Metallothionein-1+2 Deficiency Increases Brain Pathology in Transgenic Mice with Astrocyte-Targeted Expression of Interleukin 6

Neurobiology of Disease 9, 319 –338 (2002) doi:10.1006/nbdi.2002.0480, available online at http://www.idealibrary.com on

Metallothionein-1ⴙ2 Deficiency Increases Brain Pathology in Transgenic Mice with AstrocyteTargeted Expression of Interleukin 6 Mercedes Giralt,* ,1 Milena Penkowa, †,1 Joaquı´n Herna´ndez,* Amalia Molinero,* Javier Carrasco,* Natalia Lago,* Jordi Camats,* Iain L. Campbell, ‡ and Juan Hidalgo* ,2 *Instituto de Neurociencı´as and Departamento de Biologia Celular, de Fisiologı´a y de Inmunologı´a, Unidad de Fisiologı´a Animal, Facultad de Ciencias, Universidad Auto´noma de Barcelona, Bellaterra, Barcelona, Spain 08193; †Institute of Medical Anatomy, Section C, The Panum Institute, University of Copenhagen, DK-2200, Copenhagen, Denmark; and ‡ Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037 Received August 23, 2001; revised December 12, 2001; accepted January 25, 2002

Transgenic expression of IL-6 under the control of the GFAP gene promoter (GFAP-IL6 mice) in the CNS causes significant damage and alters the expression of many genes, including the metallothionein (MT) family, especially in the cerebellum. The crossing of GFAP-IL6 mice with MT-1ⴙ2 knock out (MTKO) mice provided evidence that the increased MT-1ⴙ2 expression normally observed in the GFAP-IL6 mice is an important mechanism for coping with brain damage. Thus, the GFAP-IL6xMTKO mice showed a decreased body weight gain and an impaired performance in the rota-rod test, as well as a higher upregulation of cytokines such as IL-6, IL-1␣,␤, and TNF␣ and recruitment and activation of macrophages and T cells throughout the CNS but mainly in the cerebellum. Clear symptoms of increased oxidative stress and apoptotic cell death caused by MT-1ⴙ2 deficiency were observed in the GFAP-IL6xMTKO mice. Interestingly, MT-1ⴙ2 deficiency also altered the expected frequency of the offspring genotypes, suggesting a role of these proteins during development. Overall, the results suggest that the MT-1ⴙ2 proteins are valuable factors against cytokine-induced CNS injury. © 2002 Elsevier Science (USA)

INTRODUCTION

presumably contribute importantly to the clinicopathological features of many neurological disorders, and indeed results obtained in transgenic mice with astrocyte targeted expression of cytokines such as IL-6 (GFAP-IL6 mice) (Campbell et al., 1993; Chiang et al., 1994; Heyser et al., 1997; Powell & Campbell, 1994; Steffensen et al., 1994; Castelnau et al., 1998; Brett et al., 1995; Barnum et al., 1996; Asensio et al., 1999; Nelson et al., 1999), IL-3 (Chiang et al., 1996; Powell et al., 1999a; Powell et al., 1999b; Asensio et al., 1999), IFN-␣ (Akwa et al., 1998; Campbell et al., 1999; Carr et al., 1998; Asensio et al., 1999), TNF-␣ (Stalder et al., 1998; DeLeo et al., 2000), and IL-12 (Pagenstecher et al., 2000) have demonstrated that an abnormal cytokine production may cause significant neurological disorders. On the other hand, these transgenic mice are useful animal

Cytokines are essential mediators of cell– cell communication in the CNS, being astrocytes, microglia, and macrophages major sources (Hopkins & Rothwell, 1995; Rothwell & Hopkins, 1995; Stichel & Verner Mu¨ller, 1998; McIntosh et al., 1998). In normal conditions, the cellular expression of cytokines in the CNS is very low or absent and is under tight control. Yet, in a number of neuropathological conditions the expression of some cytokines is significantly altered (see Campbell, 1998, for review). Such alterations could 1

M.G. and M.P. contributed equally to this paper. To whom correspondence and reprint requests should be addressed. Fax: ⫹34 93 581 23 90. E-mail: [email protected]. 2

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320 models for the characterization of the mechanisms underlying cytokine-induced pathological conditions in the CNS as well as for identifying potentially important genes for coping with CNS damage. One such family of genes is the metallothionein (MT) family, which we have studied in the GFAP-IL6 mice. These transgenic mice show profound effects of IL-6 in the brain, with clear symptoms of neurodegeneration, astrocytosis, microgliosis, angiogenesis, and up-regulation of several inflammatory and other host–response genes including IL-1 ␣/␤, TNF␣, GFAP, ICAM-1, the acute-phase protein EB22/5, and complement C3 protein. Moreover, concordant with these structural alterations, GFAP-IL6 mice have impaired hippocampal electrophysiology and develop a progressive learning deficit. MTs are cysteine-rich, heavy metal binding proteins, which in rodents are comprised of four isoforms (MT-1 to MT-4) (Palmiter et al., 1992; Quaife et al., 1994). Only three isoforms are expressed in the brain, namely MT-1⫹2 (which are also widely expressed, and regulated coordinately) and MT-3 (also known as Growth Inhibitory Factor). MTs bind zinc and copper and presumably function in metal ion regulation and detoxification in peripheral tissues as well as in the CNS, while more recent evidence suggest that MTs could be significant antioxidant proteins (see Aschner et al., 1997; Hidalgo et al., 1997, 2001, for review). In previous studies we have shown that MT-1⫹2 are dramatically increased in the brains of the GFAP-IL6 mice (Herna´ ndez et al., 1997; Carrasco et al., 1998), which presumably forms part of the physiological repertoire elicited in the brain for coping with tissue injury. To test this possibility, we have crossed GFAPIL6 mice with MT-1⫹2 null mice and have analyzed the outcome. The results fully support the hypothesis that MT-1⫹2 are essential proteins for coping with brain injury.

METHODS Animals Homozygous MT-1⫹2 knock-out (MTKO or MT⫺/⫺) mice generated as previously described (Masters et al., 1994a), were originally purchased from Jackson Laboratories (U.S.A). A colony has been established at the Autonomous University of Barcelona, and MTKO mice are routinely genotyped by Southern blot as previously described (Masters et al., 1994a). The MTKO mice were raised and propagated on the 129/Sv genetic background. 2002 Elsevier Science (USA) All rights reserved.

