Glutamate potentiates lipopolysaccharide–stimulated interleukin-10 release from neonatal rat spinal cord astrocytes

Neuroscience 207 (2012) 12–24

GLUTAMATE POTENTIATES LIPOPOLYSACCHARIDE–STIMULATED INTERLEUKIN-10 RELEASE FROM NEONATAL RAT SPINAL CORD ASTROCYTES E. L. WERRY,a G. J. LIU,a,b M. D. LOVELACE,a,c R. NAGARAJAHa AND M. R. BENNETTa*

Key words: astrocyte, interleukin-10, anti-inflammatory cytokine, glutamate, metabotropic receptor.

a Brain and Mind Research Institute and Bosch Institute, The University of Sydney, NSW 2006, Australia

The anti-inflammatory cytokine interleukin-10 (IL-10) can decrease the production of pro-inflammatory cytokines from activated glia (Sawada et al., 1999; Ledeboer et al., 2000, 2002). High levels of pro-inflammatory cytokines contribute to numerous central nervous system pathologies including neuropathic pain (Wieseler-Frank et al., 2005) and cellular toxicity resulting from infection (Mesples et al., 2003; Pang et al., 2005; Qian et al., 2006a,b). Inactivation of endogenous IL-10 is detrimental in these conditions (Poole et al., 1995; Amaral et al., 2008; Londono et al., 2008), whereas application of exogenous IL-10 can be beneficial. For example, intrathecal IL-10 gene therapy abolishes neuropathic pain (Milligan et al., 2006a; Ledeboer et al., 2007). Further, IL-10 decreases prenatal white matter damage from maternal Escherichia coli exposure and decreases dopaminergic and cortical neuronal degeneration from exposure to lipopolysaccharide (LPS), a component of gram-negative bacteria cell walls (Mesples et al., 2003; Pang et al., 2005; Qian et al., 2006a,b). One source of IL-10 in the central nervous system is astrocytes. In vitro and in situ brain and spinal cord astrocytes express Toll-like receptor 4 (TLR4) in inflammatory conditions (Bsibsi et al., 2002; Bowman et al., 2003; Pehar et al., 2004; Jou et al., 2006; Kigerl et al., 2007; Li et al., 2008). This receptor is bound, amongst other ligands, by gangliosides, the proteoglycan biglycan, and gram-negative bacteria (Beutler, 2000; Schaefer et al., 2005; Jou et al., 2006), and LPS is often used as a TLR4 agonist in experimental studies. Unstimulated cultured rat astrocytes express very low IL-10 mRNA levels, and IL-10 release from these cells is undetectable. When these astrocytes are exposed to LPS, IL-10 mRNA levels are upregulated and IL-10 protein release is increased (Ledeboer et al., 2002). Given that IL-10 has important anti-inflammatory effects and can be protective in inflammatory conditions, such as chronic pain and infection, and given that LPS stimulation of astrocytes is a source of endogenous IL-10, it is of interest to identify modulators of LPS-stimulated IL-10 release from astrocytes, which may provide insight into the pathomechanisms of inflammatory conditions and which may be protective in such conditions. Glutamate may be an ideal candidate modulator of LPS-stimulated IL-10 release. We recently showed that glutamate potentiates LPS-stimulated IL-10 release from spinal cord microglia (Werry et al., 2011). Astrocytes ex-

b

Life Sciences, Australian Nuclear Science and Technology Organisation, New Illawarra Road, Lucas Heights, NSW 2234, Australia c Discipline of Anatomy and Histology, School of Medical Sciences, The University of Sydney, NSW 2006, Australia

Abstract—Interleukin-10 (IL-10) has important anti-inflammatory effects and can be protective in inflammatory conditions, such as chronic pain and infection. Exploring factors that modulate IL-10 levels may provide insight into pathomechanisms of inflammatory conditions and may provide a method of neuroprotection during these conditions. Lipopolysaccharide (LPS) stimulation of astrocytes is a source of IL-10; hence, it is of interest to investigate factors that modulate this process. Glutamate is present in increased concentrations in inflammatory conditions, and astrocytes also express glutamate receptors. The present study, therefore, investigated whether glutamate modulates LPS stimulation of IL-10 release from neonatal spinal cord astrocytes. Enzymelinked immunosorbent assays (ELISAs) were used to quantify IL-10 release from cultured neonatal spinal cord astrocytes, and reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure IL-10 mRNA expression. Glutamate (1 mM) significantly increased LPS (1 ␮g/ml)-stimulated IL-10 release from astrocytes by 166% and significantly upregulated IL-10 mRNA levels. Glutamate synergistically signaled through metabotropic glutamate receptor subgroups and the phospholipase C signaling pathway. Spinal cord astrocytes may, therefore, play a larger anti-inflammatory role than first thought in situations where glutamate and a high concentration of Toll-like receptor 4 (TLR4) agonists are present. © 2012 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹61-2-9351-0872; fax: ⫹61-2-9351-0939. E-mail address: [email protected] (M. R. Bennett). Abbreviations: ActD, actinomycin D; APDC, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate; AP5, D(⫺)-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CPCCOEt, 7-hydroxyiminocyclopropan[b]chromen-1␣-carboxylic acid ethyl ester; cyclo, cycloheximide; DHPG, (S)-3,5-dihydroxyphenylglycine hydrate; DMEM⫹, supplemented Dulbecco’s modified Eagle’s medium; DMEM/BSA, DMEM stock with 2% bovine serum albumin; EGLU, (2S)-␣-ethylglutamic acid; ELISA, enzyme-linked immunosorbent assay; GFAP, glial fibrillary acidic protein; Glu, glutamate; IL-10, interleukin-10; KA, kainate; L-AP4, L-(⫹)-2-amino-4-phosphonobutyric acid; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; mGluR, metabotropic glutamate receptor; MPEP, 6-methyl-2-(phenylethynyl)pyridine; MSPG, (⫾)-␣-methyl-(4-sulfonophenyl)glycine; NMDA, N-methyl-D-aspartate; PLC, phospholipase C; PKA, protein kinase A; RpcAMPS, Rp-adenosine 3=,5=-cyclic monophosphorothioate triethylammonium salt hydrate; RT-PCR, reverse transcriptase–polymerase chain reaction; tACPD, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid; TLR4, Toll-like receptor 4. 0306-4522/12 $36.00 © 2012 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2012.01.039

