Metallothionein Treatment Reduces Proinflammatory Cytokines IL6 and TNF-α and Apoptotic Cell Death...

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Experimental Neurology 170, 1–14 (2001)doi:10.1006/exnr.2001.7675, available online at http://www.idealibrary.com on

Metallothionein Treatment Reduces Proinflammatory Cytokines IL-6and TNF-a and Apoptotic Cell Death during Experimental

Autoimmune Encephalomyelitis (EAE)Milena Penkowa* and Juan Hidalgo†

*Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200, Copenhagen, Denmark; and†Departamento de Biologıa Celular, Fisiologıa e Inmunologıa, Unidad de Fisiologıa Animal, Facultad de Ciencias,

Universidad Autonoma de Barcelona, Bellaterra, Barcelona, Spain 08193

Received October 27, 2000; accepted February 22, 2001; published online May 9, 2001

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Experimental autoimmune encephalomyelitis (EAE)is an animal model for the human autoimmune diseasemultiple sclerosis (MS). Proinflammatory cytokinessuch as interleukin-6 (IL-6) and tumor necrosis fac-tor-a (TNF-a) are considered important for inductionand pathogenesis of EAE/MS disease, which is charac-terized by significant inflammation and neuroglialdamage. We have recently shown that the exogenousadministration of the antioxidant protein zinc–metal-lothionein-II (Zn-MT-II) significantly decreased theclinical symptoms, mortality, and leukocyte infiltra-tion of the CNS during EAE. However, it is not knownhow EAE progression is regulated nor how cytokineproduction and cell death can be reduced. We here-with demonstrate that treatment with Zn-MT-II signif-icantly decreased the CNS expression of IL-6 andTNF-a during EAE. Zn-MT-II treatment could also sig-

ificantly reduce apoptotic cell death of neurons andligodendrocytes during EAE, as judged by usingUNEL and immunoreactivity for cytochrome c andaspases 1 and 3. In contrast, the number of apoptoticymphocytes and macrophages was less affected byn-MT-II treatment. The Zn-MT-II-induced decrease inroinflammatory cytokines and apoptosis during EAEould contribute to the reported diminution of clinicalymptoms and mortality in EAE-immunized rats re-eiving Zn-MT-II treatment. Our results demonstratehat MT-II reduces the CNS expression of proinflam-atory cytokines and the number of apoptotic neu-

ons during EAE in vivo and that MT-II might be aotentially useful factor for treatment of EAE/MS.

© 2001 Academic Press

Key Words: EAE/MS; neuroimmunology; cytokines;apoptosis: immunotherapy.

INTRODUCTION

Experimental autoimmune encephalomyelitis (EAE)is an animal model for the human autoimmune disease

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multiple sclerosis (MS). MS is characterized clinicallyby paralysis and histopathologically by cellular infil-trates, which show strong expression of proinflamma-tory cytokines such as interleukin-6 (IL-6) and tumornecrosis factor-a (TNF-a) (7, 8, 19, 32, 33, 52, 55, 66).IL-6 and TNF-a are considered to have important rolesin induction and pathogenesis of EAE (16, 18, 19, 21,31, 54, 60, 61, 68). Hence, genetically IL-6-deficientmice are resistant to induction of EAE (35, 38, 39, 56),and transgenic mice with overexpression of TNF-ashow increased severity of EAE and/or spontaneousdemyelinating disease (10, 11, 16, 18, 52, 68). More-over, TNF-a induces selective destruction of myelinand apoptosis of oligodendrocytes in vitro (61), andTNF-a has a role in production of reactive oxygen spe-cies (14), which per se can cause damage to oligoden-drocytes and myelin in vitro (22, 25, 26). Thus, decreas-ing the proinflammatory cytokines might be an effec-tive way to reduce the severity of EAE.

During CNS inflammation microglia/macrophagesand reactive astrocytes increase the tissue protectivefactors metallothionein I 1 II (MT-I 1 II) (4, 5, 23,43–48). MT-I 1 II are cysteine-rich, low-molecular-weight proteins, which are coordinately expressed inmost tissues. MT-I 1 II may protect cells against dam-ge from reactive oxygen species, ionizing radiationnd anticancer drugs, and these proteins can inhibitpoptotic cell death (9, 28–30, 36, 43, 50, 57, 67, 69).lso, MT-I 1 II regulate the CNS inflammatory re-ponse and cytokine production, as well as wound heal-ng and glial scar formation following traumatic brainnjury (42, 43). MT-I also reduces tissue loss, vasculardema, and improve functional outcome following focalerebral ischemia (71).With the purpose to find new therapeutic strategies

or EAE/MS, we have studied the potentially beneficialffects of MT. During EAE, MT-I 1 II are significantlyncreased during disease and remission (46). Moreover,reatment with Zn-MT-II during EAE can significantly

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2 PENKOWA AND HIDALGO

reduce the clinical symptoms, mortality and leukocyteinfiltration of the CNS (46). The present study showsthat treatment with Zn-MT-II significantly reducesproinflammatory cytokines IL-6 and TNF-a during ac-tively induced EAE, and that Zn-MT-II administrationsignificantly decreases apoptotic cell death in neuronsand oligodendrocytes in a time- and dose-dependentmanner.

