A gliotoxic factor and multiple sclerosis

Journal of Neurological Sciences 154 (1998) 209–221

A gliotoxic factor and multiple sclerosis a ,1 a,1 a,1 b ´ ´ˇ Dobransky ´ Armelle Menard , Rim Amouri , Tomas , Christiane Charriaut-Marlangue , a a c d e ´ Regine Pierig , Carmen Cifuentes-Diaz , Said Ghandour , James Belliveau , Hughes Gascan , h f g a, Fayc¸al Hentati , Olivier Lyon-Caen , Herve´ Perron , Franc¸ois Rieger * a

` INSERM, Laboratoire de Neuromodulations Interactives et Neuropathologies, 17, rue du Fer-a-Moulin , 75005 Paris, France b INSERM U 29, 123 Bd de Port-Royal, 75674 Paris cedex 14, France c ´ ´ CNRS-UPR 417, Laboratoire de Neurobiologie Ontogenetique , 5, rue Blaise Pascal, 67000 Strasbourg, France d Department of Chemistry, Providence College, Providence, RI 02918, USA e Laboratoire de Biologie Cellulaire, C.H.U. Angers, 4, rue Larrey, 49033 Angers cedex, France f ´ ´ ˆ ˆ ` , 47 -83 Bd de l’ Hopital ˆ Federation de Neurologie and INSERM U.360, Hopital de la Salpetriere , 75013 Paris, France g ´ ´ ´ d’ Italie, 69364 Lyon, France Unite´ mixte CNRS-BioMerieux , UMR 103, Ecole Normale Superieure de Lyon, 46 allee h Institut National de Neurologie, La Rabta 1007, Tunis, Tunisia Received 10 April 1997; received in revised form 29 July 1997; accepted 2 August 1997

Abstract The pathogenesis of multiple sclerosis (MS) is unknown. Searching for possible toxic factors, it was found that 3-day exposure to heat-treated cerebrospinal fluid (CSF) from MS patients caused apoptotic death of astrocytes and oligodendrocytes, but not fibroblasts, myoblasts, Schwann cells, endothelial cells and neurons, in vitro. CSFs from other inflammatory or non-inflammatory neurological diseases showed no toxicity. Exposure of these glial cells to partially purified MS CSF produced DNA fragmentation, apoptotic bodies, chromatin condensation, cell shrinkage, and changes in the levels of known cytokines. A cytotoxic factor, called gliotoxin, was characterized chromatographically as a stable 17-kDa glycoprotein. Since this protein is highly cytotoxic for astrocytes and oligodendrocytes, it may represent an initial pathogenic factor, leading to the neuropathological features of MS, such as blood–brain barrier involvement and demyelination.  1998 Elsevier Science B.V. Keywords: Multiple sclerosis; Cytotoxin; Apoptosis; Cerebrospinal fluid; Astrocytes; Oligodendrocytes

1. Introduction Multiple sclerosis (MS) is a human demyelinating disease of the central nervous system (CNS) (Waksman Abbreviations: MS, multiple sclerosis; CSF, cerebrospinal fluid; CNS, central nervous system; PLL, poly L-lysine; GFAP, glial fibrillary acidic protein; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, Tdt-mediated dUTP-biotin nick end labeling; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium; PLP, myelin proteolipid protein; GC, galactocerebroside; MBP, myelin basic protein. *Corresponding author. Tel.: 133 1 45876150; fax: 133 1 45876151. 1 These authors have contributed equally to the work. 0022-510X / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0022-510X( 97 )00231-1

and Reynolds, 1984; Martin et al., 1992). The prevailing view of the etiology of MS is that it is an immunopathological disorder, with various environmental factors functioning in a genetically susceptible host. The study of the immune system in MS has shown abnormalities in the cellular immune response and abnormalities in the T-cell populations that mediate this response (Hafler et al., 1985; Ilonen et al., 1987; Whitaker, 1994). It has been suggested that in the early stages of inflammatory demyelination, there is a cytokine-induced disturbance in blood–brain barrier function (Scolding et al., 1989). We now report the existence of a toxic protein present at significant levels in the cerebrospinal fluid (CSF) from

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relapsing–remitting MS patients at relapse, and at lower levels in most of the other MS patients. CSF from other selected neurological diseases showed no toxicity. Eighteen-hour exposure to this protein produced disruption of intermediate filament network in both primary immortalized or transformed mouse or human astrocytes. Two- to 3-day exposure induced apoptotic cell death in exponentially growing cultures of primary and immortalized cultures of both astrocytes and oligodendrocytes. The cytotoxic action on oligodendrocytes is of obvious relevance to demyelination in MS tissues.

2. Materials and methods Dulbecco’s modified Eagle’s medium (DMEM), F12, fetal calf serum (FCS), trypsin and antibiotics were purchased from Seromed (Berlin, Germany). All cell lines were maintained at 378C in a humidified atmosphere of 5% CO 2 in air.

2.1. Heat-treatment of CSF samples Patient CSF samples were individually heated for 30 min at 568C, pooled, centrifuged and dialyzed twice against PBS at 48C. Heat-treated MS CSF and control hydrocephalus CSF were added to the glial cell cultures at a dilution of 1:20.