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press all four subtypes of glutamate receptor, namely AMPA, kainate (KA), N-methyl-D-aspartate (NMDA), and metabotropic glutamate receptors (mGluRs) (Aicher et al., 1997; Silva et al., 1999; Brand-Schieber et al., 2004; Werry et al., 2006). Astrocytes are exposed to glutamate released from neurons, neighboring astrocytes, and activated microglia (Chen et al., 2005; Lalo et al., 2006; Takeuchi et al., 2006), and in inflammatory situations astrocytes are exposed to increased levels of glutamate (Benveniste et al., 1984; Rego et al., 1996; Bezzi et al., 1998; Sasaki et al., 1998; Takeuchi et al., 2006; Tilleux and Hermans, 2007). Although glutamate can be excitotoxic at high concentrations, glutamate has also been shown to have anti-inflammatory and neuroprotective effects in inflammatory conditions. Glutamate decreases class II major histocompatibility complex markers on interferon-␥-activated astrocytes (Lee et al., 1992), decreases excitotoxic or LPS-induced neuronal damage (Bruno et al., 1998; Zhou et al., 2006), and decreases cytokine-induced astrocytic nitric oxide synthase expression (Murphy et al., 1995). Given these general anti-inflammatory effects of glutamate on astrocytes, this study aimed to investigate the effect of glutamate on LPS-stimulated IL-10 release from spinal cord astrocytes.

EXPERIMENTAL PROCEDURES Preparation of astrocyte cultures All experiments were approved by the University of Sydney Animal Ethics Committee and were conducted in accordance with the Australian Government National Health and Medical Research Council code of practice for the care and use of animals for scientific purposes. The number of animals used and the suffering of these animals was minimized. Unless otherwise stated chemicals used in all experiments were purchased from Sigma-Aldrich, St. Louis, MO, USA. Protocols for isolation of spinal cord astrocytes were based on those described previously (Werry et al., 2006). Briefly, whole spinal cords were dissected from 0- to 1-dayold male and female Sprague–Dawley rats. Meninges and peripheral nerves were removed, and spinal cords were incubated in porcine trypsin (0.125%) dissolved in Hanks’ balanced salt solution (in mM: 136.9 NaCl, 5.4 KCl, 4.2 NaHCO3, 0.4 KH2PO4, 0.3 Na2HPO4, 5.6 D-glucose, pH 7.4) for 20 min at 37 °C. Subsequently the cords were washed twice in 2 ml Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% cosmic calf serum (HyClone, Logan, UT, USA) and 1% penicillin/streptomycin/glutamine (DMEM⫹). Cells were then dissociated by mechanical trituration and were plated onto poly-D-lysine (20 ␮g/ml)coated 25-cm2 flasks at 37 °C for 4 h. Cells from one spinal cord were plated per flask. Media was aspirated and flasks washed twice with 2 ml DMEM⫹. Glial cells were maintained at 37 °C in 5% CO2 in DMEM⫹, and media was changed every 3– 4 days. Sixteen days after initial plating, cultures were purified by preferentially selecting for the growth of astrocytes. This was achieved by replacing the culture medium with DMEM⫹ containing 25 mM sorbitol substituted for glucose (DMEM⫹sorb). Astrocytes contain sorbitol dehydrogenase and aldose reductase and can utilize sorbitol as an energy source. Microglia and oligodendrocytes do not have these enzymes and as a consequence many of these cells do not survive in this culture medium (Wiesinger et al., 1991). Three days after addition of DMEM⫹sorb, flasks were shaken at 400 rpm at 37 °C for 20 h to further remove microglia and oligodendrocytes (Cole and deVellis, 1997). Adherent astrocytes were removed from the flask after a 5-min treatment in

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0.125% porcine trypsin and were plated in DMEM⫹ at a density of 100,000 cells per well in a plastic 24-well tissue culture plate. At this in vitro age (21 days), there exists one predominant subtype (type 1a) of spinal cord astrocyte in culture (Black et al., 1993). The purity of final cultures was verified by immunocytochemistry, according to the procedure described in Werry et al. (2011). A small percentage of cells (8%, n⫽284 total cells analyzed) were found to be positive for the microglial marker CD11b, whereas the other cells were positive for GFAP (glial fibrillary acidic protein), suggesting the remaining cells were astrocytes and that there were no fibroblasts present.

Cell treatment protocol The effect of glutamate on LPS-stimulated IL-10 mRNA and cytokine protein levels. Eighteen hours after plating in 24-well plates, media was removed from wells, and cells were preincubated in 500 ␮l of either fresh DMEM⫹ or 1 ␮g/ml LPS from Escherichia coli 055:B5 in DMEM⫹ for 8 h to activate astrocytes. All solutions were then removed and discarded. Cells preincubated in DMEM⫹ were then incubated in 500 ␮l of either DMEM stock with 2% bovine serum albumin (DMEM/BSA) or 1 mM glutamate in DMEM/BSA for 16 h. Cells preincubated in LPS were incubated in 500 ␮l of either 1 ␮g/ml LPS in DMEM/BSA or 1 ␮g/ml LPS with 1 mM glutamate in DMEM/BSA for 16 h. Medium was then collected and centrifuged before further analysis in enzyme-linked immunosorbent assay (ELISA) and lactate dehydrogenase toxicology experiments. Cells were utilized for quantitative reverse transcriptase–polymerase chain reaction experiments. Studies using 10 ng/ml LPS elicit only weak IL-10 release from astrocytes (Bolin et al., 2005), so to ensure sufficient IL-10 release and to allow comparisons with previous work on the effect of glutamate on LPS-stimulated IL-10 release from microglia (Werry et al., 2011), LPS was used at 1 ␮g/ml in this study. LPS is known to specifically activate the TLR4 at this concentration (Kalis et al., 2003). Further characterization of the effect of glutamate on LPSstimulated IL-10 release. To examine if a preincubation in LPS was necessary for the potentiating effects of glutamate, cells were incubated in 500 ␮l of either 1 ␮g/ml LPS in DMEM/BSA or 1 ␮g/ml LPS with 1 mM glutamate in DMEM/BSA for 16 h. Medium was then collected and centrifuged before further analysis in ELISA experiments. Examination of the concentration and time dependency of the effect of glutamate. To explore if the effect of glutamate was dependent on its concentration, cells were preincubated in 1 ␮g/ml LPS for 8 h. All solutions were then removed and discarded, and 500 ␮l of either 1 ␮g/ml LPS in DMEM/BSA or 1 ␮g/ml LPS with 1 ␮M–10 mM glutamate in DMEM/BSA was added for 16 h. Medium was then collected and centrifuged before further analysis in ELISA experiments. To explore if the effect of glutamate was dependent on exposure time, cells were preincubated in 1 ␮g/ml LPS for 8 h. All solutions were then removed and discarded, and 500 ␮l of either 1 ␮g/ml LPS in DMEM/BSA or 1 ␮g/ml LPS with 1 mM glutamate in DMEM/BSA was added for 2– 48 h. Medium was then collected and centrifuged before further analysis in ELISA experiments. Antagonist and agonist experiments. In experiments involving application of antagonists (Results sections “Measurement of endotoxin . . . ,” “The enhancement of . . . ,” and “The role of translation . . . ”), cells were preincubated in 1 ␮g/ml LPS for 8 h. Seven and a half hours into the preincubation, antagonists were added for 30 min to allow pre-binding of antagonists before exposure to glutamate. After this preincubation, solutions were removed and discarded, and 500 ␮l of either 1 ␮g/ml LPS⫹antagonist in DMEM/BSA or 1 ␮g/ml LPS with 1 mM glutamate and antagonist

