Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures

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

Pergamon

PII: S0306-4522(00)00515-7

Neuroscience Vol. 102, No. 4, pp. 805±818, 2001 805 q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00

www.elsevier.com/locate/neuroscience

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. CARRASCO b and J. HIDALGO b* a

Department of Medical Anatomy, The Panum Institute, University of Copenhagen, Copenhagen, Denmark b Departamento 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 glutamate analogue inducing epileptic seizures, has been studied by means of interleukin-6 null mice. At 35 mg/kg, kainic acid induced 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 brain damage. To compare the histopathological damage caused to the brain, control and interleukin-6 null mice were administered 8.75 mg/kg kainic acid and were killed six days later. Morphological damage to the hippocampal ®eld CA1±CA3 was seen after kainic acid treatment. Reactive astrogliosis and microgliosis were prominent in kainic acid-injected normal mice hippocampus, and clear signs of increased oxidative stress were evident. Thus, the immunoreactivity for inducible nitric oxide synthase, 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-mediated deoxyuridine triphosphate-biotin nick end labeling and interleukin-1b converting enzyme/Caspase-1 stainings. In kainic acid-injected interleukin-6 null mice, reactive astrogliosis and microgliosis were reduced, while morphological hippocampal damage, oxidative stress and apoptotic neuronal death were increased. Since metallothionein-I 1 II levels were lower, and those of inducible nitric oxide synthase higher, these concomitant changes are likely to contribute to the observed increased oxidative 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¯ammatory response after kainic acid-induced seizures. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: hippocampus, gliosis, apoptosis, metallothionein, neurodegeneration.

round brain macrophages, while astrocytes show hyperplasia and hypertrophy. 17,39,61 In the case of severe brain damage or direct trauma, the brain in¯ammatory response is supplemented by blood-derived leucocytes, since monocytes, lymphocytes and neutrophil granulocytes enter the brain parenchyma. 61 These responses are regulated by a number of cytokines, among which interleukin-6 is crucial for both the glial activation and the leucocyte recruitment. 34,36,37,57 Kainic acid (KA) is a glutamate receptor agonist with excitotoxic effects with the hippocampus being one of the most sensitive areas. 40,68 During KA-induced seizures, hippocampal neurons degenerate followed by microglia activation and reactive astrogliosis. 32,53,68 A number of studies demonstrated that interleukin-6 (IL-6) appears to be involved in excitotoxicity-induced brain damage. Firstly, IL-6 production is increased during excitotoxicity, 3,14,19,30,31,50 and secondly, in vitro studies have shown that IL-6 can signi®cantly protect against glutamate- and N-methyl-d-aspartate (NMDA)-induced excitotoxicity and cell death of cultured neurons. 3,8,71,75 However, the mechanisms underlying the protective role of IL-6 during excitotoxicity in vivo have to be precisely elucidated.

Injury to the CNS elicits a characteristic in¯ammatory response. Rami®ed resting microglial cells are activated and transform into phagocytic cells with retracted processes and plump cell bodies or into amoeboid or *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 -diaminobenzidine-tetrahydrochloride; DG, dentate gyrus; FITC, ¯uorescein isothiocyanate; GFAP, glial ®brillary acidic protein; GM± CSF, granulocyte±macrophage colony stimulating factor; HE, hematoxylin-eosin; HRP, horse radish peroxidase; ICE, interleukin-1b converting enzyme; Ig, immunoglobulin; IL-6, interleukin6; INOS, inducible nitric oxide synthetase; KA, kainic acid; KO, knock-out; MDA, malondialdehyde; MMP, matrix metalloproteinase; Mn-SOD, Mn-superoxide dismutase; MOMA-1, monocytederived macrophage marker-1; MT, metallothionein; NITT, protein tyrosine nitration; NMDA, N-methyl-d-aspartate; NSE, neuronspeci®c enolase; ROS, reactive oxygen species; SS, singlestranded; StreptABComplex, streptavidin±biotin-complex; TBS, Tris-buffered saline; TNF-a, tumor necrosis factor-a; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labeling; TXRD, Texas Red. 805

806

M. Penkowa et al.

Neuronal damage and cell death during excitotoxicity and seizures are associated with generation of reactive oxygen species (ROS), 12,13,65,69 which initiate various signaling cascades leading to neuronal degeneration and apoptosis. 12,25,69 Therefore, one of the ways in which IL-6 could exert its neuroprotective effects could be by decreasing the excitotoxicity-induced oxidative stress. Metallothionein I 1 II (MT-I 1 II) and Cu/Znand Mn-superoxide dismutase (Cu/Zn- and Mn-SOD) are important antioxidant proteins, which protect against cellular damage from ROS produced during pathological conditions, 5,13,29,33,42,43,46,59,62,66 whereby MT-I 1 II, Cu/ Zn-SOD and Mn-SOD can prevent neuronal damage and cell death. 13,35,43,44,54,60,62,64 Interestingly, IL-6 is a major regulator in vivo of MT-I 1 II. 9,10,15,27,28,45,56,57 In the present report, we have studied the putative role of IL-6 on KA-induced hippocampal damage by using IL-6 null mice. 36 EXPERIMENTAL PROCEDURES

Experiments IL-6 null knock-out (KO) mice 36 were kindly provided by Dr Horst Bluethmann (CNS Department, Pharma Research Gene Technologies, F. Hoffmann-La Roche AG, CH-4070, Basel, Switzerland). In a ®rst experiment, adult normal (1295 v, n ˆ 8) and IL-6 KO (n ˆ 8) mice were injected i.p. with 35 mg/kg KA, which is a glutamate receptor agonist with excitotoxic effects. 40 The number of mice showing seizures as well as the mortality were recorded. Because of the high mortality of the IL-6 KO mice, no histopathological analysis of these brains was carried out. In a second experiment, normal and IL-6 KO mice were injected 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 KAinjected mice. The mice were housed in cages at the Animal Department of the Autonomous University of Barcelona under constant temperature and had free access to food and water. All experiments were carried out in a humane manner and were approved by the proper Ethical Committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available. Tissue processing Normal and IL-6 KO mice were killed at six days after KA or saline injection. Mice were deeply anesthetized with 10 mg/100 g Brietal (methohexital 10 mg/ml, Eli Lilly) and were transcardially 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 added 15% picric acid solution [1.2% (saturated) aqueous picric acid]. Formaldehyde was prepared shortly before use by alkaline hydrolysis 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 dehydrated according to standard procedures, embedded in paraf®n, and cut in serial, coronal 3 mm thick sections to be used for Hematoxylin-Eosin (HE) and Toluidine Blue stainings, lectin histochemistry, immunohistochemistry, immuno¯uorescent histochemistry and in situ detection of DNA fragmentation/terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labeling (TUNEL) labeling. Sections were afterwards rehydrated and incubated in 1.5% H2O2 in Tris-buffered saline (TBS)/Nonidet (TBS: 0.05 M Tris, pH 7.4, 0.15 M NaCl) for 15 min at room temperature to