2.2. Immortalized mouse CLTT 1 -1 astrocytes Immortalized mouse CLTT 1-1 astrocytes (Galiana et al., 1990) were cultured in poly L-lysine (PLL)-coated (5 mg / ml in PBS, Sigma, St. Louis, MO, USA) eight-well polystyrene chamber slides at a density of 2310 3 cells / well (LAB TEK, NUNC Inc., Naperville, IL, USA) in F12-DMEM supplemented with 10% FCS, 0.05 mg / ml gentamicin (complete medium), 36 h before experimentation.

2.3. Primary cortical cell culture Primary cell cultures of astrocytes were prepared from the spinal cord of 1-month-old C57BL / 6J mice. Spinal cords were dissected, finely minced and dissociated with 0.1% trypsin for 20 min at 378C. The cell suspension was diluted with complete medium (containing minimum essential medium (70%) and medium 199 (30%, Gibco, Paisley, UK) supplemented with 20% FCS, 10 IU penicillin, and 10 mg / ml streptomycin), and then centrifuged at 3003g for 8 min. The cell pellet was gently resuspended in complete medium. Cells were plated at a density of 4310 3 cells / cm 2 in eight-well polystyrene chamber slides (LAB TEK).

2.4. GFAP immunostaining Cells on coverslips were washed with D-PBS and fixed in fresh 4% paraformaldehyde in PBS for 20 min at 378C. Cells were preincubated with the rabbit anti-GFAP (Sigma) for 2 h at 378C, followed by anti-rabbit IgG (Sigma). All washings were carried out with PBS supplemented with 5% FCS.

2.5. Vimentin immunostaining Cells on coverslips were washed with D-PBS and fixed in acetone for 10 min at 2208C. Cells were preincubated overnight at 48C with monoclonal anti-vimentin IgM (clone VIM 13,2, Sigma), followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (R&D Systems Inc., Minneapolis, MN, USA). All washings were carried out with PBS supplemented with 5% FCS.

2.6. MTT assay Mouse cell cultures were plated in 60-mm plastic dishes at a density of 115310 3 cells / plate (Falcon, Becton Dickinson, Franklin Lakes, USA) in 2 ml of complete medium for 36 h before experimentation. Heat-treated CSF samples were directly diluted in the 2 ml of complete medium and incubation was continued for 3 days with no change in medium. The medium was then removed and 2 ml of 3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT, Sigma) (0.5 mg / ml in DMEM) were added (Mosmann, 1983). Living cells transform the tetrazolium salt into dark blue formazan crystals. After a 2 h incubation, the reaction was stopped by addition of 2 ml of 40 mM HCl in isopropanol which solubilizes the formazan product. After a 5-min centrifugation at 50003g, the optical density was measured using a test wavelength of 570 nm and a reference wavelength of 630 nm. Cytotoxicity was expressed as a percentage: % cytotoxicity5% of dead cells51002[(OD sample / OD control )3100]. Dose–response curves were generated by measuring the percentage of cytotoxicity in three replicate dishes of primary or immortalized astrocyte cultures. Hydrocephalus CSF was used as the control and did not present any toxicity.

2.7. Mouse immortalized oligodendrocyte cultures Secondary oligodendrocyte-enriched cultures derived from new-born mouse cerebral hemispheres (Feutz et al., 1995) were transfected with a plasmid containing the SV40 T-antigen gene expressed under the control of the mouse metallothionein-1 promoter (Tennekoon et al., 1987). The calcium phosphate method was used for cell transfection. Colonies were selected for their resistance to G418 (Geneticin sulfate, Gibco-BRL). After cloning by limit dilution, selected clones such as the 158N cell line

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were maintained in culture in DMEM supplemented with 10% calf serum. Immunostaining of 158N cells for myelin basic protein (MBP) and myelin proteolipid protein (PLP) was accomplished by using rabbit polyclonal antibodies directed against MBP and against tridecapeptide corresponding to the sequence 117–129 of PLP (dilution 1:50), and then revealed with FITC-conjugated goat anti-rabbit IgG (R&D Systems Inc.). A mouse monoclonal antibody directed against GC (a gift from Dr Ranscht) was used at a 1:20 dilution and then revealed with FITC-conjugated goat anti-mouse IgG (R&D Systems Inc.).

2.8. TUNEL technique Astrocyte and oligodendrocyte cells, cultured on PLLcoated slides in culture chambers (LAB TEK) were exposed to partially purified MS CSF for 2–3 days. The cells were fixed in 4% paraformaldehyde for 15 min, dehydrated in successive graded alcohol concentrations and kept at 2208C until use. The slides were exposed to 2% H 2 O 2 in PBS for 10 min. Terminal transferase (10 mM) and biotinylated dUTP (20 mM in Gibco buffer) were added (1 h, 378C). After washing, development was performed using streptavidin-peroxidase (Sigma), and diaminobenzidine (Sigma) was used as a substrate.

