Interleukin6 deficiency reduces the brain inflammatory response and increases oxidative stress and...

INTERLEUKIN-6 DEFICIENCY REDUCES THE BRAIN INFLAMMATORY

RESPONSE AND INCREASES OXIDATIVE STRESS AND

NEURODEGENERATION AFTER KAINIC ACID-INDUCED SEIZURES

M. PENKOWA,a A. MOLINERO,b J. CARRASCOb and J. HIDALGOb*aDepartment of Medical Anatomy, The Panum Institute, University of Copenhagen, Copenhagen, Denmark

bDepartamento de BiologõÂa Celular, FisiologõÂa e InmunologõÂa, Unidad de FisiologõÂa Animal, Facultad de Ciencias,Universidad AutoÂnoma de Barcelona, Bellaterra, Barcelona, 08193, Spain

AbstractÐThe role of interleukin-6 in hippocampal tissue damage after injection with kainic acid, a rigid glutamateanalogue inducing epileptic seizures, has been studied by means of interleukin-6 null mice. At 35 mg/kg, kainic acidinduced convulsions in both control (75%) and interleukin-6 null (100%) mice, and caused a signi®cant mortality (62%)only in the latter mice, indicating that interleukin-6 de®ciency increased the susceptibility to kainic acid-induced braindamage. To compare the histopathological damage caused to the brain, control and interleukin-6 null mice were administered8.75 mg/kg kainic acid and were killed six days later. Morphological damage to the hippocampal ®eld CA1±CA3 was seenafter kainic acid treatment. Reactive astrogliosis and microgliosis were prominent in kainic acid-injected normal micehippocampus, and clear signs of increased oxidative stress were evident. Thus, the immunoreactivity for inducible nitric oxidesynthase, peroxynitrite-induced nitration of proteins and byproducts of fatty acid peroxidation were dramatically increased,as was that for metallothionein I 1 II, Mn-superoxide dismutase and Cu/Zn-superoxide dismutase. In accordance, a signi®-cant neuronal apoptosis was caused by kainic acid, as revealed by terminal deoxynucleotidyl transferase-mediateddeoxyuridine triphosphate-biotin nick end labeling and interleukin-1b converting enzyme/Caspase-1 stainings. In kainicacid-injected interleukin-6 null mice, reactive astrogliosis and microgliosis were reduced, while morphological hippocampaldamage, oxidative stress and apoptotic neuronal death were increased. Since metallothionein-I 1 II levels were lower, andthose of inducible nitric oxide synthase higher, these concomitant changes are likely to contribute to the observed increasedoxidative stress and neuronal death in the interleukin-6 null mice.

The present results demonstrate that interleukin-6 de®ciency increases neuronal injury and impairs the in¯ammatoryresponse after kainic acid-induced seizures. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Key words: hippocampus, gliosis, apoptosis, metallothionein, neurodegeneration.

Injury to the CNS elicits a characteristic in¯ammatoryresponse. Rami®ed resting microglial cells are activatedand transform into phagocytic cells with retractedprocesses and plump cell bodies or into amoeboid or

round brain macrophages, while astrocytes show hyper-plasia and hypertrophy.17,39,61 In the case of severe braindamage or direct trauma, the brain in¯ammatoryresponse is supplemented by blood-derived leucocytes,since monocytes, lymphocytes and neutrophil granulo-cytes enter the brain parenchyma.61 These responsesare regulated by a number of cytokines, among whichinterleukin-6 is crucial for both the glial activation andthe leucocyte recruitment.34,36,37,57

Kainic acid (KA) is a glutamate receptor agonist withexcitotoxic effects with the hippocampus being one of themost sensitive areas.40,68 During KA-induced seizures,hippocampal neurons degenerate followed by microgliaactivation and reactive astrogliosis.32,53,68 A number ofstudies demonstrated that interleukin-6 (IL-6) appearsto be involved in excitotoxicity-induced brain damage.Firstly, IL-6 production is increased during excitotoxi-city,3,14,19,30,31,50 and secondly, in vitro studies have shownthat IL-6 can signi®cantly protect against glutamate- andN-methyl-d-aspartate (NMDA)-induced excitotoxicityand cell death of cultured neurons.3,8,71,75 However, themechanisms underlying the protective role of IL-6 duringexcitotoxicity in vivo have to be precisely elucidated.

In¯ammatory response in IL-6-de®cient mice 805

805

Neuroscience Vol. 102, No. 4, pp. 805±818, 2001q 2001 IBRO. Published by Elsevier Science Ltd

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*Corresponding author. Fax: 134-93-581-23-90.E-mail address: [email protected] (J. Hidalgo).Abbreviations: AMCA, aminomethylcalmarin; ANOVA, analysis of

variance; BBB, blood±brain barrier; CD, cluster of differentiation;Cu, Zn-SOD, Cu, Zn-superoxide dismutase; DAB, 3,3 0-diamino-benzidine-tetrahydrochloride; DG, dentate gyrus; FITC, ¯uores-cein isothiocyanate; GFAP, glial ®brillary acidic protein; GM±CSF, granulocyte±macrophage colony stimulating factor; HE,hematoxylin-eosin; HRP, horse radish peroxidase; ICE, interleu-kin-1b converting enzyme; Ig, immunoglobulin; IL-6, interleukin-6; INOS, inducible nitric oxide synthetase; KA, kainic acid; KO,knock-out; MDA, malondialdehyde; MMP, matrix metalloprotei-nase; Mn-SOD, Mn-superoxide dismutase; MOMA-1, monocyte-derived macrophage marker-1; MT, metallothionein; NITT, proteintyrosine nitration; NMDA, N-methyl-d-aspartate; NSE, neuron-speci®c enolase; ROS, reactive oxygen species; SS, single-stranded; StreptABComplex, streptavidin±biotin-complex; TBS,Tris-buffered saline; TNF-a, tumor necrosis factor-a; TUNEL,terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuri-dine triphosphate (dUTP)-digoxigenin nick end labeling; TXRD,Texas Red.

