y 195 (2008) 60–72www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolog

Japanese Encephalitis Virus infection induces IL-18 and IL-1β in microgliaand astrocytes: Correlation with in vitro cytokine responsiveness

of glial cells and subsequent neuronal death

Sulagna Das, Manoj Kumar Mishra, Joydeep Ghosh, Anirban Basu ⁎

National Brain Research Centre, Manesar, Haryana, India

Received 15 October 2007; received in revised form 7 January 2008; accepted 22 January 2008

Abstract

IL-1β and IL-18 are members of the IL-1 family of ligands, and their receptors are members of the IL-1 receptor family. Although severalbiological properties overlap for these cytokines, differences exist. In order to assess functional importance of these two cytokines in viralencephalitis, we have exploited an experimental model of Japanese Encephalitis (JE) and subsequent in vitro cell culture system. We report for thefirst time that in Japanese Encephalitis, microglia and astrocytes both produce IL-18 and IL-1β. In vitro, these two cytokines differentially activatemicroglia and astrocyte, and also alter the by stander neuronal survival following treatment with these two cytokines.© 2008 Elsevier B.V. All rights reserved.

Keywords: Japanese Encephalitis Virus; IL-18; IL-1β; Microglia; Astrocytes; Neuron

1. Introduction

Japanese Encephalitis (JE) is an acute viral infection of thecentral nervous system (CNS), caused by a mosquito-borneflavivirus called Japanese Encephalitis Virus (JEV). JEV targetsthe CNS, clinically manifesting with fever, headache, vomiting,signs of meningeal irritation and altered consciousness leadingto high mortality and neurological sequelae in some of thosewho survive (Kumar et al., 1991).The inflammation results inan increased level of cytokines such as macrophage-derivedchemotactic factor (MDF), tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), MCP-1 and IP-10 in the brain of JEVinfected animals (CSF) (Bhowmick et al., 2007; Khanna et al.,1991; Singh et al., 2000). The increased levels of inflammatorymediators appear to play a protective role or to initiate anirreversible immune response leading to cell death.

⁎ Corresponding author. National Brain Research Centre, Manesar, Haryana-122050, India. Tel.: +91 124 2338921x225; fax: +91 124 2338910/28.

E-mail address: anirban@nbrc.res.in (A. Basu).

0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jneuroim.2008.01.009

IL-18 is a pro-inflammatory cytokine that belongs to the IL-1family of ligands (Okamura et al., 1995; Ushio et al., 1996). Itwas first isolated as interferon-γ inducing factor, a mediator ofinflammatory tissue damage (Okamura et al., 1995). The roleplayed by IL-18 in CNS inflammation following any viralinfection is unknown. IL-18 is secreted as an active peptidefollowing cleavage by interleukin 1β converting enzyme ICE.The IL-18 receptors, although distinct from IL-1 receptors, alsobelong to the IL-1 receptor family. The IL-18 receptor (IL-18R)complex consists of two receptor chains: a ligand-binding chaintermed the IL-18Rα chain and a coreceptor termed IL-18Rβchain; both chains are required for signaling. IL-18 might havedirect effects on the central nervous system (CNS) or act via theproduction of other cytokines. In vitro IL-18 stimulates theproduction of IL-1β in primary astrocytes (Culhane et al., 1998)and of IL-1β and IL-8 via TNF-α in blood mononuclear cells(Puren et al., 1998). One hypothesis is that IL-18 may play arole in modulating IL-1β and thus indirectly affect the IL-1βdependent actions on the brain including inflammation,stimulation of the hypothalamic–pituitary–adrenal-(HPA), andbehavioral changes associated with diseases. In vivo IL-18

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stimulates cytotoxicity accounting for the TNF-α- and FasL-mediated tissue damage in liver (Tsutsui et al., 1997) and hasantitumor and antimicrobial activity (Kawakami et al., 1997;Micallef et al., 1997).

Despite the pathological importance of JEV, knowledge ofthe immunological mechanisms including cytokine mediatedsignaling events occurring during viral infection is limited. Therole played by different cytokines and their cross talk withmicroglia and astrocytes, in the pathogenesis of JE is still notwell defined. In this communication we have evaluated the roleof IL-18 in JE. Another purpose of the current study is toexamine the differences between IL-18 and IL-1β in activatingmicroglia and astrocytes in vitro and to further assess theirsecondary role in neuronal survival. In the present study wehave demonstrated that following JEV infection 1) there is anupregulation of IL-18 and IL-1β in brain 2) both microglia andastrocytes differentially produce IL-18 and IL-1β, 3) IL-18 andIL-1β differentially modulate microglia and astrocytes to re-lease cytokines and chemokines. We have also demonstratedfollowing IL-18 and IL-1β treatment to microglia and as-trocytes produce mediators, which are capable of modulatingneuronal survival.

2. Materials and methods

2.1. Viruses and cell

The GP78 strain of Japanese Encephalitis Virus was initiallyobtained from Dr. Sudhansu Vrati (National Institute of Im-munology, New Delhi, India) and further propagated in suck-ling BALB/c mice. Their brain tissue was harvested whensymptoms of sickness became visible. A 10% suspension of thebrain tissue was made by homogenization in the minimumessential medium (MEM). It was then centrifuged at 10,000×gto remove cellular debris and filtered through 0.22 µm sterilefilter (Millipore, MA, USA). The mouse brain derived virus wasstored at −70 °C in small aliquots and was used as the source ofvirus for all the experiments (Appaiahgari et al., 2006). Porcinestable kidney (PS) cell line and mouse neuroblastoma Neuro2a(N2a) were obtained from National Centre for Cell Science,Pune, India. Human cell line of fetal microglial origin CHME3(a gift from Dr Steve Levison, UMDNJ, New Jersey, USA),human fetal astrocyte cell line SVG (a gift from Dr Pankaj Seth,National Brain Research Centre (NBRC), India) and humanneuroblastoma cell line SK-N-SH (a gift from Dr Kakoli Gho-shal, National Institute of Immunology, New Delhi, India)were grown at 37 °C in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin. All the reagents related to cell culturewere obtained from Sigma, St. Louis, USA, unless otherwisestated.