quench endogenous peroxidase. Saline-injected and KA-injected normal and IL-6 KO mice were processed in parallel. Sections were boiled in citrate buffer, pH 9.1 or pH 6.0, for 10 min in a microwave oven for heat-induced antigen retrieval. After cooling down to room temperature, the sections were preincubated with 10% normal goat or donkey serum in TBS/Nonidet for 30 min at room temperature to block non-speci®c binding. Those sections prepared for incubation with monoclonal mouse-derived antibodies were also incubated with Blocking Solutions 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 to standard procedures. In addition, the sections were incubated overnight at 48C with biotinylated tomato lectin from Lycopersicon esculentum (Sigma, USA, code L9389), which was diluted 1:500 in 10% goat serum and used as a marker for all cells of the myelo-monocytic cell lineage, such as rami®ed and plump microglial cells and macrophages. Labeling by lectin was veri®ed using streptavidin±biotin-peroxidase complex (StreptABComplex/HRP, Dakopatts, DK, code K377) prepared at manufacturer's recommended 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 the following primary antibodies: monoclonal rat anti-mouse IL-6, diluted 1:10 (Harlan Sera Lab, UK, code MAS 584); polyclonal rabbit anti-human neuronal speci®c enolase (NSE) (as a neuronal marker) diluted 1:1000 (Dakopatts, DK, code A589); polyclonal rabbit anti-cow glial ®brillary acidic protein (GFAP; as a marker for astrocytes) diluted 1:250 (Dakopatts, DK, code Z 334); monoclonal rat anti-mouse MOMA-1 (as a monocyte-derived macrophage marker) diluted 1:20 (Hybridomus, DK, code HD-212-85OMA); polyclonal rabbit anti-human albumin (as a marker for exudation of plasma proteins through the blood±brain barrier; BBB) diluted 1:5000 (Dakopatts, DK, code A 0001); monoclonal mouse anti-rat endothelial barrier antigen/blood±brain barrier antigen (BBB antigen) (marking an endothelial protein present only in areas with an intact BBB) diluted 1:100 (Af®nity, UK, code BA1116); polyclonal rabbit anti-rat MT-I 1 II; 20,21 monoclonal 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 antimalondialdehyde (MDA; a marker for oxidative stress) diluted 1:100 (Alpha Diagnostic Int., USA, code MDA 11-S); polyclonal rabbit 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); polyclonal rabbit anti-mouse interleukin-1b converting enzyme (ICE)/ Caspase-1 1:100 (Santa Cruz, USA, code sc-1218R); polyclonal rabbit anti-human caspase-3 1:50 (Santa Cruz, USA, code sc7148); polyclonal rabbit anti-human caspase-9 1:50 (Santa Cruz, USA, code sc-8355); polyclonal rabbit anti-human cytochrome-c 1:50 (Santa Cruz, USA, code sc-7159); monoclonal mouse anti-human tumor necrosis factor-a (TNF-a) receptor 1:100 (Zymed, USA, code 330100); monoclonal mouse anticalf single-stranded (SS) DNA (IgM) 1:100 (Alexis, USA, code 804-192-R200). The primary antibodies were detected in rats and mice using biotinylated mouse anti-rabbit IgG or biotinylated speciesspeci®c sheep anti-rat IgG (as mentioned above) or biotinylated goat anti-mouse IgG 1:200 (Sigma, USA, code B8774), or goat anti-mouse-IgM 1:10 (Jackson, USA, code 115-065-020), for 30 min at room temperature, followed by StreptABComplex/ HRP (Dakopatts, DK, code K377) prepared at the manufacturer's recommended dilutions for 30 min at room temperature. The immunoreaction was visualized using DAB as a chromogen (as mentioned above).

807

In¯ammatory response in IL-6-de®cient mice Table 1. Response of 129Sv and interleukin-6-de®cient mice to kainic acid administration in Experiment 1 Strain 129Sv IL-6KO

% Seizing

% Animals with jumping

Latency time (min)

Number of convulsions

Mortality (%)

75 100

75 87.5

29.4 ^ 4.0 33.2 ^ 8.4

9.4 ^ 2.3 19.5 ^ 3.3*

0 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 of convulsions are the mean ^ S.E.M., (129Sv n ˆ 8; IL-6KO n ˆ 8). *Signi®cantly greater than 129Sv mice (P , 0.05). Means are compared with t-test and frequency data are analyzed with x 2 test.