MATERIALS AND METHODS

Experimental Procedures

For this report we used pathogen-free female Lewisrats, weighing 180–200 g, provided from the PanumInstitute in Copenhagen, Denmark. Rats were keptunder standardized conditions of light and tempera-ture and had free access to food and water. All exper-iments were carried out in accordance with the Euro-pean Communities Council Directive of November 24,1986 (86/609/EEC). All efforts were made to minimizeanimal distress and to reduce the number of animalsused.

EAE Induction

EAE was induced by intradermal (id) injection of anemulsion consisting of the following for each rat: 100mg myelin basic protein (MBP) from guinea pig (Sigma,St. Louis, MO, Code M2295), 0.1 ml saline (0.9% sa-line), and 0.1 ml complete Freund’s adjuvant (CFA)(Difco, Detroit, MI, Code 0638), and 0.2 mg of Myco-bacterium tuberculosis H37 Ra (Difco, Code 3114). Thisemulsion was injected in two halves in the medialfootpad of each hindfoot under fentanyl/fluanison(Hypnorm, Janssen Pharmaceuticals, Denmark) andmidazolam (Dormicum, F. Hoffmann-La Roche AG,Switzerland) anesthesia. In the following days, ani-mals were treated with analgesic buprenorphin(Temgesic, Schering-Plough, Denmark). On days 1 and3 after id injection of the emulsion, rats received Bor-detella pertussis vaccination intravenously (iv) (ob-tained from Statens Serum Institute, Copenhagen,Denmark). We used a total of 8 mg B. pertussis toxoiddiluted in saline for each rat. The addition of M. tuber-culosis to the emulsion and the use of B. pertussisvaccination were done in order to boost the immunesystem of the rats, which leads to a significant andsufficient EAE response.

By using this method for actively inducing EAE,70–90% of the rats developed clinical EAE symptoms,which were graded into the following categories: 1 5flaccid tail; 2 5 mild paraparesis; 3 5 moderate tosevere paraparesis; 4 5 severe paraparesis; 5 5quadriparesis associated with incontinence or mori-bund state.

Four groups of control rats were used: (a) rats in-jected only with M. tuberculosis-enriched CFA, (b) rats

receiving only B. pertussis vaccination, (c) rats injectedwith both M. tuberculosis-enriched CFA and B. pertus-sis vaccination, and (d) uninjected rats. These foursubgroups of controls were used in order to rule out ifany interfering, biological effects due to the substancesinjected per se could affect the EAE response or thegiven treatment with ZnCl or Zn-MT-II. All controlrats were included in the experiments simultaneouslywith EAE-sensitized rats. Controls were sacrificedalong with EAE-sensitized rats at different timings,ranging from 3 to 50 days after induction.

Zn-MT-II Treatment for EAE

We performed a series of separate experimentswhere both control (all groups described above) andEAE-sensitized rats were treated with ip injections ofZn-MT-II (Sigma, Code M9542). Treatment with Zn-MT-II was chosen because this was the MT isoformcommercially available that is enriched in zinc andwith a very low Cd content (less than 0.5% Cd), a heavymetal which can have neurotoxic actions. From thepublished studies, MT-II does not appear to be sub-stantially different to MT-I. Thus, in a first set ofexperiments, control and EAE-sensitized rats were di-vided randomly into three groups, which received ei-ther saline or a daily dose of 100 mg Zn-MT-II dividedin three injections or a daily dose of 7 mg Zn as ZnCl(Sigma Code Z-0152). The ZnCl control is imperativesince the Zn-MT-II protein carries that metal, whichcan have functions per se during EAE. Injections wereinitiated the first day the rats showed clinical EAEsymptoms (generally by day 10 after immunization)and until the day of sacrifice ranging from 10 to 50 daysafter induction. Accordingly, control rats were injectedwith saline three times a day from day 10 and until theday of sacrifice. Different groups of rats were killed atseveral timings for histopathological analysis.

In a second set of experiments, we further character-ized the effect of Zn-MT-II by administering differentdoses of this MT isoform 10 days after the immuniza-tion, daily and up to 50 days after EAE induction. Tothis end, different groups of EAE rats received a dailydose of 33.3, or 100, or 300 mg Zn-MT-II. Some ratswere injected with 7 mg ZnCl as an additional control.Different groups of rats were killed at several timingsfor histopathological analysis.

In a third set of experiments, we characterized theeffect of Zn-MT-II (100 mg/day) once the disease waswell developed. To this end, EAE rats were treatedwith Zn-MT-II either 13 or 17 days after the immuni-zation. Again, different groups of rats were killed atseveral timings for histopathological analysis. Thoserats sacrificed in the same day that treatment wasinitiated were killed 12 h after the first injection.

