Induction of apoptosis by cerebrospinal fluid from patients with primary-progressive multiple sclerosis in cultured neurons

Neuroscience Letters 255 (1998) 75–78

Induction of apoptosis by cerebrospinal fluid from patients with primary-progressive multiple sclerosis in cultured neurons Alberto Alca´zar a, Ignacio Regidor b, Jaime Masjuan b, Matilde Salinas a, Jose´ C. A´lvarez-Cermen˜o b , c ,* a

Departamento de Investigacio´n, Hospital Ramo´n y Cajal, Madrid, Spain b Servicio de Neurologı´a, Hospital Ramo´n y Cajal, Madrid, Spain c Department of Medicine, Universidad de Alcala´, Alcala´ de Henares, Spain Received 12 June 1998; received in revised form 25 August 1998; accepted 28 August 1998

Abstract We have studied the noxious effect of cerebrospinal fluids (CSF) from patients with primary-progressive multiple sclerosis (MS) on cultured neurons. Cells were exposed to CSF for 8 days and the possible neuronal damage was determined. Morphological studies with phase-contrast microscopy showed cellular shrinkage indicating apoptosis. CSF-induced apoptosis as evidenced by the fluorescent DNA-binding dye Hoechst 33342, as well as by the TUNEL-reaction, was only present in primary-progressive MS patients with a worsening disease. This neuron injury did not correlate with blood–brain barrier dysfunction nor with intrathecal IgG synthesis. On the contrary, CSF from either stable primary-progressive or other non-inflammatory neurological diseases, did not induce any culture damage. Undetectable or low similar tumor necrosis factor-alpha (TNF-a) levels (range to 8.7 pg/ml) were found in the CSFs tested regardless they damage cultures or not. These results suggest that soluble factors, other than TNF-a, molecules transudated from blood or IgG, present in the CSF of active primary-progressive patients with MS induce neuronal apoptosis.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Multiple sclerosis; Primary-progressive multiple sclerosis; Apoptosis; Cultured neurons

Despite extensive research in recent years, the precise mechanism causing tissue damage in multiple sclerosis (MS) remains elusive. The study of MS lesions has revealed T-cell infiltration and upregulation of several cytokines [3] which, at least in vitro, have been shown to induce myelin injury and oligodendrocyte apoptosis [13,17]. Further macrophage and resident microglia activation [7] and local production of reactive oxygen intermediates are part of an inflammatory cascade which produces myelin breakdown, local edema, and homing of new infiltrating lymphocytes to the lesion [4]. Nevertheless, magnetic resonance imaging studies indicate that severe demyelination can occur in MS patients who are asymptomatic [6]. It seems likely that in addition to a hypothetical specific attack against myelin or the oligodendrocyte, other neighboring * Corresponding author. Fax: +34 91 3369016; e-mail: [email protected]

cells, such as neurons, could be damaged by soluble mediators of inflammation in a non-specific fashion [15]. Since such events in MS are often reflected by the presence of inflammatory mediators in the cerebrospinal fluid (CSF) [14], we considered it of interest to investigate whether the CSF from patients with MS would damage cortical neurons in primary cultures. We report for the first time that CSF from active patients with primary-progressive MS induces neuronal apoptosis in cultured neuronal cells. The effect of eleven different CSF samples on cultured neurons was studied. Five of these samples were obtained from patients with clinically definite primary-progressive (PP) MS, whilst the remaining six were from patients with other non-inflammatory neurological diseases (OND). The clinical diagnosis of each of these OND patients included ischemic III and VI nerve palsies, bulbar paralysis, cervical compressive myelopathy and two thrombotic strokes. All samples were obtained by lumbar puncture (LP), performed