Neuronal damage and cell death during excitotoxicityand seizures are associated with generation of reactiveoxygen species (ROS),12,13,65,69 which initiate varioussignaling cascades leading to neuronal degenerationand apoptosis.12,25,69 Therefore, one of the ways inwhich IL-6 could exert its neuroprotective effects couldbe by decreasing the excitotoxicity-induced oxidativestress. Metallothionein I 1 II (MT-I 1 II) and Cu/Zn-and Mn-superoxide dismutase (Cu/Zn- and Mn-SOD)are important antioxidant proteins, which protect againstcellular damage from ROS produced during pathologicalconditions,5,13,29,33,42,43,46,59,62,66 whereby MT-I 1 II, Cu/Zn-SOD and Mn-SOD can prevent neuronal damageand cell death.13,35,43,44,54,60,62,64 Interestingly, IL-6 is amajor regulator in vivo of MT-I 1 II.9,10,15,27,28,45,56,57 Inthe present report, we have studied the putative role ofIL-6 on KA-induced hippocampal damage by using IL-6null mice.36

EXPERIMENTAL PROCEDURES

Experiments

IL-6 null knock-out (KO) mice36 were kindly provided by DrHorst Bluethmann (CNS Department, Pharma Research GeneTechnologies, F. Hoffmann-La Roche AG, CH-4070, Basel,Switzerland).

In a ®rst experiment, adult normal (1295 v, n� 8) and IL-6 KO(n� 8) mice were injected i.p. with 35 mg/kg KA, which is aglutamate receptor agonist with excitotoxic effects.40 The numberof mice showing seizures as well as the mortality were recorded.Because of the high mortality of the IL-6 KO mice, no histopatho-logical analysis of these brains was carried out.

In a second experiment, normal and IL-6 KO mice wereinjected i.p. with 8.75 mg/kg KA for histopathological analysis.Other normal and IL-6 KO mice were administered saline (0.9%NaCl in distilled water) i.p. and served as controls for the KA-injected mice.

The mice were housed in cages at the Animal Department ofthe Autonomous University of Barcelona under constant tem-perature and had free access to food and water. All experimentswere carried out in a humane manner and were approved by theproper Ethical Committee. All efforts were made to minimizeanimal suffering, to reduce the number of animals used, and toutilize alternatives to in vivo techniques, if available.

Tissue processing

Normal and IL-6 KO mice were killed at six days after KA orsaline injection. Mice were deeply anesthetized with 10 mg/100 gBrietal (methohexital 10 mg/ml, Eli Lilly) and were transcar-dially perfused with 0.9% saline with 0.3% heparin (15,000 IU/l)for 1 min followed by Zamboni's ®xative for 8±10 min, pH 7.4.Zamboni's ®xative consists of buffered 4% formaldehyde added15% picric acid solution [1.2% (saturated) aqueous picric acid].Formaldehyde was prepared shortly before use by alkaline hydro-lysis of paraformaldehyde.

Afterwards, all the brains were ®xed by immersion in Zamboni's®xative for 4 h, pH 7.4, at room temperature. Brains were dehy-drated according to standard procedures, embedded in paraf®n,and cut in serial, coronal 3 mm thick sections to be used forHematoxylin-Eosin (HE) and Toluidine Blue stainings, lectinhistochemistry, immunohistochemistry, immuno¯uorescent histo-chemistry and in situ detection of DNA fragmentation/terminaldeoxynucleotidyl transferase (TdT)-mediated deoxyuridinetriphosphate (dUTP)-digoxigenin nick end labeling (TUNEL)labeling. Sections were afterwards rehydrated and incubated in1.5% H2O2 in Tris-buffered saline (TBS)/Nonidet (TBS: 0.05 MTris, pH 7.4, 0.15 M NaCl) for 15 min at room temperature to

quench endogenous peroxidase. Saline-injected and KA-injectednormal and IL-6 KO mice were processed in parallel.

Sections were boiled in citrate buffer, pH 9.1 or pH 6.0, for10 min in a microwave oven for heat-induced antigen retrieval.After cooling down to room temperature, the sections were pre-incubated with 10% normal goat or donkey serum in TBS/Nonidetfor 30 min at room temperature to block non-speci®c binding.Those sections prepared for incubation with monoclonalmouse-derived antibodies were also incubated with BlockingSolutions A 1 B from HistoMouse-SP Kit (Zymed Lab., USA,code 95-9544), in order to quench endogenous mouse IgG.

Histochemistry

HE and Toluidine Blue stainings were done according tostandard procedures. In addition, the sections were incubatedovernight at 48C with biotinylated tomato lectin from Lycopersi-con esculentum (Sigma, USA, code L9389), which was diluted1:500 in 10% goat serum and used as a marker for all cells of themyelo-monocytic cell lineage, such as rami®ed and plump micro-glial cells and macrophages. Labeling by lectin was veri®ed usingstreptavidin±biotin-peroxidase complex (StreptABComplex/HRP,Dakopatts, DK, code K377) prepared at manufacturer's recom-mended dilution and incubated for 30 min at room temperature.The immunoreaction was visualized using 0.015% H2O2 in 3,3 0-diaminobenzidine (DAB)/TBS for 10 min at room temperature.