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Construction and characterization of the GFAP-IL6 transgenic mice has been described previously (Campbell et al., 1993). Briefly, an expression vector derived from the murine glial fibrillary acidic protein (GFAP) gene was used to target expression of IL-6 to astrocytes. The GFAP-IL6 mice have a mixed C57B6 ⫻ SJL genetic background. Production of GFAP-IL6-MTKO Mice For obtaining GFAP-IL6 mice with genetic deficiency of MT-1⫹2, we initiated a series of crosses as follows. First, heterozygous GFAP-IL6 mice were crossed with MT-KO mice. The offspring mice were all heterozygous for MT-1⫹2 (MT⫹/⫺), and those animals that were heterozygous for the transgenic IL-6 were identified by PCR as previously described (Penkowa et al., 2000). The GFAP-IL6⫹/⫺ MT⫹/⫺ mice were crossed again with MT-KO mice. The offspring of this second cross were genotyped by PCR and Southern blot for identifying the four possible genotypes: (a) GFAP-IL6⫹/⫺ MT⫹/⫺, (b) GFAP-IL6⫹/⫺ MT⫺/⫺, (c) GFAP-IL6⫺/⫺ MT⫹/⫺, and (d) GFAPIL6⫺/⫺ MT⫺/⫺. These F2 mice have a 75% genetic background of the 129/Sv strain, and can be compared between them directly. This experimental design was followed in two separate experiments. In some experiments we wanted to compare GFAPIL6⫹/⫺ MT⫺/⫺ mice with GFAP-IL6⫹/⫺ MT⫹/⫹ mice; in order to obtain the latter mice with a more comparable genetic background that the parental GFAP-IL6 mice, we initiated a second series of crossings as follows. GFAP-IL6⫹/⫺ MT⫺/⫺ animals of the F2 offspring described above were crossed with normal 129/Sv mice, and their offspring genotyped. F3 GFAP-IL6⫹/⫺ MT⫹/⫺ animals were then crossed again with 129/Sv mice, and their offspring genotyped to select animals that were GFAP-IL6⫹/⫺ MT⫹/⫹. These F4 mice have theoretically 93.75% genetic background of the 129/Sv strain, and thus can conservatively be compared with the F2 offspring described above. Motor Coordination Motor coordination was assayed using a rota-rod apparatus (Ugo Basile Rota-rod treadmille, Model 7600). The mice were first trained during 2 consecutive days by habituating them to the rota-rod room for 15 min, before a 5-min period in the rota-rod apparatus set at 16 rpm. After this training, the mice were subjected to the rota-rod test during 3 consecutive days.

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Metallothioneins Protect Against IL-6-Induced Injury

Each day, the animals were habituated to the rota-rod room for 15 min, subjected to the rota-rod test for 5 min at 16 rpm, returned to the animal room for 1 h, and subjected again to the rota-rod test again for 5 min but at 32 rpm. The number of drops during each of the periods was annotated. All experiments were carried out starting at 3 P.M., and 5–11 animals per group were studied. In Situ Hybridization Two-four mice per group were used for analyzing MT-1 and MT-3 expression by in situ hybridization. The mice were killed and their brains removed, frozen in liquid nitrogen, and stored at ⫺80°C. Later, serial coronal sections (20 ␮m in thickness) were obtained with a cryostat and mounted on slides coated with poly(l)lysine. The in situ hybridization analysis of the MT-1 and MT-3 isoforms was carried out as previously described (Carrasco et al., 1998), see below for details. The MT-1 and MT-2 isoforms are regulated coordinately (Yagle & Palmiter, 1985), also in the brain (Masters et al., 1994b; van Lookeren Campagne et al., 2000), and thus we assume that the results for MT-1 are representative of those of MT-2. For MT-1 mRNA studies, a mouse cDNA was used kindly provided by Dr. R. D. Palmitter, University of Washington WA. For MT-3 mRNA studies we have used a specific DNA fragment of 153 bp that contains the coding region for the terminal 15 aminoacids and the 3⬘ untranslated region until the poly G stretch of MT-3 mRNA, generously provided by Dr. G.K. Andrews, University of Kansas Medical Center, KS. Both the MT-1 and MT-3 cDNAs were labelled with [␣- 35S]UTP using a SP6/T7 transcription kit (Boehringer-Mannheim, Mannheim, Germany). After the transcription process, the DNA was digested by adding 1 ␮l of DNase and incubating for 30 min at 37°C. After DNAse treatment, the RNA was extracted with phenol and phenol: cloroform: isoamyl alcohol (25:24: 1). The upper phase (200 ␮l) was recovered and incubated overnight with 1 ␮l of tRNA (10 mg/ml), 10 ␮l of 3M sodium acetate, and 500 ␮l of 100% ethanol to precipitate the RNA. After centrifugation the precipitated probe was dissolved in 50 ␮l of RNAse free H 2O. In situ hybridization was performed using procedures described by Yuguchi et al. (1995) with some modifications: the sections were incubated with 0.1 N HCl instead of proteinase K, and we used RNAse at 10 ␮g/ml instead of 1 ␮g/ml to digest the free probe. The concentration of probe used was 1 ⫻ 10 6 dpm/90 ␮l/slide. Autoradiography was performed exposing

the film (hyperfilm-MP, Amersham) to the slides for 6 days. All sections to be compared were prepared simultaneously and exposed to the same autoradiographic film. MT-1 or MT-3 mRNA levels were semiquantitatively determined in 4 sections per brain area and animal, by measuring the optical densities and the number of pixels in defined areas with a Leica Q 500 MC system. The MT-1 and MT-3 mRNA values shown are expressed in arbitrary units (number of pixels ⫻ optic density).

RNase Protection Assay RNase protection assay for the detection of cytokine and host-gene RNA was performed with four to six animals per group as described previously (Hobbs et al., 1993; Campbell et al., 1994; Chiang et al., 1994). For the synthesis of a radiolabeled antisense RNA probe set, the final reaction mixture contained 120 ␮Ci [␣- 32P]UTP (3000 Ci/mmol, Amersham), UTP (73 pmol), GTP, ATP, CTP (2.5 mmol each), DTT (100 nmol), transcription buffer (1⫻), Rnasin (20U), T7 polymerase (10U) (all from Promega), and an equimolar pool of EcoRI-linearized templates (150 ng total). After 1 h at 37°C the mixture was treated with DNase I (1 U, Promega) for 45 min at 37°C and the probe was purified by extraction with phenol/chloroform and precipitated with ethanol. Dried probe was then dissolved (2.2 ⫻ 10 5 cpm/␮l) in hybridization buffer (HB: 80% formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM Pipes, pH 6.4) and 2 ␮l of this added to the tubes containing target RNA dissolved in 8 ␮l HB. The samples were overlaid with mineral oil, heated to 96°C, and then incubated at 56°C for 12–16 h. Singlestranded RNA was digested by addition of a mixture of RNase A (1 mg/ml) and RNase T1 (125 U/␮l: Bethesda laboratories, Bethesda, MD) in 10 mM Tris, 300 mM NaCl, 5 mM EDTA, pH 7.5. Following incubation for 45 min at 32°C, 18 ␮l of a mixture containing proteinase K (0.5 mg/ml, Beckman, Fullerton, CA), SDS (3.5%), and yeast tRNA (100 ␮g/ml, Sigma) was added and the samples incubated for a further 30 min at 37°C. The RNA duplexes were isolated by extraction/precipitation as described above, dissolved in 80% formamide and dyes, and electrophoresed in a standard 5% acrylamide/7 M urea TBE sequencing gel. Dried gels were placed on XAR film (Kodak, Rochester, NY) with intensifying screens, and were developed at ⫺70°C. The intensity of the bands was measured densitometrically and normalized with the L32 mRNA levels. ©

