Journal of Neurochemistry Raven Press, Ltd., New York D I990 International Society for Neurochemistry
Demyelination Induced in Aggregating Brain Cell Cultures by a Monoclonal Antibody Against Myelin/Oligodendrocyte Glycoprotein Nicole Kerlero de Rosbo, *Paul Honegger, ?Hans Lassmann, and Jean-Marie Matthieu Laboratoire de Neurochimie, Service de Pbdiatrie, Centre Hospitalier Universitaire Vaudois; *Institut de Physiologie de l'liniversite' de Lausanne, Latisunne, Switzerland; ?Institute for Bruin Research, Austrian Academy of Sciences: and ?Neurological Institute, University of Vienna, Vienna, Austria
Abstract: A monoclonal antibody (8- 18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures. With increasing doses of anti-MOG antibody in the presence of complement, myelin basic protein (MBP) concentrations decreased in a dose-related manner. A similar, albeit less pronounced, effect was observed on specific activity of 2',3'-cyclic nucleotide 3'phosphohydrolase. In the absence of Complement, anti-MOG antibody did not induce detectable demyelination.In contrast to the effect of anti-MOG antibody and as expected, antiMBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement. These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side ofthe myelin membrdne, is a good target antigen for antibodyinduced demyelination. Indeed, they show that a purified
anti-MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures. Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin-associated glycoprotein. These observations may have important implications regarding the possible role of antiMOG antibodies in demyelinating diseases. Key Words: Myelin/oligodendrocyte glycoprotein-Demyelination-Aggregating brain cell culture-Myelin basic protein-2',3'Cyclic nucleotide 3'-phosphohydrolase.Kerlero de Rosbo N. et al. Demyelination induced in aggregating brain cell cultures by a monoclonal antibody against myelin/oligodendrocyte glycoprotein. J. Neurochem. 55, 583-587 (1990).
Autoimmune responses to myelin and oligodendroglial components have been demonstrated in multiple sclerosis, a primary demyelinating disease (for review, see Walsh and Tourtellotte, 1983), and demyeh a t i n g activity has been linked to immunoglobulins in sera and CSF of multiple sclerosis patients (Walsh and Tourtellotte, 1983; Raine, 1984). However, the antigens responsible for induction of antibody-mediated demyelination in multiple sclerosis have not yet been identified. Sera from animals with the demyelinating form of experimental autoimmune encephalomyelitis (EAE), which are known to contain antibodies directed against myelin components such as myelin
basic protein (MBP), proteolipid protein (PLP), and galactocerebroside, are myelinotoxic in CNS cultures (Lassmann and Linington, 1987). Although polyclonal antisera raised in rabbits against purified MBP, PLP, or myelin-associated glycoprotein had no deleterious effect in such cultures (Seil et al., 1968, 1981; Seil and Agrawal, 1980), antibodies directed against myelin glycolipids initiated demyelination (Dubois-Dalcq et al., 1970; Fry et al., 1974; Raine et al., 1981; Seil and Agrawal, 1984). However, both in vitro (Lebar et al., 1976) and in vivo (Schwerer et al., 1984) studies have shown that the demyelinating activity in EAE sera is not attributable solely to detectable antiglycolipid an-
Received August 15, 1989; revised manuscript received December 10, 1989; accepted January 3, 1990. Address correspondence and reprint requests to Dr. J.-M. Matthieu at Laboratoire de Neurochimie, Service de PMiatrie, Centre Hospitalier Universitaire Vaudois, CH- I0 1 1 Lausanne, Switzerland. The present address of Dr. N. Kerlero de Rosbo is Neuroimmunology Laboratory, La Trobe University, Bundoora 3083, Victoria, Australia.
Abbreviaions used:CNP, 2',3'-cyclic nucleotide 3'-phosphohydrolase; EAE, experimental autoimmune encephalomyelitis; GS, glutamine synthetase; MBP, myelin basic protein; MOG, rnyelin/oligodendrocyte glycoprotein; PBS, phosphate-buffered saline; PLP, proteolipid protein.
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N. KERLERO DE ROSBO ET AL.