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00708- 3

76

A. Alca´zar et al. / Neuroscience Letters 255 (1998) 75–78

as part of the clinical work-up. In all cases, informed consent was obtained before the procedure. None of the patients had received immunosuppressive drugs or had been treated with corticosteroids in the preceding 6 months. CSF routine studies were performed in each case including cell count, biochemical studies and nephelometric quantification of albumin and IgG. Albumin ratio was determined as an index of blood–brain barrier (BBB) function, meanwhile IgG index value above 0.7 was considered to reflect increased intrathecal synthesis of IgG [16]. Primary neuronal cultures were obtained according to the method described previously [1]. One milliliter of cell suspension from forebrain cortex of 16- to 17-day-old rat embryos was seeded on plastic multidishes with glass coverslips, both precoated with 0.05 mg/ml poly-d-lysine, and kept at 37°C in a 5.5% CO2 atmosphere, in Dulbecco’s medium supplemented with 15% fetal calf serum and 4.5 mg/ml glucose. After 24 h, neuronal cultures were placed and maintained in serum-free medium Dulbecco’s:Ham’s F12 (1:1, v/v) medium supplemented with N2 (100 mg/ml transferrin, 100 mM putrescine, 20 nM progesterone, 30 nM sodium selenite and 5 mg/ml insulin). Two to 4-day-old neurons in culture, at a final cell density of 50–60 × 103 cells/cm2, were used in the experiments, and this was considered day 0 of the experiment. Neuronal cultures were treated with 0.1 ml of CSFs in 1 ml of serum-free medium [19] to induce the neuronal apoptosis. Control experiments, with 0.1 ml phosphate-buffered saline (PBS)/ml serum-free medium, were run in parallel for each experiment. The neuronal content, as determined by immunocytochemistry with antibodies against the neuron-specific protein b-tubulin isotype III, was found to be more than 95%. Each experiment was performed with a different batch of culture and neuronal morphology was monitored with phase-contrast invert microscopy. Nucleated cells were stained with the fluorescent DNAbinding dye Hoechst 33342 to detect the total nuclei and the characteristic features of apoptotic nuclei, such as condensed chromatin and the segmentation of chromatin into small round bodies [5]. To confirm the appearance of these apoptotic nuclear characteristics, the measurement of the Table 1 Clinical and biochemical data of CSF from primary-progressive MS patients Primary progressive disease activity

Follow-up Sex (F, female; M, male) Age (years) Disease duration (months) Albumin ratio IgG index

Active

Stable

worsening F,F,M 44 ± 5 68 ± 14 5.1 ± 1.4 1.4 ± 0.4

stable F,M 65 ± 5 138 ± 42 10.3 ± 3.0 2.2 ± 1.5

Data are expressed as the mean ± SEM. No statistical significance was found.

fragmented DNA of apoptotic cells was performed by using the TUNEL-reaction. A positive control was done by nicking the nuclear DNA with DNase I. A negative control was achieved by excluding the Terminal deoxynucleotidyl transferase (TdT) enzyme from the reaction. TUNEL positive fluorescein-dye nuclei (green) matched with condensed Hoechst-dye nuclei (intense blue). The clinical features of the five MS patients studied, all of them with primary-progressive MS, are shown in Table 1. Three of them suffered from an ‘active’ form of the disease, and their clinical situation had deteriorated by at least 0.5 points according to Kurtzke’s Expanded Disability Status Scale (EDSS) during the 6 months prior to lumbar puncture (named as active patients). The other two PP patients had an unchanged EDSS for the same period of time (named as stable patients). Morphological studies of active primaryprogressive MS CSF-treated neuronal cultures revealed features, such as cellular shrinkage, formation of membranebound bodies and break-down of the neuritic processes, indicating apoptosis (Fig. 1A,B). Induction of apoptosis was further demonstrated by intense fluorescence after staining with the DNA-binding dye Hoechst 33342 (Fig. 1C,D). Apoptosis was also analyzed by positive TUNELreaction (Fig. 1E,F). The data of quantitative analysis of the neuronal apoptosis presented in Table 2 were studied using a non-parametric ANOVA analysis. The comparison of means by the Newman-Keuls post test indicated statistical significance of active PP group versus stable PP and OND groups (P , 0.05). Microscopic evaluation of fluoresceinlabeled TUNEL positive cells revealed apoptosis and condensed DNA-binding dye Hoechst characteristic of apoptotic nuclei, confirming that most labeled cells, but not necessarily all, were dying by apoptotic mechanisms rather than cell necrosis. Scarce cell necrosis (less than 5%) was estimated by the percentage of lactate dehydrogenase (LDH) release into centrifugated culture medium over total LDH from both medium and cell lysate [12]. No significant differences between cultures treated with CSFs from active or stable PP patients and OND group were observed (P = 0.448). Tumor necrosis factor-alpha (TNFa) may induce apoptosis [18] and is implicated in the pathogenesis of MS [14]. We have measured its levels by means of an ELISA procedure for the quantitative determination of human TNF-a concentration (Genzyme, Cambridge, MA) in CSF samples. We found very low TNF-a levels (range, undetectable to 8.7 pg/ml) in all CSF samples tested without differences between OND and MS cases. Therefore, it seems very unlikely that TNF-a plays a role in the neuronal damage observed in MS. Our results show that CSF from active patients with primary-progressive MS induces neuronal apoptosis in cultured neuronal cells. Interestingly, the CSF which induced neuronal injury were obtained from patients with an aggressive primary-progressive MS, a disease category where neuronal damage is significantly higher than in patients with relapsing disease MS and little fixed clinical deficit [10].