2.9. Cytokine level determination Immortalized astrocytes (Galiana et al., 1990) were plated at 5310 3 cells / well in flat-bottomed 24-well plates (Falcon) in complete culture medium (400 ml / well). The medium was replaced after 36 h of incubation at 378C with either control heat-treated hydrocephalus CSF or with partially purified MS CSF (40 ml of toxic sample containing 52 mg of protein) diluted in fresh medium 1:10. After various incubation times (12, 24, 36, 48 and 96 h), supernatants were collected, centrifuged, and stored until tested. The levels of IL-1b, TNF-a and TGF-b1 were measured by using commercially available ELISA kits.

2.10. Alamar blue assay Immortalized astrocytes (Galiana et al., 1990) were plated at a density of 1500 cells / well in 48-well microtiter plates (Falcon) using 200 ml DMEM 10% FCS per well. After 24 h, 50 ml of cytokines or eluted fractions were added and incubation was continued for 3 days with no change of medium. The medium was then removed and 200 ml of Alamar blue (Biosource, Sacramento, CA, USA) diluted in medium (1 / 20 v / v) were added. Living cells incorporate and reduce the Alamar blue non-fluorescent substrate into a fluorescent product. After a 3-h incubation, the fluorescence of each well was directly determined in the microliter plates using the Cytofluor II Multi-well Fluorescence Plate Reader (PerSeptive Biosystems, Framingham, NA, USA). Fluorescence of cells treated with

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cytokines, or eluted fractions was compared to a control of untreated cells in an identical medium. Cytotoxicity was expressed as a percentage and was calculated according to the formula: % cytotoxicity5% of dead cells51002 [(fluorescence of sample / fluorescence of control)3100].

2.11. Partial purification of MS CSF sample All of the following steps were performed at 48C. Ten ml of heat-treated MS CSF, containing approximately 17.6 mg of proteins, were loaded onto a HiTrap Protein G column (Pharmacia, Uppsala, Sweden) and the immunoglobulin-free fraction was collected. The immunoglobulins were eluted from the protein G column with 50 mM glycine–HCl buffer at pH 3.0 and dialyzed twice against PBS. The toxic immunoglobulin-free fraction (13.4 mg of protein; toxicity recovery 98%) was then applied onto a DEAE–Sepharose CL 6B column (Pharmacia) and eluted in Tris–HCl (20 mM, pH 8.8) with steps at 120, 200 and 500 mM NaCl and dialyzed twice against PBS. The toxic fraction (9 ml, 11.8 mg protein, toxicity recovery 92%) was aliquoted and stored at 2808C. Protein concentrations were measured by the method of Bradford (1976) using bovine serum albumin as a standard. Toxicity was determined on immortalized astrocytes using the MTT method. Toxicity recovery was determined by use of the formula: % toxicity recovery5[(total protein after purification step3amount of protein needed to induce death of 50% cells before purification step) /(total protein before purification step3amount of protein needed to induce death of 50% cells after purification step)3100].

2.12. Gel filtration on Superose 12 A 1.5-ml sample of partially purified MS CSF was concentrated to 50 ml by centrifugation using UltrafreeMC (cut-off, 10 kDa, Millipore, Bedford, MA, USA). Two mg of concentrated proteins were loaded onto a Superose 12 column and eluted in PBS at 0.5 ml / min and 48C using the FPLC system (Pharmacia). Fractions of 500 ml were collected. Toxicity was analyzed in triplicate for each fraction collected from the Superose 12 column (MTT assay).

2.13. Gel filtration on Superdex 75 A 1.5-ml sample of partially purified MS CSF was concentrated to 50 ml by centrifugation using UltrafreeMC (cut-off, 10 kDa, Millipore). Thirty ml were loaded onto a Superdex 75 column and eluted in PBS at 50 ml / min and 48C using the SMART system (Pharmacia). Fractions of 100 ml were collected. Toxicity was analyzed in duplicate for each 100-ml fraction collected from the Superdex 75 column (Alamar blue assay).

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3. Results and discussion

3.1. Disruption of the intermediate filament network and induction of glial cell death by the CSF of multiple sclerosis patients at relapse MS CSF was collected from three MS patients at relapse and control CSF was collected from three patients suffering from hydrocephalus. Heat-treated CSF samples were added into the culture medium of both primary and CLTT 1-1 immortalized mouse astrocytes (Galiana et al., 1990), and both primary (Feutz et al., 1995) and 158N immortalized oligodendrocytes. Heat-treated MS CSF caused shortterm morphological changes (less than 20 h) and longerterm cell death (2–3 days). The most prevalent short-term change in primary astrocyte cultures involved the glial

fibrillary acidic protein (GFAP) cytoskeleton (Fig. 1A,C). After addition of heat-treated MS CSF, filament disruption could first be detected at about 18 h with coalescent gliofilament bundles around the nuclear membrane. The same cellular gliofilament network disorganization was observed in treated immortalized CLTT 1-1 astrocytes, which first express vimentin while dividing and GFAP at confluence (Galiana et al., 1990) (Fig. 1B,D). Most, if not all, MS CSF-treated immortalized astrocytes appeared in clusters in the culture dish and displayed signs of partial or total disruption of their vimentin network at 1 day of exposure. In confluent immortalized astrocytes which expressed GFAP and were exposed to the toxic sample, there was no gliofilament disorganization, suggesting that confluent cells were insensitive to the disorganizing factor. After 2–3 days in culture, signs of cell death were noticed