2.2. Virus titration

JEV was titrated by plaque formation on the monolayers ofporcine kidney (PS) cell line. PS cell line was seeded in 35mmdishes to give semi-confluent monolayers in about 18 h. Mono-

layers were inoculated with ten fold dilutions of virus samplemade in MEM containing 1% FCS and incubated for 1 h at37 °C with occasional shaking. The inoculum was removed byaspiration and the monolayers were further overlaid with MEMcontaining 4% FCS, 1% low melting point agarose and acocktail of antibiotic–antimyotic solution (Gibco, CA, USA)containing penicillin, streptomycin and amphotericin-B. Plateswere incubated at 37 °C for 3–4 days until plaques were visible.To allow counting of the plaques, the cell monolayer wasstained with crystal violet after fixing the cells with 10% for-maldehyde (Vrati et al., 1999a).

2.3. Virus infection of animals

We have used a previously described animal model of Jap-anese Encephalitis (Vrati et al., 1999a,b). Three to four days oldBALB/c mice of either sex were injected intra-cerebrally withapproximately 100 p.f.u (in 30 µl of 1× PBS) of JE virus ofstrain GP78. Control animals received the same amount of 1×PBS as the experimental group. Groups of three mice weresacrificed at each time point either for tissue or protein andRNA. From the third day post infection animals started to showsymptoms of JE including limb paralysis, poor pain responseand whole body tremor. On the fourth day post infection allanimals succumbed to infection. All experiments were per-formed according to the protocol approved by the InstitutionalAnimal Ethics Committee of NBRC.

2.4. Treatment of human glial cell line with virus and cytokines

CHME3 human microglial cell line and SVG humanastroglial cell line were plated at 5×105cells/well in a six wellplate. After 24 h in MEM with 10% serum, the cells wereswitched to serum free media for 12 h. Both the cell lines werethen adsorbed with JE virus (multiplicity of infection, MOI=5)for 1 h. After adsorption, unbound viruses were removedby gentle washing with phosphate-buffered saline (1× PBS,pH7.4). Fresh serum free medium was added to each well forfurther incubation for 24 h at 37 °C. Following 24 h incubation,cells were processed for immunoblot analysis as described laterin Materials and methods. Virus titration was performed withthis supernatant to further confirm the presence or absence oflive viruses.

In another set of experiments, the glial cells were treated withcytokines IL-18 (10 ng/ml) and IL-1β (5 ng/ml) as well as witha combination of IL-18 (10 ng/ml) and IL-1β (5 ng/ml) for 18 h.Both cytokines were obtained from R & D Systems, Min-neapolis, USA. Following the treatment, cells were washedonce with 1× PBS and serum free media were added to them.The cells were then incubated at 37 °C for 18 h, and followingthat supernatant was collected and stored for later use while celllysates were prepared.

2.5. Immunoblot analysis

For protein isolation, pups were sacrificed by decapitation.The brains were isolated and homogenized in buffer containing

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proteinase inhibitor (Roche, Basel, Switzerland). The homo-genate was then centrifuged and 20 µg of supernatant wasseparated on polyacrylamide gels, electrophoresed, transferredonto nitro cellulose membranes and immunoblots performedas reported earlier (Basu et al., 2002). Following incubationwith the primary antibody (IL-18 and IL-1β, 1:1000, R & DSystems), the blots were extensively washed (4×) in PBS-Tween and further incubated for 2 h at room temperature withHRP conjugated goat anti-rabbit secondary antibody (Vectorlaboratories, CA, USA). The membranes were then washedand visualized by chemiluminescence. The blots were strippedand reprobed with anti-β-tubulin (1:1000, Santa Cruz) todetermine equivalent loading. Measurements of the opticaldensities for IL-18 and IL-1β were normalized to the levels ofβ-tubulin.

CHME3 and SVG cells under different conditions werewashed twice with ice-cold 1× PBS, then lysed in buffercontaining 1% Triton-X-100, 10 mM Tris–HCl (pH 8.0),150 mM NaCl, 0.5% Nonidet P (NP-40), 1 mM EDTA, 0.2%EGTA, 0.2% sodium orthovanadate and protease inhibitorcocktail (Sigma, St. Louis, USA). DNAwas sheared using a 24-gauge needle and the lysate was incubated on a rockingplatform at 4 °C for 30 min before centrifugation at 10,000×gfor 15 min at 4 °C. Protein levels were determined by Bradfordmethod using Varioskan Flash spectral scanning multimodereader (Thermo Electron Corporation, Finland). To increasethe intracellular accumulation of IL-18 and IL-1β protein inCHME3 and SVG cells, Brefeldin A (BD Pharmingen, 1 µg/ml)was added to the culture for the last 4h of incubation beforeprotein isolation (Basu et al., 2000).

2.6. Immunohistochemistry

Animals at days 2 and 4 post infection and age matchedcontrols were perfused with 1× PBS containing 7U/ml heparin,followed by a fixative containing 2.5% para-formaldehyde inphosphate buffer. The brains were processed for cryostatsectioning and the sections were stained as described previously(Basu et al., 2002). Astrocytes were labeled with rabbit anti-GFAP (1:750, Dako, Denmark) and microglia were labeled withrat anti-mouse CD11b (1:200, BD Biosciences, NJ, USA),which were then double stained with goat anti IL-18 and IL-1β(R & D systems, Minneapolis, MN, USA) (1:100). Thefollowing secondary antibodies were used: anti-rabbit Fluor-escein (1:100; Vector, CA, USA) for GFAP, anti-rat FITC(1:200, Vector) and Alexa red (1:1000), and anti-goat AlexaRed (1:500; Molecular Probes Invitrogen, CA, USA) for IL-18.