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tibodies. It has been suggested that the CNS-specific myelin/oligodendrocyteglycoprotein (MOG)would be a good target for .antibody-induced demyelination (Lassmann and Linington, 1987), as it is located preferentially at the external surface of myelin sheaths and oligodendrocytes (Brunner et al., 1989). The potential role of anti-MOG antibodiesin demyelination has been tested in vivo: In acute EAE, where little or no demyelination is usually seen (Raine, 1984), extensive CNS demyelination can be induced by intravenous injection of a monoclonal anti-MOG antibody, clone 818C5 (Linington et al., 1984), at the time when the blood-brain bamer is breached (Schluesener et al., 1987). In addition, the possible involvement of MOG as a target antigen in demyelinating diseases is shown by the presence of anti-MOG antibodies in chronic relapsing EAE (Linington and Lassmann, 1987). In aggregating rat brain cell cultures, established and maintained in serum-free, chemically defined medium, cell development and differentiationproceed as in normal brain tissue (Honegger, 1985). Furthermore, the chemical composition of the cultured myelin membrane closely resembles that found in normal brain tissue (Matthieu et al., 1979). Thus, such a system is ideally suited to investigate the potentially neurotoxic effects of antibodies recognizing myelin and/or oligodendroglial components, avoiding the complex systemic interactions and possible artifacts inherent to in vivo demyelination assays. By combining such a culture system with the use of the purified immunoglobulin fraction of anti-MOG antibody, we show that the demyelinating effect of anti-MOG antibody is specific and dose related in the presence of complement.
PBS) in the presence or absence of complement (guinea pig serum; 25 pl/ml of culture medium). In control cultures, the same volume of PBS with or without complement (total volume, 500 pl) was added. On day 26, the aggregates of each culture flask were split into two equal parts; the volume of each flask was then made up to 8 ml by addition of 4 ml of fresh medium with or without guinea pig complement as above. Media were replenished by exchange of 5 ml of medium per flask on day 27, and the cultures were harvested on day 29.
MATERIALS AND METHODS
In aggregating fetal rat brain cell cultures, myelination starts at around culture day 16 and reaches a maximum -8 days later (Honegger and Matthieu, 1980). Thus, the potentially demyelinating effect of antimyelin antibodies on brain cell aggregates was investigated by the application of antibody preparations on culture day 25, in the presence or absence of complement (guinea pig serum). Three parameters were examined on treatment completion. The amount of myelin was assessed by measuring the content of MBP in the cultures. The effect of the treatments on dial cells was estimated by measuring changes of two specific markers: CNP for oligodendrocytes and GS for astrocytes. In the absence of complement, no demyelination was seen with the antimyelin antibodies at all concentrations used (data not shown). Complement alone had no effect on MBP content in the cultures (Table 1). However, in the presence of complement, a statistically highly significant loss of MBP was observed with anti-MOG IgG at a concentration of 62.5 pg/ml of culture (Table 1). In contrast, anti-MBP IgG at the same (Table 1) or a higher ( 125 pg/ml of culture; data not shown) concentration had no such effect. In the presence of complement, there was a significant de-
Aggregating brain cell cultures Rotation-mediated, serum-free aggregating cell cultures were prepared from mechanically dissociated fetal ( 15- 16 days of gestation) rat telencephalon (Wistar strain; Madorin AG, Fiillinsdorf, Switzerland), as previously described (Honegger, 1985; Honegger et al., 1986).
Antibodies Monoclonal antibody against rat MOG was derived from clone 8-18C5 (Linington et al., 1984); the IgG was purified from ascites fluid produced in mice by affinity chromatography using the Bio-Rad Econo-Pac protein A kit (Bio-Rad, Richmond, CA, U.S.A.). Polyclonal rabbit anti-MBP antibody was a gift from Dr. c. Bernard ( L a Trobe University, Bundoora, Victoria, Australia). It had been raised against a synthetic peptide comprising the sequence of amino acid residues 160- 167 of the I 8.5-kDa human MBP. The IgG fraction was affinity-purified from the antiserum on a protein ASepharose CL4B column as described previously (Bernard et al., 1981).
