Interleukin-1-induced neurotoxicity is mediated by glia and requires caspase activation and free...

Interleukin-1-induced neurotoxicity is mediated by glia andrequires caspase activation and free radical release

Peter Thornton, Emmanuel Pinteaux, Rosemary M. Gibson1, Stuart M. Allan and Nancy J. Rothwell

Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester, UK

Abstract

Interleukin (IL)-1 expression is induced rapidly in response to

diverse CNS insults and is a key mediator of experimentally

induced neuronal injury. However, the mechanisms of IL-1-

induced neurotoxicity are unknown. The aim of the present

study was to examine the toxic effects of IL-1 on rat cortical cell

cultures. Treatment with IL-1b did not affect the viability of pure

cortical neurones. However, IL-1 treatment of cocultures of

neurones with glia or purified astrocytes induced caspase

activation resulting in neuronal death. Neuronal cell death

induced by IL-1 was prevented by pre-treatment with the IL-1

receptor antagonist, the broad spectrum caspase inhibitor Boc-

Asp-(OMe)-CH2F or the antioxidant a-tocopherol. The NMDA

receptor antagonist dizolcipine (MK-801) attenuated cell death

induced by low doses of IL-1b but the a-amino-3-hydroxy-5-

methylisoxazole-4-propionic acid receptor antagonist 2,3-di-

hydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) had

no effect. Inhibition of inducible nitric oxide synthase with N(x)-

nitro-L-arginine methyl ester had no effect on neuronal cell

death induced by IL-1b. Thus, IL-1 activates the IL-1 type 1

receptor in astrocytes to induce caspase-dependent neuronal

death, which is dependent on the release of free radicals and

may contribute to neuronal cell death in CNS diseases.

Keywords: glia, interleukin-1, neuroinflammation, neuronal

death.

J. Neurochem. (2006) 98, 258–266.

The pro-inflammatory cytokine interleukin (IL)-1 is a keymediator of inflammation and neuronal death in acute CNSinjuries, such as stroke and brain trauma (Allan et al. 2005),and has been implicated in chronic neurodegenerativediseases such as Alzheimer’s disease and HIV-1-associateddementia (Griffin et al. 1989; Royston et al. 1992; Corasanitiet al. 2001). The IL-1 family of cytokines is composed oftwo agonists, IL-1a and IL-1b, that bind to the IL-1 type 1receptor and IL-1 receptor accessory protein, leading tosignal transduction. IL-1 activity is inhibited by the naturallyoccurring IL-1 receptor antagonist (IL-1RA) (Sims 2002).

Interleukin-1 expression is increased rapidly within thebrain following acute brain insults in rodents (e.g. focal/global cerebral ischaemia or excitotoxin administration)(Allan et al. 2005). Post-mortem examination of stroke andbrain injury victims also reveals increased IL-1 levels inbrain tissue and CSF (see Rothwell 2003). Intracerebroven-tricular injection of IL-1b significantly exacerbates braindamage whereas IL-1RA is neuroprotective in models offocal cerebral ischaemia or excitotoxic damage in rodents(Relton and Rothwell 1992). Furthermore, mice lackingIL-1a and IL-1b have smaller infarct volumes (reduced by70%) compared with wild-type controls in response totransient cerebral ischaemia (Boutin et al. 2001).

In vitro, the effects of IL-1 on neuronal survival have beenconflicting. IL-1b dose-dependently increases NMDA-induced cytosolic calcium levels in primary hippocampalcultures (Viviani et al. 2003) whereas, in primary, cortical,neuronal cultures, pre-treatment with IL-1b for 24 h signi-ficantly reduces (up to 70%) neuronal death induced byexcitotoxins (Strijbos and Rothwell 1995).

As astrocytes can modulate neuronal survival (Chen andSwanson 2003), IL-1 may act via these cells. For example,IL-1b dose-dependently inhibits glutamate uptake by astro-cytes (Hu et al. 2000). IL-1b up-regulates the death protein

Received September 27, 2005; revised manuscript received February 17,2006; accepted February 20, 2006.Address correspondence and reprint requests to Nancy J. Rothwell,

Faculty of Life Sciences, Michael Smith Building, University ofManchester, Oxford Road, Manchester M13 9PT, UK.E-mail: [email protected] present address of Rosemary M. Gibson is Health & SafetyLaboratory, Harpur Hill, Buxton SK17 9JN, UK.Abbreviations used: Boc-D-FMK, Boc-Asp-(OMe)-CH2F; IL, inter-

leukin; IL-1RA, interleukin-1 receptor antagonist; LDH, lactate dehy-drogenase; MEM, minimum essential medium; MK-801, dizolcipine;NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline; NeuN,neuronal nuclei; VAD-FMK, Val-Ala-Asp-(OMe)-CH2F.