Fig. 1. Disruption of intermediate filaments in mouse astrocytes exposed to toxic, heat-treated MS CSF samples. (A) GFAP immunostaining of primary mouse cortical cell culture exposed at 14 days post-seeding for 18 h to control heat-treated hydrocephalus CSF. (B) Vimentin immunostaining of immortalized astroglial cell culture exposed at 14 days post-seeding for 18 h to control hydrocephalus CSF. (C) GFAP immunostaining of primary mouse cortical cell culture exposed at 14 days post-seeding for 18 h to heat-treated MS CSF. (D) Vimentin immunostaining of immortalized astrocyte cell culture exposed at 14 days post-seeding for 18 h to heat-treated MS CSF. MS CSF samples caused disruption of intermediate filaments. Bar: A and C, 12 mm; B and D, 7 mm.

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in both primary and immortalized astrocyte cultures. The same results were obtained with primary and also immortalized oligodendrocytes (data not shown). Using the fluorescence method of MacCoubrey et al. (1990) to distinguish between live and dead cells, qualitative signs of extensive cell death were observed in exponentially growing, immortalized astrocytes as well as in oligodendrocytes cultures (data not shown). Quantitative evaluation of cell viability was then determined in treated cultures using the MTT method (Mosmann, 1983) in which only viable cells hydrolyze a methyl tetrazolium derivative. A dose–response study of the cell death phenomenon was performed comparing heat-treated MS CSF to heat-treated hydrocephalus CSF with both primary and immortalized astrocyte cultures (Fig. 2). Cell survival was decreased with increasing amounts of the toxic MS CSF sample, in a dose-dependent manner, and primary cells were slightly more sensitive than immortalized cells. In order to determine whether other glial cell types were sensitive to the gliotoxic activity present in MS CSF, the same type of experiments were performed using recently characterized, immortalized oligodendrocytes, immunoreactive for PLP, galactocerebroside (GC), and MBP (Fig. 3). Again, cell death was observed in actively dividing oligodendrocyte cultures and the % toxicity was clearly dose dependent (Fig. 3). This same effect was also observed in primary mouse oligodendrocyte cultures (data not shown). To assess the specificity of the gliotoxic factor to additional types of cells, immortalized fibroblasts and myoblasts (Li et al., 1993), SV40 large T antigen-immortalized Schwann cells, cerebral endothelial cells, and

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neurons were tested under the same conditions as the glial cells. The quantitative MTT assay demonstrated no toxicity on these four mouse cell types in culture. The fact that no cytotoxic effect was observed on fibroblasts and myoblasts, which express vimentin, may be understood if vimentin does not play any critical role in these cells, or if vimentin is organized differently, or if the cells do not possess a specific receptor for the toxic protein. To assess the sensitivity of the cytotoxic effect on human cells, currently available human glial cell lines (European Collection of Animal Cell Cultures, Salisbury, UK) were used. Human brain CCF-STTG1 astrocytoma (ECACC 90021502) and the A172 glioblastoma (ECACC 88062428) cell lines were sensitive to MS CSF under conditions similar to those used for the mouse cell line (data not shown). Additionally, it was found that the human GO-G-UVW astrocytoma (ECACC 86022703) cell line was resistant. These experiments strongly suggest that the cytotoxic effect is not species specific and that the conclusions made for mouse cells can be extended to cells of human origin. Nevertheless, it would be extremely interesting to directly test primary cultures of human CNS glial cells for their degree of sensitivity to exposure to the gliotoxic activity. However, current ethical rules for human tissue acquisition and experimentation make these experiments very difficult to perform.

3.2. Disease specificity of the gliotoxic effect and its correlation with active phases of multiple sclerosis In order to establish the disease specificity of the gliotoxic effect, heat-treated CSF samples were tested from 20 patients with clinically defined MS (Poser et al., 1983) and 20 patients with inflammatory or non-inflammatory neurological diseases (Table 1). CSF samples were tested on mouse CLTT 1-1 astrocyte cultures (see Section 2.2) and cytotoxicity was determined by the MTT assay. All CSF from MS patients at relapse demonstrated high toxicity. In contrast to these results, CSF samples from patients at remission and with chronic progressive MS demonstrated significantly lower toxicity. CSF samples from patients with some other selected inflammatory and non-inflammatory neurological diseases did not display any significant toxicity.

3.3. Apoptotic features of glial cell death

Fig. 2. Dose–response curves of cell death in astrocyte cultures after exposure to heat-treated MS CSF samples. Non-confluent primary or immortalized astrocytes were exposed to a diluted heat-treated MS CSF sample from patients at relapse. Cytotoxicity was evaluated by determining the surviving cells using the MTT assay and expressed as a percentage of cell death, compared to control cultures exposed to heattreated hydrocephalus CSF. Bars represent 6SEM.