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3METALLOTHIONEIN IS NEUROPROTECTIVE DURING EAE

Tissue Processing

The rats were deeply anesthetized with 10 mg/100 gBrietal (methohexital, 10 mg/ml, Eli Lilly) and weretranscardially perfused with 0.9% saline containing0.3% heparin (15000 IU/liter) for 1 min followed by 4%(w/v) formaldehyde in 0.1 M potassium phosphate-buffered saline (KPBS) (pH 7.4) for 8–10 min. The CNSwas embedded in paraffin and cut in serial, 3-mm-thicksections. Sections were processed as previously de-scribed (46) and were preincubated with 10% normalgoat or donkey serum in order to block nonspecificbinding.

Histochemistry

Hematoxylin–eosin (HE) and methyl green stainingsof brain and spinal cord sections were performed ac-cording to standard procedures.

Immunohistochemistry

The sections were incubated overnight at 4°C withone of the following primary antibodies: polyclonal rab-bit anti-rat liver MT-I 1 II 1:500 (20, 44, 47, 48);monoclonal mouse anti-rat CD11b/c (as a commonmarker for leukocytes) 1:50 (Serotec, UK, Code MCA275G); polyclonal goat anti-rat IL-6 1:50 (Santa CruzBiotechnology, Santa Cruz, CA, Code sc-1266); andpolyclonal rabbit anti-mouse TNF-a 1:50 (Biosource,Code AMC3012).

The primary antibodies were detected using biotin-ylated mouse anti-rabbit IgG 1:400 (Sigma, CodeB3275), biotinylated goat anti-mouse IgG 1:200(Sigma, Code B8774), or donkey anti-sheep/goat IgG1:20 (Amersham, UK, Code RPN 1025), and these sec-ondary antibodies were detected as previously de-scribed (46). Standard control stainings were per-formed as previously described (46). A further controlfor anti-IL-6 and anti-TNF-a antibodies was to preab-sorb the primary antibodies with their correspondingantigenic proteins. For this purpose rat IL-6 (SantaCruz, Code sc-1266 P) and rat TNF-a (Santa Cruz,Code sc-1349 P) were used.

In Situ Detection of DNA Fragmentation (TUNEL)

Terminal deoxynucleotidyl transferase (TdT)-medi-ated deoxyuridine triphosphate (dUTP)–digoxigeninnick end labeling (TUNEL) staining was performed aspreviously described (43).

Immunofluorescence and Fluorescein-Linked TUNEL

In order to determine which cells expressed cytokinesduring EAE, triple immunofluorescence for IL-6 andCD11 (both as mentioned above) and rabbit anti-cowGFAP 1:250 (Dakopatts, Denmark, Code Z334) were in-cubated overnight at 4°C. Also, triple immunofluores-

cence for TNF-a and CD11 (both as mentioned above)and goat anti-human GFAP 1:100 (Santa Cruz, Codesc-6170) were incubated overnight at 4°C. The primaryantibodies were detected by using donkey anti-goat IgG1:40 linked with aminomethylcoumarin (AMCA) (Jack-son ImmunoResearch Lab., Inc., West Grove, PA, Code705-155-147) and donkey anti-mouse IgG 1:40 linkedwith Texas red (TXRD) (Jackson ImmunoResearchLab., Inc., Code 715-075-151), and donkey anti-rabbitIgG 1:50 linked with fluorescein (FITC) (JacksonImmunoResearch Lab., Inc., Code 711-095-152).

In order to determine which cells suffer from apopto-sis, triple and double stainings for TUNEL and cellularmarkers were performed. Sections were incubated withFITC-linked TUNEL (Oncor, U.S.A., Code S7111-KIT)according to manufacturers protocol and afterward in-cubated overnight at 4°C with polyclonal rabbit anti-human neuron-specific enolase 1:1000 (Calbiochem,San Diego, CA, Code D05059) and simultaneouslymonoclonal mouse anti-human CNPase (as a markerfor oligodendrocytes) 1:50 (Biogenesis, UK, Code 2406-3006). Also, sections were incubated with FITC-linkedTUNEL and monoclonal mouse anti-rat CD11 (as men-tioned above). These primary antibodies were detectedusing TXRD-linked goat anti-rabbit IgG 1:50 (JacksonImmunoResearch Lab., Inc., Code 111-075-144) andAMCA-linked goat anti-mouse IgG 1:50 (Jackson Im-munoResearch Lab., Inc., Code 115-155-100).

In order to better characterize apoptotic cells, sec-tions were incubated with FITC-linked TUNEL (On-cor, Code S7111-KIT) (as mentioned above) and after-ward incubated overnight at 4°C with rabbit anti-mouse caspase-1/ICE 1:100 (Santa Cruz, Codesc-1218R) and goat anti-horse cytochrome c 1:100Santa Cruz, Code sc-7159) simultaneously. Other sec-ions were incubated with FITC-linked TUNEL (asentioned above) and afterward incubated overnight

t 4°C with goat anti-horse cytochrome c (as mentionedabove) and rabbit anti-human caspase-3/CPP32 1:50(Santa Cruz, Code sc-7148) simultaneously. These pri-mary antibodies were detected by using TXRD-linkeddonkey anti-rabbit IgG 1:40 (Jackson Immuno-Research Lab., Inc., Code 711-075-152) and AMCA-linked donkey anti-goat IgG 1:40 (Jackson ImmunoRe-search Lab., Inc., Code 705-155-147). The secondaryantibodies were used simultaneously for 30 min atroom temperature.

