Effect of tumor necrosis factor α and β on human oligodendrocytes and neurons in culture

~) Pergamon

Int. J. Devl Neuroscience, Vol. 13, No. 3/4,pp. 369-381,1995

0736-5748(95)00012-7

Elsevier ScienceLtd Copyright© 1995ISDN Printed in Great Britain. All rights reserved 0736-5748/95$9.50+0.00

E F F E C T OF T U M O R N E C R O S I S F A C T O R c~ A N D 13 O N H U M A N O L I G O D E N D R O C Y T E S A N D N E U R O N S IN C U L T U R E J. M c L A U R I N , S. D ' S O U Z A , J. S T E W A R T , M. B L A I N , A. B E A U D E T , J. N A L B A N T O G L U and J. P. A N T E L * Montreal Neurological Institute, McGill University, Department of Neurology and Neurosurgery, Montreal, Quebec, Canada (Received 4 September 1994; revised 6 January 1995; accepted 9 January 1995)

Abstraet--Cytokines produced by infiltrating hematogenous cells or by glial cells activated during the course of central nervous system disease or trauma are implicated as mediators of tissue injury. In this study, we have assessed the extent and mechanism of injury of human-derived CNS oligodendrocytes and neurons in vitro mediated by the cytokines tumor necrosis factor a and 13and compared these with the tumor necrosis factor independent effects mediated by activated CD4 + T-cells. We found that activated CD4 + T-cells, but not tumor necrosis factor ct or 13, could induce significant release of lactate dehydrogenase, a measure of cell membrane lysis, from oligodendrocytes within 24 hr. Neither induced DNA fragmentation as measured using a fluorescence nick-end labelling technique. After a more prolonged time period (96 hr), tumor necrosis factor ct did induce nuclear fragmentation changes in a significant proportion of oligodendrocytes without increased lactate dehydrogenase release. The extent of DNA fragmentation was comparable to that induced by serum deprivation. Tumor necrosis factor 13effects were even more pronounced. In contrast to oligodendrocytes, the extent of DNA fragmentation, assessed by propidium iodide staining, induced in neurons by tumor necrosis factor a was less than that induced by serum deprivation. In-situ hybridization studies of human adult glial cells in culture indicated that astrocytes, as well as microglia, can express tumor necrosis factor ct mRNA. Key words: cytokines, tumor necrosis factor c~and 13,lysis, apoptosis.

Cytokines are a family of soluble protein molecules initially isolated f r o m cells of the i m m u n e system, including l y m p h o c y t e s and monocytes. T h e s e molecules are i m p o r t a n t mediators of cell-cell c o m m u n i c a t i o n within the i m m u n e system, providing critical signals which regulate the i m m u n e r e s p o n s e following initial stimulation. Specific cytokine molecules, such as t u m o r necrosis factor ( T N F ) a and 13, can also function as effectors of the i m m u n e response, i.e. contribute to elimination of the target o f the response. Cells o t h e r than those of i m m u n e origin, including central n e r v o u s system (CNS) glia, are n o w s h o w n to p r o d u c e as well as r e s p o n d to a wide array of cytokines. T o be resolved is w h e t h e r and h o w cytokines p r o d u c e d by e n d o g e n o u s cells o f the CNS, as well as the i m m u n e cells which e n t e r this c o m p a r t m e n t u n d e r pathologic conditions, regulate i m m u n e reactivity within the C N S and contribute to disease-related tissue injury. T h e focus o f the current r e p o r t is on T N F a and 13 as m e d i a t o r s o f injury of h u m a n C N S - d e r i v e d oligodendrocytes ( O L s ) and neurons. Cell-mediated i m m u n e m e c h a n i s m s are postulated to underlie d e v e l o p m e n t of the h u m a n CNS demyelinating disease multiple sclerosis (MS). Such mechanisms are a r e q u i r e m e n t for active induction o r passive transfer o f the animal m o d e l of a u t o i m m u n e CNS demyelination, n a m e l y experimental allergic encephalomyelitis ( E A E ) . T h e pathologic hallmarks of b o t h the h u m a n and animal disorders, in addition to i n f l a m m a t i o n and demyelination, are reactive gliosis and p r o m i n e n t a c c u m u l a t i o n of microglia and m a c r o p h a g e s . T h e mechanisms accounting for the a p p a r e n t selective injury in these disorders of myelin a n d / o r its cell o f origin, the o l i g o d e n d r o c y t e (OL), r e m a i n to be clearly defined. T h e lack of expression o f r e q u i r e d m a j o r histocompatibility c o m p l e x ( M H C ) antigens on the O L s m a k e it unlikely that these cells are injured in a direct antigen- and M H C - r e s t r i c t e d m a n n e r . 16 Non-restricted m e c h a n i s m s o f injury could involve b o t h cell contact and soluble f a c t o r - d e p e n d e n t mechanisms, m e d i a t e d not only by the i m m u n e constituents which have b e e n recruited to the CNS, but also the e n d o g e n o u s glial cells. 4,15,21,26 *To whom all correspondence should be addressed at: Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada, H3A 2B4. 369