Journal of Neurochemistry, 2006, 98, 258–266 doi:10.1111/j.1471-4159.2006.03872.x

258 Journal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 98, 258–266� 2006 The Authors

FasL in astrocytes which may, in turn, mediate neuronalinjury (Ghorpade et al. 2003). Conditioned medium fromIL-1b-treated astrocytes can induce apoptosis of primaryneurones, although the role of potential mediators was notaddressed in this study (Deshpande et al. 2005). In addition,chlomethiazole, a neuroprotective agent, inhibits IL-1 actionson glia, implicating these cells in IL-1-induced neurodegen-eration (Simi et al. 2002). In cocultures of neurones andmicroglia, IL-1b exacerbates NMDA- and glutamate-inducedneuronal death (Ma et al. 2002).

However, the mechanism by which IL-1 induces neuronalinjury is not known. The aim of the present study wastherefore to characterize the effects of IL-1 on cortical cellsfrom rat brain and to elucidate the mechanism of IL-1-induced neurotoxicity. The role of excitotoxicity, apoptosisand free radicals in neuronal cell death is well knownand their contribution to IL-1-induced neurotoxicity wasaddressed using pharmacological inhibitors.

Materials and methods

Materials

Recombinant rat IL-1b [National Institute for Biological Standards

and Control (NIBSC), Potters Bar, UK] was diluted in vehicle [0.9%

saline/0.1% (w/v) low-endotoxin bovine serum albumin]. This

IL-1b has a bioactivity of 317 IU/lg and an endotoxin content of

0.3 fg/10 ng. Recombinant human IL-1RAwas produced by Amgen

(Thousand Oaks, CA, USA). Dizolcipine (MK-801), NMDA

and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid were

obtained from Tocris (Bristol, UK). Boc-Asp-(OMe)-CH2F (Boc-D-

FMK) and FITC-conjugated Val-Ala-Asp-(OMe)-CH2F (VAD-

FMK) were purchased from Merck Biosciences (Nottingham,

UK). Mouse anti-neuronal nuclei (NeuN) antibody was supplied

by Chemicon (Temecula, CA, USA) and A2B5 antibody was a gift

from Christine Pigott and Peter Andrews (Sheffield University, UK).

Fluorescently conjugated secondary antibodies and Vectashield

mounting medium with 4¢,6-diamidino-2-phenylindole dihydrochlo-

ride were obtained from Vector Laboratories (Burlingame, CA,

USA). Cell culture reagents and Texas Red-conjugated isolectin IB4

were purchased from Gibco (Paisley, UK), and all other reagents

were from Sigma (Poole, UK) unless stated otherwise.

Cell culture

Glial–neuronal cocultures were prepared from embryonic day 18

Sprague-Dawley rat embryos. Briefly, the cortices were isolated and

dissociated in minimum essential medium (MEM) plus Earle’s salts

(Hyclone, Logan, UT, USA), 2 mM glutamine, 10% fetal bovine

serum Gold (PAA, Somerset, UK), B27 supplement with antioxi-

dants, 100 U/mL penicillin and 100 lg/mL streptomycin. The cell

suspension was collected by centrifugation for 10 min at 250 g.Sterile plastic 48-well culture plates or 13-mm glass coverslips in

24-well plates (Corning, Hemel Hempstead, UK) were pre-coated

with poly-D-lysine and viable cells were seeded at a density of

2.5 · 105 cells/cm2. After 1 day in vitro, the medium was

exchanged for MEM without B27 supplement. Half media changes

were performed every 3 days with MEM without fetal bovine serum.

Pure cortical neuronal cultures were prepared as above with the

addition of 50 lM 5¢-fluoro-2-deoxyuridine and 50 lM uridine to

prevent glial proliferation. Neuronal cultures were maintained in

50 lM 5¢-fluoro-2-deoxyuridine and 50 lM uridine throughout all

procedures.