The sections were embedded in 20 ml fluorescentmounting (Dakopatts, Code S3023) and kept in dark-ness at 4°C. Standard control stainings were per-formed as previously described (42, 46).

Cell Counts

For statistical purposes, in some experiments cellularcounts of positively stained cells were performed in ablinded fashion in matched 0.3-mm2 bilateral areas of

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brain stem, cerebellum, and spinal cord of each rat. Amean value for these three CNS areas of each animal wascalculated and used for statistical analysis. Cell countswere carried out in at least seven rats per group.

Statistical Analysis

Cell counts were carried out in independent animalsin representative experiments as described above. Inmost cases, results were evaluated by two-way analysisof variance (ANOVA), with EAE and type of treatmentas main factors. When only two groups were compared,the Student t test was used.

RESULTS

General

In the series of experiments carried out, none of thecontrol groups evaluated showed clinical signs of EAEor weight loss (46). In contrast, in EAE-sensitized ratsweight loss was seen at 5–6 days after immunization.Maximum weight loss coincided with paralysis andreached 15–20% of total weight (46). Signs of EAE wereobserved in 70–90% of EAE-sensitized rats and beganat day 10 after induction, were increased at day 13, andpeaked at day 15–17, and recovery began at days19–21 and was completed by days 30–40 (46). EAE-sensitized rats treated with 100 or 300 mg Zn-MT-IIrom day 10 or 13 or 17 after EAE immunization showiminished EAE symptoms and have completely recov-red from EAE by days 21–25 after induction (46).ollowing is a detailed description of the new his-

FIG. 1. CD11 immunoreactivity in matched areas of brain stem ofwith saline (A) or Zn-MT-II (B) show very few CD111 leukocytes. (Cells were observed. (D) In Zn-MT-II-treated rats with EAE, a reduc

topathological analysis carried out in this experimen-tal paradigm. Since the four types of controls employed(see Materials and Methods) did not differ betweenthem, we will be showing only saline- or Zn-injectedanimals only.

EAE Infiltrates

HE or methyl green-stained sections of EAE-sensi-tized rats showed infiltrates by days 10–13, and bydays 15–17 multiple and pronounced EAE infiltrateswere seen. By days 19–21 the EAE infiltrates gradu-ally diminished, and sections were comparable to thoseof control rats by days 40–50 (46). Thus, the observedhistopathological changes correspond well with theclinical stages of EAE as well as with other EAE stud-ies (21, 46, 51). In Zn-MT-II-treated rats with EAE, theinfiltrates were reduced compared with those of salineor ZnCl-treated rats, as judged by both HE or methylgreen staining (46) and by immunoreactivity for theleukocyte common marker CD11 (Figs. 1 and 2). Byusing CD11 immunoreactivity, it was observed thatthe normal CNS contains very few CD111 leukocytes(Figs. 1A and 1B). During EAE, the number of CD111leukocytes was significantly increased (Figs. 1C, 1D,and 2). The number of CD111 leukocytes was signifi-cantly decreased in Zn-MT-II-treated rats relatively tosaline or ZnCl-treated rats during EAE (Figs. 1C, 1D,2A, and 2B), which is in agreement with studies show-ing that Zn-MT-II administration during EAE can sig-nificantly reduce the number of recruited monocytes/macrophages and lymphocytes (46).

line- and Zn-MT-II-treated rats at day 17. (A, B) Control rats injectedEAE-immunized rats treated with saline, numerous round CD111

number of CD111 leukocytes was seen. Bars (A–D), 25 mm.

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The MT-induced reduction in CD111 cells was dose-ependent (Fig. 2A). Hence, increasing the daily Zn-T-II dose from 33.3 to 100 mg made a significant

difference in decreasing the number of CD111 cells ofbrain stem, cerebellum, and spinal cord (Fig. 2A). How-ever, an additional increase in the daily dose from 100to 300 mg Zn-MT-II could only slightly reduce the num-ber of CD111 cells (Fig. 2A). In contrast, the adminis-tration of zinc alone, if anything, caused a mild in-crease in CD111 cells relatively to saline treatmentFig. 2A).

Zn-MT-II treatment was also effective when it wasnitiated once the disease had developed. Thus, a sig-ificant reduction in CD111 cells was observed when

FIG. 2. Cellular counts from brain stem, cerebellum, and spinalmean value was calculated for each animal for statistical analysis.

aline or ZnCl or Zn-MT-II from day 10 (A, C, E) or from day 13 (Bncreased the number of cell expressing CD11, IL-6, and TNF-a (P

(P , 0.001). In contrast, the administration of zinc alone was ineffdevelop, the number of cells expressing CD111 (B), IL-61 (D), and

the daily treatment of 100 mg Zn-MT-II was initiatedon day 13 (Fig. 2B) or day 17 (not shown) after EAEimmunization. This effect was noticed even at 12 hafter the first injection.

