recombinant LIF pro-tects neurons against glutamate-induced excitotoxicity[13]. In this study, we investigated the mechanism bywhich astrocytes produce and release LIF. Here we show

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Figure 5 NECA-induced leukemia inhibitory factor (LIF) expression and secretion levels in primary astrocytes are blocked by proteinkinase C (PKC) inhibitor, but not by protein kinase A (PKA) inhibitor. Primary cortical astrocytes were treated without or with NECA (1 μM)for 2 hours (for real-time PCR) or 4 hours (for Western blot and ELISA). Where indicated, astrocytes were pre-treated for 30 minutes with specificinhibitors of PKA (KT 5720, 250 nM) or PKC (Ro 31–8220, 250 nM), prior to NECA stimulation. (A) shows real-time PCR analysis of LIF geneexpression (relative to GAPDH). Data are normalized to the control and presented as Mean± SEM of three independent experiments. P< 0.05. (B)shows Western blot analysis to detect LIF protein levels. β-actin served as a loading control. (C) shows a representation of sandwich ELISAexperiment performed to detect LIF content in supernatants of cultured astrocytes. Each bar corresponds to the mean concentration of LIF intriplicate samples; error bars indicate SEM. Observations were confirmed by repeating the experiments two additional times. P< 0.05. NECA, 5′-N-ethylcarboxamide.


that glutamate-induced neuronal excitotoxicity leads toadenosine receptor-mediated increase in LIF mRNA ex-pression in cultured cortical astrocytes. We demonstratethat the upregulation of LIF mRNA and protein is ad-enosine A2B receptor-dependent, and is mediatedthrough Gq/11-PLC-PKC-MAPK-NF-κB signaling path-ways. We furthermore show that LIF is transitingthrough the Golgi and is found in recycling endosomesrather than in LDCV. Finally, LIF produced by astro-cytes can protect neurons against excitotoxicity.It has been known for more than a decade that astro-

cytes are the major source for LIF in the CNS[18,19,59,60]. However, the factors responsible for theregulation of LIF expression in these cells are still largelyunknown. It is well known that stressed neurons releasenucleotides such as ATP and adenosine [30,61]. Re-cently, it was demonstrated that astrocytes increase LIFproduction and release in response to ATP receptorstimulation [18]. In this study, the authors demonstratethat neurons during action potentials can secrete ATP,

which triggers LIF production in astrocytes. This ATP-dependent upregulation of LIF by astrocytes is respon-sible for the promotion of oligodendrocyte-mediatedmyelination around neuronal axons. ATP is also knownto be secreted by neurons during stressful conditionssuch as seizure, ischemia and hypoxia [26,27]. However,when we blocked adenosine receptors with the non-selective antagonist caffeine, or with specific A2A/A2B re-ceptor antagonists, the effect of glutamate-stressed neur-onal supernatants on LIF expression in astrocytes wasabsent, suggesting that adenosine, but not ATP, is re-sponsible for astrocytic LIF production duringglutamate-induced neuronal stress. Thus, it might behypothesized that depending on the CNS status, astro-cytic LIF expression and secretion is differentially regu-lated; during normal neuronal activity and developmentATP is involved whereas during neuronal insults, adeno-sine might enhance LIF secretion by astrocytes.Several studies have demonstrated the involvement of

adenosine A2B receptors in the regulation of IL-6

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Figure 6 Basal and NECA-induced leukemia inhibitory factor (LIF) expression and secretion levels in primary astrocytes are dependenton ERK1/2- and p38- but not on JNK-MAPK activation. Primary cortical astrocytes were treated without or with NECA (1 μM) for 2 hours (forreal-time PCR) or 4 hours (for ELISA). Where indicated, astrocytes were pre-treated for 2 hours with specific inhibitors of ERK1/2 (U 0126, 5 μM),p38 (SB 203580, 10 μM) or JNK (SP 600125, 10 μM) pathways, prior to NECA stimulation. (A) shows real-time PCR analysis of LIF gene expression(relative to GAPDH). Data are normalized to the control and presented as Mean± SEM of three independent experiments. P< 0.05. (B) shows arepresentation of sandwich ELISA experiment performed to detect LIF content in supernatants of cultured astrocytes. Each bar corresponds to themean concentration of LIF in triplicate samples; error bars indicate SEM. Observations were confirmed by repeating the experiments twoadditional times. P< 0.05. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NECA,5′-N-ethylcarboxamide.