Immunohistochemistry

Sections were incubated overnight at 48C with one of thefollowing primary antibodies: monoclonal rat anti-mouse IL-6,diluted 1:10 (Harlan Sera Lab, UK, code MAS 584); polyclonalrabbit anti-human neuronal speci®c enolase (NSE) (as a neuronalmarker) diluted 1:1000 (Dakopatts, DK, code A589); polyclonalrabbit anti-cow glial ®brillary acidic protein (GFAP; as a markerfor astrocytes) diluted 1:250 (Dakopatts, DK, code Z 334); mono-clonal rat anti-mouse MOMA-1 (as a monocyte-derived macro-phage marker) diluted 1:20 (Hybridomus, DK, code HD-212-85-OMA); polyclonal rabbit anti-human albumin (as a marker forexudation of plasma proteins through the blood±brain barrier;BBB) diluted 1:5000 (Dakopatts, DK, code A 0001); monoclonalmouse anti-rat endothelial barrier antigen/blood±brain barrierantigen (BBB antigen) (marking an endothelial protein presentonly in areas with an intact BBB) diluted 1:100 (Af®nity, UK,code BA1116); polyclonal rabbit anti-rat MT-I 1 II;20,21 mono-clonal mouse anti-bovine Cu/Zn-SOD 1:50 (Biogenesis, UK,code 8474-9702); polyclonal sheep anti-bovine Mn-SOD 1:50(Biogenesis, UK, code 8474-9550); polyclonal rabbit anti-malondialdehyde (MDA; a marker for oxidative stress) diluted1:100 (Alpha Diagnostic Int., USA, code MDA 11-S); polyclonalrabbit anti-nitrotyrosine (NITT; a marker for oxidative stress)diluted 1:100 (Alpha Diagnostic Int., USA, code NITT 12-A);polyclonal rabbit anti-mouse inducible nitric-oxide synthase(iNOS) 1:100 (Biomol Res. Lab., USA, code SA200); polyclonalrabbit anti-mouse interleukin-1b converting enzyme (ICE)/Caspase-1 1:100 (Santa Cruz, USA, code sc-1218R); polyclonalrabbit anti-human caspase-3 1:50 (Santa Cruz, USA, code sc-7148); polyclonal rabbit anti-human caspase-9 1:50 (SantaCruz, USA, code sc-8355); polyclonal rabbit anti-human cyto-chrome-c 1:50 (Santa Cruz, USA, code sc-7159); monoclonalmouse anti-human tumor necrosis factor-a (TNF-a) receptor1:100 (Zymed, USA, code 330100); monoclonal mouse anti-calf single-stranded (SS) DNA (IgM) 1:100 (Alexis, USA, code804-192-R200).

The primary antibodies were detected in rats and mice usingbiotinylated mouse anti-rabbit IgG or biotinylated species-speci®c sheep anti-rat IgG (as mentioned above) or biotinylatedgoat anti-mouse IgG 1:200 (Sigma, USA, code B8774), or goatanti-mouse-IgM 1:10 (Jackson, USA, code 115-065-020), for30 min at room temperature, followed by StreptABComplex/HRP (Dakopatts, DK, code K377) prepared at the manufacturer'srecommended dilutions for 30 min at room temperature. Theimmunoreaction was visualized using DAB as a chromogen (asmentioned above).

M. Penkowa et al.806

In order to evaluate the extent of non-speci®c binding in theimmunohistochemical experiments, control sections were incu-bated in: (i) the DAB medium alone (to examine endogenousperoxidase activity); (ii) the DAB medium and the ABComplexprepared at the manufacturer's recommended dilutions (to exam-ine endogenous biotin activity); and (iii) the absence of primaryantibody (to examine cross-reaction among IgGs of the differentspecies). Results were considered only if these controls werenegative. Comparisons of sections which were either incubatedor not incubated with Blocking Solutions A 1 B from Histo-Mouse-SP Kit were also made.

In situ detection of DNA fragmentation (TUNEL)

TUNEL staining was performed according to the manufac-turer's protocol and after tissue processing as mentioned above.

Sections were incubated with 20 mg/ml proteinase K (Sigma,USA, code P2308) for 5 min to strip off nuclear proteins. TUNELwas accomplished using the Apoptag Plus, In Situ ApoptosisDetection Kit (Oncor, USA, Code S7101-KIT). After immersionin equilibration buffer for 10 min, sections were incubatedwith TdT and dUTP-digoxigenin in a humidi®ed chamber at378C for 1 h and then incubated in the stop/wash buffer at 378Cfor 30 min to stop the reaction. Afterwards, the sections wereincubated in anti-digoxigenin-peroxidase solution for 30 min.DAB was used as chromogen (as mentioned above), and thesections were counterstained with Methyl Green. Negative controlsections were treated similarly but incubated in the absence ofTdT enzyme, dUTP-digoxigenin or anti-digoxigenin antibody.Sections were compared with positive control slides (Oncor,USA, code S7115). Furthermore, morphological criteria for apop-tosis were evaluated too, since the TUNEL may stain necroticcells.

Immuno¯uorescence histochemistry and TUNEL

In order to determine which cells underwent apoptosis, doubleTUNEL staining and staining for lectin or GFAP or NSE wereperformed. Sections were incubated with ¯uorescein-linkedTUNEL (Oncor, USA, code S7110-KIT) according to the manu-facturer's protocol. Afterwards, sections were incubated withlectin linked with Texas Red (TXRD) 1:50 (Sigma, USA, codeL9139) or anti-GFAP or anti-NSE (as mentioned above). Theanti-GFAP and anti-NSE antibodies were detected by usinggoat anti-rabbit IgG linked with TXRD 1:50 (Jackson Immuno-Research Lab., USA, code 111-075-144).