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Recent studies using magnetic resonance spectroscopy (MRS) to analyze the chemical pathology of MS lesions in vivo indicate a loss of the N-acetyl-aspartate (NAA) peak in patients with the progressive form of MS and who have developed significant neurologic disability) The N A A peak is considered to be derived from neurons and their axons in the adult human CNS. 33 A reduction in this peak would thus seem to indicate neuron/axon loss in MS, confirming earlier pathologic descriptions. Immune mediators are now also demonstrated to accumulate in the regions of neuronal loss in human degenerative diseases, such as Alzheimer's disease, and speculated to contribute to the disease process. 19 These findings underlie our aim to examine neuron-, as well as OL-directed immune-mediated injury. In the studies described in this report, we have utilized human CNS-derived dissociated cell cultures to assess the injury-producing effects of TNF a and 13on OLs and neurons and compared these effects with those induced using activated T-cells (in this study, CD4 + T-cells). Previous studies using OLs as targets indicated that CD4 + T-cell effects are not mediated via TNF. 1 We have utilized assays designed to assess the two major currently identified mechanisms of cell injury, namely direct cell membrane injury (lysis) and nucleus event-dependent processes (apoptosis). 7,34 We have also attempted to demonstrate which of the adult human glial cells are capable of producing the cytokine TNF a. EXPERIMENTAL PROCEDURES

Effector cells and molecules CD4 + T-cells were prepared from peripheral blood samples obtained from healthy volunteers and activated in vitro with anti-CD3 antibody, as previously described. 1,27 Supernatants were collected from these activated cultures for testing in the cytotoxicity assays described below. Recombinant cytokines used in these studies were obtained from commercial sources [TNF a and 1~ (Genzyme Boston MA),-~-interferon (~/-IFN) (UBI Lake Placid NY), and interleukin (IL) 113 (Intermedico Diagnostics, Markham, Ontario)].

Preparation of glial cells Adult CNS-derived cells. OLs were derived from temporal lobe tissue specimens resected from young adults undergoing surgical resection for the treatment of intractable epilepsy. The method of cell isolation, previously described in d e tail,,140 utilizes an initial trypsinization step, followed by Percoll gradient centrifugation. Dissociated cells are cultured in minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS) and 0.1% glucose. Subsequent separation of glial cell subtypes is based on their differential adhesion to uncoated Falcon tissue culture flasks (Fisher, Montreal, Canada) or Petri dishes. Enriched populations of OLs are harvested from the mixed glial preparation in the form of the non-adherent cell fraction after 24 hr and placed onto poly-L-lysine-coated Aclar coverslips (5× 104 cells per coverslip). The proportion of OLs in these cultures is estimated at 75-80% using the immunocytochemical methods described below. A microglia-enriched population (90% +) can be prepared by shaking of the residual glial cell preparation one week after the OLs are depleted. The proportion of astrocytes in the OL- and microglia-depleted fraction varies from 40 to 70% and thus cannot be considered as highly enriched. Fetal CNS-derived cells. Neural cells are derived from human fetal CNS (cerebral hemispheres) tissue obtained at 12-16 week gestation. The cultures are prepared by dissociation of the fetal CNS tissue with 0.05% trypsin and 50 ixg/ml DNase, passing the tissue through a 125 ~m nylon mesh screen and then through a 70 p,m screen. After washing with PBS, the cells are suspended in culture medium (MEM supplemented with 5% fetal bovine serum, 0.1% glucose and 1 mM sodium pyruvate) and then placed onto poly-L-lysine-coated Aclar coverslips (5 × 104 cells per coverslip). Culture dishes containing the coverslips are treated on day 4 with 1 mM 5-fluoro-2-deoxyuridine (5-FDU) to deplete astrocytes. The treatment is repeated twice over a two-week time period. Immunocytochemistry The individual neural cell types were characterized by immunostaining with mouse anti-GalC (H8H9) (derived from hybridoma supernatant) or mouse anti-MBP monoclonal antibody (mAb)