In order to obtain additional insight into the nature of cell death occurring in the astrocyte and oligodendrocyte cultures after exposure to toxic MS samples, we looked for DNA fragmentation as evidenced by terminal deoxyribonucleotide transferase-mediated dUTP biotin nick end labeling (TUNEL) staining (Gavrieli et al., 1992). Upon exposure of a partially purified MS CSF sample, immortalized astrocytes of variable TUNEL staining intensities were observed (Fig. 4B–G). In addition, various apoptotic

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Fig. 3. Dose–response curve of cell death in immortalized oligodendrocyte cultures after exposure to heat-treated MS CSF samples. A 158N oligodendrocyte cell culture was used which was immunoreactive in vitro for all three oligodendrocyte proteins: myelin proteolipid protein (PLP), myelin basic protein (MBP) and galactocerebroside (GC). Dose–response curve of cell death caused by dilutions of a heat-treated MS CSF sample (same MS CSF sample and dilutions as in Fig. 2 for astrocytes) was generated using the MTT assay. Bars represent 6SEM.

features, such as chromatin condensation, cell shrinkage and apoptotic body formation were visible (Fig. 4B–G). These morphological signs are reminiscent of most of the characteristic alterations that define programmed cell death or apoptosis (Clarke, 1990; Bursch et al., 1992). These same features were observed in treated immortalized oligodendrocyte cultures (Fig. 5B,C). Cell abnormalities developed in an asynchronous fashion in the cultures, and clusters of dying cells were often observed with apparently non-affected neighboring cells. Direct non-specific damage due to the gliotoxic factor does not seem probable, considering the lack of effect on

other cell types. Thus, cell death induced by the gliotoxic factor probably results from the activation of an endogenous cell death program. It has also been shown that cell death occurs in vitro in a dose–response manner and with a rather slow time course, compared to other existing models of cell death in vitro. These observations suggest a series of activation steps or concentration effects for the mechanism of cell death. Repeated attempts to detect DNA laddering by agarose gel electrophoresis failed. This is similar to what has been reported, in vitro, in developing retina, where TUNEL staining detected DNA fragmentation in isolating dying cells, but no DNA laddering

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Table 1 Cytotoxicity of CSF from patients with neurological disease on CLTT 1-1 astroglial cells Disease category

n

Percentage of dead cells (mean6SD)

MS, relapsing–remitting at relapse MS, relapsing–remitting at remission MS, chronic progressive Other inflammatory a neurological diseases Non-inflammatory b neurological diseases

10 5 5 10 9

41.6611.7 2.862.1 4.663.6 ,0.5 ,0.5

Cytotoxicity (percentage of dead cells) was measured by MTT staining (Mosmann, 1983) using heat-treated CSF samples. Each patient’s result is the mean of data obtained in two separate experiments, each performed in five replicate wells. Hydrocephalus CSF was used as a negative control and did not present any toxicity. The results for patients with relapsing–remitting MS at relapse vs. relapsing–remitting MS at remission, and relapsing–remitting MS at relapse vs. chronic progressive MS, were significantly different (P,0.001, Student t-test). n represents the number of tested patients. SD represents the standard deviation. a ¨ Inflammatory neurological diseases: Guillain-Barre´ (n56); Sjogren’s syndrome (n52); bacterial meningitis (n51); Lyme disease (n51). b Non-inflammatory neurological diseases: Parkinson’s disease (n52); Alzheimer’s disease (n51); normal pressure hydrocephalus (n56).

(Portera-Cailliau et al., 1994). Synchronized cultures could help to observe DNA fragmentation at particular stages of cell division.

3.4. Modulation of cytokine production by immortalized astrocytes treated with the gliotoxic factor

chronically active lesions (Wu and Raine, 1992). Thus, TNF-a expression by astrocytes in vivo, when activated by other cytokines, INF-g or IL-1b (Chung and Benveniste, 1990), may be a contributor to MS (Beck et al., 1988; Hauser et al., 1990; Sharief and Hentges, 1991).

3.5. Physicochemical properties of the gliotoxic factor A significantly increased production of interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) in astrocyte cell culture supernatants was observed when immortalized astrocyte cells were exposed to partially purified MS CSF (Fig. 6). Thus, the behavior of immortalized astrocytes exposed to high doses of various human cytokines was tested in order to detect any possible cytotoxic effect of a wide selection of cytokines. In the treated astrocyte cultures, no cytotoxic effects were observed for concentrations of 20 and 100 ng / ml (Table 2). On the contrary, a higher level of proliferation versus controls was observed for IL-1b and TNF-a at 1000 ng / ml (data not shown). Our results are in agreement with previous observations demonstrating that cytokines generally have a stimulatory effect on astrocytes in vitro (Giulian and Lachman, 1985), but no cytotoxic activity (Selmaj et al., 1991). In contrast to IL-1b and TNF-a, decreased levels of TGF-b1 were observed in the supernatants of treated cultures (Fig. 6). Even at a concentration of 1000 ng / ml, this cytokine did not induce significant cytotoxicity. The study of cytokine levels in the CSF of MS patients has shown the presence of IL-1b, TNF-a and sometimes IL-6 (Hauser et al., 1990; Maimone et al., 1991). CSF levels of TNF-a have been shown to correlate with the degree of disability in patients with progressive disease (Beck et al., 1988; Sharief and Hentges, 1991). Cytotoxic T lymphocytes which produce TNF-a are involved in the events leading to acute demyelination (Scolding et al., 1994) and TNF-a and TNF-b have been proposed to cause both disruption of myelin and permanent damage to oligodendrocytes (Selmaj et al., 1991; Wu and Raine, 1992). Immunohistochemical analysis of MS plaques have revealed that a majority of the TNF-a-positive cells are astrocytes, most frequently positioned along the edge of