Table 1Primers used for RT-PCR experiments and expected size of amplified products

Forward primer

IL-1β 5′-TGGAAAAGCGGTTTGTCT-3′IL-18 5′-GTGAACCCCAGACCAGACTG-3′Cyclophilin 5′-CCATCGTGTCATCAAGGACTTCAT-3′

IL – interleukins; RT-PCR s– Reverse transcriptase-polymerase chain reaction.

The stained slides were mounted with DAPI and observed underthe Zeiss Axioplan 2 Fluorescence microscope.

2.7. RNA isolation and semi-quantitative RT-PCR analysis

Total cellular RNA was isolated from virus treated andcontrol mice brains on second and fourth day post infectionusing TRI reagent. Total RNA (1.0 µg) was reverse-transcribedusing oligo dT. Oligonucleotide primer pairs against mouseIL-18, IL-1β and cyclophilin mRNAs were chosen, checkedfor specificity using a basic BLAST search, and prepared fromMicrosynth (Balgach, Switzerland) (Table 1). Amplification ofthe mRNA sequences of IL-18, IL-1β and cyclophilinproduced single bands that were of the size predicted fromthe reported sequences for these mRNAs. Reverse transcrip-tase-polymerase chain reaction (RT-PCR) was performedusing the onestep RT-PCR kit (Qiagen Biosciences; Hamburg,Germany) following the manufacturer's protocol and reactionswere carried out on Genius Techne thermal cycler. One mi-crogram of the total RNA was used as template in 25 µl PCRreactions, containing 5× PCR buffer (5 µl), dNTPs (1 µl),enzyme mix (1 µl) and specific forward and reverse primers(5 µM) and RNase free water. The PCR products were se-parated on 2% agarose gel, stained with ethidium bromide, andphotographed using Chemigenius Bioimaging System, Syn-gene. Measurements of the optical densities for IL-18 and IL-1β were normalized to the levels of cyclophilin.

JEV treated CHME3 and SVG cells were washed twice withice-cold 1× PBS, then total cellular RNA was isolated asdescribed earlier in methodology section. Reverse transcriptase-polymerase chain reaction (RT-PCR) for IL-18, IL-1β andcycophilin was performed using the onestep RT-PCR kit(Qiagen Biosciences).

2.8. Human cytokine and chemokine bead array

Human cytokine and chemokine bead array kit (CBA) (BDBiosciences, NJ, USA) was used to quantitatively measurecytokine and chemokine expression levels in the supernatantcollected from human microglia and astroglial cell linesfollowing IL-18 and IL-1β treatment. Using 50 µl of standardand sample dilutions, the assay was performed as described(Sharma et al., 2007) and analyzed on FACS Calibur (BectonDickinson). The beads coated either with IL-1β, IL-8, TNF-αand IL-6 or with IP-10, MCP-1, RANTES and MIG reactedwith test lysates and standards, to which fluorescence dyes werethen added. Analysis was performed using CBA software that

Reverse primer Product size (bp)

5′-ATAAATAGGTAAGTGGTTGCC-3′ 2945′-CCTGGAACACGTTTCTGAAAGA-3′ 3255′-TTGCCATCCAGCCAGGAGGTCT-3′ 192

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allows the calculation of cytokines, chemokines and apoptoticmediators in unknown lysates (Sharma et al., 2007).

2.9. TUNEL assay and human apoptosis bead array

Human neuroblastoma cell line SK-N-SH was plated at adensity of 5×104cells/well of 8 well chamber slides (Nunc,Denmark) in medium containing 10% FBS. After 24 h, cellswere grown in serum free media for 8h. Next, the medium wasreplaced by 50% of culture supernatant obtained from previousexperiment (Treatment of human glial cell line with cytokinesIL-18 and IL-1β) and 50% of fresh serum free medium. In caseof JEV infection, after virus treatment for 1 h, the media werereplaced by the conditioned media from glial cells as describedabove. After 24 h, dying cells (N2a) were identified using InSitu Cell Death Detection Kit, TMR red (Roche, Germany).Briefly, neuronal cultures (N2a) were fixed with 4% PFA in 1×PBS and blocked with 4% BSA containing 0.02% Triton-X-100. The fixed cells were then incubated in the TUNEL mix(terminal deoxynucleotidyl transferase in storage buffer andTMR red labeled-nucleotide mixture in reaction buffer) for 1 hat RT. The slides were mounted with Vectashield mountingmedia containing DAPI (Vector Laboratories Inc., CA, USA).

A different set of SK-N-SH cells from the same experimentswas used to prepare lysates for human apoptosis bead array.Human apoptosis kit (BD Biosciences) was used to quantita-tively measure the expression levels of caspase-3, PARP andBcl-2 in protein lysates collected from above experiments.

Fig. 1. Induction of pro-inflammatory IL-18 and IL-1β in the mouse brain at differenpost infected mice brains and reverse-transcribed. The levels of IL-18 and IL-1β werthree independent experiments. The increase in the levels of both IL-18 and IL-1β is efold change in the mRNA levels of IL-18 and IL-1β over control at different days postchange from control, pb0.05). C. Protein isolated from control, 2 days and 4 days poslevel of both IL-18 and IL-1β at 4 day post JE infection was observed. Data shown afold change in the protein expression levels of IL-18 and IL-1β over control at differ(⁎ significant change from control, pb0.05).

Using 50 µl of human apoptosis standard and sample dilutions,the assay was performed as described (Sharma et al., 2007) andanalyzed on FACS Calibur (Becton Dickinson).