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(Boehringer-Manheim, Laval, Quebec) for OLs, rabbit anti-GFAP Ab (Dako, Westchester, PA) for astrocytes and rabbit anti-neuron-specific enolase (NSE) Ab (Dako) or mouse anti-Tau (Sigma No. T-5530) or anti-neurofilament (SMI 33 Sternberger-Meyer) mAbs for neurons, respectively, followed by fluorescein-conjugated goat anti-mouse (TAGO, Camarillo, CA) or goat anti-rabbit Abs (Cappel, Lexington, MA) or goat anti-rabbit horseradish peroxidase (ABC Kit, Dimension Lab.) with diaminobenzadine (DAB) visualization, respectively. MBP immunostaining was preceded by fixation in 1:1 acetone:methanol for 10 min; astrocyte and neuron staining involved fixation of cells in acid:alcohol (5% glacial acetic acid 95% absolute alcohol) for 15 min. Except for fixation, which was done at -20°C, all steps were performed at room temperature. The Gal C staining was done on live cells without fixation. Cells on coverslips were incubated with all primary and secondary antibodies for 45 rain; cells were washed three times in PBS between incubations. After staining, previously non-fixed cells were fixed in 2% paraformaldehyde. Assays of neural cell injury. For these studies, effector cells or soluble molecules were added to cultures of target cells attached to coverslips placed in 24-well microtiter plates at E:T ratios or concentrations and for time durations indicated in Results. Initial studies established that CD4 + T-cell activity was dependent on the E:T ratio and that cytokine effects were concentration dependent. Most T-cell-mediated injury studies reported here were performed at a 10:1 E'T ratio, estimating that each coverslip with OLs or neurons contained 3×104 cells. After the indicated co-culture period, target cell injury was assessed either in terms of cell membrane- or nucleusdirected injury. Cell membrane-directed injury. In initial studies involving assays of 18 hr or shorter, we utilized 51chromium (Cr) release as the standard measure of cell membrane injury. 27 The 51Cr assay is, however, unsuited for long-term assays due to unacceptably high spontaneous release values. In current studies, which range in durations of up to four days, we have used release of lactate dehydrogenase (LDH) from target cells into the culture supernatants, as determined using a commercial kit (Sigma), as our measure of cell membrane injury. In this assay, supernatant was collected from cultures containing the indicated combination of effectors and targets. Sample tubes containing 0.5 ml of 2 mg/ml N A D H , 0.5 ml of 1.5 mmol per l pyruvate substrate and 100 p~l of test sample were incubated for 30 rain at 37°C. Pyruvate calibration curve tubes were set up according to the table provided in the kit (Sigma procedure No. 500 booklet). One ml of color reagent was then added to all tubes which were then left at room temperature for 20 min, after which 10 ml of 0.4 N N a O H was added to each tube to stop the reaction. Absorbency was read at 460 nm. Test sample L D H values are calculated by comparison with a curve generated using the pyruvate standards. Results are expressed as B-B units/ml (as per Sigma assay kit). Results are presented as L D H values of test samples minus L D H values of culture medium alone. Nuclear injury (apoptosis). Given that our cell targets of injury are non-dividing primary cells and are available only in relatively small cell numbers, we have utilized either the terminal transferase (TdT)-mediated dUTP-biotin nick end labelling (TUNEL) technique which'~identifies DNA fragmentation or changes in nucleus morphology seen with propidium iodide (PI) staining as our measures of nuclear injury. For the T U N E L assay, coverslips containing OLs, initially fixed and immunostained with anti-MBP antibody as described, are incubated for I hr at 37°C with 50 :p~lof nick end labelling solution containingTdT (0.3 U/ml) and biotinylated dUTP (0.01 nmol/ml) in TdT buffer (Promega). The reaction is terminated by transferring the coverslips into microwells containing Tris buffer for 15 min at room temperature. After blocking with 2% BSA for 15 min and washing, the coverslips are incubated with Streptavidin-FITC (1:20, 30 min at 37°C) (BoehringerMannheim); coverslips are then incubated with Hoechst dye 33258 (10 ~g/ml, 20 min) to identify individual nuclei. Propidium iodide staining was carried out on neuron-containing cultures by initially fixing coverslips as previously described. After rehydration for 30 min in PBS, coverslips are immunostained with anti-MBP or anti-NSE Abs. Coverslips are then stained with propidium iodide (10 ~g/ml, 20-min incubation), washed in PBS and mounted for counting. We have used serum deprivation, a commonly used means to induce apoptosis in many cell lines, as a means to determine whether our target cells will show evidence of nuclear injury in the above assays. T U N E L and PI quantitative data were obtained by counting coded slides. For OLs, between