In order to evaluate the extent of non-speci®c binding in the immunohistochemical experiments, control sections were incubated in: (i) the DAB medium alone (to examine endogenous peroxidase activity); (ii) the DAB medium and the ABComplex prepared at the manufacturer's recommended dilutions (to examine endogenous biotin activity); and (iii) the absence of primary antibody (to examine cross-reaction among IgGs of the different species). Results were considered only if these controls were negative. Comparisons of sections which were either incubated or not incubated with Blocking Solutions A 1 B from HistoMouse-SP Kit were also made. In situ detection of DNA fragmentation (TUNEL) TUNEL staining was performed according to the manufacturer'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. TUNEL was accomplished using the Apoptag Plus, In Situ Apoptosis Detection Kit (Oncor, USA, Code S7101-KIT). After immersion in equilibration buffer for 10 min, sections were incubated with TdT and dUTP-digoxigenin in a humidi®ed chamber at 378C for 1 h and then incubated in the stop/wash buffer at 378C for 30 min to stop the reaction. Afterwards, the sections were incubated in anti-digoxigenin-peroxidase solution for 30 min. DAB was used as chromogen (as mentioned above), and the sections were counterstained with Methyl Green. Negative control sections were treated similarly but incubated in the absence of TdT enzyme, dUTP-digoxigenin or anti-digoxigenin antibody. Sections were compared with positive control slides (Oncor, USA, code S7115). Furthermore, morphological criteria for apoptosis were evaluated too, since the TUNEL may stain necrotic cells. Immuno¯uorescence histochemistry and TUNEL In order to determine which cells underwent apoptosis, double TUNEL staining and staining for lectin or GFAP or NSE were performed. Sections were incubated with ¯uorescein-linked TUNEL (Oncor, USA, code S7110-KIT) according to the manufacturer's protocol. Afterwards, sections were incubated with lectin linked with Texas Red (TXRD) 1:50 (Sigma, USA, code L9139) or anti-GFAP or anti-NSE (as mentioned above). The anti-GFAP and anti-NSE antibodies were detected by using goat anti-rabbit IgG linked with TXRD 1:50 (Jackson ImmunoResearch Lab., USA, code 111-075-144). Furthermore, triple immuno¯uorescence histochemistry was performed in order to better characterize apoptotic cells. Sections were incubated with ¯uorescein-linked TUNEL (as mentioned above), and afterwards incubated overnight at 48C with rabbit anti-mouse ICE/Caspase-1 1:100 (Santa Cruz, USA, code sc1218R) and goat anti-horse cytochrome-c 1:100 (Santa Cruz, USA, code sc-7159) simultaneously. The anti-ICE antibodies were detected using donkey anti-rabbit IgG linked with TXRD 1:40 (Jackson ImmunoResearch Lab., USA, code 711-075-152), and the anti-cytochrome-c antibodies were detected using donkey anti-goat IgG linked with Aminomethylcoumarin (AMCA) 1:40 (Jackson ImmunoResearch Lab., USA, code 705-155-147). The secondary antibodies were used simultaneously for 30 min at room 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 antisera, control sections were incubated in the absence of primary antibody. Negative control sections of the TUNEL labeling were incubated in the absence of TdT enzyme. Results were considered only if these controls were negative. Comparisons of sections, which were either incubated or not incubated with Blocking Solutions A 1 B from HistoMouse-SP Kit, were also made. Furthermore, morphological criteria for apoptosis were evaluated too, since the TUNEL is known to stain necrotic as well as apoptotic cells. For the simultaneous examination and recording of the two stains, a Zeiss Axioplan2 light microscope equipped with a triple band (DAPI/FITC/TXRD) ®lter was used. Statistical analysis In addition to morphological analysis, cellular countings were carried out in a 1 mm 2 area of 3 mm thick sections of hippocampal area CA3 of both hemispheres of saline-injected and KA-injected normal and IL-6 KO mice at day 6 after injection for statistical evaluation of the results. To this end, positively stained cells, de®ned as cells with staining of the soma, or in the case of TUNEL cells with nuclear staining, were counted in the CA3 of hippocampus. Positively stained cells were counted from an area of CA3 of both hemispheres of saline-injected and KA-injected mice. Cell countings were performed in at least three mice per group. Results were evaluated by two-way analysis of variance (ANOVA), with strain and KA injection as main factors. When the interaction was signi®cant, it was interpreted to be the consequence of a speci®c effect of the IL-6 de®ciency during the in¯ammation. This was veri®ed by post-hoc Student's t-test. When only two groups were compared (i.e. IL-6 immunostaining), the Student's t-test was used. Frequency data were analysed with the x 2 test. The probability level was set at P , 0.05. RESULTS

Susceptibility to kainic acid Saline-injected mice of normal and IL-6 KO genotypes showed similar behavioral patterns. No spontaneous convulsions were seen in these mice. The administration of 35 mg/kg KA produced convulsions in both normal and IL-6 KO mice (Table 1), but the percentage of mice seizing and the number of convulsions were higher in the latter (P , 0.05). Moreover, ®ve out of eight (62.5%) of the IL-6 KO mice died in the following few hours after the KA administration, while none 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 mice but 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

808

M. Penkowa et al.

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

comparable stainings for neuronal and glial structure by HE, 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). After KA injection, hippocampal morphological damage was

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

Fig. 2. Interleukin-6 and albumin immunoreactivity of the hippocampal ®eld CA3 of saline-injected and kainic acid-injected mice at six 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 in microglia/macrophages and reactive astrocytes throughout the hippocampus after KA treatment of normal mice. (C) After KA treatment, IL-6 expression is still absent from IL-6 KO mice. (D) Albumin immunostaining in saline-injected normal mice with intact BBB properties. (E) Albumin immunostaining in saline-injected IL-6 KO mice with intact BBB properties. (F) Albumin immunostaining in KA-injected normal mice showing that the BBB properties are still intact. (G) Albumin immunostaining in KAinjected IL-6 KO mice showing intact BBB properties. Scale bars ˆ 44 mm (A±C, in C), 36 mm (D±G, in G).

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

809

Fig. 3. Glial ®brillary acidic protein immunohistochemistry of hippocampus of normal and interleukin-6 KO mice. (A) GFAP immunoreactivity of saline-injected normal mice hippocampal ®eld CA3. (B) GFAP immunoreactivity of saline-injected IL-6 KO mice 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) Higher magni®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 that reactive astrogliosis is decreased in IL-6 KO mice compared to that of normal mice. Scale bars ˆ 44 mm (A±D, in B, D), 22 mm (E, F, in F).

Interleukin-6 expression As expected, IL-6 expression was only observed in normal mice (Fig. 2A±C). In normal saline-injected mice IL-6 immunostaining was only seen weakly in a few hippocampal cells (Fig. 2A), while after KA treatment the number of IL-6-immunostained cells was increased (Fig. 2B). IL-6 immunostaining was observed in microglia/macrophages and reactive astrocytes throughout the hippocampus. In IL-6 KO mice, IL-6 expression was absent in all mice (Fig. 2C).

hardly showed albumin immunostaining in the brain parenchyme, indicating that KA treatment does not affect the BBB properties (Fig. 2D±G). In agreement with the albumin data were immunostainings for an endothelial barrier antigen/BBB antigen, which is an endothelial protein present only in areas with an intact BBB. In all saline-injected and KA-injected mice, the vasculature was stained positively for BBB antigen (data not shown), indicating that the BBB properties remained intact after KA.