The most toxic CSF samples from 10 patients with relapsing–remitting MS at relapse were pooled to prepare a partially purified MS CSF sample (see Section 2.11.). This partially purified MS CSF was analyzed by onedimensional (1D) SDS–PAGE (Fig. 7A). The proteins contained in serial 2-mm thick gel slices were recovered and SDS was removed by competition with 1% Triton X-100 followed by desalting exclusion chromatography on a NAP25 column. The cytotoxicity was essentially recovered in the 17-kDa gel region obtained for MS CSF. No toxicity was observed in fractions of control hydrocephalus CSF analyzed by an identical 1D SDS–PAGE procedure. A second analysis by two-dimensional (2D) SDS–PAGE showed that the 1D SDS–PAGE MS 17-kDa band can be resolved into three main spots corresponding to different isoelectric points in the 6.5–7.5 pI range (Fig. 7B). Elution from the gel and SDS removal demonstrated that two of the three spots were toxic (pI 6.5 and 7.0), suggesting two isoforms of the 17-kDa gliotoxic protein. In order to independently confirm the molecular weight of the gliotoxic factor, a sample of partially purified MS CSF was analyzed by Superdex 75 gel filtration (fractionation range 3000–70 000, Pharmacia-SMART system). The main toxic fraction was indeed recovered in a 17-kDa peak (Fig. 8). Additionally, a minor toxicity was observed in the exclusion volume, which, by Superose 12 gel filtration, gave a MW of 110 kDa (see Section 2.12.). The heat stability of this protein in the partially purified MS CSF sample was studied. No decrease in toxic activity was found between 56 and 728C. At 768C, the biological activity started showing a time-dependent decrease. A 2-min heat treatment at 1008C completely abolished the toxic activity. The heat stability may be related to the

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Fig. 4. DNA fragmentation following exposure of individual immortalized CLTT 1-1 astrocytes to the gliotoxic factor, and visualization by the TUNEL technique and phase-contrast microscopy. (A) Control culture. (B,C) High magnification of TUNEL-positive nuclei (curved arrows) showing apoptotic bodies (small arrows). (D) Another field showing two apoptotic nuclei (curved arrows), and nuclei with intermediate stages of apoptosis with the nuclear membrane still visible (straight arrows) and two necrotic cells (open arrows) with nuclear membrane disappearance and cytoplasic vacuoles. (E–G) Details of various stages of apoptotic cell death. Note the condensed cytoplasm remnants and apoptotic bodies in (G). Bar: 10 mm.

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Fig. 5. DNA fragmentation following exposure of gliotoxic factor to immortalized oligodendrocytes. (A) Control culture. (B) High magnification of TUNEL positive nuclei. Note the presence of numerous small vacuoles reminiscent of type II apoptosis (Clarke, 1990). (C) High magnification of a TUNEL positive nucleus. Note the intense staining and reduced nuclear size with the formation of two apoptotic bodies. Bar: 10 mm.

glycoprotein nature of the factor, as shown by its retention on Concanavalin A Sepharose (not shown). The full characterization of the gliotoxic factor is presently hampered by the very low amounts present in the MS biological fluids where it has been detected. An attempt to purify sufficient amounts of this gliotoxic factor for amino acid microsequencing from 1.5 l of CSF from MS patients in the active stages of the disease yielded only about 1 mg of the purified protein. Using this purified protein, proteolytic cleavage was performed directly in the gel with endoproteinase lys C, and the resulting peptides were separated on a DEAE–HPLC column linked to a C18 reverse-phase column eluted with a 0–50% acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. However, no amino acid identification resulted from classical NH 2 terminal amino acid sequencing analysis on a gas-phase sequencer. A subsequent procedure involved using an aliquot of this purified protein on an Immobilon P membrane with trypsin digestion and analysis by peptide mass fingerprinting with a MALDI-TOF mass spectrometer (Cottrell, 1994). A series of peptides was obtained

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Fig. 6. Effect of gliotoxic MS CSF on cytokine production by immortalized astrocytes. Partially purified MS CSF stimulated the production of IL-1b (A) and TNF-a (B), and decreased TGF-b 1 levels (C) in the supernatant of treated immortalized astrocytes (circles) as compared to the heat-treated hydrocephalus CSF control (squares). Each point is the mean6SD of 10 determinations from two separate experiments.