A. Alca´zar et al. / Neuroscience Letters 255 (1998) 75–78

CSF from patients with stable primary-progressive disease did not damage cultures, nor did incubation with PBS (data not shown), or CSF from patients with other non inflammatory neurological diseases. These findings seem to agree that

77

there exist disease variants related to different pathogenic mechanisms of tissue damage in MS [9]. Our findings may explain as well the decreased amount of neuronal markers, reflecting neuron damage, found in locations without in-

Fig. 1. Induction of neuronal apoptosis by cerebrospinal fluid from active primary- progressive multiple sclerosis patients. Neurons cultured on glass coverslips were maintained in 1 ml of cell culture medium. On days 0, 2 and 4, 100 ml of CSF from patients with non-inflammatory diseases (A,C,E) and active primary-progressive MS (B,D,F) were added, and the cultured was maintained until day 8. After treatment, the medium was aspirated, the cells were fixed with 4% formaldehyde for 25 min and permeabilized in 0.2% Triton X-100 in PBS for 5 min on ice. Afterwards, the cells were washed three times in PBS, and TdT incubation with fluorescein-12-dUTP was carried out for 1.5 h (TUNEL assay). The reaction was terminated by transferring the coverslips to 2× SSC for 15 min, followed by extensive washing in PBS and in deionized water at room temperature. The coverslips were then mounted on slides in glycerol-buffer containing p-phenylenediamine and 30 mM bis-benzimide (Hoechst 33342) for nuclear staining. Cells were visualized under phase-contrast (A,B). Ultraviolet (C,D) and fluorescein-labeled (positive TUNEL reaction) (E,F) nuclei were visualized with fluorescent microscope of the same area. Scale bar, 54 mm.

78

A. Alca´zar et al. / Neuroscience Letters 255 (1998) 75–78

Table 2 Effect of CSF from primary-progressive MS patients on nuclear morphology Disease (No. of patients)

% of cells with apoptotic nuclei

Active primary-progressive MS (3) Stable primary-progressive MS (2) Other non-inflammatory diseases (6)

45 ± 7.0** 26 ± 2.0 24 ± 1.6

Cultured neurons on glass coverslips in appropriate medium, were treated with CSF from patients and processed as described in Fig. 1. Neurons were analyzed with fluorescent microscopy (objective 20×) to count the apoptotic nuclei, green (TUNEL assay), versus total number of nuclei, blue (nuclear DNA-binding dye Hoechst 33342). The nuclear stained images for each field were digitized with CCD camera (760 × 570 pixel resolution) and counted with an image analyzer. Four cardinal points per coverslip, with an area of 138, 125 mm2 each, were counted. Three-four experiments with duplicate coverslips were run for each patient. Data are the mean ± SEM. Statistical differences between the groups were studied using a Kruskal-Wallis ANOVA test. Pairwise comparisons were made using the NewmanKeuls post-hoc test (**P , 0.01) versus OND group.

flammation in magnetic resonance spectroscopy studies in MS [11]. The inflammatory mediators present in CSF [14] could induce neuron apoptosis distant from inflammatory regions. And they do seem to be neither blood components transudated across a damaged BBB nor IgG, since CSF from stable PP patients, with a BBB breakdown (an albumin ratio of 10.3 ± 3.0) and a high IgG index (2.2 ± 1.5), did not cause tissue injury in cultures. These findings are in agreement with the fact that, in some pathological studies of MS, leakage of serum proteins reflecting BBB disruption were present in the central nervous system in the absence of active demyelination and tissue damage [2,8]. In conclusion, regardless of the precise underlying mechanism, soluble factors present in the CSF of active PP patients with MS induce neuronal apoptosis. Present work have not addressed the identification of the possible mediators of cell apoptosis in our cultures. Further research is warranted to characterize the responsible molecules. The identification of these molecules in MS patients could have therapeutic implications, since it would open up the possibility of finding specific inhibitors. We are grateful to M. Go´mez-Calcerrada and J. Cha´fer for their technical and editorial assistance. I.R. acknowledges a fellowship from ‘Fundacio´n Lair’. This work was supported by FIS grants 96/2125 and 97/2114 and by DGES grant PM97-0071 from the Spanish Administration. [1] Alca´zar, A., Rivera, J., Go´mez-Calcerrada, M., Mun˜oz, F., Salinas, M. and Fando, J.L., Changes in the phosphorylation of eukaryotic initiation factor 2a, initiation factor 2B activity and translational rates in primary neuronal cultures under different physiological growing conditions, Mol. Brain Res., 38 (1996) 101–108.