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100 and 300 cells per slide were counted, with at least duplicate slides for each test condition being used. For neurons, 400 cells per slide were counted. TNF e~ effects on OL nuclei were confirmed using electron microscopy (800 nM sections of 2% gluteraldehyde fixed coverslips). In some studies, an Mq-T assay was applied to TNF e~-or [3-treated OLs and results quantitated by microscopy. Detection of T N F o~m R N A in glial cells in vitro To establish which glial cell population expressed TNF o~ mRNA in mixed astrocyte-microglia cultures, we have used a combined immunoperoxidase--in-situ hybridization approach. For these studies, cells were plated onto slides and incubated for seven days to allow morphological development. Cultures were then treated with a combination of lipopolysaccharide (LPS) (5 ixg/ml) and IFN ~ (100 units/ml) for 24 hr. Slides were washed in PBS before fixation of cells in 2% PFA for 4 min, followed by treatment with 0.03% Triton X-100 in PBS. Immunostaining with an anti-GFAP antibody was carried out using the Vectastain kit (Dimension Laboratories, Mississauga, Ontario) according to the manufacturer's recommendations. For in-situ hybridization, the same slides were treated with 0.2 N HCI for 20 min, washed and then incubated with pronase (60 p~g/ml) for 20 min, followed by a post-fixation step to remove excess pronase. The samples were acetylated for 10 min in 0.25% acetic acid (in 0.1 M ethanolamine) and washed in PBS prior to prehybridization for 3-8 hr at 37°C in 1 × salts solution [0.3 M NaC1, 0.01 M Tris-HC1, 0.01 M NaH2PO4 (pH 6.8)], containing 5 mM EDTA, 0.02% Ficol1400, 0.02% polyvinylpyrolidone,0.02% bovine serum albumin and 100 txg/ml total yeast RNA. The sense and anti-sense TNF a RNA probes were labelled with 35S-UTP using the in-vitro transcription system of Ambion (Austin, Texas). Samples were hybridized overnight at 50°C in the presence of 1×106 cpm probe in hybridization buffer [1 Xsalts solution containing 50% formamide, 10% dextran sulfate, 10 mM DTT]. Slides were treated with RNase (20 Ixg/ml) for 30 min at 37°C to remove excess unhybridized probe followed by several washes of increasing stringency with the last wash consisting of 0.1 x SSC (0.03 M NaCI, 0.015 M sodium citrate) at room temperature. After dehydration, slides were dipped in Kodak NTB2 emulsion and exposed for 10-20 days. The slides were developed with Kodak D-19 developer. For most studies, sister cultures were immunostained for GFAP by the previously described florescence technique; these studies provided morphologic data to identify astrocytes which did or did not hybridize in our in-situ hybridization studies.