2.10. Statistical analysis

All the experiments performed and the data generated wereanalyzed statistically by paired two-tailed Student's t-test.

3. Results

3.1. Induction of IL-18 and IL-1β following JE

Previous work from our lab has shown that JEV induces arobust neuroinflammatory response across different brain areasand a massive increase in the levels of some of the pro-inflammatory molecules. We therefore investigated the expres-sion profile of IL-1 family of cytokines, IL-18 and IL-1β,following JE infection in 3–4 days old BALB/c mice. The levelof mRNA transcripts of these two cytokines was determinedusing RT-PCR. Compared to uninfected controls, the mRNAlevels of these cytokines increased with progressive infection(Fig. 1A, B). Although the expression of IL-18 was comparableto control levels at 2 days post infection, it was enhanced by 2.5folds at 4 days post infection over control ( pb0.05). IL-1β alsoshowed 2 fold and 2.2 fold increase in expression on the 2and 4 days post infection respectively with respect to control( pb0.05).

t days post JE infection. A. Total mRNAwas isolated from control, 2 and 4 dayse determined using semi-quantitative RT-PCR. Data shown are representative ofvident with gradual infection with respect to controls. B. The graph represents theinfection. Values represent mean±SEM from 3 mice in each group (⁎ significantt infected mice brains were analyzed by immunoblot. A significant increase in there representative of three independent experiments. D. The graph represents theent days post infection. Values represent mean±SEM from 3 mice in each group

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The protein levels of these cytokines were also assessed atsimilar time points using Western blot. Both cytokines showedsimilar increase in their expression following infection (Fig. 1C,D). While levels of IL-18 increased to 2 folds at 4 day postinfection over control, that of IL-1β increased to 2.5 folds at4 day post infection ( pb0.05). Thus, both the cytokines showeda gradual increase in their expression levels with progressive JEinfection.

Fig. 2. Release of IL-18 from reactive astrocytes and microglia following JE in mousdouble stained for GFAP and IL-18 in one set and for CDllb and IL-18 in another setsections that do not stain for either GFAP or IL-18. Panel B: The infected brain secolocalisation of GFAP (FITC, green) and IL-18 (Alexa Fluor 594, red), white arrowIL-18 expression. Panel D: The JE infection section however is enriched with react(FITC, green) as indicated by their colocalisation (yellow, white arrows). (For interpreweb version of this article.)

3.2. Both astrocytes and microglia express IL-18 and IL-1βfollowing JEV

Activation of astrocytes and microglia is the hallmark ofneuroinflammation and has been characterized in JEV infectionalso (Ghoshal et al., 2007; Mishra et al., 2007; Swarup et al.,2007). Immunohistochemistry performed on 4 days postinfected brain sections revealed that indeed there is induction

e brain. Cryostat sections from control and 4 day JE infected BALB/c mice wereand mounted using Vectashield containing DAPI. Panel A represents the controlctions show marked presence of astrocytes releasing IL-18 as indicated by thes. Panel C: The control brain sections do not exhibit any CD11b positive cells orive microglia, Cd11b positive cells (Alexa Fluor 594, red) which express IL-18tation of the references to colour in this figure legend, the reader is referred to the

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of IL-18 following JE and that the release of this cytokineoccurs from both activated astrocytes as well as activatedmicroglia. Colocalisation of GFAP (marker for activatedastrocytes) and IL-18 was noted in 4 day infected brains(Fig. 2, Panel B), clearly indicating that astrocytes release IL-18in JE infection. Double staining of CDllb (a marker for activatedmicroglia/macrophages) with IL-18 revealed that activatedmicroglia also secrete IL-18 robustly (Fig. 2, Panel D). Thecontrol sections however showed no such expression for either

Fig. 3. Release of IL-1β from reactive astrocytes and microglia following JE infec(reactive astrocytes marker) as well as IL-1β. Panel B: Prominent staining for GFAsections and marked colocalisation of these two markers was also observed, shown bobserved in control brain sections. Panel D: The JE infected sections showed CD11bCD11b with IL-1β was also conspicuous (yellow, white arrows). (For interpretationversion of this article.)

activated astrocytes, or microglia or the cytokine IL-18 (Fig. 2,Panels A and C).

IL-1β, which has already been explored in great detail invarious disease states, is also upregulated in JE and the primarycells releasing IL-1β are activated astrocytes (colocalisation ofGFAP with IL-1β) (Fig. 3, Panel B) and activated microglia(colocalisation of CD11b with IL-1β) (Fig. 3, Panel D). Whilethese double positive cells were clearly evident in the 4 dayinfected brain, no such cells were identified in the control brain

tion in mouse brain. Panel A: The control brain sections do not express GFAPP (FITC, green) and IL-1β (Alexa Fluor 594, red) was observed in JE infectedy white arrows. Panel C: No detectable fluorescence for CD11b and IL-1β waspositive cells (red) as well as IL-1β expression (green) and the colocalisation ofof the references to colour in this figure legend, the reader is referred to the web

Fig. 5. IL-18 and IL-1β stimulated production of pro-inflammatory cytokinesfrom astrocytic and microglial cell lines. A. The graph represents the increase in

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(Fig. 3, Panels A and C). These results clearly substantiate theexpression of IL-18 and IL-1β in the diseased brain and identifythe cellular sources of the two cytokines following JEVinfection.

To further confirm the above in vivo findings, an in vitro cellculture model of JE infection was utilized. Human astrocytic cellline SVG and human microglial cell line CHME3 were mockand JE infected. RNAwas isolated from these cells after 24 h ofJE infection. RT-PCR analysis revealed a 2 fold increase in IL-18mRNA levels in JE infected CHME3 cells and a 2.5 fold increasein JE infected SVG cells over mock-infected ones (Fig. 4A andB). Western blot analysis was also done to quantify the proteinlevels of these cytokines in the two cell types.While the levels ofIL-18 were upregulated to 2.5 folds in microglia post JEinfection, its levels in the astrocytes showed 1.8 fold increase ascompared to mock-infected controls (Fig. 4A and B).