To obtain astrocyte–neuronal cocultures, mixed glial cultures were

prepared from the cortices of 0–3-day-old Sprague-Dawley rats as

described previously (McCarthy and de Vellis 1980; Pinteaux et al.2002). Briefly, cells from the cortices were dissociated in MEM (with

10% fetal bovine serum) and collected by centrifugation for 10 min at

250 g. Glia were seeded onto sterile plastic culture plates (Corning) ata density of two cortical hemispheres/75 cm2. The media were

replaced every 3 days. Monolayers of pure astrocytes were prepared

from the mixed glial cultures as described previously (Pinteaux et al.2002). Astrocyte–neuronal cocultures were prepared by seeding

neurones with 50 lM 5¢-fluoro-2-deoxyuridine and 50 lM uridine (as

above) onto the monolayer of astrocytes (after 7–10 days in vitro).Astrocyte–neuronal cocultures were maintained in 50 lM 5¢-fluoro-2-deoxyuridine and 50 lM uridine throughout all procedures.

Cultures were grown at 37�C in a 5% CO2 humidified

atmosphere and were used when neurones had been cultured for

12–13 days in vitro. Experiments conformed to the Animals

(Scientific Procedures) Act, UK 1986.

Treatment of cell cultures

Cultures were treated with vehicle or IL-1b (0.01–100 ng/mL ¼0.00317–31.7 IU/mL) for up to 72 h (in duplicate wells). Some

cultures were pre-treated for 2 min with IL-1RA (1–100 lg/mL) or

for 30 min with Boc-D-FMK (100 lM), a-tocopherol (10 lM),MK-801 (10 lM), N(x)-nitro-L-arginine-methylester (100 lM) or

2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX)

(50 lM). Compounds were used at concentrations above their

respective in vitro ED50 values. NMDA (500 lM) or a-amino-3-

hydroxy-5-methylisoxazole-4-propionic acid (100 lM) served as

positive controls that induced maximal neuronal cell death. Heat-

inactivated IL-1b had been heated at 95�C for 30 min. Quantifica-

tion of cell death was carried out 48–72 h after treatment.

Quantification of cell death

Basal cell death (following vehicle treatment) was measured by

calculating the percentage of trypan blue-stained neurones in 10

fields of view per culture, from three independent cultures (images

not shown). Basal cell death was 8 ± 1%, 13 ± 1% and 5 ± 1% in

glial–neuronal cocultures, pure neuronal cultures and astrocyte–

neuronal cocultures, respectively (data not shown).

The CytoTox 96� Non-Radioactive Cytotoxicity Assay (Pro-

mega, Southampton, UK) was used to measure lactate dehydrogen-

ase (LDH) release into the medium of treated cultures. Following

IL-1b treatment, 50 lL of supernatant was collected from duplicate

wells and the assay performed as stated in the manufacturer’s

instructions. Neuronal cell death was expressed as a fold increase

over basal LDH release (in response to vehicle).

Immunostaining

Cells were seeded on 13-mm glass coverslips in 24-well plates.

Astrocytes and neurones were fixed in acetone/methanol (1 : 1) and

blocked in 3% bovine serum albumin in phosphate-buffered saline.

Astrocytes were detected with rabbit anti-glial fibrillary acidic

IL-1-induced neurotoxicity 259

� 2006 The AuthorsJournal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 98, 258–266

protein antibody (1 : 2000, 1 h at 23�C) and FITC-conjugated goat

anti-rabbit antibody (1 : 200, 1 h at 23�C) or mouse Cy3-conjugated

anti-glial fibrillary acidic protein antibody (1 : 2000, 1 h at

23�C). Neurones were detected with mouse anti-NeuN antibody

(1 : 200, 1 h at 23�C) and Texas Red-conjugated anti-mouse

antibody (1 : 200, 1 h at 23�C). Antibodies were diluted in 0.1%

bovine serum albumin/phosphate-buffered saline unless stated

otherwise.

Microglia were detected with FITC- or Texas Red-conjugated

anti-Griffonia simplicifolia lectin 1 isolectin B4 (1 : 200 in MEM,

30 min, 37�C) and oligodendrocyte precursor cells (O2A progenitor

cells) with A2B5 antibody (1 : 2 in MEM, 30 min, 37�C). Microglia

and O2A progenitors were then fixed in 4% paraformaldehyde in

phosphate-buffered saline for 15 min and O2A progenitors detected

with anti-mouse Texas Red-conjugated antibody.