IL-6 Expression

In all control rats, including rats injected with zincalone, IL-6 immunoreactivity of the CNS was barelyobserved (Figs. 3A and 3B). During EAE all the ratsshowed clearly increased IL-6 expression throughoutbrain stem, cerebellum, and spinal cord (Figs. 2 and 3).Treatment with Zn-MT-II during EAE decreased sig-nificantly the number of IL-61 cells in a dose-depen-

d of CD111 cells (A, B), IL-61 cells (C, D), and TNF-a1 cells (E, F).ults shown are means 6 SE (n 5 at least 7). Rats were injected with, F) after EAE immunization. Two-way ANOVA revealed that EAE.001) and that Zn-MT-II decreased it in a dose-dependent mannerve. When Zn-MT-II was administered once the EAE had started toF-a1 (F) was also decreased (P , 0.001).

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dent manner, while Zn alone was without effect (Figs.2C, 3D, and 3F). As for CD11 staining, Zn-MT-II treat-ment was also effective regarding IL-6 expressionwhen it was initiated once the disease had developed.Thus, a significant reduction in IL-61 cells was ob-served when the daily treatment of 100 mg Zn-MT-IIwas initiated on day 13 (Fig. 2D) or day 17 (not shown)after EAE immunization.

Both CD111 leukocytes and GFAP1 astrocytes wereexpressing IL-6 in the EAE immunized rats (Figs. 7A

FIG. 3. IL-6 immunoreactivity in matched areas of brain stemontrol rats injected with saline (A) or Zn-MT-II (B) show very few

saline, several stellate and round cells were expressing IL-6 inZn-MT-II-treated rats with EAE, a reduced number of IL-61 cellEAE-immunized rats treated with saline, several cells expressed IL-number of IL-61 cells was seen in the spinal cord. Bars, (A, B) 38 m

and 7B). The CD111 leukocytes were both macro-phages and lymphocytes as judged by morphology andcell size.

TNF-a Expression

In saline- and zinc-injected control rats, TNF-a im-munoreactivity of the CNS was barely observed (Figs.4A and 4B). During EAE all the rats showed increasedTNF-a expression throughout brain stem, cerebellum,

spinal cord of saline and Zn-MT-II-treated rats at day 17. (A, B)1 cells in the brain stem. (C) In EAE-immunized rats treated withbrain stem. Most cells were seen in the white matter. (D) In

as seen in both white and gray matter of the brain stem. (E) Inthe spinal cord. (F) In Zn-MT-II-treated rats with EAE, a reduced

(C, D) 50 mm; (E, F) 25 mm.

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and spinal cord (Figs. 2 and 4). In line with the previ-ous results, treatment with Zn-MT-II decreased signif-icantly the number of TNF-a1 cells in a dose-depen-

ent manner, while zinc alone was ineffective (Figs.E, 4D, and 4F). Also in accordance with CD11 andL-6 stainings, Zn-MT-II treatment caused a signifi-ant reduction in IL-61 cells was observed when theaily treatment of 100 mg Zn-MT-II was initiated once

the disease had developed, namely, day 13 (Fig. 2F) orday 17 (not shown) after EAE immunization.

FIG. 4. TNF-a immunoreactivity in matched areas of brain stemontrol rats injected with saline (A) or Zn-MT-II (B) show some TNaline, several cells increased TNF-a in the brain stem. (D) In Zn-MT

in the brain stem. (E) In EAE-immunized rats treated with saline, mats with EAE, a reduced number of TNF-a1 cells was seen in the

Both CD111 leukocytes and GFAP1 astrocytes wereexpressing TNF-a in the EAE immunized rats; how-ever, the main source of TNF-a was CD111 leukocytes(not shown).

Apoptotic Cell Death

In saline- and zinc-injected control rats, TUNEL-labeled cells of the CNS were barely observed (Figs. 5Aand 5B). During EAE an increase in the number of cells

d spinal cord of saline and Zn-MT-II-treated rats at day 17. (A, B)1 cells in the brain stem. (C) In EAE-immunized rats treated with-treated rats with EAE, a reduced number of TNF-a1 cells was seeny cells expressed TNF-a in the spinal cord. (F) In Zn-MT-II-treatedal cord. Bars, (A–D) 50 mm; (E, F) 25 mm.

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labeled by TUNEL was observed in brain stem, cere-bellum, and spinal cord of all the rats (Figs. 5C–5F and6). Zn-MT-II decreased the number of TUNEL1 cellsin a dose-dependent manner, while zinc alone slightlyincreased them (Fig. 6A). Zn-MT-II treatment was alsoeffective when administered once EAE was developed,namely, at day 13 (Fig. 6B) or day 17 (not shown)following the immunization.