RESULTS O L-directed injury These studies were conducted using 2- to 4-week-old process-bearing OLs as target cells (Fig. 1A). As shown in Fig. 2 and Table 1, after 96 hr of TNF et or [3 exposure, a significant proportion of the cells were labelled by the TUNEL technique (Fig. 1B); at this time there was still no significant increase in LDH release over control values (Fig. 2). The extent of TNF B-induced DNA fragmentation was greater than that of TNF e~;the latter extent was comparable to that induced by serum deprivation (Table 1). Other cytokines tested, IFN ~ and IL-1, did not induce significant DNA fragmentation. The delayed TNF et effects on OL nuclei were confirmed by electron microscopy (Fig. 3). MTT assays indicated a decrease in viable cells following the prolonged TNF e~or [3 exposure. We found neither significant LDH release nor DNA fragmentation in cultures of OLs that were serum-deprived or treated with TNF a for 24 hr (Table 1). In contrast to the TNF e~and [3effects, anti-CD3 activated CD4 + T-cells induced significant LDH release from OLs within 24 hr. Previous studies indicated that no significant lysis was seen at 5 hr or with non-activated cellsJ As shown in Table 2, the T-cell-mediated lysis was not inhibited by addition to the cultures of 5 mM EGTA which blocks calcium dependent cell lysis or 10 ~g/ml cycloheximide which blocks new protein synthesis. At the 24-hr time point following co-culture with activated CD4 + T-cells, we did not observe a significant increase in proportion of OLs with DNA-fragmented nuclei compared to control cultures (Fig. 2).

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Fig. 1. Four-week-old human adult OLs maintained in dissociated cell culture and then exposed to 1000 units TNF ct for 96 hr. Panels A and D are TNF-treated and non-treated (control) cultures, respectively, immunostained with anti MBP antibody. Panels B and E are the corresponding treated and control cultures labelled with T U N E L technique. Panels C and F are the corresponding treated and control cultures stained with Hoechst dye to demonstrate nuclei. Magnification= 300×.

Neuron-directed injury These studies were conducted on cultures enriched for neurons by treatment of the initial mixed culture with 5-FDU. Neurons are identified by immunostaining with neuron-specific enolase antibody, as illustrated in Fig. 4, or with anti-Tau or anti-neurofilament antibodies. Using an H P L C assay (Paul Matthews, Alan Sherwin, MNH), we could detect N A A in the neuron-enriched cultures and not in purified fetal astrocyte cultures (data not shown). In parallel with our OL data, we observed that serum deprivation induced apoptosis in the neuron-enriched cultures as assessed using PI staining (Table 1, Fig. 5). The proportion of apoptotic nuclei increased over the time period tested (2~ 48 hr). Longer time intervals were difficult to evaluate because significant numbers of cells were lifting off from the cover slips. Neither TNF ~ nor IFN ~ were as effective

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-6O Fig. 2. Comparison of mechanisms of human OL injury mediated by CD4 + T-cells (10:1 E:T ratio) and TNF ta (1000 units/ml). (A) Cell membrane injury as measured by LDH release (units/ml). (B) Nuclear injury as measured by percent of cells showing DNA fragmentation by the fluorescence nick end labelling TUNEL technique. CD4 + T-cell effects were assayed at 24 hr (a); TNF e~effects were assayed at 24 (b) or 96 hr (c). Each bar represents the result of an individual experiment in which LDH and TUNEL assays were. applied to the same target cells exposed to the indicated effector condition. Open bars in [A(a)] indicate LDH in CD4 + T-cell: OL co-cultures; hatched bars indicate LDH in cultures containing only CD4 + T,cells. LDH release by OLs alone did not differ significantly from LDH values found in serumcontaining culture medium alone.

Table 1. Induction of DNA fragmentation in human OLs and neurons by TNF ct and I~ and serum deprivation % Target cells with fragmented DNA OLs (TUNEL assay) Test conditions

24 hr

Control cultures

4+2% n =3 5-+1 n =3 5-+1 n=3

Serum deprivation TNF ct--1000 units TNF 13--1000 units T-cell supernatant INF ~--100 units ILl 13---100units

96 hr

Neurons (P.I. stain) 24 hr

48 hr

5.7-+0.9% n =9 66.1---3.6 n =7 64.7-+3.3 n=7 85.3+_3.3 n=3

10-+4% n =2 24

6±0% n =3 39

10

22

6_+0.3 n=2 4.3+0.3 n=2

9

42 17

Data indicate results of e x p e r i m e n t s (mean+_S.E.M.) in which D N A fragmentation of nuclei of OLs and neurons was determined using either the TUNEL assay or PI staining, respectively, in response to exposure to single doses of cytokines indicated or to serum deprivation. n = number of experiments performed under each condition. Data on neurons are derived from two separate experiments, one analyzed at 24 hr, the other at 48 hr. The T-cell supernatant was tested by substituting the supernatant for the usual culture medium.