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322 Histochemistry and Immunohistochemistry Animals were studied at different ages up to 1 year old; typically 5–10 animals per group were used, and in some stainings cell counts were carried out in a blinded manner in the cerebellum in four animals per group for statistical purposes. Tissue processing. Mice were deeply anesthetized with 10 mg/100 g body weight of Brietal (Methohexital 10 mg/ml, Eli Lilly) and were transcardially perfused with 0.9% saline with 0.3% heparin (15000 IU/ liter) for 3–5 min followed by perfusion with 0.1% Na 2S in 0.1 M PBS, pH 7.4 for 5 min, followed by perfusion with Zamboni’s fixative (buffered 4% formaldehyde added 15% picric acid solution from a 1.2% saturated aqueous picric acid solution) for 8 –10 min, pH 7.4. Afterwards, all the brains were fixed by immersion in Zamboni’s fixative for 4 h, pH 7.4, at room temperature. Brains were dehydrated according to standard procedures, embedded in paraffin, and cut in serial, coronal 3-␮m-thick sections. Sections were rehydrated, and for heat-induced antigen retrieval, sections were boiled in citrate buffer, pH 9.1 or 6.0 in a microwave oven for 10 min. After cool down to room temperature, the sections were incubated in 1.5% H 2O 2 in Tris-buffered saline (TBS)/Nonidet (TBS: 0.05 M Tris, pH 7.4, 0.15 M NaCl; with 0.01% Nonidet P-40) (Sigma-Aldrich, code N-6507) for 15 min at room temperature to quench endogenous peroxidase. Afterwards, sections were incubated with 10% normal goat serum (In Vitro, DK, code 04009-1B) in TBS/Nonidet for 30 min at room temperature in order to block nonspecific binding. Histochemistry. Sections were incubated overnight at 4°C with biotinylated tomato lectin from Lycopersicon esculentum (Sigma-Aldrich, code L9389) 1:500, which was used as a marker for cells of the myelomonocytic cell lineages, such as microglia/macrophages, as well as a marker for vessels. The lectin was developed using streptavidin-biotin-peroxidase complex (StreptABComplex/HRP, Dako, DK, code K377) prepared at manufacturer’s recommended dilutions for 30 min at room temperature. The lectin staining was visualized using 0.015% H 2O 2 in 3,3-diaminobenzidine-tetrahydrochloride (DAB)/TBS for 10 min at room temperature. Besides, hematoxyline eosine stainings of brain sections were performed according to standard procedures. Immunohistochemistry. The sections were incubated overnight at 4°C with one of the following antibodies: rat anti-mouse IL-6 1:50 (Harlan Seralab, 2002 Elsevier Science (USA) All rights reserved.

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code MAS584); mouse anti-human IL-1␤ 1:50 (Biogenesis, code 5375-4329); rabbit anti-mouse TNF-␣ 1:100 (Biosource, code AMC 3012); rabbit anti-rat MT-1⫹2 1:500 (Gasull et al., 1993, 1994; Penkowa et al., 1999); rabbit anti-bovine GFAP 1:250 (Dako, DK, code Z 334) (as a marker for astrocytes); mouse anti-rat CD3 1:50 (Serotec, UK, code KD MCA 772) (as a marker for all T lymphocytes); rabbit anti-MDA 1:100 (Alpha Diagnostic Int., code MDA 11-S) (a marker for malondialdehyde (MDA) produced as a byproduct of fatty acid peroxidation, which monitors oxidative stress); rabbit anti-NITT 1:100 (Alpha Diagnostic Int., code NITT 12-A) (a marker for peroxynitrite-induced nitration of tyrosine residues, which monitors oxidative stress); rabbit anti-human activated caspase-3 1:50 (Cell Signaling Technology Inc., code 9661) (as a marker for apoptotic signaling); mouse anti-calf ssDNA 1:25 (Alexis Biochemicals, code 804-192-R200) (as a marker for single stranded DNA in apoptotic cells). These primary antibodies were detected using biotinylated goat anti-rat IgG 1:1500 (Amersham, UK, code 1005), or biotinylated mouse anti-rabbit IgG 1:400 (SigmaAldrich, code B3275), or biotinylated goat anti-mouse IgG 1:200 (Sigma-Aldrich, code B8774), or biotinylated goat anti-mouse IgM (␮-chain-specific) 1:10 (Jackson ImmunoRes. Lab. Inc., code 115-065-020), by incubating the sections for 30 min at room temperature followed by StreptABComplex/HRP (as above). Afterwards, sections were incubated with biotinylated tyramide and streptavidin-peroxidase complex (NEN, Life Science Products, code NEL700A) prepared following manufacturer’s recommendations. The immunoreaction was visualized using DAB as a chromogen (as above). In order to evaluate the extent of non-specific binding of the antisera in the immunohistochemical experiments, the primary antibody step was omitted. A further control for some anti-CD and anti-cytokine antibodies was to preabsorb the primary antibodies with their corresponding antigenic proteins before the immunohistochemical reaction was carried out. For this purpose the following blocking peptides were used: mouse IL-6 peptide (RD Systems, UK, code 406ML-005); mouse IL-1␤ peptide (Santa Cruz Biotech. Inc., code sc-1251P); mouse TNF-␣ peptide (RD Systems, UK, code 410-MT-010); and mouse CD3 peptide (Santa Cruz Biotech. Inc., code sc-1127P). Results were considered only if these controls were negative. In situ detection of DNA fragmentation. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labeling (TUNEL) staining was performed according to

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manufacturer’s protocol and after tissue processing as mentioned above. Sections were incubated with 20 mg/ml Proteinase K (Sigma-Aldrich, code P2308) for 5 min to strip off nuclear proteins. TUNEL was accomplished using the Apoptag Plus, In Situ Apoptosis Detection Kit (Oncor, Code S7101-KIT). After immersion in equilibration buffer for 10 min, sections were incubated with TdT and dUTP-digoxigenin in a humified chamber at 37°C for 1 h and then incubated in the stop/wash buffer at 37°C 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 from (Oncor, code S7115). Furthermore, TUNEL stainings were compared with stainings for apoptotic markers (ssDNA and activated caspase-3). In addition, the morphologic criteria for apoptosis (cell shrinkage, formation of apoptotic bodies, membrane blebbing, no loss of cellular integrity, compaction of chromatin into uniformly dense masses) were evaluated. Triple labeling. In order to determine which cells suffered from oxidative stress and/or apoptosis, sections were were incubated with fluorescein (FITC)linked TUNEL (Oncor, code S7111-KIT) according to manufacturers protocol, and afterwards incubated overnight at 4°C with mouse anti-human NF 1:250 (Dako, DK, code M762) (as a neuronal marker) or instead with mouse anti-porcine vimentin 1:50 (Dako, DK, code M0725) (as a marker for both reactive astrocytes and macrophages) and simultaneously with either rabbit anti-NITT, or rabbit anti-MDA (as above). The monoclonal antibodies were detected by using goat anti-mouse IgG linked with TexasRed (TXRD) 1:50 (Southern Biotechnology Ass., code 1030-07), and the polyclonal antibodies were detected by using goat anti-rabbit IgG linked with aminomethylcoumarin (AMCA) 1:20 (Jackson ImmunoResearch Lab. Inc., code 111-155-144). The sections were embedded in 20 ␮l Fluorescent mounting (Dako, DK, code S3023) and kept in darkness at 4°C. In order to evaluate the extent of nonspecific binding of the antisera in the fluorescence stainings, control sections were incubated in the absence of primary antibody. Results were considered only if these controls were negative. For the simultaneous examination and recording of the three stains, a

Zeiss Axioplan2 light microscope equipped with a tripleband (FITC/TXRD/AMCA) filter was used. Cell counts. For statistical evaluation of the results, positively stained cells were counted from matched, 0.35 mm 2 areas of 3-␮m-thick sections of cerebellum of the different groups. Positively stained cells were defined as those cells with cytoplasmic staining, except from the TUNEL- and ssDNA-positive cells, which were defined as cells with nuclear staining. Cell counts were performed in a blinded manner in four mice per group. Statistical Analysis Results were analyzed with the Student t test, the Mann–Whitney U test, or with two-way ANOVA.