which unfortunately did not correspond to any known peptide mass fingerprint in available databases. Sera from 25 MS patients were analyzed for the presence of this gliotoxic factor. It was found that the cytotoxic activity is about 10 times weaker than in corresponding CSFs (data not shown). Using a Superose 12 purification procedure on 10 ml of concentrated serum from two MS patients at relapse, it was found that this gliotoxic factor in serum also had an apparent MW of 17 kDa. Thus, these results suggest the existence of a protein in both CSF and serum from MS patients possessing a novel biological activity which causes the death of astrocytes and oligodendrocytes in vitro. Gliotoxicity was also observed with a CSF sample which had been treated at 728C for 20 min and from which immunoglobulins had been removed by HiTrap Protein G column chromatography (Pharmacia), thus excluding heat-sensitive complement proteins and

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218 Table 2 Effect of cytokines on cell survival

% survival6SEM 20 ng / ml a

Tumor necrosis factor-a Tumor necrosis factor-b b Transforming growth factor-b1 c Interferon-g c Interleukin-1a c Interleukin-1b a Interleukin-2 d Interleukin-4 d Interleukin-5 d Interleukin-6 c Interleukin-7 d Interleukin-8 c Interleukin-10 d Interleukin-11 e Interleukin-12 e Interleukin-13 d Brain-derived neurotrophic factor f Neurotrophin-3 f Neurotrophin-4 / 5 f Epidermal growth factor c Granulocyte CSF g Granulocyte / monocyte CSF e Platelet-derived growth factor aa c FLT3 ligand (stem cell factor)c Oncostatin M c Prolactin g MS toxic sample

101.1 99.2 96.6 96.0 103.7 112.3 91.2 111.5 103.6 109.5 100.1 96.2 97.7 111.6 98.7 93.6 105.9 97.6 100.2 96.9 94.7 103.6 108.1 106.3 101.4 98.5 24.8

100 ng / ml 1.6 0.9 2.5 2.3 3.5 6.5 3.3 2.4 6.1 5.6 6.3 1.2 0.6 5.7 0.6 4.1 4.7 1.2 2.0 3.6 4.5 9.9 4.0 0.5 12.6 2.4 13.4

97.7 103.5 102.1 95.2 107.6 106.5 107.6 104.0 109.1 105.1 96.0 99.1 98.9 101.3 98.6 95.0 102.3 113.2 99.7 92.7 94.5 96.3 91.0 109.4 104.0 91.7 /

2.8 1.0 1.5 4.0 2.7 3.5 5.4 3.2 1.0 1.3 3.2 1.9 5.3 2.8 1.1 3.1 1.1 2.8 1.2 1.6 1.1 4.9 3.7 1.9 5.2 3.4 /

Cell survival (%) was determined using the sensitive fluorescent Alamar blue vital live cell assay (Page´ et al., 1993) after a 3-day incubation with purified human cytokines to a final concentration of 20 or 100 ng / ml in culture medium. Each result was expressed as the mean of three separate experiments (standard error the mean (SEM)), performed in duplicate and gave reproducible results. The % survival of all cytokines was compared to a control of cells in medium. A relapsing–remitting MS CSF toxic sample at relapse (approximately 100 ng of proteins) was used as a toxic reference. Results over 100% are indicative of a higher survival / proliferative effect with cytokine treatment compared to control cultures. CSF, colony-stimulating factor. The cytokines were obtained from a Boehringer Mannheim, b R&D Systems, c Genzyme, d DNAX Research Institute, e Genetics Institute, f Regeneron and from g our laboratories, respectively.

toxic immunoglobulins as possible candidates for the gliotoxic factor (data not shown). Only a very small quantity of the toxic protein, a few ng / ml, is required to cause death of 50% of treated cells in vitro, under our assay conditions, in 3 days, which demonstrates the high biological activity of this protein. Such an efficiency may be understood if only a few receptors are able to transduce some intracellular cascade of signalization with amplification effects such as those observed for second messenger systems, or if the toxic molecule is a protein with enzymatic activity. The time lag of 2 or 3 days before observing actual cell death and decreased cell number in vitro has no straightforward explanation. Since astrocytes and oligodendrocytes in our culture conditions possess a cell division period of about 1

day, one simple hypothesis for such delayed death is that the factor’s action results in progressive cell depletion of a substance which inhibits cell death. The source of the gliotoxic protein has not yet been discovered. An intrathecal or intracerebral synthesis could make sense, although a ´ peripheral origin (see Menard et al., 1997) cannot be rejected. We would like to designate this toxic protein specific for glial cells, gliotoxin.