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E. L. Werry et al. / Neuroscience 207 (2012) 12–24

Table 1. List of agonists and antagonists. All agonist incubations (except LPS) were for 16 h. All antagonists were added 7.5 h into the 8 h LPS preincubation. At the 8-h timepoint, solutions were removed and LPS and/or glutamate added with antagonists for 16 h. All drugs were obtained from Sigma-Aldrich, USA, except APDC, EGlu, and kainate, which were obtained from Tocris, Bristol, UK Drug name

Agonists Trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (S)-3,5-dihydroxyphenylglycine hydrate (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate L-(⫹)-2-amino-4-phosphonobutyric acid ␣-amino-3-hydroxy-5-methylisoxazole-4-proprionate N-methyl-D-aspartate Kainate Glutamate Lipopolysaccharides (purified) from Escherichia coli 055: B5 Antagonists Actinomycin D Cycloheximide 7-Hydroxyiminocyclopropan[␤]chromen-1␣-carboxylic acid ethyl ester (2S)-␣-ethylglutamic acid (⫾)-␣-methyl-(4-sulfonophenyl)glycine 6-Methyl-2-(phenylethynyl)pyridine D(—)-2-amino-5-phosphonopentanoic acid 6-Cyano-7-nitroquinoxaline-2,3-dione Rp-adenosine 3=, 5=-cyclic monophosphorothioate triethylammonium salt hydrate N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride 1-[6-[((17␤)-3-methoxyestra-1,3,5[10]-trien-17 yl)amino]hexyl]-1Hpyrrole-2,5-dione 1-[6-[((17␤)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-2,5pyrrolidinedione Thapsigargin

in DMEM/BSA was added for 16 h. Medium was then collected and centrifuged before further analysis in ELISA experiments. In experiments involving application of agonists (Results section “The enhancement of . . . ”), cells were preincubated in 1 ␮g/ml LPS for 8 h. Solutions were then removed and discarded, and 500 ␮l of either 1 ␮g/ml LPS⫹agonist in DMEM/BSA or 1 ␮g/ml LPS with 1 mM glutamate and agonist in DMEM/BSA was added for 16 h. Medium was then collected and centrifuged before further analysis in ELISA experiments. A list of antagonists and agonists used can be found in Table 1. All antagonists and agonists were dissolved in DMEM apart from trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD) and (2S)-␣-ethylglutamic acid (EGLU), which were dissolved in NaOH (maximum final concentration of 1 mM), and 7-hydroxyiminocyclopropan[b]chromen-1␣-carboxylic acid ethyl ester (CPCCOEt), U73122, U73323, and thapsigargin, which were dissolved in DMSO (maximum concentration 0.1%). At 1 mM NaOH did not affect solution pH, and 0.1% DMSO did not affect LPS⫹glutamate-stimulated IL-10 release. All antagonists and agonists were used at concentrations that were within one order of magnitude above their EC50 or IC50 (Dragland-Meserve et al., 1985; Foster and Wong, 1987; Andreasen et al., 1989; Whittemore and Koerner, 1989; Manzoni et al., 1991; Sather et al., 1992; von Stebut et al., 1994; Wieprecht et al., 1994; Wyllie and Cull-Candy, 1994; Boxall et al., 1996; Schoepp et al., 1996; Davis et al., 1998; Seebeck et al., 1998; Brauner-Osborne et al., 1999; Vetter et al., 1999; De Colle et al., 2000; Noda et al., 2000; Shiraishi et al., 2001; Kato

Abbreviated name

Dose

Receptor/signaling system drug is active at

tACPD DHPG APDC L-AP4 AMPA NMDA KA Glu LPS

10 ␮M–1 mM 500 ␮M 5 ␮M 1 mM 100 ␮M 500 ␮M 100 ␮M 1 ␮M-10 mM 1 ␮g/ml

mGluRI and II mGluRI mGluRII mGluRIII AMPA receptor NMDA receptor Kainate receptor All glutamate receptor subtypes TLR4

ActD Cyclo CPCCOEt EGLU MSPG MPEP AP5 CNQX Rp-cAMPS

1 ␮g/ml 1 ␮g/ml 75 ␮M 500 ␮M 1 mM 1 ␮M 200 ␮M 20 ␮M 10 ␮M

H-89

300 nM

Transcription inhibitor Translation inhibitor mGluR1 mGluRII mGluRII/III mGluR5 NMDA receptor AMPA/Kainate receptors membrane permeable inhibitor of cAMP-dependent protein kinase activation PKA inhibitor

U73122

5 ␮M

PLC inhibitor

U73343

5 ␮M

Inactive analog of U73122

—

1 ␮M

Endoplasmic reticulum Ca2⫹dependent ATPase inhibitor

et al., 2005; Barker et al., 2006; Chiocchetti et al., 2006; Abe and Hiraki, 2009).

Measurement of IL-10 levels ELISA. Levels of IL-10 protein in the medium were quantified using a BD OptEIA Rat IL-10 ELISA Set (BD Biosciences, San Diego, CA, USA), with a minimum reliable detection level of 31.25 pg/ml. Levels of IL-1␤ in the medium were quantified using a Quantikine® Rat IL-1␤ Immunoassay (R&D Systems, Minneapolis, USA), with a minimum reliable detection level of 31.25 pg/ml. Assays were conducted as per the manufacturers’ protocol. A Fluostar Galaxy Multiplate reader (BMG Labtechnologies, Offenburg, Germany) was used to measure the absorbance of media samples. Each experimental condition was tested on at least two different culture batches with at least two repetitions of measurement per batch giving a minimum n⫽4. Values in time and concentration experiments, agonist and antagonist experiments were normalized to the average level of LPS-stimulated IL-10 release, as detailed in Werry et al. (2011). In most cases, results were presented as mean concentration of IL-10 (pg/ml) or mean ratio⫾SEM. Quantitative reverse transcriptase–polymerase chain reaction. Astrocytes were cultured as described previously in the text, but were plated on to the base of poly-D-lysine (20 ␮g/ml)-coated 35 mm cell culture dishes at a density of 6.77⫻105 cells per dish and were maintained for 1 day before experimentation. After an 8-h