RESULTS MT-1ⴙ2 Deficiency Alters the Expected Frequency of the Offspring Genotypes We carried out two separate series of crossings. In both cases, the F1 offspring showed a normal frequency of the offspring, being about 50% of each sex. Also as expected, about 50% of the offspring were GFAP-IL6⫹/⫺ (data not shown). A different pattern emerged in the F2 offspring. Table 1 shows the data of the two separate series of crossings. As expected, about 50% of the offspring were of each sex. Also, about 50% of males and females were GFAP-IL6⫹/⫺. But when the frequencies of MT⫺/⫺ and MT⫹/⫺ genotypes were examined, it was observed that in contrast to the expected 50% frequency within each group, the GFAP-IL6⫹/⫺ females had a significantly smaller frequency of the MT⫺/⫺ genotype. A similar but milder tendency was observed in the GFAPIL6⫹/⫺ males. MT-1ⴙ2 Deficiency Potentiates Neurological Disease in Mice Expressing GFAP-IL6 Transgene Mice expressing GFAP-IL6 transgene developed the expected hunched posture, tremor, ataxia, and hindlimb weakness. In an attempt to identify an effect of MT-1⫹2 deficiency on the neurological disease induced by transgenic IL-6, the motor coordination of mice from all groups was evaluated with a rota-rod apparatus at 6 months (n ⫽ 5–11) and 12-month age (n ⫽ 7–10). As expected, animals dropped more times at 32 rpm than at 16 rpm, and at 12-month than at ©

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TABLE 1 Frequencies of Gender and Genotypes of the F2 Offspring Males (Exp. 1/2: n ⫽ 85/154) GFAP-IL6⫹/⫺ (n ⫽ 46/77)

Females (Exp. 1/2: n ⫽ 99/148)

GFAP-IL6⫺/⫺ (n ⫽ 39/77)

GFAP-IL6⫹/⫺ (n ⫽ 54/71)

GFAP-IL6⫺/⫺ (n ⫽ 45/77)

MT⫹/⫺

MT⫺/⫺

MT⫹/⫺

MT⫺/⫺

MT⫹/⫺

MT⫺/⫺

MT⫹/⫺

MT⫺/⫺

23/43

23/34

19/ND

20/ND

43/45

11/26

26/ND

19/ND

Note. Two separate crossings were carried out as described under Materials and Methods (Exp. 1/2). The main conclusion is that the Mendelian distribution is not maintained when the mice have concomitantly the GFAP-IL6 transgene and MT-1⫹2 deficiency, especially the females. ND, not determined.

6-month age. The expression of the GFAP-IL6 transgene tended to increase the number of droppings in 6-month-old mice (P ⬍ 0.084), and this number decreased (P ⬍ 0.001) in consecutive trials similarly in all groups (data not shown); MT-1⫹2 deficiency did not affect the motor coordination at that age. In contrast, in 12-month-old animals the number of droppings at 16 rpm did not decrease in consecutive trials, and motor coordination was impaired by the GFAP-IL6 transgene in the MTKO mice but not in MT⫹/⫺ mice (Fig. 1). MT-1ⴙ2 Deficiency Decreases Body Weight Gain As expected (Campbell et al., 1993), old GFAP-IL6 mice tended to weigh less than littermate controls. In contrast, it was unexpected to find that body weight gain was significantly decreased by MT-1⫹2 defi-

ciency in both GFAP-IL6⫹/⫺ mice and littermate controls (Table 2). MT-3 Expression Is Not Affected by MT-1ⴙ2 Deficiency in GFAP-IL6 Mice As expected (Herna´ ndez et al., 1997; Carrasco et al., 1998), the transgenic expression of IL-6 altered MT-1 and MT-3 expression in a brain area-specific manner, with the cerebellum the area most affected. The IL6 encoded by the transgene was associated with a significant upregulation of both MT isoforms in the cerebellum, being the induction much more prominent for MT-1 than for MT-3 (Table 3). The response of the latter in GFAP-IL6 MT⫺/⫺ mice was similar to that in GFAP-IL6 MT⫹/⫺ mice, indicating that MT-3 expression was not affected by MT-1⫹2 deficiency. MT-1⫹2 protein determination by immunohistochemistry con-

FIG. 1. Rota-rod performance of the F2 offspring. Motor control of the F2 offspring was evaluated with a rota-rod apparatus. The figure shows the performance (number of falls/5 min, mean ⫾ SE, n ⫽ 7–10) at 16 rpm of the 12-month-old male mice. Motor coordination was impaired by the GFAP-IL6 transgene in the MTKO mice but not in MT⫹/⫺ mice.

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Metallothioneins Protect Against IL-6-Induced Injury TABLE 2 Body Weight Gain of the F2 Offspring.

Genotype GFAP-IL6⫹/⫺ MT⫹/⫺ GFAP-IL6⫹/⫺ MT⫺/⫺ GFAP-IL6⫺/⫺ MT⫹/⫺ GFAP-IL6⫺/⫺ MT⫺/⫺

Females, 6 months

Males, 12 months

29.5 ⫾ 1.30 (n ⫽ 10) 26.0 ⫾ 1.21* (n ⫽ 5) 29.7 ⫾ 0.94 (n ⫽ 11) 28.2 ⫾ 0.89* (n ⫽ 9)

44.5 ⫾ 1.74 (n ⫽ 9) 39.4 ⫾ 1.75** (n ⫽ 10) 46.5 ⫾ 1.22 (n ⫽ 8) 42.5 ⫾ 1.67** (n ⫽ 7)

Note. Body weight (g) attained at 6 and 12 months of age is shown. Results (mean ⫾ SE) were evaluated with two-way ANOVA with GFAP-IL6 transgene and MT-1⫹2 deficiency as main factors. MT⫺/⫺ mice weighed less than MT⫹/⫺ mice (*P ⬍ 0.05; **P ⬍ 0.025).

firmed the MT-1 mRNA in situ results (data not shown). MT-1ⴙ2 Deficiency Increases GFAP-IL6-Induced Cytokine Expression Figure 2 shows IL-6 immunostaining results of the cerebellum. In GFAP-IL6⫺/⫺ MT⫹/⫺ and GFAPIL6⫺/⫺ MT⫺/⫺ mice IL-6 immunoreactivity was only seen in the meninges, while intraparenchymal cells of the CNS were hardly IL-6 positive (Figs. 2A and 2B). In all the GFAP-IL6⫹/⫺ mice IL-6 immunoreactivity increased throughout the CNS but especially in the cerebellum (Fig. 2). IL-6 immunoreactivity was mainly seen in round or amoeboid microglia/ macrophages, and in some lymphocytes and astrocytes of the cerebellum white and grey matter. IL-6 immunoreactivity was significantly higher in GFAPIL6⫹/⫺ MT⫺/⫺ mice than in either GFAP-IL6⫹/⫺