Vectashield (with 4¢,6-diamidino-2-phenylindole dihydrochlo-

ride) was used to mount all preparations and cells were examined

using a BX51 fluorescence microscope (Olympus, Middlesex, UK)

with METAVUE imaging software (Nikon, Kingston upon Thames,

UK). The proportion of individual cell types was calculated as a

percentage of total cell number (4¢,6-diamidino-2-phenylindole

dihydrochloride-stained) in five 40· fields (approximately 50 cells

per field) from three independent cultures.

To confirm neuronal caspase activation in glial–neuronal cocul-

tures in response to IL-1b, cells were costained with anti-NeuN

antibody and FITC-conjugated VAD-FMK (an in situ marker of

activated caspases; diluted 1 : 300 in MEM); 20 min later cells were

washed in phosphate-buffered saline and processed for NeuN

staining (as above). Some cultures received a pre-treatment of Boc-

D-FMK (100 lM) to block caspase activation and serve as a

negative control. The number of caspase-positive neurones was

determined by scoring the percentage of cells labelled with FITC-

VAD-FMK in 10 fields of view per culture (approximately 20

neurones per field) from three independent cultures. Some cells were

costained with FITC-VAD-FMK and mouse anti-glial fibrillary

acidic protein antibody or isolectin IB4 to assess the extent of

caspase activation in astrocytes and microglia, respectively.

Statistical analysis

Data were expressed as mean (± SEM) from at least three

independent cultures and analysed using ANOVA with a Tukey

post-hoc test. p < 0.05 was considered to be statistically significant.

Results

Characterization of cortical cultures

Characterization of the cultures as assessed by immunocyt-ochemistry is shown in Fig. 1. Glial–neuronal coculturesconsisted of 52 ± 3% neurones, 35 ± 4% astrocytes, 9 ± 1%microglia and 5 ± 1% O2A progenitor cells. Pure neuronal

Fig. 1 Characterization of cortical cell cultures. Immunofluorescent

staining for neuronal NeuN (i–iii), astrocytic glial fibrillary acidic protein

(GFAP) (iv–vi), microglial IB4 (vii–ix) and O2A progenitor cell A2B5 (x–

xii) (with 4¢,6-diamidino-2-phenylindole dihydrochloride costain). Scale

bar, 20 lm. Five fields of view were analysed per culture and images

are representative of three independent cultures.

260 P. Thornton et al.

Journal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 98, 258–266� 2006 The Authors

cultures consisted of 98 ± 0% neurones and 2 ± 1% astro-cytes. Astrocyte–neuronal cocultures were composed of57 ± 3% neurones and 43 ± 3% astrocytes (< 1% microglia).

Interleukin-1b-induced neuronal cell death

Interleukin-1b treatment for 48 h did not affect the viabilityof pure neuronal cultures (Fig. 2a) or mixed glial cultures(data not shown) as assessed by the release of LDH into theculture medium. In contrast, treatment with NMDA for 48 hincreased neuronal cell death (Fig. 2a). In glial–neuronalcocultures, treatment with IL-1b (or IL-1a, data not shown)for 48 h dose-dependently increased neuronal cell death(Fig. 2b). This effect was also observed in astrocyte–neuronal cocultures (lacking microglia and O2A progenitors)(Fig. 2c). Significant neuronal cell death in glial–neuronalcocultures was observed at low concentrations (0.1 ng/mL)of IL-1b (Fig. 2b). A maximal 3.5-fold increase in neuronalcell death was observed in response to 100 ng/mL IL-1b(Fig. 2b). Pre-treatment of glial–neuronal or astrocyte–neur-onal cocultures with IL-1RA (1 lg/mL) prevented IL-1b

(1 ng/mL)-induced neuronal cell death (89 or 60% inhibitionin each culture, respectively) (Figs 2b and c). Similarly, pre-treatment with IL-1RA (100 lg/mL) caused a 68% inhibitionof neuronal cell death evoked by 100 ng/mL IL-1b in glial–neuronal cocultures (data not shown). Heat inactivation ofIL-1b completely abolished neurotoxicity (Figs 2b and c).Neuronal cell death peaked after 48 h of IL-1b (1 ng/mL)treatment (Fig. 2d).