E. L. Werry et al. / Neuroscience 207 (2012) 12–24 preincubation in either DMEM⫹ or 1 ␮g/ml LPS in DMEM⫹ and a subsequent 16-h incubation in DMEM with 2% bovine serum albumin, 1 ␮g/ml LPS, or 1 ␮g/ml LPS⫹1 mM glutamate, total RNA was extracted from astrocytes using a GenEluteTM Mammalian Total RNA Miniprep Kit, according to the manufacturer’s protocol. Before purified RNA was eluted, the sample was treated with amplification grade DNase I (Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature to remove any contaminating DNA. The quantity of isolated RNA was determined spectroscopically using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Extracted total RNA was reverse transcribed to cDNA using a SuperscriptTM III First-Strand Synthesis System and oligo-dT primer (Invitrogen) according to the manufacturer’s protocol. Residual DNA contamination was tested for by replacing the reverse transcriptase enzyme with RNase free water. The cDNA was stored at ⫺20 °C until the polymerase chain reaction (PCR). Aliquots of first strand cDNA (40 ng) were amplified in 10 ␮l of Platinum SYBR-Green qPCR SuperMix-UDG (Invitrogen) with 0.8 ␮l of forward primer, 0.8 ␮l of reverse primer, made up to a total volume of 20 ␮l with diethylpyrocarbonate-treated H2O. Primers were as follows: 18S forward primer: 5=-GCCGCTAGAGGTGAAATTCTTG-3=; 18S reverse primer: 5=-AAAACATTCTTGGCAAATGCTTT-3=; IL-10 forward primer: 5=-TGCCTTCAGTCAAGTGAAGAC-3=; IL-10 reverse primer: 5=-AAACTCATTCATGGCCTTGTA-3=. All were synthesized by Invitrogen. PCR was carried out using the Rotor-Gene 3000 system (Corbett Life Science, Sydney, NSW, Australia). Incubation conditions were as follows: 2 min at 50 °C, 2 min at 95 °C, then thermo-cycling for 40 cycles of 20 s at 95 °C, 20 s at 60 °C, then 20 s at 72 °C. The threshold cycle number and reaction efficiencies of each condition were identified with Rotor-Gene 3000 v3.1 Software (Corbett Life Science). Relative quantification was carried out using the relative expression software tool (Pfaffl et al., 2002), whereby IL-10 mRNA expression was standardized by the house-keeping gene 18S rRNA expression. Mean relative mRNA levels were determined with at least three separate reactions per condition. Values are expressed as mean relative IL-10 mRNA expression⫾SEM.

Endotoxin measurement To test for the presence of potentially contaminating endotoxins in aqueous solutions of agonists (apart from LPS), a QCL-1000® Endpoint Chromogenic Limulus Amebocyte Lysate Assay was used (Lonza, Basel, Switzerland) and the assay was performed as per the manufacturer’s protocol. The minimum reliable detection level of the assay was 0.1 Endotoxin Units/ml.

Lactate dehydrogenase toxicology assay Cell membrane integrity was assessed with a lactate dehydrogenase (LDH)-based In Vitro Toxicology Assay Kit (Sigma-Aldrich) as per the manufacturer’s protocol. Each experimental condition was tested on at least two different culture batches and at least two repetitions of measurement per batch giving a minimum n⫽4.

Statistical analyses Differences between means in initial experiments examining the effect of glutamate on LPS-stimulated IL-10 protein and mRNA production (in Methods section “The effect of glutamate . . . ”/ Results section “Glutamate enhances the . . . ”) were examined using one-way ANOVA followed by Tukey’s multiple comparison post-test. All other experiments examining differences between means in multiple conditions were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison posttest. The results of Levine’s test showed that variances did not significantly differ between each condition on which an ANOVA was per-

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formed. Examination of whether glutamate potentiated LPS-stimulated IL-10 release without a preincubation (Methods section “Further characterization of . . . ” and Results section “Further characterization of . . . ”) and whether glutamate potentiated LPSstimulated IL-1␤ release was conducted using a Student’s twosample t-test (in Methods “The effect of glutamate . . . ” and Results section “Glutamate enhances the . . . ”). In all these conditions, P⬍0.05 was considered as statistically significant. To examine if means in time-dependency experiments (Methods section “Examination of the . . . ”/Results section “Verification of intact membrane integrity after exposure to LPS . . . ”) were different from a ratio of 1, one-sample t-tests with Bonferroni correction for multiple comparisons were conducted, with a P-value ⬍0.008 regarded as statistically significant.

RESULTS Glutamate enhances the ability of LPS to stimulate IL-10 protein release and mRNA levels in spinal cord astrocytes The influence of glutamate on the ability of LPS to stimulate IL-10 release from spinal cord astrocytes was determined, and significant differences between groups were found (F⫽87.7, P⬍0.0001). Over 16 h, both unstimulated astrocytes (astrocytes not exposed to LPS; n⫽57) and astrocytes exposed to 1 mM glutamate (n⫽10; Fig. 1A) did not elicit IL-10 release above the minimum reliable reading level of the assay. We then compared these results with those obtained from LPS-preincubated astrocytes. To activate astrocytes, cells were preexposed to 1 ␮g/ml LPS for 8 h. A period of 8 h was chosen, as this length of LPS exposure did not cause a significant IL-10 release in brain astrocytes (Ledeboer et al., 2002). After this 8-h preincubation in LPS, a further 16-h exposure to LPS stimulated 561⫾45 pg/ml IL-10 release (n⫽65 culture batches), whereas a 16-h exposure to LPS⫹1 mM glutamate stimulated significantly more IL-10 release (872⫾62 pg/ml; n⫽65; P⬍0.001; Fig. 1A). The average percentage increase of IL-10 release in the presence of glutamate was 166⫾6% (n⫽65). These results suggest, therefore, that glutamate can potentiate LPS-stimulated IL-10 release from spinal cord astrocytes. To investigate whether glutamate also increased LPSstimulated IL-10 mRNA levels, reverse transcriptase–polymerase chain reaction (RT-PCR) was used to examine the levels of IL-10 mRNA in LPS and glutamate-treated astrocytes. This treatment induced significant differences between groups (F⫽10.19, P⬍0.05). Glutamate on its own stimulated a 1.3⫾1.6-fold increase in mRNA levels over those found in unstimulated astrocytes (n⫽3). LPS stimulated a 6.2⫾2.7-fold increase (n⫽3), whereas LPS⫹glutamate significantly increased expression levels beyond this LPS-stimulated level to 30.3⫾7.9-fold above basal expression (n⫽3; P⬍0.05; Fig. 1B). To investigate if glutamate potentiated the release of an LPS-stimulated pro-inflammatory cytokine, levels of IL-1␤ were measured in response to a 16-h incubation in 1 ␮g/ml LPS and compared with levels stimulated after a 16-h incubation in 1 ␮g/ml LPS⫹1 mM glutamate. Both 16-h incubations occurred after an 8-h preincubation in 1 ␮g/ml LPS. After this preincubation, 16-h exposure to 1 ␮g/ml LPS stimulated a release of