MT⫹/⫺ or GFAP-IL6⫹/⫺ MT⫹/⫹ mice, and increased progressively with age (Figs. 2C–2H). For statistical evaluation, cell counts were carried out in the cerebellum (see Fig. 9). We also carried out immunostainings for IL-1␤ and TNF␣. Similarly to IL-6, in GFAP-IL6⫺/⫺ and MT⫹/⫺ and GFAP-IL6⫺/⫺ MT⫺/⫺ mice immunoreactivity for both cytokines was only seen in the meninges and choroid plexus, while intraparenchymal cells of the CNS were hardly expressing IL-1␤ and TNF␣ (not shown). In GFAP-IL6⫹/⫺ mice, an increased number of IL-1␤ and TNF␣ positive cells were seen throughout the CNS but especially in the cerebellum, but such increase was significantly higher in GFAP-IL6⫹/⫺ MT⫺/⫺ mice (where a progressive increase was in addition observed with aging), than in either GFAP-IL6⫹/⫺ MT⫹/⫹ (see Fig. 3 for 8- to 10-month-old animals) or GFAP-IL6⫹/⫺ MT⫹/⫺ mice (see Fig. 9 for cell counts). Staining was observed in some neurons, astrocytes, microglia/macrophages, and lymphocytes. Accordingly, these immunohistochemistry results suggested that the expression of proinflammatory cytokines of the cerebellum was increased when MT1⫹2 were absent, and this pattern was observed throughout the CNS. In order to confirm the immunohistochemistry data, cytokine mRNA levels were measured in the cerebellum of 4-month-old animals by a specific ribonuclease protection assay that measures a number of cytokines simultaneously (Fig. 4). Densitometric quantitation and normalization of the cytokine mRNA with the L32 mRNA levels demonstrated that MT-1⫹2 deficiency potentiated GFAPIL-6-induced IL-6, IL-1␣, IL-1␤, and TNF␣ expression, while not altering the other cytokines analyzed (Table 4).

TABLE 3 Metallothionein Expression in Normal and Transgenic GFAP-IL6 Mice 129/Sv (n ⫽ 3)

Purkinje Granular Molecular

MT-KO (n ⫽ 2)

GFAP-IL6 MT⫹/⫺ (n ⫽ 4)

GFAP-IL6 MT⫺/⫺ (n ⫽ 4)

MT-1

MT-3

MT-1

MT-3

MT-1

MT-3

MT-1

MT-3

807 ⫾ 55 225 ⫾ 50 13 ⫾ 4

690 ⫾ 81 252 ⫾ 80 124 ⫾ 27

-

680 ⫾ 5 324 ⫾ 63 81 ⫾ 14

1842 ⫾ 79*** 1317 ⫾ 164*** 211 ⫾ 92*

1057 ⫾ 65*** 548 ⫾ 107** 194 ⫾ 56

-

1070 ⫾ 52*** 535 ⫾ 58** 149 ⫾ 22

Note. In situ hybridization analysis for MT-1 and MT-3 mRNA were carried out as described under Materials and Methods. All samples were processed in parallel and exposed to the same autoradiographic film. Results (arbitrary units; mean ⫾ SE) were evaluated with the Student t test or the Mann–Whitney U test (MT-1), or with two-way ANOVA with GFAP-IL6 transgene and MT-1⫹2 deficiency as main factors (MT-3). MT-1 was significantly upregulated in the cerebellar areas analyzed (*P ⬍ 0.05; ***P ⬍ 0.005 versus 129/Sv mice). The GFAP-IL6 transgene also increased MT-3 expression, but to a much lower extent than the MT-1 isoform (**P ⬍ 0.025; ***P ⬍ 0.005 versus normal mice).

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FIG. 2. IL-6 immunoreactivity in the cerebellum. (A, B) In GFAP-IL6⫺/⫺ MT⫹/⫺ mice (A) and GFAP-IL6⫺/⫺ MT⫺/⫺ (B) mice, IL-6 immunoreactivity was hardly seen in the CNS parenchyme. (C, D) 3-month-old mice. (E, F) 6-month-old mice. (G, H) 8- to 10-month-old mice. (C, E) GFAP-IL6⫹/⫺ MT⫹/⫺ mice. (G) GFAP-IL6⫹/⫺ MT⫹/⫹ mice. (D, F, H) GFAP-IL6⫹/⫺ MT⫺/⫺ mice. IL-6 was seen in round or amoeboid microglia/ macrophages and some lymphocytes in the cerebellum white and grey matter in all mice, but the number of IL-6 positive cells was clearly higher in the GFAP-IL6⫹/⫺ MT⫺/⫺ mice. Scale bars: A–H, 20 ␮m. 2002 Elsevier Science (USA) All rights reserved.

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FIG. 3. Expression of proinflammatory cytokines IL-1␤ and TNF-␣ in cerebellum of 8 –10 month old mice. (A, B) IL-1␤ expression is lower in GFAP-IL6⫹/⫺ MT⫹/⫹ mice (A) when compared to that of GFAP-IL6⫹/⫺ MT⫺/⫺ mice (B). (C, D) Also TNF-␣ immunoreactivity was significantly lower in the GFAP-IL6⫹/⫺ MT⫹/⫹ mice (C) when compared to that of GFAP-IL6⫹/⫺ MT⫺/⫺ mice (D). Thus, the levels of IL-1␤ and TNF-␣ are decreased in the CNS by MT expression. Scale bars: A–H, 20 ␮m.

MT-1ⴙ2 Deficiency Increases Microglia/Macrophage and T Lymphocyte Recruitment and Activation Figure 5 shows lectin staining, a marker of microglia/macrophages and blood vessels. In all the GFAPIL6⫺/⫺ mice, lectin staining was mainly observed in vessels in the cerebellum and throughout the CNS, although some ramified microglial cells were also stained (Figs. 5A and 5B). In all the mice with transgenic IL-6 expression, recruitment/activation of microglia/macrophages was observed throughout the CNS, but the most pronounced response was observed in the cerebellum, where increased numbers of round or amoeboid or bushy microglia/macrophages were seen (Figs. 5C–5H). However, in GFAP-IL6⫹/⫺ MT⫺/⫺ mice of all ages studied, the number and activation (judging by cell morphology) or microglia/ macrophages was significantly increased relatively to that seen in GFAP-IL6⫹/⫺ mice with MT-1⫹2 expression (either ⫹/⫺ or ⫹/⫹) (see Figs. 5 and 9). In accordance with lectin stainings, Mac1 mRNA levels also tended to be higher in the cerebellum of GFAP-IL6⫹/⫺ MT⫺/⫺ than in GFAP-IL6⫹/⫺ MT⫹/⫹ mice (Table 4).