The TUNEL1 apoptotic cells were both NSE1 neu-rons, CNPase1 oligodendrocytes, and CD111 leuko-ytes, as judged by using triple and double immunoflu-rescence for TUNEL and cell markers (Figs. 6C, 7C,nd 7D). However, the neurons were the predominantell suffering of apoptotic cell death during EAE, asell as those having the higher beneficial effects of MT

reatment relatively to glial and blood-derived cells

FIG. 5. TUNEL and methyl green staining in matched areas of bA, B) Control rats injected with saline (A) or Zn-MT-II (B) show som

In EAE-immunized rats treated with saline, several cells displayed TZn-MT-II-treated rats with EAE, a reduced number of TUNEL1 cellaline, many TUNEL1 nuclei were seen in the spinal cord. (F) In Zn-

observed in the spinal cord. Bars, (A, B) 38 mm; (C, D) 30 mm; (E, F

Fig. 6C). Supporting the notion of apoptotic cell death,he TUNEL1 cells were also immunoreactive forpoptotic signals caspase-1/ICE and caspase-3/CPPnd showed cytochrome c dispersed in the cytoplasmFigs. 7E and 7F).

DISCUSSION

The present study shows the potential roles for treat-ment with ip-injected Zn-MT-II during actively in-duced EAE in adult Lewis rats. The injected MTreaches the CNS tissue across the EAE-induced dis-ruption of the blood–brain barrier (BBB) after 15–45min (46)

During EAE, the numbers of CD111 leukocytes in-creased throughout brain stem, cerebellum, and spinal

stem and spinal cord of saline and Zn-MT-II-treated rats at day 17.ethyl green-stained cells, but TUNEL1 nuclei are barely seen. (C)EL1 nuclei in both white and gray matter of the brain stem. (D) Inas seen in the brain stem. (E) In EAE-immunized rats treated with-II-treated rats with EAE, a reduced number of TUNEL1 nuclei was

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cord of all rats. Also, the expression of proinflammatorycytokines IL-6 and TNF-a was significantly increased

uring EAE. IL-6 and TNF-a were detected in CD111leukocytes and GFAP1 astrocytes, and their expres-sion pattern followed the clinical EAE symptoms.Moreover, TUNEL-labeled apoptotic cells appearedduring EAE. In addition, apoptotic signals such asimmunoreactivity for caspase-1/ICE, caspase-3/CPP,and cytoplasmic cytochrome c (34) were increased inTUNEL1 cells. The latter is important since TUNELmay label both apoptotic and necrotic cells. However,TUNEL1 cells also displayed the morphologic criteriafor apoptosis. By using triple and double immunofluo-rescence, it appeared that mainly neurons, but alsooligodendrocytes and leukocytes were suffering fromapoptosis.

Daily treatment with Zn-MT-II during EAE had sig-nificant dose- and time-dependent decreasing effectson the recruitment of CD111 leukocytes, the numberof cells expressing IL-6 or TNF-a, and interestingly on

FIG. 6. Cellular counts from brain stem, cerebellum, and spinalcord of TUNEL1 cells showing dose and time dependency of Zn-

T-II treatment. A mean value was calculated for each animal fortatistical analysis. Results shown are means 6 SE (n 5 at least 7,xcept the bottom panel, where n 5 3 for neurons and oligodendro-ytes). (A) TUNEL1 cells of EAE-sensitized rats injected with saline,nCl, or different doses of Zn-MT-II (33.3, 100, or 300 mg of Zn-MT-I) from day 10 after EAE immunization. As shown, TUNEL1 cellsas decreased by Zn-MT-II treatment in a dose-dependent manner

elatively to saline or ZnCl treatment (P , 0.001). (B) TUNEL1ells of EAE-sensitized rats injected with either saline or 100 mg

Zn-MT-II from day 13 after EAE immunization. The number ofTUNEL1 cells was also decreased by Zn-MT-II treatment after

isease onset (P , 0.001). (C) The MT-induced reduction in apopto-ic cells was most pronounced in neurons and oligodendrocytes, whilehe MT-induced decrease in apoptotic leukocytes was less pro-ounced (P at least ,0.025).

the number of TUNEL1 apoptotic cells. The use ofnCl as a control treatment is important since MTarries Zn, which has many biological actions (62), andhus it is essential to discriminate between the effectsf metallothionein from those of the metal per se.Interestingly, Zn-MT-II treatment during EAE was

ollowed by decreased numbers of CD111 leukocytes,which were reduced by 50% by the optimal Zn-MT-IIdose initiated by day 10 after EAE immunization.Moreover, the number of IL-61 cells was significantlyreduced in Zn-MT-II-treated rats during EAE rela-tively to saline or ZnCl treatment. Thus, IL-6 expres-sion of Zn-MT-II-treated rats was reduced three to fivetimes of that seen in saline or ZnCl-treated rats. Also,the number of TNF-a1 cells was reduced significantlyby Zn-MT-II treatment, which decreased TNF-a duringEAE to 30–40% of the expression seen in saline- orZnCl-treated rats. In agreement with this, mice withgenetical MT-I 1 II deficiency have increased levels ofboth IL-6 and TNF-a mRNA and protein in the CNSfollowing traumatic brain injury (42). Thus, it is likelythat MT has direct inhibiting roles on IL-6 and TNF-aexpression during CNS pathological conditions, whichindicates a role for endogenous MT-I 1 II production inregulating the inflammatory response, at least in thepresent circumstances. Hence, the decreased numberof recruited CD111 leukocytes in Zn-MT-II-treatedrats during EAE could be explained by the reducedexpression of IL-6 and TNFa.