Blood±brain barrier

Reactive astrogliosis

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

GFAP immunostaining was comparable in all salineinjected mice and was seen in a few hippocampal

810

M. Penkowa et al.

Fig. 4. Lectin histochemistry of hippocampus of normal and interleukin-6 KO mice. (A) Lectin staining of saline-injected normal mice hippocampal ®eld CA3, showing that mainly vessel walls are stained. (B) Lectin staining of saline-injected IL-6 KO mice hippocampal ®eld CA3, showing that mainly vessel walls are stained. (C) Microglia/macrophages of normal mice hippocampal area CA3, showing rami®ed or amoeboid cells. Curved arrow depicts the area magni®ed in E. (D) Microglia/macrophages of IL-6 KO mice hippocampal area CA3, showing mainly amoeboid or round cells. Curved arrow depicts the area magni®ed in F. (E) Higher magni®cation of depicted area in C. (F) Higher magni®cation of depicted area in D. As seen in E, F, microglia/macrophages are increased in number and have more rami®ed morphology in normal mice compared to those of IL-6 KO mice after KA treatment. Scale bars ˆ 44 mm (A±D, in B and D), 22 mm (E, F, in F).

astrocytes (Fig. 3A, B). After KA treatment, GFAP-positive astrocytes appeared in increased numbers in both normal and IL-6 KO mice hippocampus CA1±CA3 (Figs 3, 8). Reactive astrocytes with swollen cell bodies were seen next to degenerated neurons, such as those of the hippocampal area CA1, CA3 and DG. However, the GFAP-positive reactive astrogliosis was reduced in IL-6 KO mice compared to that of normal mice (Figs 3C±F, 8). Microglia/macrophage activation Lectin staining was comparable in all saline-injected mice and was seen in vessel walls (Fig. 4A, B). After KA treatment, lectin-positive microglia/macrophages appeared in increased numbers in all the mice (Figs 4, 8). Activated, bushy or stout microglia/macrophages were seen in CA1±CA3 and DG of normal KA-injected

mice (Fig. 4C, E). Only a small amount of round macrophages appeared compared to the number of bushy lectin-positive cells, which is in agreement with other studies of rats. 2,32 In KA-injected IL-6 KO mice, the microglia/macrophages of CA1±CA3 and DG were less bushy and more plump or amoeboid (Fig. 4D, F). However, in the normal mice, activated microglia/ macrophages were clearly increased compared to those of IL-6 KO mice (Figs 4C±F, 8). It is likely that the lectin-positive cells observed in the mice after KA are merely derived from resident microglia. In agreement with this are immunohistochemical stainings for MOMA-1, which stains monocyte-derived macrophages, in that MOMA-1-positive cells were hardly detected (not shown). In addition, the BBB properties appeared intact, which further supports that the lectin-positive macrophages were microglia derived.

Fig. 5 (Caption overleaf).

812

M. Penkowa et al.

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 cells expressed immunostaining for MT-I 1 II, Cu/Zn-SOD or Mn-SOD in the hippocampus (Figs 5A, B, 8), and the number of these cells was comparable in normal and 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, which were reactive astrocytes and microglia/macrophages (Figs 5, 8). However, the number of MT-I 1 II immunostained cells was signi®cantly higher in normal mice compared to that in IL-6 KO mice (Figs 5C±H, 8). The normal mice showed many MT-I 1 II expressing cells in CA1, CA3 and DG (Fig. 5C, E, G), which are areas containing oxidative stress and neuronal damage (see below). In IL-6 KO mice, a decreased number of astrocytes and microglia/macrophages was immunostained for MT-I 1 II after KA treatment (Fig. 5D, F, H). However, those MT-I 1 II-positive cells seen in IL-6 KO mice were mainly situated in areas showing oxidative stress and neuronal damage, such as in CA1, CA3 and DG. The number of Cu/Zn-SOD and Mn-SOD-immunostained cells after KA was comparable in all mice examined (Figs 5I, J, 8). At six days after KA injection, the numbers of Cu/Zn-SOD and Mn-SOD immunostained cells were increased in all the mice. Mainly reactive astrocytes and microglia/macrophages of CA1, CA3 and DG, but also some surviving neurons showed Cu/ Zn-SOD and Mn-SOD immunoreactivity. Hence, IL-6 de®ciency under the present experimental conditions did not in¯uence the levels of these antioxidants. Oxidative stress MDA, NITT and iNOS immunoreactivity was comparable in all saline-injected mice and was seen in a very few cells of the hippocampus (Figs 6A±C, 8). After KA injection, increases in MDA, NITT and iNOS immunostaining were observed in all mice examined (Figs 6, 8). However, IL-6 KO mice showed a higher increase in all these markers than did the normal mice injected with KA (Figs 6D±I, 8). The increased immunoreactivity for MDA, NITT and iNOS was observed in neurons of CA1, CA3 and DG. Apoptosis TUNEL staining was comparable in the hippocampus

of all saline-injected mice, but was hardly seen in the hippocampus (Fig. 8). In addition, in all saline-injected mice, stainings for ICE/caspase-1, caspase-3, caspase-9, cytochrome-c, TNF-a-receptor and ssDNA were comparable and showed low or absent expression of these markers (not shown). At six days after KA treatment, hippocampal cell death, of an apoptotic nature to a great extent as judged by TUNEL staining, was increased in all mice examined (Fig. 8). Supporting the TUNEL data, immunoreactivity for ICE/caspase-1, caspase-3, caspase-9, cytochrome-c, TNF-a-receptor and ssDNA was also increased in all mice examined after KA treatment (data not shown). The dying cells were mainly located to areas containing oxidative stress, such as CA1, CA3 and DG (not shown). In addition, hippocampal cell death was detected in neurons mainly, as veri®ed using double ¯uorescence histochemistry for TUNEL and NSE (Fig. 7A, B). Supporting the notion of apoptotic cell death are expression of cytochrome-c outside the mitochondria, and the increased expression of ICE/caspase-1, which both were seen in most of the TUNEL labeled cells (Fig. 7C±F). By comparing 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 results suggest that dying neurons were undergoing apoptotic cell death after KA treatment in both normal and IL-6 KO mice. As expected, the IL-6 KO mice showed an increased number of dying neurons compared to that of normal mice (Figs 7, 8). DISCUSSION

The IL-6 null mice are a unique tool for analysing the role of this cytokine on KA-induced hippocampal damage. The results clearly show that the IL-6 mice are more susceptible to KA-induced seizures, with a higher percentage of animals seizing and a greater mortality compared to control mice. Associated with this increased susceptibility, the IL-6 mice showed an impaired glial response to KA-induced seizures, an unbalanced antioxidant pro®le that led to an increased oxidative stress, and an increased neuronal death at least in part through the activation of apoptosis. KA is a glutamate receptor agonist with excitotoxic effects. 7,40,68 The IL-6 null mice displayed a greater susceptibility to KA; to obtain some insight into the putative mechanisms involved, a detailed histopathological analysis 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 KO mice hippocampus. In saline-injected normal (A) and IL-6 KO (B) mice, a few cells of the hippocampal ®eld CA3 express MTI 1 II. (C) In KA-treated normal mice, MT-I 1 II expression in astrocytes and microglia/macrophages is increased in the DG granular (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 astrocytes and microglia/macrophages of the CA3 area of KA-treated normal mice. The arrow depicts the area magni®ed in (G). (F) MT-I 1 II expressing 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 and microglia/macrophages expressing MT-I 1 II. (H) Higher magni®cation of depicted area in (F) showing the decreased number of MT-I 1 II expressing cells. (I) Cu/Zn-SOD expression in hippocampal area CA3 of normal mice. (J) Cu/Zn-SOD expression in hippocampal 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 ˆ 44 mm (A±F, I, J, in B, D, F, J), 22 mm (G, H, in H).