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E. L. Werry et al. / Neuroscience 207 (2012) 12–24

assay was performed. The amount of LDH found in the extracellular medium as a percentage of total intracellular LDH did not significantly differ in non-LPS-stimulated astrocytes over 16 h (3.78⫾0.86%; n⫽4) as compared with astrocytes that were stimulated with LPS (1 ␮g/ml) and glutamate (1 mM) for 16 h after an 8-h LPS preincubation (4.42⫾0.29%; F⫽0.1355; n⫽4; P⬎0.05). Measurement of endotoxin content of agonists and antagonists To verify that the potentiation of LPS-stimulated IL-10 release from astrocytes was not a result of endotoxin contamination of the glutamate stocks used, a Chromogenic Endpoint Limulus Amebocyte Lysate Assay was used. The endotoxin level in glutamate, and the other agonists and antagonists used for experiments, were below the minimum reliable reading level of the assay. The enhancement of LPS-stimulated IL-10 release by glutamate is concentration and time dependent

Fig. 1. (A) Glutamate significantly potentiates LPS-stimulated IL-10 release from spinal cord astrocytes. Treatments of astrocytes were as follows: basal—no preincubation in LPS and no addition of agonists (n⫽10); Glu—no preincubation in LPS but a 16 h exposure to 1 mM glutamate (n⫽10); LPS— 8 h preincubation in LPS (1 ␮g/ml) after which the media was removed and replaced with LPS for 16 h (n⫽65); LPS⫹Glu— 8 h preincubation in LPS after which the media was removed and replaced with LPS⫹1 mM glutamate for 16 h (n⫽65). (B) IL-10 mRNA expression standardized to 18S rRNA expression after the treatments described in (A). n⫽3 in each condition. Values are mean⫾SEM. * P⬍0.05 and *** P⬍0.001 when LPS⫹Glu is compared with LPS.

53⫾16 pg/ml IL-1␤ (n⫽4), whereas a 16-h exposure to LPS⫹1 mM glutamate did not significantly further potentiate IL-1␤ release (62⫾26 pg/ml; n⫽4; t⫽0.318; P⬎0.05).

Glutamate potentiated LPS-stimulated IL-10 release in a dose-dependent manner with an EC50 of 400 ␮M (Fig. 2). n⫽4 for all conditions except 1 mM glutamate where n⫽65. To ensure a concentration above EC50 was used, all subsequent experiments with glutamate used a concentration of 1 mM. To examine if the potentiation of glutamate on LPSstimulated IL-10 release was dependent on the duration of LPS and glutamate co-application, LPS and glutamate were added for various times after a constant 8-h preincubation in LPS. The level of IL-10 release measured in these conditions was compared with the level stimulated from exposure to LPS for the same amount of time (Fig. 3). The relative increase of LPS⫹glutamate-stimulated IL-10 release beyond that stimulated by LPS alone was largest when agonists were added for 16 h (P⬍0.001 compared with a ratio of 1, t⫽14.31; n⫽65), but were also significantly higher than a ratio of 1 when agonists were added for 8 h (t⫽7.91; n⫽6) and 24 h (t⫽8.25; n⫽6). As the time course demonstrated a peak at 16 h, agonists were ap-

Further characterization of the potentiating effect of glutamate To explore if an 8-h preincubation in LPS was necessary for the potentiating effect of glutamate, agonists were applied with different preincubation conditions. Without a preincubation in LPS, it was found a 16-h application of 1 ␮g/ml LPS stimulated 211⫾12 pg/ml IL-10 release (n⫽8). When 1 mM glutamate was co-applied with LPS, this release significantly increased to 240⫾8 pg/ml (n⫽8; t⫽2.031; P⬍0.05). As the highest levels of IL-10 release were seen with an 8-h preincubation in LPS, subsequent experiments were performed with an 8-h preincubation. Verification of intact membrane integrity after exposure to LPS and glutamate To verify the effect of LPS and glutamate on IL-10 release did not reflect compromised membrane integrity, an LDH

Fig. 2. The modulation of LPS-stimulated IL-10 release by glutamate is dose-dependent. Astrocytes were preincubated in 1 ␮g/ml LPS for 8 h. Media was removed and LPS with various concentrations of glutamate (Glu) were then added. The EC50 was 400 ␮M. Values are mean release of IL-10⫾SEM. For each condition, n was at least 4.

E. L. Werry et al. / Neuroscience 207 (2012) 12–24

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Glutamate acts on multiple metabotropic glutamate receptor subtypes to potentiate LPS-stimulated IL-10 release

Verification of intact membrane integrity after exposure to actinomycin D and cycloheximide

As spinal cord astrocytes express all four subtypes of glutamate receptor, agonists and antagonists were used to examine which subtype glutamate acted on to bring about the potentiation of LPS-stimulated IL-10 release. tACPD, an mGluRI and II receptor agonist, increased LPS-stimulated IL-10 release across a range of concentrations. The ratio of IL-10 release in the presence of 10 ␮M tACPD⫹LPS compared with LPS alone was 1.25⫾0.08 (n⫽6). This ratio for 100 ␮M tACPD was 1.58⫾0.06 (n⫽6; Fig. 5A) and for 1 mM tACPD was 1.84⫾0.03 (n⫽6). Surprisingly, application of agonists for individual mGluR groups such as (S)-3,5-dihydroxyphenylglycine hydrate (DHPG; 500 ␮M; mGluRI agonist), (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC; 5 ␮M; mGluRII agonist), and L-(⫹)-2-amino-4-phosphonobutyric acid (L-AP4; 1 mM; mGluRIII agonist) produced no potentiation of LPS-stimulated IL-10 release (Fig. 5A; n⫽6 for each condition). Kainate (100 ␮M), AMPA (100 ␮M), and NMDA (500 ␮M) also showed little potentiation of LPS-stimulated IL-10 release (Fig. 5A; n⫽6 for each condition). All agonists elicited a ratio of IL-10 release that was significantly lower than the ratio of IL-10 release stimulated by glutamate⫹LPS (F⫽12.15, P⬍0.001), apart from 100 ␮M and 1 mM tACPD, which were not significantly different from the glutamate ratio. CPCCOEt (75 ␮M; mGluR1 antagonist; n⫽13), 6methyl-2-(phenylethynyl)pyridine (MPEP; 1 mM; mGluR5 antagonist; n⫽8), and EGLU (500 ␮M; mGluRII antagonist; n⫽9) did not significantly block the potentiating effect of glutamate on LPS-stimulated IL-10 release (Fig. 5B). A significant decrease in the potentiation of LPS-stimulated IL-10 release was seen, however, on application of mGluR antagonists that concurrently block multiple receptor groups (F⫽5.34, P⬍0.001), such as (⫾)-␣-methyl-(4-sulfonophenyl)glycine (MSPG; 1 mM; mGluRII and III antag-