By reducing proinflammatory cytokines, MT may ex-ert neuroprotective actions, in that IL-6 and TNFacarry the potential to induce and/or exacerbate EAE(16, 18, 21, 31, 32, 35, 38, 39, 52, 54, 56, 60, 68). DuringEAE these proinflammatory cytokines can be injuriousto brain tissue by enhancing the inflammatory re-sponse including reactive oxygen species secretion,which can lead to apoptotic cell death and myelin dam-age (14, 19, 22, 25, 26, 32, 59, 61, 63–65). In thisregard, we have already shown that Zn-MT-II de-creased oxidative stress (46) (see below for furtherdiscussion), and we herewith show that Zn-MT-IItreatment during EAE significantly reduced the num-ber of apoptotic cells as judged by using TUNEL label-ing and immunoreactivity for caspase-1/ICE, caspase-3/CPP, and cytochrome c. MT-induced inhibition ofapoptotic cell death was dose dependent; increasingthe daily dose of Zn-MT-II beyond 100 mg did not addmuch further to the response of this particular exper-iment, suggesting a saturated mechanism. The cellsbenefiting the most from MT-induced decreases inapoptosis were neuronal cells, as judged by double andtriple immunofluorescence. However, daily treatmentwith Zn-MT-II also reduced the number of apoptoticCNPase1 oligodendrocytes and CD111 leukocytes.

However, apoptotic cell death could be both detri-mental and protective during EAE (58). Hence, apopto-tic cell death of neurons and oligodendrocytes is likely

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detrimental, while apoptotic cell death of autoreactiveT cells might be beneficial by eliminating infiltrating Tcells (58). Thus, the role for proapoptotic factors suchas TNFa as well as for antiapoptotic factors such as MTcould be dual during EAE. In this regard, it is inter-esting that the observed antiapoptotic effect of MTduring EAE is less pronounced in leukocytes when

FIG. 7. Triple fluorescence stainings of saline- and Zn-MT-II-injeCD111 leukocytes (red), GFAP1 astrocytes (green), and IL-6 (blue).n many CD111 leukocytes (pink) and in GFAP1 astrocytes (turquoi

is seen in a few CD111 leukocytes (pink) and in very few GFAP1 asCNPase1 oligodendrocytes (blue), and TUNEL (green). (C) TUNEimmunized rats injected with saline. Also, some other cells are labeleof Zn-MT-II-treated EAE-immunized rats. Also, some other cells arerespectively, showing triple immunofluorescence for caspase-1/ICEcaspase-1/ICE and cytochrome c (pink) is seen in the cytoplasm ofCoexpression of caspase-1/ICE and cytochrome c (pink) is seen inZn-MT-II. However, some nonapoptotic cells are also expressing cas

compared to the antiapoptotic effects observed in neu-rons and oligodendrocytes. In this regard, it is alsoimportant to realize that the beneficial effects of MTduring EAE are not limited to its antiapoptotic effects,but MT may be a valuable tool in the treatment ofEAE/MS due to its functions in the prevention andinhibition of EAE development as well as the MT-

d rats by day 17 after EAE induction. (A, B) Immunofluorescence forIn EAE-immunized rats injected with saline, IL-6 expression is seen. (B) In EAE-immunized rats treated with Zn-MT-II, IL-6 expressionytes (turquoise). (C, D) Triple fluorescence for NSE1 neurons (red),is seen in many neurons and in some oligodendrocytes of EAE-y TUNEL. (D) TUNEL is seen in a few neurons and oligodendrocyteseled by TUNEL. (E, F) Neighboring sections to those of (C) and (D),d), cytochrome c (blue), and TUNEL (green). (E) Coexpression ofe TUNEL1 cells of EAE-immunized rats injected with saline. (F)cytoplasm of TUNEL1 cells of EAE-immunized rats treated withe-1/ICE (red). Bars, (A, B) 10 mm; (C–F) 15 mm.

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induced inhibition of leukocyte recruitment and ex-pression of proinflammatory cytokines. Some of theputative mechanisms underlying such role(s) are dis-cussed below.