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Fig. 3. Electron micrograph of human adult OLs maintained in dissociated cell culture under control conditions (A) or exposed to TNF tx 1000 units/ml (B) for 72 hr. TNF c~-exposed cells show nuclear condensation, cell volume reduction and membrane blebbing. Magnification = 10,000×.

inducers of apoptosis of neurons as was serum deprivation. In contrast, T-cell supernatant induced apoptosis to an extent similar to serum deprivation values.

Expression of TNF ol m R N A by glial cells As illustrated in Fig. 6, when mixed LPS- and IFN ~/-activated adult glial cell cultures were probed using a TNF a anti-sense probe, a positive hybridization signal was found over some but not all G F A P + cells and, even more strongly, over almost all microglia (defined by combination of morphology and non-reactivity with GFAP). No specific signal was detected using the sense probe control. In previous studies, we demonstrated that human microglia-produced TNF oL can be detected by bioassay.1

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J. McLaurin et aL Table 2. Effects of calcium depletion and protein synthesis inhibition on CD4 + T-cellmediated injury of OLs--24-hr assay LDH release (units/ml) +Culture medium OLs alone OLs+CD4 + T-cells CD4 + T-cells alone

271---94 682_+105 94-+40

+EGTA (5 raM)

+Cycloheximide (10 ~g/ml)

155+--157 505+44

-63_+16 505_ + 106

Data indicate mean_S.E.M. LDH release by cultures containing either OLs alone or OLs and activated CD4 + T-cells (10:1 E:T ratio) and to which are added either EGTA or cycloheximide. Results are derived from three separate experiments performed under each condition. All cultures were performed in FBS-containing medium. LDH content of medium alone was subtracted from total LDH values obtained.

Fig. 4. Two-week-old cultures of 5-FDU-treated human fetal CNS cultures enriched for neurons. Culture is immunostained with anti-NSE antibody using a peroxidase reaction. Magnification=400×. DISCUSSION B o t h c e l l - c o n t a c t - d e p e n d e n t a n d s o l u b l e - f a c t o r m e d i a t o r s a r e i m p l i c a t e d as p o t e n t i a l m e d i a t o r s o f tissue i n j u r y as m a y occur d u r i n g t h e c o u r s e o f i n f l a m m a t o r y a n d d e g e n e r a t i v e d i s o r d e r s o f t h e C N S o r f o l l o w i n g C N S t r a u m a . W e h a v e u t i l i z e d in-vitro assays to c o m p a r e t h e s u s c e p t i b i l i t y o f human OLs and neurons, to TNF-dependent and TNF-independent effector mechanisms. In o u r initial studies using a d u l t C N S - d e r i v e d O L s , w e f o u n d t h a t t h e s e cells w e r e s u s c e p t i b l e t o n o n - M H C - r e s t r i c t e d cell m e m b r a n e i n j u r y m e d i a t e d b y a c t i v a t e d C D 4 + a n d C D 8 + T-cells, as m e a s u r e d using a 51Cr r e l e a s e assay. 1,27 T h i s effect c o u l d n o t b e r e p r o d u c e d using T-cell s u p e r n a t a n t o r with r e c o m b i n a n t T N F a o r [3. A n t i - T N F a n t i b o d i e s d i d n o t b l o c k t h e effect. N e i t h e r a c t i v a t e d m a c r o p h a g e s n o r m i c r o g l i a , cell t y p e s s h o w n to s e c r e t e T N F a , i n d u c e d significant cell m e m b r a n e

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Fig. 5. Propidium iodide staining of a two-week-old neuron enriched fetal human CNS culture which had been serum deprived for 72 hr. Examples of apoptotic nuclei are indicated by arrows. Magnification =800 x.