Furthermore, triple immuno¯uorescence histochemistry wasperformed in order to better characterize apoptotic cells. Sectionswere incubated with ¯uorescein-linked TUNEL (as mentionedabove), and afterwards incubated overnight at 48C with rabbitanti-mouse ICE/Caspase-1 1:100 (Santa Cruz, USA, code sc-1218R) and goat anti-horse cytochrome-c 1:100 (Santa Cruz,USA, code sc-7159) simultaneously. The anti-ICE antibodieswere detected using donkey anti-rabbit IgG linked with TXRD1:40 (Jackson ImmunoResearch Lab., USA, code 711-075-152),and the anti-cytochrome-c antibodies were detected using donkeyanti-goat IgG linked with Aminomethylcoumarin (AMCA) 1:40(Jackson ImmunoResearch Lab., USA, code 705-155-147). Thesecondary antibodies were used simultaneously for 30 min atroom temperature.

The sections were embedded in 20 ml ¯uorescent mounting(Dakopatts, DK, code S3023) and kept in darkness at 48C. In

order to evaluate the extent of non-speci®c binding of the anti-sera, control sections were incubated in the absence of primaryantibody. Negative control sections of the TUNEL labeling wereincubated in the absence of TdT enzyme. Results were consideredonly if these controls were negative. Comparisons of sections,which were either incubated or not incubated with BlockingSolutions A 1 B from HistoMouse-SP Kit, were also made.Furthermore, morphological criteria for apoptosis were evaluatedtoo, since the TUNEL is known to stain necrotic as well as apop-totic cells.

For the simultaneous examination and recording of the twostains, a Zeiss Axioplan2 light microscope equipped with a tripleband (DAPI/FITC/TXRD) ®lter was used.

Statistical analysis

In addition to morphological analysis, cellular countings werecarried out in a 1 mm2 area of 3 mm thick sections of hippo-campal area CA3 of both hemispheres of saline-injected andKA-injected normal and IL-6 KO mice at day 6 after injectionfor statistical evaluation of the results. To this end, positivelystained cells, de®ned as cells with staining of the soma, or inthe case of TUNEL cells with nuclear staining, were counted inthe CA3 of hippocampus. Positively stained cells were countedfrom an area of CA3 of both hemispheres of saline-injected andKA-injected mice. Cell countings were performed in at least threemice per group.

Results were evaluated by two-way analysis of variance(ANOVA), with strain and KA injection as main factors. Whenthe interaction was signi®cant, it was interpreted to be the conse-quence of a speci®c effect of the IL-6 de®ciency during thein¯ammation. This was veri®ed by post-hoc Student's t-test.When only two groups were compared (i.e. IL-6 immunostain-ing), the Student's t-test was used. Frequency data were analysedwith the x2 test. The probability level was set at P , 0.05.

RESULTS

Susceptibility to kainic acid

Saline-injected mice of normal and IL-6 KO geno-types showed similar behavioral patterns. No spon-taneous convulsions were seen in these mice. Theadministration of 35 mg/kg KA produced convulsionsin both normal and IL-6 KO mice (Table 1), but thepercentage of mice seizing and the number of convul-sions were higher in the latter (P , 0.05). Moreover, ®veout of eight (62.5%) of the IL-6 KO mice died in thefollowing few hours after the KA administration, whilenone of the control mice died (P , 0.05). At 8.75 mg/kg,KA also produced convulsions, albeit more moderate,which seemed to be present in all the IL-6 KO micebut only in about half of the normal mice (not shown).None of the mice died at this dosage of KA.

General

Saline-injected normal and IL-6 KO mice showed


Table 1. Response of 129Sv and interleukin-6-de®cient mice to kainic acid administration in Experiment 1

Strain % Seizing % Animals with jumping Latency time (min) Number of convulsions Mortality (%)

129Sv 75 75 29.4^ 4.0 9.4^ 2.3 0IL-6KO 100 87.5 33.2^ 8.4 19.5^ 3.3* 62.5*

Animals were males of 6±7 month-old. Mice were injected intraperitoneally with KA at 35 mg/kg body weight. Latency time and number ofconvulsions are the mean^S.E.M., (129Sv n� 8; IL-6KO n� 8).*Signi®cantly greater than 129Sv mice (P , 0.05). Means are compared with t-test and frequency data are analyzed with x2 test.

comparable stainings for neuronal and glial structure byHE, Toluidine Blue, lectin, MDA, NITT, iNOS, NF,TUNEL, GFAP, MOMA-1, CD3, albumin, BBB antigen,MT-I 1 II, Cu/Zn-SOD and Mn-SOD (see Figs 1±7). AfterKA injection, hippocampal morphological damage was

observed and consisted of neuronal cell loss and pyknoticneurons in CA1, CA3 and dentate gyrus (DG) as veri®edby HE (Fig. 1A, C) and Toluidine Blue stainings. Hippo-campal morphological damage was higher in IL-6 KOmice than in normal mice at six days after KA (Fig. 1B, D).


Fig. 1. Hematoxylin±Eosin stainings of hippocampal ®eld CA3 of saline-injected mice (A, B) and kainic acid-injected mice (C, D) atsix days after KA. (A, B) HE stainings of saline-injected normal (A) and IL-6 KO (B) mice showing intact hippocampal neurons. (C,D) HE stainings of normal (C) and IL-6 KO (D) mice at six days after KA. (C) The morphology of hippocampal neurons is affectedwith cell loss and appearance of pyknotic neurons in normal mice. (D) In IL-6 KO mice after KA treatment, the morphologyof hippocampal neurons is more damaged than that of normal mice, and an increased neuronal cell loss is seen. Scale bar� 33 mm

(A±D, in D).