Similar expression profile of IL-1β was also detected on bothastrocytic and microglial cells. IL-1β mRNA levels wereupregulated by 3 folds in JE infected CHME3 cells and 2 foldsin that of SVG cells with respect tomock-infected controls. IL-1βdetected by immunoblot also showed elevated levels, 1.3 fold and1.8 fold increase in astrocytic and microglial cell linesrespectively following JE infection as compared tomock-infectedcells (Fig. 4A and B). These results clearly indicate that bothastrocytes and microglia are potent producers of the cytokinesIL-18 and IL-1β upon JEV infection, albeit at different levels.

3.3. Regulation of pro-inflammatory cytokine production byIL-18 and IL-1β from microglia and astrocytes

We next investigated the contribution of the IL-1 family ofcytokines in initiation of neuroinflammation, manifested in

Fig. 4. JE induces the release of both IL-18 and IL-1β following co-culture withmicroglial cell line CHME3 and astrocytic cell line SVG. Human microglial cellCHME3 and human astrocytic cell SVG were co-cultured with (MOI-5) andwithout JE virus for 1 h at 37 °C. After 24 h, RNA and protein were extractedfrom the cells for RT-PCR and immunoblot respectively. A. The expression ofboth IL-18 and IL-1β in CHME3 cells showed a significant increase in mRNAlevels following treatment with JEV. Similar level of induction was also noticedfor protein levels of IL-18 and IL-1β following virus treatment in CHME3 cells.B. SVG cells post infection with the virus also demonstrate elevated expressionof IL-18 as compared to mock-infected in mRNA as well as protein levels. Theother cytokine, IL-1β levels also increase markedly in these cells following JEtreatment as detected by semi-quantitative RT-PCR and immunoblot.

the levels of the various pro-inflammatory cytokines like IL-1β, IL-8, TNF-α,IL-6 (measured using Human CBA kit) and IL-18 (densitometric quantificationobtained from immunoblotting) following treatment of human microglialCHME3 cell lines post treatment with IL-18 (10 ng/ml) and IL-1β (5 ng/ml)individually and also in concert for 18 hour duration. Values represent mean±SEM from 3 experiments in each condition (⁎ significant change from control,pb0.01). B. The graph represents the increase in the levels of the various pro-inflammatory cytokines like IL-1β, IL-8, IL-6 (measured using Human CBAkit) and IL-18 (densitometric quantification obtained from immunoblotting)following treatment of human astrocytic SVG cell lines with IL-18 (10 ng/ml),IL-1β (5 ng/ml) and combined treatment with IL-18 and IL-1β for 18 h. TNF-αlevels however were not detected in SVG samples post treatment with thecytokines. Values represent mean±SEM from 3 experiments in each condition(⁎ significant change from control, pb0.05).

terms of elevated levels of various pro-inflammatory cytokinesand chemokines. The IL-1 family of cytokines has establishedroles in induction of other pro-inflammatory molecules as wellas stimulates their own production via an auto-feedback loop.Both SVG and CHME3 cell lines were treated with IL-18(10 ng/ml) and IL-1β (5 ng/ml) individually and in conjunctionalso for 18 h and cell lysates were then processed for cytokinebead array for IL-1β, IL-8, TNF-α, IL-6 and immunoblottingfor IL-18 (Fig. 5).

IL-18 treatment to CHME3 cells induced the production ofcytokines like IL-1β (3 folds over control), IL-8 (8 folds overcontrol), TNF-α (2 folds over control) and IL-6 (11 folds over

Fig. 6. Induction of pro-inflammatory chemokines by IL-18 and IL-1β fromastrocytic and microglial cell lines. A. The graph represents the increase in thelevels of the various pro-inflammatory chemokines like IP-10, MCP-1, MIG andRANTES following treatment of human microglial cell line CHME3 posttreatment with IL-18 (10 ng/ml) and IL-1β (5 ng/ml) individually and also inconcert for 18 h. Values represent mean±SEM from 3 experiments in eachcondition (⁎ significant change from control, pb0.01; # significant change fromcontrol pb0.05). B. The graph represents the increase in the levels of the variouspro-inflammatory chemokines like IP-10, MCP-1, and MIG following 18 hourtreatment of human astrocytic SVG cell lines with IL-18 (10 ng/ml), IL-1β(5 ng/ml) and combined treatment with IL-18 and IL-1β. RANTES levelshowever were not detected in SVG samples post treatment with the cytokines.Values represent mean±SEM from 3 experiments in each condition (⁎

significant change from control, pb0.01; # significant change from controlpb0.05).

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control) ( pb0.01) as well as its own production (3 folds overcontrol) ( pb0.05) (Fig. 5A). IL-1β, the other IL-1 familymember, also elevates the levels of all these cytokines upontreatment in microglial cells. In an auto-feedback loop itupregulated its own level by 5 folds over control cells and alsothat of the other cytokines like IL-8 (15 folds over control),TNF-α (6 folds over control), IL-6 (10 folds over control) andIL-18 (2 folds over control) ( pb0.01). Upon combinedtreatment of the microglial cells with both the IL-1 familycytokines, a synergistic action was noted in terms of productionof IL-1β, which was elevated to 8 folds over control, and IL-6showing 22 fold increase ( pb0.01) over control. TNF-α, IL-8and IL-18 however intriguingly did not show the synergisticincrease and respective 4 fold, 17 fold and 3 fold increase in itslevels over control was observed (Fig. 5A).