Results for CD3⫹ T lymphocytes were similar to those observed for macrophages: they were rarely seen in the CNS parenchyma of GFAP-IL6⫺/⫺ animals, their number increased significantly in all the GFAP-IL6⫹/⫺ mice but especially in the cerebellum, and the GFAP-IL6⫹/⫺ MT⫺/⫺ mice were the ones with the highest T cells content (data not shown). MT-1ⴙ2 Deficiency Alters Astrocyte/Bergmann Glia Reactivity GFAP immunostainings of the cerebellum of GFAP-IL6⫺/⫺ mice showed a faint staining in white and grey matter (Figs. 6A and 6B). In GFAPIL6⫹/⫺ mice with MT-1⫹2 expression (either MT⫹/⫺ or MT⫹/⫹), GFAP stainings increased progressively with age (Figs. 6C, 6E, and 6G), an effect also observed in other CNS areas albeit in a more moderate manner. Interestingly, in the 3-month-old GFAP-IL6⫹/⫺ MT⫺/⫺ mice the GFAP immunostaining of the molecular layer was increased, while that of the white matter was slightly decreased, in comparison to that observed in GFAP-IL6⫹/⫺ MT⫹/⫺ mice (Figs. 6C and 6D). In contrast, in other CNS areas no significant differ©

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FIG. 4. Analysis of cytokine expression. Cerebellum mRNA levels of a number of cytokines of 4-month-old mice were measured by RNase protection assay. As expected, only some of the cytokines were upregulated in the GFAP-IL6⫹/⫺ MT⫹/⫹ mice. MT⫺1⫹2 deficiency caused a further upregulation of some cytokines (TNF␣, IL-1␣,␤, and IL-6), as shown by the densitometric analysis of the bands (see Table 4).

ences were observed. In contrast to the 3-month-old animals, in the 6- and 8- to 10-month-old GFAPIL6⫹/⫺ MT⫺/⫺ mice a significant reduction of

GFAP stainings was observed in comparison to animals with MT-1⫹2 expression (Figs. 6E– 6H). Although the most prominent response was observed

TABLE 4 Analysis of Cerebellum Cytokine (See Fig. 4) and Host-Gene Expression by RNase Protection Assay in 4-Month-Old-Mice

IL-6 IL-1␣ IL-1␤ TNF␣ GFAP Mac1 ICAM EB22

GFAPIL6⫺/⫺ MT⫹/⫹

GFAPIL6⫺/⫺ MT⫺/⫺

GFAPIL6⫹/⫺ MT⫹/⫹

GFAPIL6⫹/⫺ MT⫺/⫺

ND ND ND ND 17.3 ⫾ 4.20 0.43 ⫾ 0.08 0.26 ⫾ 0.14 6.09 ⫾ 2.31

ND ND ND ND 71.3 ⫾ 12.6 † 0.99 ⫾ 0.51 1.02 ⫾ 0.18 † 13.32 ⫾ 1.67 †

18.9 ⫾ 2.81 ND 2.77 ⫾ 0.30 ND 157.8 ⫾ 24.1*** 6.41 ⫾ 1.01*** 6.13 ⫾ 1.36*** 82.7 ⫾ 8.25***

37.4 ⫾ 5.16** 0.96 ⫾ 0.27 5.09 ⫾ 0.56** 1.04 ⫾ 0.17 205.3 ⫾ 43.6*** 9.13 ⫾ 1.64*** 9.68 ⫾ 3.48*** 89.6 ⫾ 22.1***

Note. Results (arbitrary units; mean ⫾ SE; n ⫽ 4–6) were evaluated with two-way ANOVA with GFAP-IL6 transgene and MT-1⫹2 deficiency as main factors. When only two groups were compared, the Student t test was employed. ND, not detectable. ***P ⬍ 0.005 versus respective GFAP-IL6⫺/⫺ mice. **P ⬍ 0.025 vs GFAPIL6⫹/⫺ MT⫹/⫹ mice. †P ⬍ 0.025 vs GFAPIL6⫺/⫺ MT⫹/⫹ mice.

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Metallothioneins Protect Against IL-6-Induced Injury

FIG. 5. Lectin stainings in the cerebellum. (A, B) In GFAP-IL6⫺/⫺ MT⫹/⫺ mice (A) and GFAP-IL6⫺/⫺ MT⫺/⫺ (B) mice, lectin staining is seen in vessel walls mainly. (C, D) 3-month-old mice. (E, F) 6-month-old mice. (G, H) 8- to 10-month-old mice. (C, E) GFAP-IL6⫹/⫺ MT⫹/⫺ mice. (G) GFAP-IL6⫹/⫺ MT⫹/⫹ mice. (D, F, H) GFAP-IL6⫹/⫺ MT⫺/⫺ mice. Significant activation of microglia/macrophages is seen in the white and grey matter in all mice, but it was significantly higher in the MT-1⫹2-deficient mice; the highest number of activated, round, or amoeboid microglia/macrophages is seen in the 8- to 10-month-old GFAP-IL6⫹/⫺ MT⫺/⫺ mice (H). Scale bars: A–H, 30 ␮m.

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FIG. 6. GFAP immunoreactivity in the cerebellum. (A, B) GFAP staining was seen in some astrocytes of the cerebellar white (large asterisk) and grey (small asterisk) matter of GFAP-IL6⫺/⫺ MT⫹/⫺ (A) and GFAP-IL6⫺/⫺ MT⫺/⫺ (B) mice. (C, D) Reactive astrogliosis was seen in white (large asterisk) and grey (small asterisk) matter of the 3-month-old GFAP-IL6⫹/⫺ MT⫹/⫺ (C) and GFAP-IL6⫹/⫺ MT⫺/⫺ (D) mice. As shown, the GFAP-IL6⫹/⫺ MT⫺/⫺ mice (D) show increased astrogliosis in the grey matter but slightly reduced astrogliosis in the white matter relatively to the GFAP-IL6⫹/⫺ MT⫹/⫺ mice (C). (E, F) The 6-month-old GFAP-IL6⫹/⫺ MT⫹/⫺ (E) and GFAP-IL6⫹/⫺ MT⫺/⫺ (F) mice also show significant reactive astrogliosis in the white (large asterisk) and grey (small asterisk) matter of cerebellum. In the 6-month-old mice, the GFAP⫹ reactive astrogliosis is increased in the GFAP-IL6⫹/⫺ MT⫹/⫺ mice (E) relatively to the GFAP-IL6⫹/⫺ MT⫺/⫺ mice (F). (G, H) The 8- to 10-month-old GFAP-IL6⫹/⫺ MT⫹/⫹ (G) and GFAP-IL6⫹/⫺ MT⫺/⫺ (H) mice also show significant reactive astrogliosis in the white (large asterisk) and grey (small asterisk) matter of cerebellum. In the GFAP-IL6⫹/⫺ MT⫹/⫹ mice (G), the GFAP⫹ reactive astrogliosis is dramatically increased in both white and grey matter relatively to the GFAP-IL6⫹/⫺ MT⫺/⫺ mice (H). Scale bars: A–H, 60 ␮m.