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

813

Fig. 6. Oxidative stress in normal and interleukin-6 KO mice hippocampal area CA3. In saline-injected normal mice, MDA (A) and NITT (B) and iNOS (C) immunostaining are hardly seen. At six days after KA treatment, normal mice increase the immunoreactivity for 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 glial cells, and the number of cells showing MDA, NITT and iNOS immunoreactivity is higher in IL-6 KO mice than in normal mice. Scale bars ˆ 44 mm (A±E, in B, C, E), 28 mm (F, G, in G), 14 mm (H, I, in I).

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 in normal 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 the hippocampal areas CA1, CA3 and DG. 2,7,32,68 In IL-6 KO mice, the number of GFAP-positive and lectin-positive cells was signi®cantly decreased, indicating an impaired gliosis. These results are consistent with previously

814

M. Penkowa et al.

Fig. 7. Cell death observed in the hippocampal CA3 area after kainic acid treatment of normal and interleukin-6 KO mice. NSE immuno¯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) In neighboring 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 by TUNEL or cytochrome-c were expressing ICE/caspase-1, and these were probably glial cells. Scale bars ˆ 25 mm (A±D, in D), 10 mm (E, F, in F).

published studies with IL-6 KO mice, which showed a decreased gliosis after transection of the facial nerve 34 and after a cortical freeze lesion. 57 Only astrocytes and some neurons express IL-6 receptors, 34 and thus it seems likely that the impaired microgliosis is an indirect effect of IL-6 de®ciency. Klein et al. 34 proposed that the diminished astrogliosis observed in IL-6 KO mice could lead to a decreased production of astrocyte growth factors, which could be important for the activation of microglia. We indeed observed a signi®cant reduction in granulocyte-macrophage colony stimulating factor expression of astrocytes in IL-6 KO mice, 57 and it has been shown that this growth factor is a potent microglial/ macrophage mitogen. 22±24 In normal mice, lectin-positive microglia/macrophages were swollen and had short, thick processes, while in

IL-6 KO mice the lectin-positive cells were plump and rather amoeboid with fewer cell processes. Even though blood monocytes and spleen macrophages can develop cell rami®cations and a microglia-like morphology when they encounter astrocytes, they hardly do so within six days. 67,74 Accordingly, it is likely that the bushy lectinpositive cells seen after KA treatment are microglia derived. In agreement with this, MOMA-1-positive monocyte-derived macrophages were hardly observed after KA injections, and accordingly, the BBB to plasma albumin was intact, supporting that no or only mild monocyte transmigration has occurred. 55,63,73 The observation that lectin-positive cells in IL-6 KO mice are less rami®ed and more amoeboid than those of normal mice after KA could be explained by the relatively increased tissue damage seen in IL-6 KO mice after KA (see

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

815

Fig. 8. Immunohistochemical cell counts in hippocampal area CA3 of saline- and kainic acid-injected normal (1295 v) and interleukin-6 KO mice at six days after injection (cells/mm 2). Counts were carried out in both hemispheres of one section per animal in a blind manner. The main morphological features of these cells are shown in Figs 1±7 in representative animals. Results are mean ^S.E.M. (n ˆ 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.

below), and thereby microglia/macrophages are expected to be in a higher activation state accompanied by the amoeboid or round morphology. 58,61 We next evaluated the oxidative stress caused by KA. It is well known that the activation of glutamate receptors may increase free radicals, which may then lead to further receptor activation, a self-perpetuating cycle that contributes signi®cantly to neuronal death. 6,12,13,69 Thus, it was not surprising to ®nd that KA-induced seizures caused a dramatic increase in the number of cells immunostained for MDA (which re¯ects lipid peroxidation) and NITT (protein tyrosine nitration, which re¯ects peroxynitrite formation by increased nitric oxide and superoxide production). KA caused an increased expression of the prooxidant enzyme iNOS, which undoubtedly contributed to the increased oxidative stress. As could be expected, presumably adaptive responses were elicited, since the numbers of cells immunostained for the antioxidant proteins MT-I 1 II,

Cu/Zn-SOD and Mn-SOD were signi®cantly increased in the animals injected with KA. The results obtained in the IL-6 KO mice clearly indicated that they had an increased oxidative stress, since they had an increased number of MDA and NITT-immunostained cells relative to normal mice. This appears to be due to an unbalanced production of prooxidant and antioxidant factors, since KA-injected IL-6 KO mice had an increased number of iNOS (prooxidant) and a decreased number of MT-I 1 II (antioxidant) immunostained cells. Cu/Zn-SOD and MnSOD are signi®cant neuroprotective factors. Transgenic overexpression of Cu/Zn-SOD can attenuate glutamateinduced neuronal toxicity and swelling. 13 It has also been shown that transgenic Mn-SOD overexpression prevents neuronal apoptosis, 33 and that reduced Mn-SOD activity in neurons may exacerbate glutamate neurotoxicity. 46 However, the expression of Cu/Zn-SOD and Mn-SOD was somewhat surprisingly unaffected by IL-6 de®ciency, since it has been reported, at least in yeast, that

816

M. Penkowa et al.

Cu/Zn-SOD and MT are co-regulated 11 and that MTI 1 II can functionally substitute for Cu/Zn-SOD. 70 It could be argued that the observed decrease in MTI 1 II expression in IL-6 KO mice after KA treatment is simply the consequence of the reduced numbers of microglia/macrophages and astrocytes, as reactive astrocytes and microglia/macrophages are the major cell types expressing MT-I 1 II. 5,9,10,29,54,55,57 However, these cells also express Cu/Zn-SOD and Mn-SOD, but the number of cells stained positively for Cu/Zn-SOD and Mn-SOD in IL-6 KO mice was similar to that of normal mice, which indicates that factors other than the number of astrocytes and microglia contribute to the decreased MT-I 1 II. It is likely that the absence of IL-6 expression per se could also be responsible for the reduced MTI 1 II immunoreactivity, since IL-6 is a major regulator of their synthesis. 9,27,28,45,57 As expected, 19,26,38,41,48,49,51,52,71,75 the impaired gliosis and the increased oxidative stress of the KA-injected IL6 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 by apoptosis. KA has also been shown to produce apoptosis as judged by DNA fragmentation. 18 Regardless of the direct versus indirect mechanisms, the IL-6 de®ciency signi®cantly potentiated neuronal apoptosis caused by KA-induced seizures. It is feasible that in addition to the impaired and gliosis and increased oxidative stress,