To assess the responsiveness of astrocytes to IL-1 family ofcytokines, similar treatment as above was done to humanastrocytic SVG cell lines. IL-18 was potent in inducing therelease of various cytokines from astrocytes though the levelswere different from that than in microglia (Fig. 5B). Upontreatment of astrocytes with IL-18, it stimulated its ownproduction by 2 folds over control, while the levels of IL-1βincreased to 1.7 folds over control, and that of IL-8 was elevatedto 2.5 folds over control ( pb0.05). Interestingly, there was nonoticeable change in the level of TNF-α in astrocytes followingtreatment with IL-18. The treatment of astrocytes with IL-1βalso yielded 2.1 fold, 3 fold, 1.8 fold and 1.76 fold increase overcontrol in the levels of cytokines IL-1β, IL-8, IL-6, and IL-18respectively ( pb0.05). In astrocytes however, the synergisticeffect of IL-18 and IL-1β was not prominent. While IL-8 levelsincreased to 4 folds upon treatment with the two cytokines,those of IL-1β, IL-6 and IL-18 remained comparable to those ofthe individual cytokine treatment. The detected fold increases inIL-1β, IL-6 and IL-18 were 2.4 folds, 2 folds and 2.3 folds overcontrol following combined treatment of IL-18 and IL-1β inSVG cells (Fig. 5B).

The findings signify the responsiveness of both the glial celltypes – microglia and astrocytes to the IL-18 and IL-1β and thedifferential production of cytokines by these cells with treat-ment of IL-1 family of cytokines.

3.4. Regulation of chemokine production by IL-18 and IL-1βfrom microglia and astrocytes

The chemokines constitute another group of pro-inflamma-tory mediators, which are upregulated in any inflammatorycondition. Both IL-18 and IL-1β have the ability to induce thesecretion of chemokines both from astrocytes and microglia invitro (Fig. 6). IL-18, being IFN-γ inducing factor, stimulatesmicroglia to secrete the chemokine IP-10 to almost 6 foldsover control (Fig. 6A). The other chemokines upregulatedin CHME3 cells include MCP-1 (3 folds over control), MIG(3 folds over control) and RANTES (6 folds over control)( pb0.01). IL-1β also induces the release of all these che-mokines from CHME3 cells, though IP-10 and RANTES showonly 1.6 fold increase each with respect to controls ( pb0.05).While MCP-1 increases 2 folds ( pb0.05), MIG levels are

enhanced to only 1.3 folds following IL-1β treatment tomicroglia. Upon treatment with both IL-18 and IL-1β, thesignificant synergistic action in chemokine secretion howeverwas not observed. IP-10 maintained elevated levels at 3.5 foldsover control, while that of MCP-1, MIG and RANTES weremeasured as 2 folds, 1.8 and 3 folds respectively (Fig. 6A).

Interesting observations were made after treatment of IL-18and IL-1β to astrocytes in vitro. Astrocytes responded better toIL-18 and IL-1β in terms of chemokine production thanmicroglial cells, though RANTES production was not observedin them. With IL-18 treatment there was a dramatic 100 foldincrease in MCP-1, while the levels of IP-10 and MIG weremeasured at 5 ( pb0.01) and 1.2 folds over control respectively(Fig. 6B). IL-1β however showed a variation in the induction

68 S. Das et al. / Journal of Neuroimmunology 195 (2008) 60–72

Fig. 8. Induction of apoptotic molecules in SK-N-SH as part of bystander effectof IL-18 and IL-1β. A. The graph represents the alteration in the levels ofvarious apoptotic markers in human neuroblastoma SK-N-SH cells whencultured in presence of supernatant from, control, IL-18, IL-1β and both IL-18and IL-1β treated CHME3 cell lines. While levels of pro-apoptotic caspase-3and cleaved PARP increased, that of anti-apoptotic Bcl-2 decreased in the abovetreatment conditions with respect to control treatment. B. The graph representsthe increase in levels of caspase-3 and cleaved PARP and decrease in anti-apoptotic Bcl-2 in SK-N-SH cells after growing them in supernatant from IL-18,IL-1β and IL-18+ IL-1β treated SVG cells. Values represent mean±SEM from3 experiments in each condition (⁎ significant change from control, pb0.05).

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profile of chemokines from the astrocytes. While the levels ofMCP-1 increased to 10 folds, a massive 130 fold increase overcontrols in MIG was noted upon IL-1β treatment to astrocytes( pb0.01). IP-10 maintained its expression at 3 fold increaseover control ( pb0.05). The synergistic action of IL-18 and IL-1β in chemokine production from astrocytes was however notsignificant. IP-10 continued its levels at 4 fold increase overcontrol, while that of MCP-1 and MIG showed elevated levelsof 102 and 28 fold increase over control ( pb0.01) upon thecombined treatment of IL-18 and IL-1β on astrocytes.

These results clearly indicated that both microglia andastrocytes upon stimulation with two members of IL-1 family ofcytokines, IL-18 and IL-1β, secrete an array of chemokines andthus potentiate the inflammatory cascade.

3.5. Bystander neuronal death by IL-18 and IL-1β

We next investigated whether the activation of astrocytes andmicroglia following treatment with IL-18 and IL-1β and theresulting release of pro-inflammatory mediators can causebystander damage to neurons. Human microglial (CHME3) andastrocytic (SVG) cell lines were treated with IL-18 and IL-1βfor 18 h and were cultured for 18 h in fresh serum free media.The supernatant collected from them post 18 h was used to treathuman neuroblastoma cell line SK-N-SH and TUNEL stainingwas performed to detect apoptosis in them (Fig. 7). Whiletreatment of SK-N-SH with supernatant from CHME3 treatedwith IL-18 resulted in 4 fold increase in TUNEL positive cellswith respect to supernatant from control CHME3 cells, that withsupernatant from IL-1β treated CHME3 yielded more apoptoticcells (5 folds over mock-infected) ( pb0.05). However super-natant collected after combined cytokine treatment to CHME3did not result in increase TUNEL positive cells, thus negatingany synergistic effect (Fig. 7, Panels A, D).