injury in our 24-hr assay system. Our current data, using L D H release as a measure of cell membrane injury, suggest that the CD4 + T-cell mediated cell membrane injury is not associated with preceding D N A fragmentation in nuclei. The effect is also not inhibited by treatment of the OLs with either E G T A or cycloheximide, indicating that it is independent of calcium and new protein synthesis. These results differ from most reports regarding CD4 + T-cell-mediated lysis of target cells in which D N A fragmentation, measured by morphologic or radiolabelling techniques, is demonstrated to precede subsequent cell membrane lysis as measured by 51Cr or L D H release. 1°,25,42 In many of these studies, proliferating cell lines were used as targets, whereas our target cells are post-mitotic. We cannot exclude the fact that the small percent of non-OL cells (microglia, astrocytes and fibroblasts) in our "OL-enriched" cultures also sustained T-cell-mediated injury and contributed to the L D H release. In agreement with previous studies18, 3° performed using rodent or bovine OLs or an O L precursor cell line, we do find that TNF c~ and 13can induce D N A fragmentation or apoptosis of human adult OLs in vitro as assessed morphologically. Conflicting data exist as to whether OLs will form " D N A ladders" as they undergo nuclear injury.5,24 The TNF effects are delayed relative to the membrane lysis effect induced by activated T-cells. The effects of a single dose of 1000 units/ml of TNF a are similar to those we observe using serum deprivation as a means to induce D N A fragmentation. TNF 13 effects are more pronounced than those of TNF o~, in agreement with previous observations. 30 We have also found that TNF ~ treatment results in a decreased detectability in OLs of statin, a molecular complex whose expression is characteristic of cells in the GO phase of the cell cycle .2,23,35 These data would be consistent with the postulate that apoptosis in terminally differentiated cells may reflect an abortive attempt at mitosis) 4 Although this study has focussed on TNF ot and 13, one need consider that other T-cell or microglia/macrophage cytokines and non-cytokine soluble molecules may also effect OLs. Neither IFN 3' nor IL-113 induced DNA fragmentation in OLS in

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Fig. 6. Expressionof TNF a mRNA in a dissociatedadult human CNS microglia-astrocyteenriched culture stimulated in vitro with IFN ~/and lipopolysaccharide(LPS). Cultures were immunostained with anti GFAP antibody-peroxidaseand hybridized with either a TNF a anti-sense or sense probe. ~ indicates astrocytes with a positive hybridization signal; A indicates a non-hybridizingastrocyte; I~ indicates a microglial cell with a positive hybridizationsignal. No significanthybridizationsignal was seen in sister cultures probed with the sense TNF probe.

this study. Previously, we did not observe any effect of IL2 on statin expression by human OLs in vitro; 23 this cytokine is reported both to induce O L proliferation and to be cytotoxic in rodent systems.6,11,21 Distinguishing the mechanisms and mediators of O L cell injury has potential therapeutic implications. Louis et al. reported that T N F a-induced apoptotic killing of an O L precursor cell line was inhibited by pretreatment of the cells with ciliary neurotrophic factor (CNTF). 18 Our data, however, indicate that TNF-independent effector mechanisms need also to be considered if therapies aimed at protecting OLs from immune-mediated injury are to be developed. One need further consider additional mechanisms whereby cytokine effects on OLs may modulate their response to injury. IL-1, a microglia- or macrophage-produced cytokine, but not T N F a directly induces a stress (heat shock) protein response in OLs. 9 Whether this response confers protection from injury or enhances susceptibility to immune effector cells, such as ~//~ T-cells, remains speculative. 13,29 Cytokine effects may also modulate the O L response to injury via their effects on the glial cells in the environment of the OLs. For example, astrocytes in vitro can protect OLs from free radical-mediated injury;22 whether this capacity is modified by cytokine actions on the astrocytes remains to be established.