To determine whether neuronal apoptosis was responsiblefor the subsequent LDH release in glial–neuronal cocultures,apoptotic cells were labelled with FITC-conjugated VAD-FMK (a broad spectrum caspase inhibitor that binds toactivated caspases) and NeuN (a neuronal specific marker).Treatment of glial–neuronal cocultures with 1 ng/mL IL-1b(or 100 ng/mL, data not shown) for 48 h induced shrinkageof neurones as assessed by light microscopy and immuno-staining with anti-NeuN antibody (Fig. 3a, v and vi). Thiscolocalized with areas of caspase activation (Fig. 3a, vii–viii). Neuronal shrinkage and caspase activation wereinhibited by pre-treatment with IL-1RA (1 lg/mL) (Fig. 3a,

(a) (b)

(c) (d)

Fig. 2 Cell death in cortical cell cultures in response to interleukin (IL)-

1b treatment for 48 h (a–c) or 72 h (d) as measured by LDH release.

IL-1b did not affect cell viability of pure neuronal cultures (a). IL-1b dose-

dependently induced neuronal cell death in glial–neuronal cocultures.

Cell death was inhibited by IL-1 receptor antagonist (IL-1RA) and heat

inactivation of IL-1b (b). A similar response was seen in astrocyte–

neuronal cocultures (c). LDH release was quantified 24, 48 and 72 h

after treatment with IL-1b (d). NMDA (500 lM) served as a positive

control that induced maximal neuronal cell death. Data are expressed

as fold increase over basal cell death and are from at least three

independent cultures. (b) #p < 0.01 vs. vehicle, *p < 0.001 vs.

vehicle, **p < 0.001 vs. IL-1 (1 ng/mL); (c) *p < 0.05 vs. vehicle,

#p < 0.001 vs. vehicle; (d) *p < 0.0001 vs. vehicle.



(a)

(b) (c)

Fig. 3 Interleukin (IL)-1b induces neuronal caspase activation in

glial–neuronal cocultures. Cells were treated with vehicle (a, i–iv; b,

i–iii and vii–ix), IL-1b (1 ng/mL) (a, v–viii; b, iv–vi and x–xii), IL-1b

(1 ng/mL) with IL-1 receptor antagonist (IL-1RA) (1 lg/mL) pre-

treatment (a, ix–xii), NMDA (500 lM) (a, xiii–xvi) or IL-1b (1 ng/mL)

with Boc-D-FMK (100 lM) pre-treatment (a, xvii–xx). At 48 h after

treatment, cells were stained with FITC-conjugated VAD-FMK (a, iii,

vii, xi, xv and xix; b, ii, v, viii and xi), the neuronal marker NeuN (a,

ii, vi, x, xiv and xviii), the astrocyte marker glial fibrillary acidic

protein (GFAP) (b, i and iv) or the microglial marker IB4 (b, vii and

x) and visualized using fluorescent microscopy. Bright field images

were also captured (a, i, v, ix, xiii and xvii) (a and b). The per-

centage of caspase-positive neurones was calculated by scoring the

number of VAD-FMK-labelled cells following treatment with IL-1b for

48 h (c). Scale bar, 20 lm. Data are from three independent cul-

tures. *p < 0.001 vs. vehicle.



x–xii). Treatment of cells with the broad spectrum caspaseinhibitor Boc-D-FMK (prior to IL-1b addition) also blockedcaspase activation (Fig. 3a, xix). NMDA (500 lM) treatmentcaused marked neuronal shrinkage with some caspaseactivation (Fig. 3a, xiv–xvi). In cells treated with 1 ng/mLIL-1b (or 100 ng/mL, data not shown), FITC-VAD-FMKstaining did not localize with astrocytes or microglia, asassessed by immunostaining with anti-glial fibrillary acidicprotein antibody or isolectin IB4, respectively (Fig. 3b). Inresponse to treatment for 48 h, the percentage of caspase-positive neurones increased from 2 ± 1% in vehicle-treated

cells to 71 ± 5% in IL-1b-treated cells (1 ng/mL) and92 ± 2% in cells treated with the higher concentration(100 ng/mL) of IL-1b (Fig. 3c).