expression in various cell types in vitro [38,47,48,62,63]as well as in vivo [64], suggesting that A2B receptorsmight also be essential in the regulation of other IL-6-type cytokines. Our results show that adenosine-dependent LIF regulation is mediated through the A2B

receptor, since no increase in LIF expression was foundin cultured astrocytes from A2B receptor deficient mice.Instead NECA caused a down-regulation of LIF mRNAafter 8 and 24 hours in these cells, indicating that knock-ing out A2B receptors may have unmasked an inhibitoryeffect on LIF mRNA expression of an unidentified ad-enosine receptor. Whether or not this might explain thevery short-lived effect of NECA on LIF mRNA expres-sion in wild-type astrocytes is at the moment unclearand a subject of future investigations. We furthermoredemonstrated that A2B-mediated LIF expression isdependent on the PKC, but not the PKA pathway. These

data are in line with the study of Aloisi and colleagues,which demonstrated that LIF modulation by pro-inflammatory cytokines in human astrocytes wasmediated through PKC activation [59]. Moreover, PKChas also been shown to be essential in IL-6 regulation[47,48,62,65], revealing a prominent role for PKC in thesignaling pathway controlling LIF gene expression.MAPKs have been reported to be involved in adeno-

sine A2B receptor-mediated regulation of IL-6 gene ex-pression in astrocytoma cells [48]. In our experiments,both basal as well as NECA-induced LIF gene expressionand release in cultured astrocytes were inhibited by spe-cific inhibitors of p38 and ERK1/2, but not JNK-MAPKs.In line with our findings, it has been shown that LIF ex-pression in Schwann cells is mediated through PKCpathway-induced ERK1/2 activation [49]. Furthermore,we show here that adenosine-dependent LIF expression

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Figure 7 Basal and NECA-induced leukemia inhibitory factor (LIF) expression and secretion levels in primary astrocytes are dependenton NF-κB activation. (A) shows Western blot analysis of primary cortical astrocytes treated without or with NECA (1 μM; for 0.5, 1, 2 and 4 hours)to detect phosphorylation at Ser536 of NF-κB p65 (RelA) proteins. Where indicated, cells were pre-treated with selective inhibitor of NF-κB (BAY11–7082, 10 μM) for 2 hours prior to NECA stimulation. β-actin served as a loading control. Subsequently, astrocytes were treated without or withNECA (1 μM) for 2 hours (for real-time PCR) and 4 hours (for Western blot and ELISA), in presence or absence of BAY 11–7082 (10 μM, added2 hours prior to NECA stimulation). (B) shows real-time PCR analysis of LIF gene expression (relative to GAPDH). Data are normalized to thecontrol and presented as Mean± SEM of three independent experiments. P< 0.05. (C) shows Western blot analysis to detect LIF protein levels.β-actin served as a loading control. (D) shows a representation of sandwich ELISA experiment performed to detect LIF content in supernatants ofcultured astrocytes. Each bar corresponds to the mean concentration of LIF in triplicate samples; error bars indicate SEM. Observations wereconfirmed by repeating the experiments two additional times. P< 0.05. NECA, 5′-N-ethylcarboxamide.


in astrocytes is regulated through the NF-κB transcrip-tion factor. This observation is in line with several stud-ies showing an NF-κB-dependent regulation of IL-6 geneby this transcription factor in several cell types[38,50,51,65,66]. It has been shown that NECA-inducedNF-κB activation and the resultant IL-6 gene expressionwas abolished by inhibitors of MAPK pathways [65]. Inour study, preliminary observations indicate that NECA-induced activation of the NF-κB pathway is reduced byselective inhibitors of p38 and ERK1/2 pathways (datanot shown), suggesting that these pathways might playas upstream mediators in NF-κB-dependent LIF expres-sion in astrocytes.Recent evidence indicates that, depending on the cell

type, different secretory pathways are employed for cyto-kine release [67]. For example, T cells use two differentrelease mechanisms: IL-2 and IFN-γ are secreted at theimmunological synapse whereas CCL3 and TNF-α aresecreted multidirectionally, suggesting different secretorypathways [68]. In neurons or neuron-like cells, secretorygranules called LDCVs are the organelles used forthe selective secretion of IL-6, TGF-β2 and CCL21[53,54,69]. The same organelles are also used in immunecells such as mast cells and neutrophils [67]. Here we

show that LIF protein is transported through Golgi butits secretion by astrocytes is not mediated by secretorygranules. Instead, LIF co-localizes with Rab11, a knownmarker of recycling endosomes [57,58]. Moreover, weobserved a partial co-localization of LIF with clathrin,which also associates with recycling endosomes where itis implicated in protein sorting [56]. Recycling endo-somes have now been shown to be responsible for cyto-kine secretion in several cell types. For example, IFN-γand TNF-α secretion from natural killer cells requireRab11 [70]. Recycling endosomes are also responsiblefor the constitutive secretion of IL-6 and TNF-α inmacrophages [71]. Further studies will be needed to bet-ter understand LIF sorting, trafficking and release bythese vesicles.Interestingly, our data indicate that LIF is constitu-

tively released from astrocytes. Indeed constant levels ofLIF were present in the supernatants of untreated astro-cytes when measured by ELISA. Similar data wereobserved in human astrocyte cultures [59]. Whether thisobservation is representative of the physiological behav-ior of astrocytes in vivo or is due to the culture condi-tions remains to be determined. We further show thatby blocking the early secretory pathway with BFA, the