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

The present report demonstrates that IL-6 de®ciency impairs signi®cantly the gliosis induced by KA-induced seizures, and increases the oxidative stress and the neuronal death, processes where the partially blunted MTI 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) for his continuous support and for the generous gift of the IL-6 KO mice. The superb excellent technical assistence of Hanne Hadberg, Pernille S. Thomsen, Mette Sùberg, Grazyna Hahn, Birgit Risto and Keld Stub is gratefully acknowledged. These studies were supported by Hjerteforeningen (MP), Kong Christian X's Fond (MP), Boldings Fond (MP), Fonden til Lñgevidenskabens fremme (MP), Dir. Ejnar Jonasson og Hustrus 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 Selskabs Forskningsfond (MP), Haensch's Fond (MP), Mimi og Victor Larsens Fond (MP), Diabetesforeningen (MP), Foreningen Vñrn Om Synet (MP), and by PSPGC PM98-0170, Comissionat per a Universitats i Recerca 1999SGR 00330, and FundacioÂn ªLa Caixaº 97/102-00 (JH). JC is a fellow of CIRIT FI 96/2613.

REFERENCES

1. Abdel-Mageed A. and Agrawal K. (1997) Antisense down-regulation of metallothionein induces growth arrest and apoptosis in human breast carcinoma cells. Cancer Gene Ther. 4, 199±207. 2. Akiyama H., Tooyama I., Kondo H., Ikeda K., Kimura H., McGeer E. G. and McGeer P. L. (1994) Early response of brain resident microglia to kainic acid-induced hippocampal lesions. Brain Res. 635, 257±268. 3. Ali C., Nicole O., Docagne F., Lesne S., MacKenzie E. T., Nouvelot A., Buisson A. and Vivien D. (2000) Ischemia-induced interleukin-6 as a potential endogenous neuroprotective cytokine against NMDA receptor-mediated excitotoxicity in the brain. J. cereb Blood Flow Metab. 20, 956±966. 4. Apostolova M. D., Ivanova I. A. and Cherian M. G. (1999) Metallothionein and apoptosis during differentiation of myoblasts to myotubes: protection against free radical toxicity. Toxicol. appl. Pharmac. 159, 175±184. 5. Aschner M. (1998) Metallothionein (MT) isoforms in the central nervous system (CNS): regional and cell-speci®c distribution and potential functions as an antioxidant. Neurotoxicology 19, 653±660. 6. Bains J. and Shaw C. (1997) Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res. Rev. 25, 335±358. 7. Ben-Ari Y. (1985) Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience 14, 375±403. 8. Carlson N. G., Wieggel W. A., Chen J., Bacchi A., Rogers S. W. and Gahring L. C. (1999) In¯ammatory cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha impart neuroprotection to an excitotoxin through distinct pathways. J. Immun. 163, 3963±3968. 9. Carrasco J., Hernandez J., Bluethmann H. and Hidalgo J. (1998) Interleukin-6 and tumor necrosis factor-alpha type 1 receptor de®cient mice reveal a role of IL-6 and TNF-alpha on brain metallothionein-I and -III regulation. Brain Res. Molec. Brain Res. 57, 221±234. 10. Carrasco J., Hernandez J., Gonzalez B., Campbell I. L. and Hidalgo J. (1998) Localization of metallothionein-I and -III expression in the CNS of transgenic mice with astrocyte-targeted expression of interleukin 6. Expl Neurol. 153, 184±194. 11. Carri M. T., Galiazzo F., Ciriolo M. R. and Rotilio G. (1991) Evidence for co-regulation of Cu,Zn superoxide dismutase and metallothionein gene expression in yeast through transcriptional control by copper via the ACE 1 factor. Fedn Eur. biochem. Socs Lett. 278, 263±266. 12. Cassarino D. S. and Bennett J. P. Jr (1999) An evaluation of the role of mitochondria in neurodegenerative diseases: mitochondrial mutations and oxidative pathology, protective nuclear responses, and cell death in neurodegeneration, Brain Res. Rev. 29, 1-25. 13. Chan P. H., Chu L., Chen S. F., Carlson E. J. and Epstein C. J. (1990) Reduced neurotoxicity in transgenic mice overexpressing human copperzinc-superoxide dismutase. Stroke 21, 80±82. 14. de Bock F., Dornand J. and Rondouin G. (1996) Release of TNF alpha in the rat hippocampus following epileptic seizures and excitotoxic neuronal damage. NeuroReport 7, 1125±1129. 15. De S. K., McMaster M. T. and Andrews G. K. (1990) Endotoxin induction of murine metallothionein gene expression. J. biol. Chem. 265, 15,267±15,274. 16. Deng D. X., Cai L., Chakrabarti S. and Cherian M. G. (1999) Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein. Toxicology 134, 39±49. 17. Eddleston M. and Mucke L. (1993) Molecular pro®le of reactive astrocytesÐimplications for their role in neurological disease. Neuroscience 1, 15±36.