Fig. 2. Interleukin-6 and albumin immunoreactivity of the hippocampal ®eld CA3 of saline-injected and kainic acid-injected mice atsix days after injection. (A) IL-6 is only seen weakly in a few cells of normal saline-injected mice. (B) IL-6 expression is increased inmicroglia/macrophages and reactive astrocytes throughout the hippocampus after KA treatment of normal mice. (C) After KAtreatment, IL-6 expression is still absent from IL-6 KO mice. (D) Albumin immunostaining in saline-injected normal mice withintact BBB properties. (E) Albumin immunostaining in saline-injected IL-6 KO mice with intact BBB properties. (F) Albuminimmunostaining in KA-injected normal mice showing that the BBB properties are still intact. (G) Albumin immunostaining in KA-

injected IL-6 KO mice showing intact BBB properties. Scale bars� 44 mm (A±C, in C), 36 mm (D±G, in G).

Interleukin-6 expression

As expected, IL-6 expression was only observed innormal mice (Fig. 2A±C). In normal saline-injectedmice IL-6 immunostaining was only seen weakly in afew hippocampal cells (Fig. 2A), while after KA treat-ment the number of IL-6-immunostained cells wasincreased (Fig. 2B). IL-6 immunostaining was observedin microglia/macrophages and reactive astrocytesthroughout the hippocampus. In IL-6 KO mice, IL-6expression was absent in all mice (Fig. 2C).

Blood±brain barrier

The BBB permeability to serum albumin was compar-able in all saline-injected and KA-injected mice, which

hardly showed albumin immunostaining in the brainparenchyme, indicating that KA treatment does not affectthe BBB properties (Fig. 2D±G). In agreement with thealbumin data were immunostainings for an endothelialbarrier antigen/BBB antigen, which is an endothelialprotein present only in areas with an intact BBB. In allsaline-injected and KA-injected mice, the vasculaturewas stained positively for BBB antigen (data notshown), indicating that the BBB properties remainedintact after KA.

Reactive astrogliosis

GFAP immunostaining was comparable in all saline-injected mice and was seen in a few hippocampal


Fig. 3. Glial ®brillary acidic protein immunohistochemistry of hippocampus of normal and interleukin-6 KO mice. (A) GFAPimmunoreactivity of saline-injected normal mice hippocampal ®eld CA3. (B) GFAP immunoreactivity of saline-injected IL-6 KOmice hippocampal ®eld CA3. (C) Reactive astrogliosis in hippocampal area CA3 of normal mice. The star marks the area shown in(E). (D) Reactive astrogliosis in hippocampal area CA3 of IL-6 KO mice. The star marks the area shown in (F). (E) Highermagni®cation of the area in (C) marked by a star. (F) Higher magni®cation of the area in (D) marked by a star. It is clear thatreactive astrogliosis is decreased in IL-6 KO mice compared to that of normal mice. Scale bars� 44 mm (A±D, in B, D), 22 mm (E,

F, in F).

astrocytes (Fig. 3A, B). After KA treatment, GFAP-posi-tive astrocytes appeared in increased numbers in bothnormal and IL-6 KO mice hippocampus CA1±CA3(Figs 3, 8). Reactive astrocytes with swollen cell bodieswere seen next to degenerated neurons, such as those ofthe hippocampal area CA1, CA3 and DG. However, theGFAP-positive reactive astrogliosis was reduced in IL-6KO mice compared to that of normal mice (Figs 3C±F, 8).

Microglia/macrophage activation

Lectin staining was comparable in all saline-injectedmice and was seen in vessel walls (Fig. 4A, B). AfterKA treatment, lectin-positive microglia/macrophagesappeared in increased numbers in all the mice (Figs 4, 8).

Activated, bushy or stout microglia/macrophageswere seen in CA1±CA3 and DG of normal KA-injected

mice (Fig. 4C, E). Only a small amount of roundmacrophages appeared compared to the number ofbushy lectin-positive cells, which is in agreement withother studies of rats.2,32 In KA-injected IL-6 KO mice,the microglia/macrophages of CA1±CA3 and DG wereless bushy and more plump or amoeboid (Fig. 4D, F).However, in the normal mice, activated microglia/macrophages were clearly increased compared to thoseof IL-6 KO mice (Figs 4C±F, 8).

It is likely that the lectin-positive cells observed in themice after KA are merely derived from resident micro-glia. In agreement with this are immunohistochemicalstainings for MOMA-1, which stains monocyte-derivedmacrophages, in that MOMA-1-positive cells werehardly detected (not shown). In addition, the BBB prop-erties appeared intact, which further supports that thelectin-positive macrophages were microglia derived.


Fig. 4. Lectin histochemistry of hippocampus of normal and interleukin-6 KO mice. (A) Lectin staining of saline-injected normalmice hippocampal ®eld CA3, showing that mainly vessel walls are stained. (B) Lectin staining of saline-injected IL-6 KO micehippocampal ®eld CA3, showing that mainly vessel walls are stained. (C) Microglia/macrophages of normal mice hippocampal areaCA3, showing rami®ed or amoeboid cells. Curved arrow depicts the area magni®ed in E. (D) Microglia/macrophages of IL-6 KOmice hippocampal area CA3, showing mainly amoeboid or round cells. Curved arrow depicts the area magni®ed in F. (E) Highermagni®cation of depicted area in C. (F) Higher magni®cation of depicted area in D. As seen in E, F, microglia/macrophages areincreased in number and have more rami®ed morphology in normal mice compared to those of IL-6 KO mice after KA treatment.