Incubation in actinomycin D and cycloheximide caused astrocyte processes to enlarge. An LDH assay was performed to verify this morphological observation did not reflect compromised cell health. The amount of LDH found in the extracellular medium as a percentage of total intracellular LDH did not significantly differ in astrocytes incubated in actinomycin D with LPS⫹glutamate (4.51⫾0.33%; F⫽ 0.1344; n⫽4) from the percentage found in medium of unstimulated astrocytes (3.78⫾0.86%; n⫽4) or astrocytes incubated in LPS⫹glutamate (4.42⫾0.29%; n⫽4). Similarly, incubation in cycloheximide with LPS⫹glutamate did not generate significantly different amounts of released LDH (4.35⫾0.06%; n⫽4) compared with unstimulated or LPS⫹glutamate-stimulated astrocytes. This is not surprising as another study has shown that cultured astrocytes, incubated for 24 h in higher concentrations of cycloheximide to what was used here, do not undergo apoptosis (Tsuchida et al., 2002). Similarly, higher concentrations of actinomycin D than that used here do not cause cell death in some other CNS cell types (Park et al., 1997).

Fig. 4. Upregulation of IL-10 mRNA is necessary to bring about the potentiating effects on LPS-stimulated IL-10 release. The effect of incubation in actinomycin D (ActD; transcription inhibitor; 1 ␮g/ml) and cycloheximide (Cyclo; translation inhibitor; 1 ␮g/ml) on LPS (1 ␮g/ml) and LPS plus glutamate (Glu; 1 mM)-stimulated IL-10 release (16 h), after an 8 h preincubation in LPS. n⫽14 for actinomycin D experiments and n⫽10 for cycloheximide experiments. *** P⬍0.001.

Fig. 3. The modulation of LPS-stimulated IL-10 release by glutamate is time-dependent. Cells were preincubated in 1 ␮g/ml LPS for 8 h. All solutions were then removed and discarded, and 500 ␮l of either 1 ␮g/ml LPS in DMEM/BSA or 1 ␮g/ml LPS with 1 mM glutamate in DMEM/BSA was added for 2– 48 h. Values are expressed as a mean ratio of LPS⫹Glu release to LPS release (⫾SEM indicated). The peak potentiation occurred when glutamate was applied with LPS for 16 h. For each condition, n was at least 4. *** P⬍0.001 when compared with a ratio of 1.

plied for 16 h in subsequent experiments to ensure maximal IL-10 release. The role of translation and transcription in the effect of glutamate To further investigate whether IL-10 mRNA upregulation was necessary for the potentiating effects of glutamate, astrocytes were incubated in actinomycin D to block mRNA transcription and cycloheximide to block mRNA translation. Significant differences were seen between conditions (F⫽18.03, P⬍0.0001). Actinomycin D (1 ␮g/ml) did not significantly decrease LPS-stimulated IL-10 release (n⫽14) but significantly decreased LPS⫹glutamate-stimulated IL-10 release (n⫽14; P⬍0.001), indicating a role for mRNA transcription in the potentiating effects of glutamate (Fig. 4). Blocking translation with cycloheximide (1 ␮g/ml) ablated both LPS-stimulated IL-10 release (n⫽10) and LPS⫹glutamate-stimulated IL-10 release (n⫽10; Fig. 4).

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onist; n⫽11), co-exposure to MPEP⫹EGLU (n⫽6) and co-exposure to MPEP, EGLU and CPCCOEt (n⫽6). The ionotropic glutamate receptor antagonists D(⫺)-2amino-5-phosphonopentanoic acid (AP5; 200 ␮M; NMDA receptor antagonist; n⫽6) and 6-cyano-7-nitroquinoxaline2,3-dione (CNQX; 20 ␮M; AMPA and kainate receptor antagonist; n⫽6) had no significant effect on the potentiation by glutamate (Fig. 5B). Taken together, these results suggest glutamate acts synergistically and exclusively through metabotropic glutamate receptor subtypes to bring about its potentiating effects. Signaling pathways involved in the potentiation by glutamate of LPS-stimulated IL-10 release Signaling pathways linked to metabotropic glutamate receptors include inhibition of cAMP through mGluRIIs and IIIs with possible subsequent decrease in cAMP-dependent protein kinase A (PKA) activity and activation of phospholipase C (PLC) by mGluRIs, with subsequent release of internal Ca2⫹ stores (Bettler and Mulle, 1995; Mori and Mishina, 1995; Pin and Duvoisin, 1995). Activators and antagonists were used to investigate the role of these signaling pathways in the potentiation of LPS-stimulated IL-10 release by glutamate. Inhibition of cAMP with 10 ␮M Rp-adenosine 3=,5=cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS) did not significantly enhance LPS-stimulated IL-10 release (n⫽6), neither did inhibition of PKA with 300 nM H-89 (n⫽6). Conversely, inactivation of elements of the PLC pathway significantly reduced the effects of glutamate (F⫽15.07, P⬍0.001). Inhibition of PLC by 5 ␮M U73122 significantly reduced the effect of glutamate on LPS-stimulated IL-10 release (n⫽4; Fig. 5C), whereas U73343, the inactive analog of U73122, did not significantly reduce the effect of glutamate (5 ␮M; n⫽4; Fig. 5C). Incubation in 1 ␮M thapsigargin to deplete endoplasmic reticulum Ca2⫹ stores ameliorated the effect of glutamate (n⫽6; Fig. 5C). These results suggest that glutamate requires a PLC/internal Ca2⫹ pathway to bring about potentiation of LPS-stimulated IL-10 release.