4. Gliotoxicity and demyelination in multiple sclerosis In acute MS, demyelination is associated with severe destruction of all elements of the CNS parenchyma. Oligodendrocytes as well as astrocytes are reduced in number within the lesions and the empty extracellular space is filled with numerous macrophages (Hauser et al., 1990; Lassmann et al., 1994). During chronic progressive MS, selective demyelination is associated with nearly complete preservation of oligodendrocytes and there is only very little damage to other elements of the CNS parenchyma such as axons and astrocytes (Lassmann et al., 1994). These observations may tentatively be explained in the light of our results showing less toxic activity in CSF from chronic progressive than relapsing–remitting MS patients at relapse (Table 1). The most commonly postulated theory of the evolution of MS lesions includes an initial systemic event, the most likely being the activation of cell mechanisms by environmental factors (Rudge, 1991), which could include the recently characterized retrovirus MSRV (Perron et al., 1991, 1993, 1997). These initial events are followed by abnormalities in immune function, occurring within and outside the CNS. Sensitized and activated T cells directed against endogenous or cross-reactive exogenous antigens circulate, adhere to endothelial cells, and transmigrate into the CNS. The initial lymphocytes mediating these events are T-helper cells (Hafler et al., 1985). Astrocytes seem to contribute to inflammation and demyelination by secreting various cytokines. The new fact that primary or immortalized oligodendrocytes are sensitive targets of the gliotoxin is directly relevant to MS, since demyelination is an obvious consequence of oligodendrocyte death. The additional fact that astrocytes are sensitive targets, with early gliofilament disruption and subsequent cell death, potentially leads, in vivo, to abnormalities, at least wherever astrocytes are known to be important, such as the maintenance of a functional blood–brain barrier (Janzer and Raff, 1987) and of a functional node of Ranvier. This present study demonstrated that TNF-a is released by gliotoxinexposed astrocytes in vitro. If this occurs in vivo, it offers an additional mechanism of cell damage and death for oligodendrocytes through a cascade of cellular events, possibly including free radical synthesis and NO release as suggested by recent studies in vivo (Bo¨ et al., 1994; Bagasra et al., 1995) and in vitro (Xiao et al., 1996). These

´ A. Menard et al. / Journal of Neurological Sciences 154 (1998) 209 – 221

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Fig. 7. (A) Protein composition from a 50-mg sample of partially purified MS CSF analyzed on a 1D 15% SDS–PAGE gel (Laemmli, 1970). (B) Protein composition from a 50-ml sample of partially purified MS CSF analyzed on a 2D 15% SDS–PAGE gel showing the resolution of the 17-kDa band into three spots of differing pI values (6.5–7.5 range). The first dimension of the 1D SDS–PAGE was isoelectric focusing using Ampholine carrier ampholytes in the 3.5–9.5 pH range (Pharmacia). Proteins were visualized by silver nitrate staining. Arrows represent the MW markers: albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and a-lactalbumin, sequentially.

different pathological mechanisms should be further studied in vivo, since an animal model for MS derived from gliotoxin infusion is now available (Rieger et al., 1996). In addition to an ‘open’ blood–brain barrier, inflammation and demyelination are observed a few weeks after a single intraventricular injection of a partially purified gliotoxic fraction (submitted for publication). Glial cell death should also be looked for in MS brain after autopsy, using the available histological techniques combining specific labeling of each glial cell type and TUNEL (see note below). Finally, studies should be carried out to ascertain the diagnostic and / or prognostic value of the cytotoxic activity found in MS patients, with the ensuing potential for new therapeutic approaches to MS. We

recently showed that monocyte / macrophage culture supernatants from MS patients containing MSRV-specific RNA and reverse transcriptase activity also contain such a toxin which induces death of primary mouse cortical glial cells ´ (Menard et al., 1997), thus strongly suggesting some link with the new MS related retrovirus, MS RV. This gliotoxin could represent a major pathogenic factor in the neuropathology of MS.

Acknowledgements We are indebted to Dr P. Rouget for supplying the CLTT 1-1 astroglial cell line, to Dr M. Pinc¸on-Raymond for the

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´ A. Menard et al. / Journal of Neurological Sciences 154 (1998) 209 – 221

Fig. 8. Gel filtration of partially purified MS CSF on Superdex 75. Chromatogram of a concentrated, partially purified MS CSF sample eluted with PBS on a Superdex 75 column. The absorbance of eluted proteins was measured at 280 and 254 nm. The first open arrow designates a minor toxic fraction eluting in the exclusion volume. The second open arrow indicates the elution of the major toxic fraction in the 17-kDa range. Toxicity of the eluted fractions was determined in duplicate for each eluted fraction using immortalized astrocytes (see Section 2.10). The small arrows, labeled 1–5, represent the elution of MW markers: dextran blue, ovalbumin, chymotrysinogen, ribonuclease A, and acetone, respectively.

immortalized mouse Schwann cells, to D. Reoutov for photographic artwork and to K. Belliveau for editing the manuscript. We are grateful to Dr J.S. Cottrell and his colleagues at Finnigan Ltd for performing laser-assisted matrix description experiments on our material, Dr C. Geny, Dr L. Degos and Dr E. Schuller for making available CSF samples from MS patients and Dr J.P. Boursier (Pharmacia, Orsay) for access to the SMART system. We thank Dr E. Schuller for valuable discussions, ´ Drs B. Mandrand, B. Pozet and B. Gilly at BioMerieux SA for interest and some critical discussions, H. Tourtet and Dr C. Geny for help in checking some patient files. This work has been partially supported by the French National Multiple Sclerosis Associations, ARSEP and NAFSEP and the INSERM-Tunisia exchange program. This research project has been performed under a formal agreement with the Comite´ Consultatif de Protection des Personnes dans la ˆ ` ˆ ´ Recherche Biomedicale (La Pitie´ Salpetriere Hopital, Paris). Note added in proof Massive glial cell death is indeed reprinted in a recent paper by Dowling et al. (1997), although the use of autopsy material requires further careful confirmation.

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