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The in-vitro data regarding neuron-directed injury were generated using fetal CNS-derived tissue. We have not been able to sustain neuron survival using cells derived from our adult material. Although our method to prepare enriched neuron cultures requires the use of an anti-metabolite (5-FDU), only a small proportion of the cells showed signs of apoptosis under serum-supplemented culture conditions. The extent of D N A fragmentation observed in neurons following exposure to 1000 units/ml of TNF a was less than that observed under conditions of serum deprivation, in contrast to the results using OLs. These results would seem consistent with those of Guilian et al. who, in a rodent system, found that soluble factor-mediated injury of neurons was not TNF-dependent, whereas OL-directed lysis was dependent o n T N F . 15 The neurons were highly susceptible to nuclear injury when cultured in the presence of activated T-cell supernatant. We have not yet established whether this effect is dependant on specific effector molecules or on non-specific effects. Under in-vivo pathologic conditions, multiple effector mechanisms mediated by one or multiple cell types will likely be operative at the same time. The array of cytokines shown to be produced by glial cells continues to expand, with production likely to vary in concert with the state of activation of the glial cells. In our highly enriched adult human microglia cultures, using ELISA and bioassays on supernatant from these cultures, we can document that these cells produce an array of cytokines, including TNF oc. 1,36 We found that myelin ingestion by such microglia resulted in increased secretion of cytokines IL-1, IL-6 and TNF ct. 37 Since our adult "astrocyte" cultures contain a variable proportion of microglia and astrocytes, we have attempted to assess cytokine "production" using in-situ hybridization. Our current data indicate that TNF ot mRNA can be detected in some astrocytes derived from adult human CNS tissue and activated in vitro, although the proportion was less than that for microglia. The re-sected tissue from which the cultures were derived did show evidence of reactive gliosis on pathologic examination. Astrocyte production of TNF a has been previously reported in "purified" rodent and human fetal astrocyte-enriched cultures, although the question of microglia contamination in such cultures persists. 17,28,31 Although the focus of this report is on the role of cytokines as direct effectors of OL and neuron injury, their role as regulators of immune and glial cell activity within the CNS should also be recognized. 12,37,38 Under pathologic conditions in which inflammatory cells infiltrate the CNS, cytokines are likely be important mediators of glial-immune interactions. In our in-vitro human tissue culture systems, we have demonstrated effects of T-cell-derived cytokines on astrocyte properties including proliferation and expression of surface molecules involved in the immune response such as major histocompatibility complex (MHC) and adhesion molecules. 41 Donor age and species prove to be important variables in these studies. In this regard we identified IFN -y as a principal mediator of T-cell supernatant-mediated proliferative effect on human astrocytes. In contrast, this cytokine inhibits proliferation of rodent astrocytes in vitro and in vivo. Specific cytokines such as IL-10 and transforming growth factor (TGF) t3 can also inhibit proliferation and down-regulate surface antigen expression. TGF 13 has now reached the clinical trial stage for multiple sclerosis. These regulatory cytokines can be derived from both T-cells and glial cells, indicating the presence of complex paracrine and autocrine regulatory networks within the CNS. The discussion above has focussed on the injury-promoting aspects of cytokine-glial interactions. Their potential converse effects as protective or regeneration-promoting agents should also be considered. 8,32 The OL-protective effects of CNTF, a molecule usually considered as a neurotrophin but with homologies with the cytokine IL-6, was referred to previously. 18 As regards regeneration, soluble factors contained in microglia or macrophage supernatant are noted to enhance neurite outgrowth. 8 Neurons themselves may utilize cytokines to signal glial cell responses.

CONCLUSION Cytokines, whether arising from endogenous glial cells or from infiltrating hematogenous cells, can exert multiple effects within the CNS. Deleterious effects include direct ones as mediators of tissue injury or indirect ones via up-regulation of glial- or immune-mediated effector processes. Positive effects include protection of target cells from injury, down-regulation of effector processes and promotion of regeneration. A continuing challenge is to define the precise cytokines mediating specific positive or negative events within the CNS and the molecular mechanisms which regulate

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t h e m . C u r r e n t k n o w l e d g e a n d a v a i l a b i l i t y o f c y t o k i n e s has a l r e a d y p e r m i t t e d u s e o f t h e s e m o l e c u l e s t h e m s e l v e s o r m o l e c u l e s w h i c h m o d u l a t e t h e i r f u n c t i o n in t h e r a p e u t i c s i t u a t i o n s . T h e r a p e u t i c u s e f o r C N S i n d i c a t i o n s will n e e d t o c o n s i d e r t h e e f f e c t s o f t h e s e m o l e c u l e s n o t o n l y o n C N S s t r u c t u r e s , but also on extra-CNS structures, including the immune system.

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