Effects of Boc-D-FMK, a-tocopherol, N(x)-nitro-L-

arginine-methylester, MK-801 and NBQX on interleukin-

1b-induced neuronal cell death

Pre-treatment with the pan-caspase inhibitor Boc-D-FMK(100 lM) caused a 70 or 87% inhibition of death induced by1 or 100 ng/mL IL-1b, respectively (Fig. 4a). The freeradical scavenger a-tocopherol (10 lM) inhibited death

(a) (b)

(d)(c)

(e)

Fig. 4 Effects of antagonists on interleukin (IL)-1b-induced neuronal

cell death in glial–neuronal cocultures. Cell death was measured by

quantifying LDH release. Cultures were pre-treated with Boc-D-FMK

(100 lM), a-tocopherol (10 lM), N(x)-nitro-L-arginine-methylester

(L-NAME) (100 lM), MK-801 (10 lM) or NBQX (50 lM) for 30 min

followed by IL-1b (1 or 100 ng/mL) for 48 h. Data are expressed as

fold increase over basal cell death and are from at least three

independent cultures. (a–e) +p < 0.001 vs. vehicle; (e) *p < 0.01 vs.

vehicle; (a and b) *p < 0.001 vs. IL-1 (1 ng/mL); (a) #p < 0.01 vs.

IL-1 (100 ng/mL); (b) **p < 0.001 vs. IL-1 (100 ng/mL); (d) *p < 0.05

vs. IL-1 (1 ng/mL). AMPA, a-amino-3-hydroxy-5-methylisoxazole-

4-propionic acid.



induced by 1 ng/mL IL-1b by 75% and completely abolisheddeath induced by 100 ng/mL IL-1b (Fig. 4b). Inhibition ofinducible nitric oxide synthase with N(x)-nitro-L-arginine-methylester (100 lM) had no effect on IL-1b-inducedneuronal cell death (Fig. 4c). The NMDA receptor antagonistMK-801 (10 lM) caused 83% inhibition of neuronal celldeath induced by 1 ng/mL IL-1b but did not affect deathinduced by 100 ng/mL IL-1b (Fig. 4d). The a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptorantagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)qui-noxaline (NBQX) (50 lM) did not affect neuronal cell deathinduced by 1 or 100 ng/mL IL-1b (Fig. 4e). Treatment withBoc-D-FMK, a-tocopherol, N(x)-nitro-L-arginine-methyl-ester, MK-801 or NBQX alone had no effect on basalneuronal cell death (Figs 4a–e).

Discussion

Previous studies have demonstrated neurotoxic actions ofIL-1 in in vitro and in vivo models of excitotoxicity,inflammation and energy deprivation (Allan et al. 2005;Fogal et al. 2005; Hailer et al. 2005). The aim of this studywas to investigate the mechanisms of IL-1-induced neuro-toxicity. We demonstrate that IL-1b (0.1–100 ng/mL)induced significant neuronal death when applied to glial–neuronal cocultures. Glia are critical for IL-1b-inducedneuronal cell death as IL-1b had no effect on the viabilityof pure neuronal cultures. This study demonstrates that theeffects of IL-1b are mediated by astrocytes, as similarneuronal death was observed in astrocyte–neuronal cocul-tures. Neurotoxicity was blocked by IL-1RA or heatinactivation of IL-1b. As caspase activation colocalized withneuronal staining, we believe that IL-1b-induced LDHrelease in glial–neuronal cocultures is neurone specific anda result of secondary necrosis occurring after apoptosis.

Previously, IL-1b was shown to exacerbate neuronal celldeath in mouse glial–neuronal cocultures in response to avariety of insults (Fogal et al. 2005). In contrast, our study isthe first to demonstrate that IL-1b induces significantneuronal death when administered to naive glial–neuronalcocultures, suggesting that the response is not triggered byinjury. Neuronal cell death was maximal 48 h after treatmentand this delayed response suggests that IL-1 induces asecondary neurotoxic factor(s). Our results indicate thatastrocytes are the primary mediator of IL-1-induced neuronalcell death. This is consistent with data demonstrating theabsence of IL-1 actions at the IL-1 type 1 receptor inmicroglia (Pinteaux et al. 2002). Astrocytes are an importantsource of pro-inflammatory molecules, some of which can beneurotoxic (John et al. 2005). In our cultures, the astrocytesand neurones are in direct physical contact. Thus, it ispossible that a juxtacrine mechanism of IL-1-inducedneuronal death exists. In contrast, the neuronal cell deathmay be triggered by a factor that is secreted from astrocytes.