Treatment of brain cell aggregates On culture day 25, brain cell aggregates were treated with varying concentrations of purified anti-MOG or anti-MBP IgG in phosphate-buffered saline (PBS; 1 mg of IgG/ml of
J. Neurochem., Vof.55, No. 2, 1990
Analytical procedures For biochemical analyses, the aggregates of each flask were washed twice with 5 ml of ice-cold PBS and homogenized in 0.5 ml of 2 mM potassium phosphate containing 1 mM EDTA (pH 6.8) using 2-ml glass-glass homogenizers (Bellco, Vineland, NJ, U.S.A.). The homogenates were sonicated briefly and stored at -80°C as aliquots for the different assays. MBP content was measured by radioimmunoassay (Burgisser, 1983). 2’,3’-Cyclic nucleotide 3‘-phosphohydrolase (CNP; EC 3.1.4.37) activity was determined by the method of Sogin (1976). Glutamine synthetase (GS;EC 6.3.1.2) activity was assayed by a modification of the method of Pishak and Phillips (1 979), using L-[ l-’4C]glutamicacid (New England Nuclear-DuPont, Boston, MA, U.S.A.) as the precursor and phosphoenolpyruvate/pyruvate kinase as the ATP-regenerating system (Pate1 et al., 1982). Protein concentrations were measured by the Folin phenol method (Lowry et al., 1951) using bovine serum albumin as the standard.
Statistics Statistical significance was assessed using Student’s t test. Differences with p < 0.05 were considered as significant.
RESULTS
ANTI-MOG-IIVDUCED DEMYELINA TION IN CELL CUL TUKE TABLE 1. Complement (C)-mediated efect oJantibodies to MBP and MOG on oligodendrocyte and myelin markers
Treatment
MBP (pg/mg of total protein)
CNP (rmol of 2'-NADP/min/ mg of total protein)
None C Anti-MBP C Anti-MOG C
1.18 f 0.39 1.01 k 0 . 1 4 1.25 f 0.17 0.25 ? O.lOb
1.81 f 0.33 3.09 f 0.57"
+ +
3.23 f 0.45" 1.88 & 0.45
Guinea pig serum and purified IgG were added to aggregating cell cultures at a concentration of 25 p1 and 62.5 pgjml, respectively. Data are mean f SD values for four culture flasks. Significant differences by Student's t test from untreated cultures were indicated: " p < 0.0 I , ' p < 0.001.
crease in CNP activity in cultures treated with antiMOG IgG (62.5 pg/ml of culture; Table I). Anti-MBP antibody had no deleterious effect on CNP activity in the presence of complement; in fact, the effect was quite the opposite, showing a twofold increase as compared with control cultures. This stimulation appears to be imputable to a trophic effect of guinea pig serum, because the same effect was observed in cultures treated with guinea pig serum alone (Table 1). A similar increase was observed on GS activity, an astrocytic marker, with complement and in the presence or absence of anti-myelin IgG (data not shown). Increasing concentrations of anti-MOG antibody in the cultures led to increasing losses of MBP (Fig. 1j. At the highest concentration tested (62.5 pg of IgG/ ml), the levels of MBP detected were -25% of those measured in control cultures (Fig. 1 and Table 1j. Similar observations were made for CNP activity, except that the decrease was less drastic than that observed for MBP. Indeed, a maximal decrease of CNP specific activity of 40% was already reached with antiMOG antibody concentrations of 12.5 pg of IgG/ml. However, it is possible that further loss in CNP specific activity was masked by the trophic effect observed in cultures treated with guinea pig serum alone (Table 1). DISCUSSION
One can postulate that epitopes relevant for demyelination should be exposed on the external surface of myelin and/or oligodendrocytes. They may not be the ones recognized by antisera raised against purified myelin proteins. In contrast, antisera raised against myelin membranes or CNS homogenates are more likely to recognize epitopes externally exposed (Seil and Agrawal, 1980; Seil et al., 1981). Although the MOG epitope recognized by the monoclonal 8- 18C5 antibody is still unknown, this antibody was not obtained from immunization with myelin membranes or CNS homogenates but with a glycoprotein fraction isolated from cerebellum (Linington et al., 1984). It should be noted that EAE sera devoid of reactivity against MBP, PLP, and cerebrosides but containing antibodies against the CNS myelin glycoprotein M2 have been shown to demyelinate CNS cultures (Lebar et al., 1976, 1979). Additional studies have shown that M2 seems to be the same antigen as MOG (Lebar et al., 1986; Glynn and Linington, 1989). Induction of demyelination