Scale bars� 44 mm (A±D, in B and D), 22 mm (E, F, in F).

Fig. 5 (Caption overleaf).

Expression of the antioxidants metallothionein-I 1 II,Cu/Zn superoxide dismutase and Mn superoxide dismutase

In all the saline-injected mice, only a few cellsexpressed immunostaining for MT-I 1 II, Cu/Zn-SODor Mn-SOD in the hippocampus (Figs 5A, B, 8), andthe number of these cells was comparable in normaland IL-6 KO mice. At six days following KA treatment,all the mice examined increased the numbers of MT-I 1 II,Cu/Zn-SOD and Mn-SOD-immunostained cells, whichwere reactive astrocytes and microglia/macrophages(Figs 5, 8). However, the number of MT-I 1 II immuno-stained cells was signi®cantly higher in normal micecompared to that in IL-6 KO mice (Figs 5C±H, 8). Thenormal mice showed many MT-I 1 II expressing cells inCA1, CA3 and DG (Fig. 5C, E, G), which are areascontaining oxidative stress and neuronal damage (seebelow).

In IL-6 KO mice, a decreased number of astrocytesand microglia/macrophages was immunostained forMT-I 1 II after KA treatment (Fig. 5D, F, H). However,those MT-I 1 II-positive cells seen in IL-6 KO mice weremainly situated in areas showing oxidative stress andneuronal damage, such as in CA1, CA3 and DG.

The number of Cu/Zn-SOD and Mn-SOD-immuno-stained cells after KA was comparable in all mice exam-ined (Figs 5I, J, 8). At six days after KA injection, thenumbers of Cu/Zn-SOD and Mn-SOD immunostainedcells were increased in all the mice. Mainly reactiveastrocytes and microglia/macrophages of CA1, CA3and DG, but also some surviving neurons showed Cu/Zn-SOD and Mn-SOD immunoreactivity. Hence, IL-6de®ciency under the present experimental conditionsdid not in¯uence the levels of these antioxidants.

Oxidative stress

MDA, NITT and iNOS immunoreactivity wascomparable in all saline-injected mice and was seen ina very few cells of the hippocampus (Figs 6A±C, 8).After KA injection, increases in MDA, NITT and iNOSimmunostaining were observed in all mice examined(Figs 6, 8). However, IL-6 KO mice showed a higherincrease in all these markers than did the normal miceinjected with KA (Figs 6D±I, 8). The increased immuno-reactivity for MDA, NITT and iNOS was observed inneurons of CA1, CA3 and DG.

Apoptosis

TUNEL staining was comparable in the hippocampus

of all saline-injected mice, but was hardly seen in thehippocampus (Fig. 8). In addition, in all saline-injectedmice, stainings for ICE/caspase-1, caspase-3, caspase-9,cytochrome-c, TNF-a-receptor and ssDNA werecomparable and showed low or absent expression ofthese markers (not shown).

At six days after KA treatment, hippocampal celldeath, of an apoptotic nature to a great extent as judgedby TUNEL staining, was increased in all mice examined(Fig. 8). Supporting the TUNEL data, immunoreactivityfor ICE/caspase-1, caspase-3, caspase-9, cytochrome-c,TNF-a-receptor and ssDNA was also increased in allmice examined after KA treatment (data not shown).

The dying cells were mainly located to areas contain-ing oxidative stress, such as CA1, CA3 and DG (notshown). In addition, hippocampal cell death was detectedin neurons mainly, as veri®ed using double ¯uorescencehistochemistry for TUNEL and NSE (Fig. 7A, B).Supporting the notion of apoptotic cell death are expres-sion of cytochrome-c outside the mitochondria, and theincreased expression of ICE/caspase-1, which both wereseen in most of the TUNEL labeled cells (Fig. 7C±F). Bycomparing sections stained for TUNEL and NSE (Fig.7A, B) with neighboring sections stained for TUNEL,ICE/caspase-1 and cytochrome-c (Fig. 7C, D), the resultssuggest that dying neurons were undergoing apoptoticcell death after KA treatment in both normal and IL-6KO mice. As expected, the IL-6 KO mice showed anincreased number of dying neurons compared to that ofnormal mice (Figs 7, 8).

DISCUSSION

The IL-6 null mice are a unique tool for analysing therole of this cytokine on KA-induced hippocampaldamage. The results clearly show that the IL-6 miceare more susceptible to KA-induced seizures, with ahigher percentage of animals seizing and a greatermortality compared to control mice. Associated withthis increased susceptibility, the IL-6 mice showed animpaired glial response to KA-induced seizures, anunbalanced antioxidant pro®le that led to an increasedoxidative stress, and an increased neuronal death atleast in part through the activation of apoptosis.