DISCUSSION Glutamate potentiates LPS-stimulated IL-10 release by IL-10 mRNA upregulation

Fig. 5. Glutamate potentiates LPS-stimulated IL-10 release by coactivating metabotropic glutamate receptor groups. (A) The effect on LPS (1 ␮g/ml; 16 h)-stimulated IL-10 release of glutamate receptor agonists glutamate (Glu; 1 mM), tACPD (mGluRI and II agonist, 100 ␮M), DHPG (mGluRI agonist, 500 ␮M), APDC (mGluRII agonist, 5 ␮M), L-AP4 (mGluRIII agonist, 1 mM), KA (100 ␮M), AMPA (100 ␮M), and NMDA (500 ␮M). n⫽6 for each condition. Cells were preincubated in LPS for 8 h. Values are expressed as mean ratio of LPS⫹agonist release to LPS release (⫾SEM indicated). (B) The effect of glutamate receptor antagonists MPEP (mGluR5 antagonist, 1 ␮M; n⫽8), CPCCOEt (mGluR1 antagonist, 75 ␮M; n⫽13), EGLU (mGluRII antagonist, 500 ␮M; n⫽9), MSPG (mGluRII and III antagonist, 1 mM; n⫽11), MPEP (1 ␮M)⫹EGLU (500 ␮M; n⫽6)), MPEP (1 ␮M)⫹EGLU (500 ␮M)⫹CPCCOEt (75 ␮M; n⫽6), AP5 (NMDA receptor antagonist, 200 ␮M; n⫽6), and CNQX (AMPA and kainate receptor antagonist, 20 ␮M;

This study shows for the first time that glutamate potentiates high concentration LPS-stimulated IL-10 release from

n⫽6) on glutamate⫹LPS-stimulated IL-10 release (16 h incubation, with an 8 h preincubation in LPS). Values are expressed as mean ratio of LPS⫹Glu⫹antagonist to LPS⫹antagonist (⫾SEM indicated). (C) The effect of intracellular signaling molecule antagonists U73122 (PLC inhibitor, 5 ␮M; n⫽4), U73343 (inactive analog of U73122, 5 ␮M; n⫽4), and thapsigargin (endoplasmic reticulum Ca2⫹-dependent ATPase inhibitor, 1 ␮M) on LPS⫹Glu-stimulated IL-10 release (16 h incubation, with an 8 h preincubation in LPS; n⫽6). Values are expressed as mean ratio of LPS⫹Glu ⫹antagonist to LPS⫹antagonist (⫾SEM indicated). * P⬍0.05, ** P⬍0.01, *** P⬍0.001 when compared with glutamate’s ratio.

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neonatal rat cultured spinal cord astrocytes. In comparison with the level of IL-10 mRNA stimulated by LPS alone, co-exposure to glutamate greatly increased IL-10 mRNA expression. Additionally, maximum potentiation of IL-10 release occurred after 16 h, which is within the time required for IL-10 mRNA production (Ledeboer et al., 2002). Glutamate had no effect on LPS-stimulated IL-10 release when transcription was prevented. Taken together, this suggests the potentiating effect of glutamate requires upregulation of IL-10 mRNA expression. Regulation of IL-10 secretion can also occur through post-transcriptional mechanisms (Powell et al., 2000). As we found prevention of translation completely attenuated LPS-stimulated IL-10 release, the effect of glutamate on post-transcriptional IL-10 regulation could not be examined. It can be inferred, however, that glutamate may have minimal impact on these post-transcriptional processes given that blockade of transcription completely prevents the potentiating effect of glutamate. Stimulation of astrocytes with LPS elicits the release of IL-1␤ (Lin et al., 2008; Delbro et al., 2009). As we found that glutamate does not potentiate LPS-stimulated IL-1␤ release, it is unlikely that IL-1␤ release could contribute to the potentiation of LPS-stimulated IL-10 release by glutamate. We cannot rule out the possibility that other proinflammatory cytokines may contribute to the potentiating effect of glutamate. Receptor involvement: synergistic activation of metabotropic glutamate receptor subtypes is necessary for the potentiating effect of glutamate Glutamate evoked potentiation of LPS-stimulated IL-10 release exclusively and synergistically through activation of group I, II, and possibly III metabotropic glutamate receptors. Ionotropic glutamate receptor agonists did not enhance LPS-stimulated IL-10 release, and antagonists to ionotropic receptors did not decrease the potentiating effect of glutamate, implying ionotropic glutamate receptors are not involved in the potentiating effects of glutamate. Large increases in LPS-stimulated IL-10 release were only seen when mGluRI and II were co-stimulated, and the potentiating effect of glutamate was only reversed when antagonists to different mGluR groups were co-applied. This is congruent with reports of the expression of mGluRI subunits (mGluR 1 and 5) and mGluRII subunits (mGluR 2 and 3) on spinal cord astrocytes by Silva et al. (1999) and Werry et al. (2006). Although no reports exist of mGluRIII on unperturbed spinal cord astrocytes, expression has been identified in astrocytes in pathological conditions such as multiple sclerosis (Geurts et al., 2005). Cell signaling pathways involved in the potentiation by glutamate Ligation of mGluRI leads to activation of PLC and release of Ca2⫹ from intracellular stores by inositol triphosphate (IP3) (Pin and Duvoisin, 1995; Servitja et al., 2003). The potentiating effect of glutamate was dependent on this pathway, as inhibiting PLC and depleting intracellular Ca2⫹ stores attenuated the effects of glutamate.

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There is evidence that mGluR-mediated Ca2⫹ increases are less powerful in astrocytes, compared with neurons. A group I and II mGluR agonist elicits Ca2⫹ increases in neurons with an EC50 that is eightfold more potent than in astrocytes (Ogata et al., 1994; Phenna et al., 1995). Often the EC50 for astrocytic effects of glutamate mediated by mGluR-stimulated Ca2⫹ increases, such as glutamate stimulation of inositol phosphate turnover, arachidonic acid release, and ATP release from astrocytes, are in the range of several hundred micromolars (Stella et al., 1994; Werry et al., 2006). The key role that mGluRmediated Ca2⫹ increases play in the potentiation of LPSmediated IL-10 release by glutamate may explain why the EC50 of glutamate for this effect is also in the range of hundreds of micromolars. mGluRII and III receptor activation is classically associated with a decrease in cAMP and PKA activity, although in the present work decreases in cAMP and PKA activity were not sufficient to potentiate LPS-stimulated IL-10 release. Stimulation of the mGluRII class of metabotropic receptor has also been shown to potentiate mGluRI-stimulated inositol triphosphate accumulation in hippocampal and cerebral slices without itself effecting inositol triphosphate accumulation (Mistry et al., 1998; Cho et al., 2002). Similarly, mGluRII activation and PKA inhibition enhances mGluR5-stimulated Ca2⫹ mobilization in rat perirhinal cortex (Mistry et al., 1998; Cho et al., 2002). This suggests that mGluRII and III receptors may make a synergistic contribution to the effect of glutamate by further potentiating the PLC pathway. In astrocytes, glutamate-stimulated increases in activity in the PLC pathway results in phosphorylation and increased activity of extracellular signalrelated kinase, possibly through mGluR (Schinkmann et al., 2000; D’Onofrio et al., 2001; Peavy et al., 2001). Activation of extracellular signal-related kinase renders the IL-10 promoter accessible to relevant transcription factors and greatly increases the ability of LPS to enhance IL-10 mRNA and protein levels, without affecting IL-10 levels in the absence of LPS (Lucas et al., 2005). IL-10 release from astrocytes— comparisons with microglia We have recently shown that glutamate also potentiates high concentration LPS-stimulated IL-10 release from neonatal spinal cord microglia (Werry et al., 2011). There are a number of differences between the effect of glutamate on LPS-stimulated IL-10 release in microglia and astrocytes, as illustrated in Fig. 6. When the same concentrations of glutamate and LPS are added to astrocytes, they release threefold more IL-10 than microglia. In contrast to the selective effect on mGluR in astrocytes, glutamate acts through all glutamate receptor subtypes to exert its potentiating effect on microglial IL-10 release. Further, glutamate does not act through the PLC pathway to potentiate LPS-stimulated IL-10 release in microglia (Werry et al., 2011). As cultures of astrocytes in the present study contained a small amount of microglia, the heterogeneity in mechanisms of potentiation between microglia and astrocytes in the present study provide assurance