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

817

18. Froesch D. (1997) DNA fragmentation and prolonged expression of c-fos, c- jun, and hsp70 in kainic acid-induced neuronal cell death in transgenic mice overexpressing human CuZn-superoxide dismutase. J. cereb. Blood Flow Metab. 17, 241±256. 19. Gadient R. A. and Otten U. H. (1997) Interleukin-6 (IL-6)Ða molecule with both bene®cial and destructive potentials. Prog. Neurobiol. 52, 379±390. 20. Gasull T., Giralt M., Hernandez J., Martinez P., Bremner I. and Hidalgo J. (1994) Regulation of metallothionein concentrations in rat brain: effect of glucocorticoids, zinc, copper, and endotoxin. Am. J. Physiol. 266, E760±767. 21. Gasull T., Rebollo D. V., Romero B. and Hidalgo J. (1993) Development of a competitive double antibody radioimmunoassay for rat metallothionein. J. Immun. 14, 209±225. 22. Gehrmann J. (1995) Colony-stimulating factors regulate programmed cell death of rat microglia/brain macrophages in vitro. J. Neuroimmun. 63, 55±61. 23. Giulian D. and Ingemann J. (1988) Colony-stimulating factors as promoters of ameboid microglia. J. Neurosci. 8, 4707±4717. 24. Giulian D., Li J., Li X., George J. and Rutecki P. (1994) The impact of microglia-derived cytokines upon gliosis in the CNS. Devl Neurosci. 16, 128±136. 25. Grilli M. and Memo M. (1999) Possible role of NF-kappaB and p53 in the glutamate-induced pro-apoptotic neuronal pathway. Cell Death Differ. 6, 22±27. 26. Gruol D. L. and Nelson T. E. (1997) Physiological and pathological roles of interleukin-6 in the central nervous system. Molec. Neurobiol. 15, 307±339. 27. HernaÂndez J. and Hidalgo J. (1998) Endotoxin and intracerebroventricular injection of IL-1 and IL-6 induce rat brain metallothionein-I and -II. Neurochem. Int. 32, 369±373. 28. HernaÂndez J., Molinero A., Campbell I. L. and Hidalgo J. (1997) Transgenic expression of interleukin 6 in the central nervous system regulates brain metallothionein-I and -III expression in mice. Brain Res. Molec. Brain Res. 48, 125±131. 29. Hidalgo J., Castellano B. and Campbell I. L. (1997) Regulation of brain metallothioneins. Curr. Topics Neurochem. 1, 1±26. 30. Higuchi M., Ito T., Imai Y., Iwaki T., Hattori M., Kohsaka S., Niho Y. and Sakaki Y. (1994) Expression of the alpha 2-macroglobulin-encoding gene in rat brain and cultured astrocytes. Gene 141, 155±162. 31. Hopkins S. and Rothwell N. (1995) Cytokines and the nervous system. I: Expression and recognition. Trends Neurosci. 18, 83±88. 32. Jùrgensen M. B., Finsen B. R., Jensen M. B., Castellano B., Diemer N. H. and Zimmer J. (1993) Microglial and astroglial reactions to ischemic and kainic acid-induced lesions of the adult rat hippocampus. Expl Neurol. 120, 70±88. 33. Keller J. N., Kindy M. S., Holtsberg F. W., St Clair D. K., Yen H. C., Germeyer A., Steiner S. M., Bruce-Keller A. J., Hutchins J. B. and Mattson M. P. (1998) Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J. Neurosci. 18, 687±697. 34. Klein M., MoÈller J., Jones L., Bluethmann H., Kreutzberg G. W. and Raivich G. (1997) Impaired neuroglial activation in interleukin-6 de®cient mice, Glia 19, 227-233. 35. Kondo Y., Rusnak J., Hoyt D., Settineri C., Pitt B. and Lazo J. (1997) Enhanced apoptosis in metallothionein null cells. Molec. Pharmac. 52, 195±201. 36. Kopf M., Baumann H., Freer G., Freudenberg M., Lamers M., Kishimoto T., Zinkernagel R., Bluethmann H. and KoÈhler G. (1994) Impaired immune and acute-phase responses in interleukin-6-de®cient mice. Nature 368, 339±342. 37. Kopf M., Ramsay A., Brombacher F., Baumann H., Freer G., Galanos C., Gutierrez-Ramos J. C. and Kohler G. (1995) Pleiotropic defects of IL6-de®cient mice including early hematopoiesis, T and B cell function, and acute phase responses. Ann. N.Y. Acad. Sci. 762, 308±318. 38. Kossmann T., Hans V., Imhof H. G., Trentz O. and Morganti-Kossmann M. C. (1996) Interleukin-6 released in human cerebrospinal ¯uid following traumatic brain injury may trigger nerve growth factor production in astrocytes. Brain Res. 713, 143±152. 39. Kreutzberg G. W. (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312±318. 40. Kristensen B. W., Noraberg J., Jakobsen B., Gramsbergen J. B., Ebert B. and Zimmer J. (1999) Excitotoxic effects of non-NMDA receptor agonists in organotypic corticostriatal slice cultures. Brain Res. 841, 143±159. 41. Kushima Y., Hama T. and Hatanaka H. (1992) Interleukin-6 as a neurotrophic factor for promoting the survival of cultured catecholaminergic neurons in a chemically de®ned medium from fetal and postnatal rat midbrains. Neurosci. Res. 13, 267±280. 42. Lazo J. S., Kondo Y., Dellapiazza D., Michalska A. E., Choo K. H. and Pitt B. R. (1995) Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice de®cient in metallothionein I and II genes. J. biol. Chem. 270, 5506±5510. 43. Lazo J. S., Kuo S. M., Woo E. S. and Pitt B. R. (1998) The protein thiol metallothionein as an antioxidant and protectant against antineoplastic drugs. Chem. Biol. Interact. 111±112, 255±262. 44. Lazo J. S. and Pitt B. R. (1995) Metallothioneins and cell death by anticancer drugs. A. Rev. Pharmac Toxicol. 35, 635±653. 45. Lee D. K., Carrasco J., Hidalgo J. and Andrews G. K. (1999) Identi®cation of a signal transducer and activator of transcription (STAT) binding site in the mouse metallothionein- I promoter involved in interleukin-6-induced gene expression. Biochem. J. 337, 59±65. 46. Li Y., Copin J. C., Reola L. F., Calagui B., Gobbel G. T., Chen S. F., Sato S., Epstein C. J. and Chan P. H. (1998) Reduced mitochondrial manganese-superoxide dismutase activity exacerbates glutamate toxicity in cultured mouse cortical neurons. Brain Res. 814, 164±170. 47. Liu J., Liu Y., Habeebu S. S. and Klaassen C. D. (1998) Metallothionein (MT)-null mice are sensitive to cisplatin-induced hepatotoxicity. Toxicol. appl. Pharmac. 149, 24±31. 48. Loddick S. A., Turnbull A. V. and Rothwell N. J. (1998) Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat. J. cereb. Blood Flow Metab. 18, 176±179. 49. Maeda Y., Matsumoto M., Hori O., Kuwabara K., Ogawa S., Yan S. D., Ohtsuki T., Kinoshita T., Kamada T. and Stern D. M. (1994) Hypoxia/ reoxygenation-mediated induction of astrocyte interleukin 6: a paracrine mechanism potentially enhancing neuron survival. J. exp. Med. 180, 2297±2308. 50. Minami M., Kuraishi Y. and Satoh M. (1991) Effects of kainic acid on messenger RNA levels of IL-1 beta, IL-6, TNF alpha and LIF in the rat brain. Biochem. biophys. Res. Commun. 176, 593±598. 51. Munoz-Fernandez M. A. and Fresno M. (1998) The role of tumour necrosis factor, interleukin 6, interferon-gamma and inducible nitric oxide synthase in the development and pathology of the nervous system. Prog. Neurobiol. 56, 307±340. 52. Murphy P. G., Borthwick L. S., Johnston R. S., Kuchel G. and Richardson P. M. (1999) Nature of the retrograde signal from injured nerves that induces interleukin-6 mRNA in neurons. J. Neurosci. 19, 3791±3800. 53. Niquet J., Ben-Ari Y. and Represa A. (1994) Glial reaction after seizure induced hippocampal lesion: immunohistochemical characterization of proliferating glial cells. J. Neurocytol. 23, 641±656. 54. Penkowa M., Carrasco J., Giralt M., Moos T. and Hidalgo J. (1999) CNS wound healing is severely depressed in metallothionein I- and IIde®cient mice. J. Neurosci. 19, 2535±2545. 55. Penkowa M., Giralt M., Moos T., Thomsen P. S., HernaÂndez J. and Hidalgo J. (1999) Impaired in¯ammatory response to glial cell death in genetically metallothionein-I- and -II-de®cient mice. Expl Neurol. 156, 149±164. 56. Penkowa M and Hidalgo J. (2000) IL-6 de®ciency leads to reduced metallothionein-I 1 II expression and increased oxidative stress in the brain stem after 6-aminonicotinamide treatment. Exp. Neurol. 163, 72±84.