Similar set of experiments was carried out with supernatantsfrom SVG cells under the above-mentioned treatment condi-tions. There was a significant 3 fold increase in TUNEL positivecells upon treatment with supernatant from SVG treated withIL-18 and IL-1β individually as compared to supernatant fromcontrol SVG cells ( pb0.05). The combined cytokine treatmentalso yielded 3.7 folds more apoptotic cells than supernatantfrom control SVG ( pb0.05). However the difference inapoptotic population when cultured in presence of supernatantfrom individual IL-18 and IL-1β treatment or combinedtreatment to SVG was not significant (Fig. 7, Panels B, E).This indicated that the two cytokines did not induce differentialbystander damage to the neurons and moreover synergisticeffect of cytokines in causing death was also not observed.

Fig. 7. IL-18 and IL-1β cause bystander damage to the neurons: TUNEL staining wapresence of conditioned media obtained from CHME3 and SVG cells treated withmerged with DAPI (blue), and the double positive cells (pink) from 4 different conditIL-1β treated CHME3 cells. Panel B represents the TUNEL positive cells after treaPanel C represents the TUNEL positive cells in mock-infected, JEV infected and JEVtreated SVG and CHME3 cells. D, E and F. The graph represents the percentage of Twith the mentioned conditioned media from human microglial CHME3 and astrocyticeach condition (⁎ significant change from control, pb0.05; # significant change frofigure legend, the reader is referred to the web version of this article.)

However an interesting observation made was that microgliaproduced cytotoxins post IL-18 and IL-1β treatment was morepotent in inducing neuronal death via apoptosis than astrocytesunder similar treatment conditions.

To further investigate whether the bystander damage by theIL-1 family of cytokines contribute to further neuronal loss,SK-N-SH cells, after JEV infection, were grown in conditioned

s performed in human neuroblastoma SK-N-SH cell lines after culturing them inIL-18 and IL-1β. Panel A represents the TUNEL positive (red) apoptotic cellsions – supernatant from control, IL-18 treated, IL-1β treated and both IL-18 andtment with supernatant from SVG cells under the above-mentioned conditions.infected ones cultured in presence of conditioned media from IL-18 and IL-1β

UNEL positive cells as counted from 5 different fields in 3 different experimentsSVG cell lines respectively. Values represent mean±SEM from 3 experiments inm JEV infected, pb0.05). (For interpretation of the references to colour in this

70 S. Das et al. / Journal of Neuroimmunology 195 (2008) 60–72

media from IL-18 and IL-1β treated SVG and CHME3 cells(Fig. 7, Panel C). It was found that JEV induced massive neuronalloss (60% apoptotic cells) as part of the direct neuronal death(Fig. 7F) ( pb0.01). Interestingly, when JEV infected neuronswere grown in conditioned media from IL-18 and IL-1β treatedSVG cells, no significant increase in apoptotic death wasobserved. However, when these neurons were grown in condi-tioned media from IL-18 and IL-1β treated CHME3 cells, indeedrobust apoptotic death was observed (70% TUNEL positive cells)( pb0.05). Thus it clearly shown that both direct neuronal killingalong with the bystander death mediated by the cytokine activatedmicroglia causes a pronounced neurotoxicity in JEV infection.

3.6. Induction of apoptotic molecules in neurons as part ofbystander damage by IL-18 and IL-1β

The signature of apoptosis includes decreased expression ofBcl-2 and elevated active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) expression. The expression of theseapoptotic molecules was determined in SK-N-SH cell line aftergrowing them in presence of supernatant from CHME3 andSVG cells under IL-18 and IL-1β treatment conditions asmentioned before. In correlation with the TUNEL results, asignificant 1.7 fold increase in caspase 3 expression wasdetected when supernatant from individual IL-18 and IL-1βtreated CHME3 was used. The levels of cleaved PARP andBcl-2 also demonstrated significant 2 fold increase and 1.7fold decrease respectively following treatment with individualcytokine treated supernatant from microglial cells ( pb0.05)(Fig. 8A). However, together IL-18 and IL-1β treated super-natant from CHME3 cells did not exert any synergistic effectin induction of these various apoptotic markers. While thelevels of caspase 3 and cleaved PARP demonstrated significant1.9 fold and 2.3 fold increase, Bcl-2 levels also decreased to 1.5folds compared to control CHME3 supernatant treatment.

In another set of experiments it was noted that caspase 3 andcleaved PARP were upregulated by 1.75 folds and Bcl-2downregulated by 1.3 folds with supernatant from individualIL-18 and IL-1β treated SVG cells as compared to control SVGsupernatant ( pb0.05). However no synergistic effect withsupernatant from combined IL-18 and IL-1β treated SVG wasobserved (Fig. 8B).

4. Discussion

The present study was undertaken to better understand 1) thecontributions of IL-18 in the development of neuropathologyobserved in JE and 2) to differentiate the contribution of IL-18and IL-1β in glial activation and subsequent neurodegenerationfollowing JE. The results presented in this paper clearlydemonstrated that 1) infection with JEV upregulate the levelof IL-18 and IL-1β in brain of infected animal 2) microglia andastrocytes produce IL-18 and IL-1β, though in different extent,3) IL-18 and IL-1β differentially modulate microglia andastrocytes to release other pro-inflammatory cytokines andchemokines, and 4) recombinant IL-18 and IL-1β treatedhuman microglia and astrocytes produce mediators those are

capable in modulating neuronal survival, indicating a criticalrole of bystander mechanism of neuronal death in JE.