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Figure 8 Leukemia inhibitory factor (LIF) secretion in primary astrocytes is constitutive and independent of NECA stimulation. (A)illustrates LIF immunocytochemistry in cultured cortical astrocytes, where LIF is found in vesicle-like structures throughout the cytoplasm. Scalebar corresponds to 10 μm. (B) shows complete co-localization of LIF with Giantin, a marker of Golgi apparatus, when cultured astrocytes weretreated with Brefeldin A (BFA, 5 μg/mL for 1 hour). Scale bar corresponds to 10 μm. (C) shows a representation of sandwich ELISA experimentperformed to detect LIF content in supernatants of cultured astrocytes that were treated without or with NECA (1 μM) for 4 hours. Whereindicated, cells were pre-treated with BFA (5 μg/mL) for 1 hour prior to NECA stimulation. Each bar corresponds to the mean concentration of LIFin triplicate samples; error bars indicate SEM. Observations were confirmed by repeating the experiments two additional times. P< 0.05. NECA, 5′-N-ethylcarboxamide.


LIF concentration in the culture supernatant was notincreased upon NECA stimulation. The inhibitory effectof BFA indicates that LIF passes through the Golgiprior to its secretion, and thus does not follow non-conventional secretory pathways that by-pass the Golgiand is typically insensitive to BFA, which has recentlybeen reported to be used by other cytokines [72]. Im-portantly, the inhibitory effect of BFA suggests thatNECA-stimulated release of LIF by astrocytes requires

de novo LIF synthesis, and does not involve a ready-releasable post-Golgi pool of LIF.It is now clear that one of the major roles of LIF is

directed toward cell protection. Indeed, it has beenshown that LIF is up regulated in astrocytes and neuronsafter cerebral ischemia [21] as well as in astrocytes aftercortical brain injury [24], suggesting a role of LIF inneuronal repair or protection. In line with these data,treatment of rat with LIF prevented loss of motoneurons

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Figure 9 Leukemia inhibitory factor (LIF) co-localizes with clathrin and Rab11 but not with large dense-core vesicle markers.Immunostaining performed in cultured astrocytes revealed that LIF does not co-localize with pHogrin (A) and Chromogranin A & B (B), which areboth markers for large dense-core vesicles. However, we observed a massive co-localization of LIF with clathrin (C) and a partial co-localizationwith Rab11 (D). Scale bars correspond to 10 μm.


after peripheral nerve injury [73,74] and protection ofretinal ganglia cells was compromised in LIF knock-outmice after lens injury [12]. Finally, LIF was shown to limitdemyelination in an experimental autoimmune enceph-alomyelitis mouse model [16] and has become a promin-ent therapeutic candidate for multiple sclerosis [17]. Wehave previously shown that LIF can protect cortical aswell as hippocampal neurons against glutamate-inducedexcitotoxicity [13]. Here we show that LIF coming fromthe supernatant of NECA-treated astrocytes has the sameprotective effect. Indeed, astrocytes produce several othercytokines and neurotrophic factors including IL-6, NGF,brain-derived neurotrophic factor, neurotrophin-3, S-100β protein and TGFβ [75], that might help neurons tocope with excitotoxic stress. Accordingly, conditionedmedia from astrocyte cultures protected cortical neuronsagainst glutamate (data not shown). In order to confinethe neuroprotective effect of astrocytic factors that arereleased in response to NECA treatment, we had to re-fresh astrocyte culture medium prior to NECA treatmentand testing supernatant on glutamate-stressed neurons.This, together with LIF neutralization, indicates thatLIF produced by astrocytes after adenosine receptorstimulation is necessary to witness neuronal protec-tion. Our results provide further evidence for a roleof adenosine in neuronal protection. Indeed, it hasbeen shown that adenosine can protect neurons dur-ing hypoxia [76,77], ischemia [78,79] and excessiveneuronal activity [80,81]. This adenosine protection isoften mediated through the A1 receptor subtype [82-84], but here we show that an indirect protection ofadenosine through the stimulation of A2B receptor onastrocytes leading to LIF upregulation exists. This A2B

receptor activation might be related to an anti-inflammatory process as observed previously byothers [85-87].

ConclusionsWe demonstrate a protective role of LIF against glutam-ate neurotoxicity and we provide clear evidence that ad-enosine is required for an increased production of LIFby astrocytes. These data further confirm a neuroprotec-tive role of adenosine in the brain.