[2] Bru¨ck, W., Bitsch, A., Kolenda, H., Bru¨ck, Y., Stiefel, M. and Lassmann, H., Inflammatory central nervous system demyelination: correlation of magnetic resonance imaging findings with lesion pathology, Ann. Neurol., 42 (1997) 783–793. [3] Canella, B. and Raine, C.S., The adhesion molecule and cytokine profile of multiple sclerosis lesions, Ann. Neurol., 37 (1995) 424–435. [4] Cuzner, M.L. and Norton, W.T., Biochemistry of demyelination, Brain Pathol., 6 (1996) 231–242. [5] Earnshaw, W.C., Nuclear changes in apoptosis, Curr. Opin. Cell. Biol., 7 (1995) 337–343. [6] Filippi, M., Paty, D.W., Kappos, L., Barkhof, F., Compston, D.A., Thompson, A.J., Zhao, G.J., Wiles, C.M., McDonald, W.I. and Miller, D.H., Correlations between changes in disability and T2-weighted brain MRI activity in multiple sclerosis: a follow-up study, Neurology, 45 (1995) 255–260. [7] Hartung, H.-P., Immune-mediated demyelination, Ann. Neurol., 33 (1993) 563–567. [8] Kwon, E.E. and Prineas, J.W., Blood–brain barrier abnormalities in longstanding multiple sclerosis lesions: an immunohistochemical study, J. Neuropath. Exp. Neurol., 53 (1994) 625– 636. [9] Lucchinetti, C.F., Brueck, W., Rodriguez, M. and Lassmann, H., Distinct patterns of multiple sclerosis pathology indicates heterogeneity in pathogenesis, Brain Pathol., 6 (1996) 259–274. [10] Matthews, P.M., Pioro, E., Narayanan, S., De Stefano, N., Fu, L., Francis, G., Antel, J., Wolfson, C. and Arnold, D.L., Assessment of lesion pathology in multiple sclerosis using quantitative MRI morphometry and magnetic resonance spectroscopy, Brain, 119 (1996) 715–722. [11] Narayanan, S., Fu, L., Pioro, E., De Stefano, N., Collins, D.L., Francis, G.S., Antel, J.P., Matthews, P.M. and Arnold, D.L., Imaging of axonal damage in multiple sclerosis: spatial distribution of magnetic resonance imaging lesions, Ann. Neurol., 41 (1997) 385–391. [12] Papasssotiropoulos, A., Ludwig, M., Naib-Majani, W. and Rao, G.S., Induction of apoptosis and secondary necrosis in rat dorsal root ganglion cell cultures by oxidized low density lipoprotein, Neurosci. Lett., 209 (1996) 33–36. [13] Selmaj, K., Raine, C.S., Farooq, M., Norton, W.T. and Brosnan, C.F., Cytokine cytotoxicity against oligodendrocytes: apoptosis induced by lymphotoxin, J. Immunol., 147 (1991) 1522–1529. [14] Sharief, M.K. and Thompson, E.J., In vivo relationship of tumor necrosis factor-a to blood–brain barrier damage in patients with active multiple sclerosis, J. Neuroimmunol., 38 (1992) 27–34. [15] Storch, M. and Lassmann, H., Pathology and pathogenesis of demyelinating diseases, Curr. Opin. Neurol., 10 (1997) 186– 192. ¨ hman, S., Principles of albumin and [16] Tibbling, G., Link, H. and O IgG analyses in neurological disorders. 1. Establishment of reference values, Scand. J. Clin. Lab. Invest., 37 (1977) 385– 390. [17] Vartanian, T., Li, Y., Zhao, M. and Stefansson, K., Interferongamma-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis, Mol. Med., 1 (1995) 732– 743. [18] Wallach, D., Cell death induction by TNF: a matter of self control, Trends Biochem. Sci., 22 (1997) 107–109. [19] Xiao, B.-G., Zhang, G.-X., Ma, C.-G. and Link, H., The cerebrospinal fluid from patients with multiple sclerosis promotes neuronal and oligodendrocyte damage by delayed production of nitric oxide in vitro, J. Neurol. Sci., 142 (1996) 114–120.