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Metallothioneins Protect Against IL-6-Induced Injury

FIG. 7. MDA and NITT immunoreactivity in the cerebellum of 8- to 10-month-old mice. (A, B) In GFAP-IL6⫹/⫺ MT⫹/⫹ (A) and GFAP-IL6⫹/⫺ MT⫺/⫺ (B) mice, the oxidative stress marker MDA was increased. However, the levels of MDA were significantly increased in the GFAP-IL6⫹/⫺ MT⫺/⫺ mice (B) relatively to the GFAP-IL6⫹/⫺ MT⫹/⫹ mice (A). (C, D) The GFAP-IL6⫹/⫺ MT⫹/⫹ (C) and GFAP-IL6⫹/⫺ MT⫺/⫺ (D) mice also show increased oxidative stress when judged by NITT immunostainings, which were significantly increased in the GFAP-IL6⫹/⫺ MT⫺/⫺ relatively to the GFAP-IL6⫹/⫺ MT⫹/⫹ mice. Scale bars: A–D, 30 ␮m; E, F, 20 ␮m.

in the cerebellum, such effect of MT-1⫹2 deficiency was seen throughout the CNS. GFAP mRNA levels were also measured by RPA in 4-month-old mice (Table 4), and as could be anticipated, the GFAP-IL6⫹/⫺ MT⫺/⫺ mice tended to have higher cerebellar GFAP mRNA levels than the GFAP-IL6⫹/⫺ MT⫹/⫹. Somewhat surprisingly, the GFAP-IL6⫺/⫺ MT⫺/⫺ mice had higher GFAP mRNA levels than those animals expressing MT-1⫹2, which could not be verified by GFAP immunohistochemistry. MT-1ⴙ2 Deficiency Increases Oxidative Stress and Apoptosis In littermate animals not expressing transgenic IL-6, MDA, and NITT immunoreactivity was hardly detected in the CNS (data not shown). In contrast, in GFAP-IL6⫹/⫺ mice with MT-1⫹2 expression MDA and NITT stainings were obvious in the CNS, but mainly in the cerebellum (Fig. 7). These markers of oxidative stress were further dramatically increased in GFAP-IL6⫹/⫺ MT⫺/⫺ mice at all ages studied (Figs.

7A–7D). Moreover, MDA and NITT immunoreactivity were increased in GFAP-IL6⫹/⫺ MT⫹/⫺ relatively to GFAP-IL6⫹/⫺ MT⫹/⫹ mice (data not shown). Both oxidative stress markers were detected in neuronal cells mainly but also in some astrocytes and microglia/macrophages. In littermate animals not expressing transgenic IL-6, TUNEL-positive cells were hardly detected in the CNS as judged by using TUNEL and immunoreactivity for ssDNA and activated caspase-3 (data not shown). In GFAP-IL6⫹/⫺ mice with MT-1⫹2 expression, apoptotic cells were observed scarcely; in contrast, their number was dramatically increased in the cerebellum of GFAP-IL6⫹/⫺ MT⫺/⫺ mice (Figs. 8A– 8F). The TUNEL-positive cells were mainly neurons, but some astrocytes were also positive.

DISCUSSION The present study intended to give insight into the physiological reasons for brain MT-1⫹2 induction in the GFAP-IL6 mice. The results clearly show that such ©

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FIG. 8. TUNEL and immunoreactivity for ssDNA and activated caspase-3 in the cerebellum of 8- to 10-month-old mice. (A, B) TUNEL⫹ apoptotic cells were increased in GFAP-IL6⫹/⫺ MT⫺/⫺ (B) in comparison to GFAP-IL6⫹/⫺ MT⫹/⫹ (A) mice. (C, D) Neighboring sections to those seen in (A, B) showing immunoreactivity for ssDNA in GFAP-IL6⫹/⫺ MT⫺/⫺ (D) in comparison to GFAP-IL6⫹/⫺ MT⫹/⫹ (C) mice. As shown, ssDNA colocalizes with TUNEL. (E, F) Neighboring sections to those seen in (C, D) showing immunoreactivity for activated caspase-3 in GFAP-IL6⫹/⫺ MT⫺/⫺ (F) in comparison to GFAP-IL6⫹/⫺ MT⫹/⫹ (E) mice. Activated caspase-3 is primarily observed in the cells that are positive for TUNEL and ssDNA, which strongly supports the notion of apoptotic cell death. Scale bars: A–F, 20 ␮m.

upregulation is important in terms of tissue protection, presumably by controlling the inflammatory response and oxidative stress in the brain. However, this study also provides significant information of other two aspects of the putative MT functions that we will first discuss, namely (a) a role during development, and (b) a role in the control of body weight. The generation of MTKO mice in two different genetic backgrounds (Michalska & Choo, 1993; Masters et al., 1994a) was undoubtedly a major advancement for understanding the roles of these MT isoforms. These pioneering studies showed that the MTKO mice were viable, and lived and aged normally in normal 2002 Elsevier Science (USA) All rights reserved.

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laboratory conditions, which in principle is indicative of a lack of effect of MT-1⫹2 in the development process. However, our results suggest otherwise, especially for females, since fewer female mice were obtained than expected from a normal Mendelian distribution. Such effect of MT-1⫹2 deficiency was dramatic in mice with transgenic expression of IL-6, while it was very modest in controls. To characterize developmental effects of MTs was not the aim of this study, so we have no explanation for these results. Nevertheless, they do suggest that MT-1⫹2 are important during the developmental process when IL-6 expression is not physiologically regulated. The GFAP-IL6 mice

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Metallothioneins Protect Against IL-6-Induced Injury

FIG. 9. Cell counts carried out for IL-6, IL-1␤, TNF␣, and lectin stainings in the cerebellum of the different genotypes shown in Figs. 2, 3, and 5. Results are mean ⫾ SE, n ⫽ 4. *P at least ⬍0.05 vs either GFAP-IL6⫹/⫺ MT⫹/⫺ or GFAP-IL6⫹/⫺ MT⫹/⫹ mice.

have increased brain IL-6 mRNA levels compared with control littermates even at 7 days postnatally, an effect that potentiates with aging and peaks at 3 months of age and which causes significant neurological damage and early mortality depending on the amount of IL-6 expression (Campbell et al., 1993; Chiang et al., 1994). Although the generation of IL-6deficient mice has ruled out an essential role of IL-6 in

embryogenesis (Kopf et al., 1994), it is still possible that this cytokine could have some role(s) in specific conditions. Indeed, IL-6 is expressed in the embryonic brain, and has been shown to have many effects in cultured neurons and astrocytes, but these are complex, and even opposite actions (survival versus degeneration) have been shown for neurons (see Mun˜ ozFerna´ ndez & Fresno, 1998, for review). The present ©

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334 results suggest a sex-dependent deleterious effect of IL-6 during development that manifests only with concomitant MT-1⫹2 deficiency. These proteins are generally assumed to be stress/acute phase proteins in the adult animal that could provide significant antioxidant protection, hence providing a rationale for the striking control of cytokines of their expression (Cousins & Leinart, 1988; Karin et al., 1985; De et al., 1990; Lee et al., 1999; Herna´ ndez et al., 2000). The present results suggest that MT-1⫹2 are also significant protective proteins during development under conditions of targeted astrocyte IL-6 expression. It is also worth to mention that a reproductive advantage is also provided by MT-1⫹2 during maternal dietary zinc deficiency (Dalton et al., 1996; Andrews & Geiser, 1999). A second, unexpected result, worth to discuss is that the deficiency of MT-1⫹2 tended to decrease body weight in the F2 offspring of the crosses carried out. The results indicate that the effect is initially higher in the GFAP-IL6⫹/⫺ mice than in their GFAP-IL6⫺/⫺ littermates, but at 12 month age it is clear that the MT-1⫹2 deficiency on its own seem to predominate. Thus, we may conclude that, in this genetic background, the deficiency of MT-1⫹2 is detrimental for the control of body weight, and caused a decreased body weight gain. This is in contrast to the MT-1⫹2 null mice raised in mixed 129/Ola and C57BL/6J genetic background, where a large obesity of these mice was consistently observed (Beattie et al., 1998). Also, the MT-1⫹2 null mice in the 129/SvJ genetic background tend to show a slight obesity, and the putative mechanisms underlying such effects are currently being explored. Thus, although the evidence of the relation of MT-1⫹2 with energy balance and metabolism is mounting (Rofe et al., 1996; Beattie et al., 1996, 1998; Jiang et al., 1998; Hesketh et al., 1998; Apostolova et al., 2001), the present results demonstrate a strong and significant effect of the genetic background and call for extreme care regarding the proper controls to be used. Regarding the importance of MT-1⫹2 deficiency on IL-6-induced brain pathology, the results demonstrate a significant role of these proteins. As expected (Campbell et al., 1993), the GFAP-IL6 mice showed clear symptoms of neurological damage, showing hunched posture, tremor, ataxia and hindlimb weakness. The expected histopathological features were also clearly observed, namely neurodegeneration, microgliosis, astrocytosis, angiogenesis and up-regulation of several inflammatory and other host-response genes including IL-1 ␣/␤, TNF␣, GFAP, ICAM-1, and 2002 Elsevier Science (USA) All rights reserved.