818

M. Penkowa et al.

57. Penkowa M., Moos T., Carrasco J., Hadberg H., Molinero A., Bluethmann H. and Hidalgo J. (1999) Strongly compromised in¯ammatory response to brain injury in interleukin-6-de®cient mice. Glia 25, 343±357. 58. Perry V., Bell M., Brown H. and Matyszak M. (1995) In¯ammation in the nervous system. Curr. Opin. Neurobiol. 5, 636±641. 59. Pitt B. R., Schwarz M., Woo E. S., Yee E., Wasserloos K., Tran S., Weng W., Mannix R. J., Watkins S. A., Tyurina Y. Y., Tyurin V. A., Kagan V. E. and Lazo J. S. (1997) Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury. Am. J. Physiol. 273, L856±865. 60. Przedborski S., Khan U., Kostic V., Carlson E., Epstein C. J. and Sulzer D. (1996) Increased superoxide dismutase activity improves survival of cultured postnatal midbrain neurons. J. Neurochem. 67, 1383±1392. 61. Raivich G., Bohatschek M., Kloss C. U., Werner A., Jones L. L. and Kreutzberg G. W. (1999) Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res. Rev. 30, 77±105. 62. Reaume A. G., Elliott J. L., Hoffman E. K., Kowall N. W., Ferrante R. J., Siwek D. F., Wilcox H. M., Flood D. G., Beal M. F., Brown R. H. Jr, Scott R. W. and Snider W. D. (1996) Motor neurons in Cu/Zn superoxide dismutase-de®cient mice develop normally but exhibit enhanced cell death after axonal injury. Nat. Genet. 13, 43±47. 63. Rosenberg G. A., Dencoff J. E., Correa N. Jr, Reiners M. and Ford C. C. (1996) Effect of steroids on CSF matrix metalloproteinases in multiple sclerosis: relation to blood±brain barrier injury. Neurology 46, 1626±1632. 64. Rothstein J. D., Bristol L. A., Hosler B., Brown R. H. Jr and Kuncl R. W. (1994) Chronic inhibition of superoxide dismutase produces apoptotic cell death of spinal neurons. Proc. natn. Acad. Sci. USA 91, 4155±4159. 65. Savolainen K. M., Loikkanen J., Eerikainen S. and Naarala J. (1998) Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress: glutamate and lead. Toxicol. Lett. 102±103, 363±367. 66. Schwarz M. A., Lazo J. S., Yalowich J. C., Allen W. P., Whitmore M., Bergonia H. A., Tzeng E., Billiar T. R., Robbins P. D., Lancaster J. R. Jr and Pitt B. R. (1995) Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide. Proc. natn. Acad. Sci. USA 92, 4452±4456. 67. Sievers J., Parwaresch R. and Wottge H. U. (1994) Blood monocytes and spleen macrophages differentiate into microglia-like cells on monolayers of astrocytes: morphology. Glia 12, 245±258. 68. Sperk G. (1994) Kainic acid seizures in the rat. Prog. Neurobiol. 42, 1±32. 69. Sun A. Y. and Chen Y. M. (1998) Oxidative stress and neurodegenerative disorders. J. Biomed. Sci. 5, 401±414. 70. Tamai K. T., Gralla E. B., Ellerby L. M., Valentine J. S. and Thiele D. J. (1993) Yeast and mammalian metallothioneins functionally substitute for yeast copper±zinc superoxide dismutase. Proc. natn. Acad. Sci. USA 90, 8013±8017. 71. Toulmond S., Vige X., Fage D. and Benavides J. (1992) Local infusion of interleukin-6 attenuates the neurotoxic effects of NMDA on rat striatal cholinergic neurons. Neurosci. Lett. 144, 49±52. 72. Tsangaris G. T. and Tzortzatou-Stathopoulou F. (1998) Metallothionein expression prevents apoptosis: a study with antisense phosphorothioate oligodeoxynucleotides in a human T cell line. Anticancer Res. 18, 2423±2433. 73. Westland K. W., Pollard J. D., Sander S., Bonner J. G., Linington C. and McLeod J. G. (1999) Activated non-neural speci®c T cells open the blood±brain barrier to circulating antibodies. Brain 122, 1283±1291. 74. Wilms H., Hartmann D. and Sievers J. (1997) Rami®cation of microglia, monocytes and macrophages in vitro: in¯uences of various epithelial and mesenchymal cells and their conditioned media. Cell Tissue Res. 287, 447±458. 75. Yamada M. and Hatanaka H. (1994) Interleukin-6 protects cultured rat hippocampal neurons against glutamate-induced cell death. Brain Res. 643, 173±180. 76. Zheng H., Liu J., Choo K. H., Michalska A. E. and Klaassen C. D. (1996) Metallothionein-I and -II knock-out mice are sensitive to cadmiuminduced liver mRNA expression of c-jun and p53. Toxicol. appl. Pharmac. 136, 229±235. (Accepted 30 October 2000)