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Fig. 6. Schematic illustrating the differences between the potentiation of LPS-stimulated IL-10 release by glutamate in spinal cord microglia and astrocytes. In many inflamed states, such as chronic pain and infection, astrocytes and microglia are exposed to TLR4 agonists and increased levels of glutamate. Based on the present study and data in Werry et al. (2011), we propose a model whereby glutamate binds to all subtypes of glutamate receptor on microglia, resulting in potentiation (indicated by “⫹”) of TLR4-stimulated IL-10 mRNA upregulation and IL-10 release, through mechanisms that do not involve the phospholipase C (PLC)/intracellular Ca2⫹ pathway ([Ca2⫹]i). The signaling mechanism from TLR4 activation which leads to IL-10 transcription (dashed arrow) in microglia and astrocytes is presently unknown, but likely involves activation of the IL-10 promoter by transcription factors and/or regulatory elements. In contrast, in astrocytes under the same conditions, glutamate selectively and synergistically acts through the mGluRI, II and possibly III subtypes of receptor, resulting in PLC activation (indicated by “⫹”) and [Ca2⫹]i increases. This results in a potentiation of TLR4-mediated IL-10 mRNA levels and IL-10 release. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

that microglia did not contribute to the release of IL-10 described in the present study. Implications of the glutamate-mediated potentiation of high concentration LPS-stimulated IL-10 release from astrocytes In conditions such as gram-negative bacterial infection, chronic pain and glutamate excitotoxicity IL-10 has antiinflammatory and neuroprotective outcomes (Mesples et al., 2003; Milligan et al., 2006b; Qian et al., 2006a,b; Londono et al., 2008, Nathaniel et al., 2010), and it is effective in decreasing pro-inflammatory cytokine levels at IL-10 concentrations measured in the present study (Mizuno et al., 1994; Benveniste et al., 1995; Sawada et al., 1999; Ledeboer et al., 2002). Glutamate and LPS (or

other TLR4 agonists) are also present in these conditions (Beutler, 2000; Tanga et al., 2005; Jou et al., 2006). Considering this, and also that glutamate does not elevate LPS-stimulated release of the pro-inflammatory IL-1␤, we hypothesize that the increased levels of IL-10 stimulated from astrocytes by the dual action of TLR4 agonists and glutamate in these conditions may lead to a negative feedback anti-inflammatory effect on pro-inflammatory mediators such as interleukin-6 and nitric oxide, whose kinetics of release overlap with that of IL-10 (Nakamura et al., 1999). It remains to be shown, however, whether in vivo concentrations of glutamate and LPS at the astrocyte cell surface in the aforementioned conditions reach those that brought about the in vitro potentiation in this study.

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The EC50 for the glutamate potentiation of LPS-stimulated IL-10 release was 400 ␮M, which is below the level that induces significant excitotoxicity (Zou and Crews, 2005). The concentration of glutamate at the astrocytic surface in basal conditions is not known, however, the concentration of glutamate at synapse-surrounding glia in the cerebellum is 190 ␮M (Dzubay and Jahr, 1999), slightly lower than the EC50 in the present study. This suggests that glutamate’s potentiation of LPS-stimulated IL-10 release may primarily occur when glutamate levels are elevated, for example, conditions involving an inflammatory response such as gram-negative bacterial infection, chronic pain, and ischemia (Benveniste et al., 1984; Rego et al., 1996; Sasaki et al., 1998; Ko et al., 2003; Takeuchi et al., 2006). In these inflammatory conditions glutamate may rise to concentrations that potentiate LPS-induced IL-10 release. At a concentration of 0.1 ng/ml, LPS can bring about pro-inflammatory effects like stimulation of IL-6 and IL-1␤ release from whole human blood (DeForge et al., 1992). LPS at 10 ng/ml, however, elicits only weak IL-10 release from astrocytes (Bolin et al., 2005). Considering this, to ensure sufficient IL-10 release and to allow comparisons with previous work on the effect of glutamate on LPSstimulated IL-10 release from microglia (Werry et al., 2011), LPS was used at 1 ␮g/ml in the present study. LPS has been shown to reach concentrations of 1 ␮g/ml in inflammatory conditions in vivo, for example, cerebrospinal fluid concentrations of LPS during gram-negative meningitis average 1.3 ␮g/ml, and may rise up to 11.3 ␮g/ml (Berman et al., 1976; Arditi et al., 1989). The concentration of LPS at the astrocytic surface in such conditions, however, is unknown. Further studies to clarify the levels of glutamate and LPS at the astrocyte surface in vivo during inflammation or pain would increase our understanding of the capacity of spinal cord astrocytes to beneficially modulate these diseases. In summary, in the first study investigating the effect of glutamate on astrocytic LPS-stimulated IL-10 release, we have demonstrated IL-10 release stimulated by a high concentration LPS from neonatal spinal cord astrocytes is enhanced by glutamate. This potentiation exclusively involves synergistic activation of mGluR subgroups, the PLC signaling pathway and upregulation of IL-10 mRNA levels. Acknowledgments—This work was supported by an NSW Spinal Cord Project Grant to M.R.B. and an Australian Federation of University Women Fellowship to E.L.W. This work was also funded by the Charles D. Kelman Postdoctoral Scholar Award from the International Retinal Research Foundation (IRRF, USA) to M.D.L., who was also supported by grants from International Science Linkages (CG130097) to Tailoi Chan-Ling (Discipline of Anatomy and Histology, and Bosch Institute, University of Sydney). We thank Donna Lai (Bosch Institute, University of Sydney) for assistance with RT-PCR experiments in this manuscript.

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(Accepted 20 January 2012) (Available online 28 January 2012)