The notion of MT as an inhibitor of apoptotic celldeath in the CNS has been suggested in other reports(1, 3, 17, 27, 70), and apoptotic cell death in the injuredCNS is significantly increased in vivo during genetical

T-I 1 II absence (12,43). However, the exact mecha-isms of action of MT during apoptotic cell death areot elucidated, but being an antioxidant, MT may af-ect levels of oxidative stress by neutralization of reac-ive oxygen species (28, 29, 37, 42, 57,69). Reactivexygen species are detrimental factors, which are ele-ated during EAE/MS and have important roles in theathophysiology of EAE/MS (6, 7, 22, 25, 26, 32, 41, 55,9, 63). Hence, EAE can be reduced by antioxidantatalase and by inhibition of inducible nitric oxide syn-hase (iNOS) (15, 55). Reactive oxygen species are ma-or inducers of apoptotic cell death in the CNS (13, 65),nd therefore, it is likely that MT mediates antiapop-otic functions by reducing oxidative stress due toAE-induced production of reactive oxygen species. In-eed, administration of Zn-MT-II during EAE reducesignificantly the immunoreactivity for oxidative stressarkers iNOS, MDA (malondialdehyde), and NITT

nitrotyrosine) relatively to saline or ZnCl administra-ion (46). Thus, we suggest that the role of MT inodulation of apoptosis during EAE may be related to

ts antioxidant properties. In agreement with the lat-er, a study showed a direct association between in-reased MT levels and decreased apoptosis and freeadical formation in vitro (3). Although there are manyeports showing an antiapoptotic function of MT initro (1, 3, 17, 27–30, 57, 70) and in vivo (12, 43), theechanisms of action are still unclear.It is well known that Zn supplementation can protect

gainst apoptosis (72), which is likely due to an inhi-ition of calcium-activated endonucleases, and more-ver, Zn can inhibit the apoptotic protease caspase-349). However, in this study ZnCl treatment failed tonhibit apoptosis during EAE. In any case the mecha-isms of action of MT are likely different. It has beenhown that transfecting human breast carcinoma cellsith MT antisense phosphothiolate oligomers inhibits

ell growth, reduce c-myc expression and increase c-fosnd p53 transcript levels followed by increased apopto-is (1). In addition, cells with genetical MT deficiencyhow increased apoptosis (27), and in vivo, mice withenetical MT deficiency show increased apoptosis inhe CNS during pathological conditions (12, 43), sug-esting that MT has a direct role in apoptotic celleath. In addition, another study showed a specificnteraction between MT and the p50 subunit of nuclearactor kB (NFkB) (2), which indeed is involved in EAE

during peak of disease (24). However, whether or notthe antiapoptotic effects of MT in the CNS are related

to an interaction between MT and NFkB remains to beelucidated. Nevertheless, in mice with genetical MTdeficiency, the levels of NFkB were clearly increasedrelatively to normal mice following excitotoxicity (12).On the other hand, the MT-induced reduction in proin-flammatory cytokines per se may also contribute to theobserved reduction in apoptosis during EAE, in partbecause TNFa may directly trigger apoptosis throughthe activation of the TNFa-receptor (14, 25, 32, 53, 59,63–65). Nevertheless, the role of IL-6 and TNFa inneurodegeneration is still a matter of discussion, and itis likely that these cytokines have opposite actions atdifferent time points during CNS inflammation (37,40). The fact that these cytokines could induce otherdownstream cytokines cannot be underestimated.

Our results together with the data present in theliterature suggest that the neuroprotective effect of MTcould be achieved by both prevention of free radicalformation and apoptosis, taking place mainly in neu-ronal cells, and reduction of inflammatory cytokineproduction principally involving inflammatory cells.Thus, direct prevention of neuronal death by blockadeof free radicals and apoptosis and indirect neuropro-tection by reducing proinflammatory cytokines areprobably acting in concert to prevent neurodegenera-tion. Our results consistently show that Zn-MT-II, butnot zinc alone, is responsible for the effects observed.Nevertheless, this does not necessarily mean that it isthe protein moiety and not the zinc that carries theimportant factor, since one could envisage that zincassociated to the MT protein could still be of biologicalsignificance in this experimental paradigm. Clearlyfurther work on this subject is warranted.

In conclusion, MT has a role in CNS inflammationand degeneration during EAE. However, further stud-ies are necessary to thoroughly address the mecha-nisms of MT actions, but regardless of the exact mech-anisms of action, we suggest that MT represents apromising therapeutic approach to reduce neuronaldamage during EAE/MS.

ACKNOWLEDGMENTS

This study was supported by Scleroseforeningen, Warwara Lars-ens Fond, Dir. Ejnar Jonasson’s Fond, The Novo Nordisk Fonden,Statens Sundhedsvidenskabelige Forskningsråd (SSVF); Dir. JacobMadsen’s Fond, Fonden af 17.12.1981, Dansk Epilepsi SelskabsForskningsfond, Haensch’s Fond, Kong Christian X’s Fond, Fondentil Lægevidenskabens Fremme, Mimi og Victor Larsens Fond, Bern-hard og Marie Kleins Legat, Foreningen Værn Om Synet, Hjerte-foreningen, Toyota Fonden, Marshalls Fond, Lily Benthine LundsFond, Øster-Jørgensens Fond, Eva og Robert Voss Hansens Fond,Leo Nielsens Fond, Diabetesforeningen, PSPGC PM98-0170, Comis-sionat per a Universitats i Recerca 1999SGR 00330, and Fundacion“La Caixa” 97/102-00. The excellent technical assistance of HanneHadberg, Pernille S. Thomsen, Mette Søberg, Julie Sørensen, Gra-zyna Hahn, Birgit Risto, and Keld Stub is gratefully acknowledged.


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