KA is a glutamate receptor agonist with excitotoxiceffects.7,40,68 The IL-6 null mice displayed a greatersusceptibility to KA; to obtain some insight into the puta-tive mechanisms involved, a detailed histopathologicalanalysis was carried out in animals injected with a KA


Fig. 5. Metallothionein-I 1 II (A±H) and Cu/Zn-superoxide dismutase (I, J) immunohistochemistry in normal and interleukin-6 KOmice hippocampus. In saline-injected normal (A) and IL-6 KO (B) mice, a few cells of the hippocampal ®eld CA3 express MT-I 1 II. (C) In KA-treated normal mice, MT-I 1 II expression in astrocytes and microglia/macrophages is increased in the DGgranular (open star) and molecular layer. (D) MT-I 1 II expression in KA-treated IL-6 KO mice is decreased in the granular(open star) and molecular layer of the DG compared to that of normal mice. (E) MT-I 1 II expression is increased in astrocytesand microglia/macrophages of the CA3 area of KA-treated normal mice. The arrow depicts the area magni®ed in (G). (F) MT-I 1 IIexpressing astrocytes and microglia/macrophages of the CA3 area of IL-6 KO mice are decreased compared to those of normal mice.Curved arrow depicts the area magni®ed in (H). (G) Higher magni®cation of depicted area in (E) showing reactive astrocytes andmicroglia/macrophages expressing MT-I 1 II. (H) Higher magni®cation of depicted area in (F) showing the decreased number ofMT-I 1 II expressing cells. (I) Cu/Zn-SOD expression in hippocampal area CA3 of normal mice. (J) Cu/Zn-SOD expression inhippocampal area CA3 of IL-6 KO mice. (I±J) show that Cu/Zn-SOD levels are similar in normal and IL-6 KO mice. Scale

bars� 44 mm (A±F, I, J, in B, D, F, J), 22 mm (G, H, in H).

dosage that did not cause mortality of the animals. We®rst evaluated the glial response to KA-induced seizures.In agreement with previous studies, KA induced innormal mice a signi®cant astrogliosis and microgliosis,as judged by both the morphological changes of these

cells and the GFAP and lectin stainings, mainly in thehippocampal areas CA1, CA3 and DG.2,7,32,68 In IL-6 KOmice, the number of GFAP-positive and lectin-positivecells was signi®cantly decreased, indicating an impairedgliosis. These results are consistent with previously


Fig. 6. Oxidative stress in normal and interleukin-6 KO mice hippocampal area CA3. In saline-injected normal mice, MDA (A) andNITT (B) and iNOS (C) immunostaining are hardly seen. At six days after KA treatment, normal mice increase the immunoreactivityfor MDA (D) and NITT (F) and iNOS (H) in hippocampal neurons and in some adjacent glial cells. At six days after KA treatment,IL-6 KO mice also increase the immunoreactivity for MDA (E) and NITT (G) and iNOS (I) in hippocampal neurons and some glialcells, and the number of cells showing MDA, NITT and iNOS immunoreactivity is higher in IL-6 KO mice than in normal mice.

Scale bars� 44 mm (A±E, in B, C, E), 28 mm (F, G, in G), 14 mm (H, I, in I).

published studies with IL-6 KO mice, which showed adecreased gliosis after transection of the facial nerve34

and after a cortical freeze lesion.57 Only astrocytes andsome neurons express IL-6 receptors,34 and thus it seemslikely that the impaired microgliosis is an indirect effectof IL-6 de®ciency. Klein et al.34 proposed that the dimin-ished astrogliosis observed in IL-6 KO mice could lead toa decreased production of astrocyte growth factors,which could be important for the activation of micro-glia. We indeed observed a signi®cant reduction ingranulocyte-macrophage colony stimulating factor expres-sion of astrocytes in IL-6 KO mice,57 and it has beenshown that this growth factor is a potent microglial/macrophage mitogen.22±24

In normal mice, lectin-positive microglia/macrophageswere swollen and had short, thick processes, while in

IL-6 KO mice the lectin-positive cells were plump andrather amoeboid with fewer cell processes. Even thoughblood monocytes and spleen macrophages can developcell rami®cations and a microglia-like morphology whenthey encounter astrocytes, they hardly do so within sixdays.67,74 Accordingly, it is likely that the bushy lectin-positive cells seen after KA treatment are microgliaderived. In agreement with this, MOMA-1-positivemonocyte-derived macrophages were hardly observedafter KA injections, and accordingly, the BBB to plasmaalbumin was intact, supporting that no or only mildmonocyte transmigration has occurred.55,63,73 The obser-vation that lectin-positive cells in IL-6 KO mice are lessrami®ed and more amoeboid than those of normal miceafter KA could be explained by the relatively increasedtissue damage seen in IL-6 KO mice after KA (see


Fig. 7. Cell death observed in the hippocampal CA3 area after kainic acid treatment of normal and interleukin-6 KO mice. NSEimmuno¯uorescence histochemistry (red) and ¯uorescein-linked TUNEL (green) of KA-injected normal (A) and IL-6 KO (B) mice,showing that the number of TUNEL labeled cells is increased in IL-6 KO mice compared to that of normal mice. (C±D) Inneighboring sections to those seen in (A, B), a triple ¯uorescence staining was performed of TUNEL (green), ICE/caspase-1(red) and cytochrome-c (blue). (E, F) Higher magni®cations of (C) and (D), respectively. In normal (C, E) and in IL-6 KO (D,F) mice, the TUNEL labeled cells were containing ICE/caspase-1 and cytochrome-c in the cytoplasm. Some cells not labeled byTUNEL or cytochrome-c were expressing ICE/caspase-1, and these were probably glial cells. Scale bars� 25 mm (A±D, in D),

10 mm (E, F, in F).

below), and thereby microglia/macrophages are expectedto be in a higher activation state accompanied by theamoeboid or round morphology.58,61

We next evaluated the oxidative stress caused by KA.It is well known that the activation of glutamate receptorsmay increase free radicals, which may then lead tofurther receptor activation, a self-perpetuating cyclethat contributes signi®cantly to neuronal death.6,12,13,69