A recent study supports the hypothesis that unidentifiedfactors released from IL-1-treated astrocytes are responsiblefor the death of human fetal neurones (Deshpande et al.2005).

To identify the glial factor(s) induced by astrocytes inresponse to IL-1 that triggers neuronal death, we haveinvestigated the involvement of mediators known to beneurotoxic using specific inhibitors. The inducible nitricoxide synthase inhibitor N(x)-nitro-L-arginine-methylesterdid not affect the death induced by IL-1b. This is inagreement with a previous study demonstrating that indu-cible nitric oxide synthase inhibition does not affect IL-1b/interferon-c-induced neuronal cell death (Downen et al.1999). However, the antioxidant a-tocopherol completelyprevented IL-1b-induced neuronal cell death. The involve-ment of free radicals in IL-1-induced responses is known, inparticular the activation of NF-jB (Schreck et al. 1991).Thus, a-tocopherol may repress IL-1b-induced signalling.

The broad spectrum caspase inhibitor Boc-D-FMK com-pletely abolished neuronal cell death. In situ labelling ofneurones with FITC-conjugated VAD-FMK (following treat-ment with IL-1b) demonstrated caspase-positive neurones(and not astrocytes or microglia) with cellular shrinkage,characteristic of apoptosis. Further studies are in progress todetermine whether caspase activation is induced via theextrinsic or intrinsic mitochondrial pathway of apoptosis(Li et al. 1997; Lipton 1999).

We found that NMDA receptor antagonist MK-801inhibited neuronal cell death induced by low doses ofIL-1b (1 ng/mL), which suggests that IL-1 may induce therelease of an NMDA receptor agonist, such as glutamate,that triggers neuronal death. However, the a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antag-onist NBQX did not modify IL-1b-induced death, suggestinga non-glutamatergic mechanism of death. IL-1 may induce afactor that modulates NMDA receptor currents, such astissue plasminogen activator. IL-1 is known to induce therelease of tissue plasminogen activator from glia and, at highconcentrations, tissue plasminogen activator becomes neu-rotoxic by increasing NMDA receptor currents (Flavin et al.2000). Interestingly, when IL-1b was increased to 100 ng/mL, MK-801 did not affect neuronal cell death. At thishigher concentration, it appears that IL-1 may utilize adifferent pathway to induce neuronal cell death. It is possiblethat a parallel mechanism independent of NMDA receptorcurrents exists that contributes to IL-1b-induced neuronalcell death.

The precise nature of the glial-derived neurotoxic factor(s)induced by IL-1b is currently unknown. IL-1b-primed gliacan release IL-6, transforming growth factor-b and tumournecrosis factor-a (Benveniste et al. 1990; Chung andBenveniste 1990; da Cunha et al. 1993) which may beneurotoxic. However, many studies suggest that IL-6 andtransforming growth factor-b have neuroprotective qualities



(Meucci and Miller 1996; Allan 2002; Peng et al. 2005).Only when coapplied with interferon-c does IL-1 induceneurotoxic quantities of tumour necrosis factor-a (Chung andBenveniste 1990; Downen et al. 1999). Even under theseconditions, blocking tumour necrosis factor-a reduces neuro-nal cell death by less than half (Downen et al. 1999). Whenapplied at doses that activate signal transducer and activatorof transcription-3 (STAT3) (100 and 1000 U/mL), the pro-inflammatory cytokine interferon-c did not affect neuronalcell viability in our glial–neuronal coculture system (data notshown), suggesting that the effect observed in this study wasspecific to IL-1.

In summary, we show for the first time that IL-1 inducesneuronal cell death in naive glial–neuronal cocultures,through actions on astrocytes. The effect is dependent onthe activation of IL-1 type 1 receptor and the release of freeradicals with subsequent activation of caspases and neuronalcell death.

Acknowledgements

This work was supported by the Biotechnology and Biological

Sciences Research Council (UK), the Medical Research Council

(UK) and Biogen Idec (Cambridge, MA, USA).

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Interleukin-1-induced neurotoxicity is mediated by glia and requires caspase activation and free...

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