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Giralt et al.

the acute-phase protein EB22/5, being the cerebellum the most affected area (Campbell et al., 1993; Chiang et al., 1994). The reasons for the higher expression of the IL-6 transgene in the cerebellum are unclear. We have observed that transgenic expression of a number of other cytokines under the control of the GFAP promoter (see Introduction) is typically higher in this brain area. Therefore, this might reflect a developmental control of the GFAP gene. The GFAP-IL6⫹/⫺ MT⫺/⫺ mice showed an impaired performance in the rota-rod test, suggesting an impaired motor control, and a decreased body weight gain, in comparison to GFAP-IL6 mice expressing MT-1⫹2. Both effects presumably indicate an increased neurological damage. The molecular and histopathological analysis of the cerebellum revealed that MT-1⫹2 deficiency further increased the upregulation of IL-6, IL-1 ␣/␤ and TNF␣ normally seen in the GFAP-IL6 mice. Such increased upregulation of these proinflammatory cytokines could likely potentiate the inflammatory response in the cerebellum and other brain areas (Rothwell & Hopkins, 1995; Benveniste, 1998; Campbell, 1998) and thus be responsible for the impaired motor control showed by the GFAP-IL6⫹/⫺ MT⫺/⫺ mice. Support for this assumption is provided by the increased microgliosis/macrophage recruitment and activation and T cell recruitment and by the higher oxidative stress (as determined by MDA and NITT) and apoptosis observed in the cerebellum of the GFAP-IL6⫹/⫺ MT⫺/⫺ mice. A higher astrogliosis was also observed transiently, but it was decreased at older ages. Interestingly, these effects caused by MT-1⫹2 deficiency are remarkably similar to those observed in the cortex of MTKO mice (129/SvJ genetic background) subjected to a cryolesion (Penkowa et al., 1999, 2000, 2001b). Moreover, transgenic mice overexpressing MT-1 show a completely reversed pattern of responses compared to MTKO mice in the cryolesion brain injury model (Giralt et al., 2001). MT-1⫹2 have also been demonstrated to be significant CNS protective proteins during experimental autoimmune encephalomyelitis (EAE) (Penkowa & Hidalgo, 2000, 2001; Penkowa et al., 2001a). Altogether, these results suggest that MT-1⫹2 are involved in the control of the inflammatory response in the CNS; the mechanisms underlying this control are unknown, but it is noteworthy to mention that binding sites for MT-1⫹2 have been described in T cells, B cells, macrophages and astrocytes which affect them functionally in in vitro studies (Leibbrandt & Koropatnick, 1994; Leibbrandt et al., 1994; Lynes et al., 1990, 1993; Youn et al., 1995;

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Borghesi et al., 1996; Borghesi & Lynes, 1996; El Refaey et al., 1997; Carrasco et al., 1999). For instance, exogenous MTs appear to potentiate the capacity of macrophages to produce increased levels of oxygen radicals and to kill engulfed yeast cells (Youn et al., 1995), to induce lymphocyte proliferation (Lynes et al., 1990) and to promote astrocyte migration following a scratch to the monolayer (Carrasco et al., 1999). Recent reports suggest that MT could in contrast be inhibitors of the humoral immune function (Crowthers et al., 2000; Canpolat & Lynes, 2001). Regardless of the mechanism, it is clear that the increased numbers of microglia/macrophages and T cells and the decreased astrogliosis caused by the MT-1⫹2 deficiency will contribute to the increased oxidative stress and apoptosis observed in the aged GFAP-IL6⫹/⫺ MT⫺/⫺ mice since these cells are key factors controlling free radical production, antioxidant mechanisms and apoptosis pathways (Hartung et al., 1992; Mattson & Scheff, 1994; Stichel & Verner Mu¨ ller, 1998; Eddleston & Mucke, 1993; Ridet et al., 1997). It is also likely that MT-1⫹2 per se are important as antioxidant and antiapoptotic factors (Thornalley & Vasak, 1985; Hidalgo et al., 1988; Sato & Bremner, 1993; Lazo et al., 1995; Kondo et al., 1997; Sakurai et al., 1999; Pearce et al., 2000; van Lookeren Campagne et al., 2000; Andrews, 2000). The results also demonstrate that MT-3, which was discovered as an inhibitory neuronal growth factor (in vitro) that appeared to be decreased in Alzheimer’s Disease brains (Uchida et al., 1991), was regulated in GFAPIL6⫹/⫺ MT⫺/⫺ mice similarly than in their control littermates, which strongly suggests that this MT isoform is not responsible for the neurological disturbances discussed above. In summary, the present results provide a rationale for the control of the MT-1⫹2 genes by IL-6 in the CNS, and demonstrate that these proteins are important factors for coping with cytokine-induced CNS injury.

ACKNOWLEDGMENTS The authors are grateful to Jordi Canto, Josep Graells, H. Hadberg, P. S. Thomsen, M. Søberg, G. Hahn, and K. Stub for valuable help. These studies were supported by Fundacio´ n “La Caixa” 97/ 102-00, PSPGC PM98-0170, and Direccio´ General de Recerca 2001SGR 00203 (J.H.); by Statens Sundhedsvidenskabelige Forskningsråd (SSVF), Novo Nordisk Fonden, Nordisk Forsknings Komite´ , University of Copenhagen/SVF Fond, The Danish Medical Association Research Fund, Schrøoders Fond, Holger Rabitz Mindelegat,

Dir. Ib Henriksens Fond, Dir. Jacob Madsen’s Fond, Fonden af 17.12.1981, Kong Christian IX og Dronning Louises Jubilæumslegat, Fonden til Lægevidenskabens Fremme, Gerda og Aage Haensch’s Fond, Eva og Henry Frænkels Mindelegat, Toyota Fonden, Marshalls Fond, Lily Benthine Lunds Fond, Øster-Jøorgensens Fond, Karen A. Tolstrups Fond, Leo Nielsens Fond, Eva og Robert Voss Hansens Fond, Warwara Larsens Fond, Dansk Parkinsonforening, Ragnhild Ibsens Legat for Medicinsk Forskning, Øjenfonden, Læge Eilif Trier-Hansen og Hustru Ane Trier-Hansens Legat (M.P.); and by NIH Grants MH 50426 and DA 12444 (I.L.C.).

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