IL-18 was originally described as a factor for IFN-γproduction of T lymphocytes in the presence of IL-12. Recentstudies have demonstrated that IL-18 has various otherbiological activities, including proliferation of T lymphocytesand NK cells, stimulation of their cytotoxic activity andenhancement of a Th1-mediated immune response (Akira,2000; Dinarello, 1999; Kohno et al., 1997). On the other hand,IL-18 acts as a pro-inflammatory cytokine and increases theseverity of diseases with lethal endotoxaemia (Dinarello, 2000;Lauw et al., 1999; Netea et al., 2000). Our results also suggestthat JEV infection induces the level of IL-18, and bothastrocytes and microglia produce that. In contrast, there arereports indicating the antiviral activity of IL-18. Importantly,IL-18 elicits antiviral activity in the acute phase of infection. Inthe case of vaccinia virus infection, IL-18 is involved in varioushost defence mechanisms, including NK cells and CTLs(Tanaka-Kataoka et al., 1999). In fact, virus-induced IL-18and IFN-γ enhance Fas-ligand expression on NK cells (Tsutsuiet al., 1996) and Fas molecules on virus-infected cells(Takizawa et al., 1993).

The host response to infection is central to the effectivecontrol and ultimate clearance of invading pathogens orremoval of infected cells. The response to cerebral infectionwith JEV in mice is characterized at the pathological level bysignificant recruitment and extravasation of immunoinflamma-tory cells, predominantly macrophages, T cells, and neutrophilsto the sites of viral replication in the brain (Chaturvedi et al.,1979; Mathur et al., 1988; Mathur et al., 1992). The in-flammatory response observed within the CNS in viral en-cephalitis is partly mediated by chemokines, which are releasedby various cells of the CNS (Ishiguro et al., 1997). Here, inthis study we have found that both IL-18 and IL-1β are capableof inducing pro-inflammatory cytokines and chemokines fromhuman microglia and astroglia. These pro-inflammatory cyto-kines and chemokines again recruit peripheral cells to the siteof infection, and causes further damage. Neuroinflammationis thought to develop as a consequence of two processesthat follow sequentially. The first process is the activation ofmicroglia and resident perivascular/parenchymal macrophagesfollowed closely by the mobilization and recruitment of pe-ripheral inflammatory cells into the site of damage. The sec-ond involves the production of a number of pro-inflammatorymediators from activated microglia, that further increases thecompetence of the cerebral endothelium to recruit peripheralleukocytes, primarily polymorphonuclear leukocytes and mo-nocyte/macrophage, into the damaged brain (del Zoppo et al.,2000).

IL-18 and IL-1β share several similarities in their properties,including protein folding, synthesis as inactive precursors thatlack a signal peptide, and activation by caspase-1. Even theyact via related receptor complexes to induce similar signal-ling pathways (Fantuzzi and Dinarello, 1999; Fitzgerald andO'Neill, 2000). However, considerable differences in theexpression, regulation and actions of these two cytokinesexist. IL-1β is present only at very low levels in normal brain

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tissue, whereas IL-18 is expressed constitutively at high levelsin the brain (Culhane et al., 1998). IL-1β is upregulated within4h of focal ischaemia, but IL-18 is upregulated much later, attime points associated with infiltration of peripheral immunecells (Jander et al., 2002). Similarly, we also found that IL-18potently induces the release of various chemokines from bothastrocytes and microglia and perhaps to a greater extent than IL-1β. Further clarification of the mechanisms underlying theprocessing of IL-1β and IL-18 and also their signaling eventswould allow us to understand how these pro-inflammatorycytokines regulate the generation of neuroinflammatory attackfollowing any insult.

IL-1β has been implicated in various disease states of thebrain in activation of various inflammatory processes (Allanet al., 2005). IL-1β however does not directly induce neuronaldeath, but a bystander damage has been shown. Previous reportssuggest that conditioned media from IL-1β treated astrocytesare capable of causing neuronal apoptosis (Deshpande et al.,2005), however the mediators inducing this damage were notidentified. Here we have addressed how IL-1β treatedastrocytes and microglia can induce bystander neuronal deathvia the various pro-inflammatory cytokines and chemokines.Though neurons express IL-18 receptors, but the role of IL-18in neurodegeneration is not clearly indicated. IL-18, howeveractivates Fas-ligand expression on astrocytes and microglia,which subsequently leads to Fas-mediated neuronal apoptosisunder inflammatory conditions (Felderhoff-Mueser et al.,2005). Besides, the massive upheaval in levels of inflammatorymolecules induced by IL-18, detrimental effects on the neuronalsurvival are also exerted, as reported in this study.

Although the net effect of the pro-inflammatory mediators isto kill infectious organisms and infected cells as well as tostimulate the production of molecules that amplify the mountingresponse to damage, it is also evident that in a nonregeneratingorgan such as brain, a dysregulated innate immune responsewould be deleterious. Our findings suggest that both IL-18 andIL-1β have the ability to activate aspects of the microglial andastroglial response themselves. One possible mechanism forsuch a destructive cycle might be the result of a dysregulatedfeed-forward stimulation of microglia and astrocytes by pro-inflammatory cytokines. In JE, the tight regulation of glialactivation appears to be disturbed, resulting in an autotoxic loopof microglial and astroglial activation with dire consequences.Obviously, more research is required to determine the regulationand roles of IL-18 and IL-1β in glial activation and the possibleimplication in progressive neurodegeneration in JE.

Acknowledgements

This work was supported by grant no. BT/PR/5799/MED/14/698/2005 from the Department of Biotechnology to A.B. S.D.and M. K.M. is a recipient of Senior Research Fellowshipfrom University Grants Commission, Government of India.The authors thank Mr. Kanhaiya Lal Kumawat for excellenttechnical assistance. We thank Prof. Vijayalakshmi Ravin-dranath, Director NBRC for her continuous support andencouragement.

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