AbbreviationsA2B KO: A2B receptor knock-out; BFA: Brefeldin A; bp: base pairs;ChAT: choline acetyltransferase; CNS: central nervous system; CNTF: ciliaryneurotrophic factor; CT-1: cardiotrophin-1; DMEM: Dulbecco’s modifiedEagle’s medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraaceticacid; EPAC: Exchange Protein Activated by cAMP; ERK: extracellular signal-regulated kinase; FCS: fetal calf serum; HBSS: Hank’s balanced salt solution;HEPES: hydroxyethyl piperazineethanesulfonic acid; HRP: horseradishperoxidase; IL: interleukin; IFN: interferon; JNK: c-Jun N-terminal kinase;LDCV: large dense-core vesicles; LIF: leukemia inhibitory factor; LME: L-leucinemethyl ester; MAP2: Microtubule-associated protein 2; MAPK: mitogen-activated protein kinase; NECA: 5′-N-ethylcarboxamide; NGF: nerve growthfactor; NNT-1: novel neurotrophin-1; NT: nuclear transcription factor;OBB: Odyssey™ Blocking Buffer; OSM: oncostatin M; PBS: phosphate-bufferedsaline; PBS-T: phosphate-buffered saline plus 0.1 % Tween 20; PKA: proteinkinase A; PKC: protein kinase C; PLC: phospholipase C; PVDF: polyvinylidenefluoride; qPCR: real-time PCR; rmLIF: recombinant mouse LIF;TGFβ: transforming growth factor β.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsSM performed the majority of experiments including cell culturing, ELISAand Western blotting. JV did the immunofluorescent part of the study aswell as all the microscopy and statistical analysis. Both SM and JV wereinvolved in the redaction of the manuscript. EW did the qPCR experiments.JB took part in ELISA experiments. CHS performed the toxicity and the A2BKO experiments. SCDI and PB were both involved in the secretion part of

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Figure 10 Effect of untreated and NECA-treated astrocyte supernatants on survival of cultured cortical neurons against excitotoxicity.Primary cortical neurons were treated without or with recombinant mouse Leukemia inhibitory factor (rmLIF, 0.1 ng/mL), NECA (1 μM) orastrocyte supernatants (untreated or treated with 1 μM NECA for 4 hours; diluted 1:1 in neuronal culture medium) for 24 hours. Where indicated,rmLIF and NECA-treated astrocyte supernatants were treated with an anti-LIF neutralizing antibody (AF449; 100 ng/mL for 1.5 hours) beforeapplying them to cultured neurons. Subsequently, the neurons were treated without or with glutamate (Glut.; 50 μM for 1 hour) and cell survivalassessed 24 hours after glutamate treatment by a colorimetric MTT assay. The optical densities were measured at 570 nm, with a 630 nm andblank correction. Data are normalized to percent of control and presented as Mean± S.E.M of four independent experiments. P< 0.05, whencompared to glutamate-treated condition. NECA, 5′-N-ethylcarboxamide.


the study. They gave precious antibodies, were essential in the design of theexperiments and participated actively in writing the manuscript. HWGMBand KPHB were both involved in the conception and design of the study aswell as in the manuscript redaction. All authors read and approved the finalmanuscript.

AcknowledgementsThis study was supported financially by the school of Behavioral andCognitive Neurosciences (S. Moidunny) and by the DeutscheForschungsgemeinschaft (K. Biber), grant number: FOR1336 and CA 115/5-4.

Author details1Department of Neuroscience, Section Medical Physiology, University MedicalCenter Groningen, University of Groningen, A. Deusinglaan 1, 9713 AV,Groningen, The Netherlands. 2Department of Rheumatology and ClinicalImmunology, University Medical Center Groningen, University of Groningen,A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands. 3Department ofPsychiatry and Psychotherapy, Section Molecular Psychiatry, University ofFreiburg, Hauptstrasse 5, 79104, Freiburg, Germany. 4Department of CellBiology, Section Membrane Cell Biology, University Medical CenterGroningen, University of Groningen, A. Deusinglaan 1, 9713 AV, Groningen,The Netherlands. 5Department of Cell Biology and Morphology, University ofLausanne, Rue du Bugnon 9, 1005, Lausanne, Switzerland.

Received: 19 March 2012 Accepted: 1 August 2012Published: 16 August 2012

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doi:10.1186/1742-2094-9-198Cite this article as: Moidunny et al.: Adenosine A2B receptor-mediatedleukemia inhibitory factor release from astrocytes protects corticalneurons against excitotoxicity. Journal of Neuroinflammation 2012 9:198.

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RESEARCH Open Access Adenosine A 2B receptor-mediated … · 2017. 8. 25. · RESEARCH Open Access Adenosine A 2B receptor-mediated leukemia inhibitory factor release from astrocytes