Thus, it was not surprising to ®nd that KA-inducedseizures caused a dramatic increase in the number ofcells immunostained for MDA (which re¯ects lipidperoxidation) and NITT (protein tyrosine nitration,which re¯ects peroxynitrite formation by increased nitricoxide and superoxide production). KA caused anincreased expression of the prooxidant enzyme iNOS,which undoubtedly contributed to the increased oxidativestress. As could be expected, presumably adaptiveresponses were elicited, since the numbers of cellsimmunostained for the antioxidant proteins MT-I 1 II,

Cu/Zn-SOD and Mn-SOD were signi®cantly increasedin the animals injected with KA. The results obtainedin the IL-6 KO mice clearly indicated that they had anincreased oxidative stress, since they had an increasednumber of MDA and NITT-immunostained cells relativeto normal mice. This appears to be due to an unbalancedproduction of prooxidant and antioxidant factors, sinceKA-injected IL-6 KO mice had an increased number ofiNOS (prooxidant) and a decreased number of MT-I 1 II(antioxidant) immunostained cells. Cu/Zn-SOD and Mn-SOD are signi®cant neuroprotective factors. Transgenicoverexpression of Cu/Zn-SOD can attenuate glutamate-induced neuronal toxicity and swelling.13 It has also beenshown that transgenic Mn-SOD overexpression preventsneuronal apoptosis,33 and that reduced Mn-SOD activityin neurons may exacerbate glutamate neurotoxicity.46

However, the expression of Cu/Zn-SOD and Mn-SODwas somewhat surprisingly unaffected by IL-6 de®-ciency, since it has been reported, at least in yeast, that


Fig. 8. Immunohistochemical cell counts in hippocampal area CA3 of saline- and kainic acid-injected normal (1295 v) andinterleukin-6 KO mice at six days after injection (cells/mm2). Counts were carried out in both hemispheres of one section peranimal in a blind manner. The main morphological features of these cells are shown in Figs 1±7 in representative animals. Resultsare mean ^S.E.M. (n� 4±5 mice). Results were evaluated with two-way ANOVA with KA and strain as main factors. Post-hoc

comparisons of the means were done with the Student's t-test. OP , 0.05 vs saline-injected mice. * P , 0.05 vs normal mice.

Cu/Zn-SOD and MT are co-regulated11 and that MT-I 1 II can functionally substitute for Cu/Zn-SOD.70 Itcould be argued that the observed decrease in MT-I 1 II expression in IL-6 KO mice after KA treatmentis simply the consequence of the reduced numbers ofmicroglia/macrophages and astrocytes, as reactive astro-cytes and microglia/macrophages are the major cell typesexpressing MT-I 1 II.5,9,10,29,54,55,57 However, these cellsalso express Cu/Zn-SOD and Mn-SOD, but the numberof cells stained positively for Cu/Zn-SOD and Mn-SODin IL-6 KO mice was similar to that of normal mice,which indicates that factors other than the number ofastrocytes and microglia contribute to the decreasedMT-I 1 II. It is likely that the absence of IL-6 expressionper se could also be responsible for the reduced MT-I 1 II immunoreactivity, since IL-6 is a major regulatorof their synthesis.9,27,28,45,57

As expected,19,26,38,41,48,49,51,52,71,75 the impaired gliosisand the increased oxidative stress of the KA-injected IL-6 KO mice led to signi®cantly increased neuronal death.As judged by the TUNEL, ICE/Caspase-1, caspase-9,ssDNA and cytochrome-c stainings, most of these cells(in hippocampal areas CA1, CA3 and DG) were dying byapoptosis. KA has also been shown to produce apoptosisas judged by DNA fragmentation.18 Regardless of thedirect versus indirect mechanisms, the IL-6 de®ciencysigni®cantly potentiated neuronal apoptosis caused byKA-induced seizures. It is feasible that in addition tothe impaired and gliosis and increased oxidative stress,

the partially blunted MT-I 1 II response could contributeper se to the observed increased neuronal apoptoticdeath, since the evidence that these proteins are anti-apoptotic factors is mounting.1,4,16,35,47,54,72,76

CONCLUSIONS

The present report demonstrates that IL-6 de®ciencyimpairs signi®cantly the gliosis induced by KA-inducedseizures, and increases the oxidative stress and the neuro-nal death, processes where the partially blunted MT-I 1 II expression may have a relevant role.

AcknowledgementsÐWe are indebted to Dr Horst Bluethmann(CNS Department, Pharma Research Gene Technologies,F. Hoffmann-La Roche AG, CH-4070, Basel, Switzerland) forhis continuous support and for the generous gift of the IL-6 KOmice. The superb excellent technical assistence of HanneHadberg, Pernille S. Thomsen, Mette Sùberg, Grazyna Hahn,Birgit Risto and Keld Stub is gratefully acknowledged. Thesestudies were supported by Hjerteforeningen (MP), KongChristian X's Fond (MP), Boldings Fond (MP), Fonden tilLñgevidenskabens fremme (MP), Dir. Ejnar Jonasson ogHustrus Fond (MP), Dir. Jacob Madsens og Hustrus Fond(MP), Fonden af 17.12.1981 (MP), Novo Nordisk Fonden(MP), Warwara Larsens Fond (MP), Dansk Epilepsi SelskabsForskningsfond (MP), Haensch's Fond (MP), Mimi og VictorLarsens Fond (MP), Diabetesforeningen (MP), ForeningenVñrn Om Synet (MP), and by PSPGC PM98-0170,Comissionat per a Universitats i Recerca 1999SGR 00330, andFundacioÂn ªLa Caixaº 97/102-00 (JH). JC is a fellow of CIRIT FI96/2613.

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(Accepted 30 October 2000)


Interleukin6 deficiency reduces the brain inflammatory response and increases oxidative stress and...

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