by anti-MOG antibody in acute EAE (Schluesener et al., 1987), aggravation of the disease in T cell-induced EAE (Lassmann et al., 1988; Linington et al., 1988), and in vivo demyelination in normal animals following intrathecal injection of anti-MOG antibody (Lassmann and Linington, 1987) suggest that anti-MOG-induced demyelination may occur in primary demyelinating diseases. The role of complement in antibody-mediated demyelination in vitro appears to be linked to the generation of complement membrane attack complexes (Liu et al., 1983j, following complement fixation and activation by myelin-specific antibodies. Complement alone had no demyelinating action in the aggregating brain cell cultures; similar findings have been reported in CNS cultures (Liu et al., 1983).The possibility exists that myelin isolation procedures cause unmasking of
1.3
1
2 1.0
en i
As previously described in other CNS culture systems
(Raine, 1984), anti-MBP antibodies did not cause demyelination in brain cell aggregates. In contrast to polyclonal antisera raised against purified MBP, PLP, or myelin-associated glycoprotein (Seil et al., 1968, 1981; Seil and Agrawal, 1980), the monoclonal antiMOG antibody had a strong demyelinating effect. This effect, observed with a purified anti-MOG antibody preparation, was dose related and required the presence of complement. These results bring further support to the suggestion that anti-MOG antibodies have a potentially demyelinating role (Lassmann and Linington, 1987).
585
0.8
0
I
0.6 \ m
3
0.4
n
5"
0.2 0
l
0
I
I
I
(
I
I
I
10 20 30 LO 50 60 70 anti-MOG IgG l u g / m l i
FIG. 1. Demyelination response to increasing concentrations of anti-MOG antibody in aggregating brain cell cultures in the presence of complement. Aggregating brain cell cultures were treated from day 25 with increasing concentrations of anti-MOG antibody, and levels of MBP and CNP activity were measured as indicated in Materials and Methods.
J, Neurochem., Val. 55, No. 2. 1990
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binding sites for complement on myelin (Vangun and Shin, 1986), whereas such binding sites are only made available on binding by myelin-specific antibodies in cultures. The highly increased specific activity of the enzymes CNP and GS observed in cultures treated with guinea pig serum can be explained by the presence of several hormones and trophic factors in guinea pig serum. Indeed, we have shown that addition of normal serum to culture medium increases oligodendrocyte differentiation and myelin production (Honegger and Matthieu, 1980). The decrease in CNP specific activity on treatment of the cultures with anti-MOG antibody did not parallel the dose-related loss of MBP content in the same cultures. Such a differentiated effect of the anti-MOG antibody on MBP, a myelin constituent, and CNP, which is mostly localized in oligodendrocytes, suggests that myelin can be degraded yet oligodendrocytes could survive. Whichever process operates, discontinuation of exposure to anti-MOG antibody should allow remyelination. This hypothesis is currently being tested in our laboratory. Acknowledgment: We would like to express o u r gratitude to Dr. Christopher Linington and Dr. Walter Fierz for providing us with the 8-18C5 clone and monoclonal antibody used i n this study and to Dr. Claude Bernard for purified anti-MBP antibody. T h e technical help of Mr. V a n T h a n h Nguyen is gratefully acknowledged. We thank Dr. M. Gardinier for critically reading the manuscript. This work was supported by grants from t h e Multiple Sclerosis Society of Switzerland and the Swiss National Science Foundation (grants 3.601.87 a n d 3 1-9400.88).
REFERENCES Bernard C. C. A., Randell V. B., Horvath L. B., Carnegie P. R., and Mackay 1. R. ( I 98 I ) Antibody to myelin basic protein in extracts of multiple sclerosis brain. Immiinology 43, 447-457. Brunner C., Lassmann H., Waehneldt T. V., Matthieu J.-M., and Linington C. ( 1989) Differential ultrastructural localization of myelin basic protein, myelin/oligodendroglial glycoprotein, and 2’,3%yclic nucleotide 3’-phosphodiesterase in the CNS of adult rats. J. Neurochem. 52, 296-304. Biirgisser P. (1983) Mise au point d’un dosage radioimmunologique de la prottine basiquc de la my&linedans le liquide ckphalorachidien et dans le tissu nerveux [M.D. thesis]. Universitk de Lausanne, Lausanne, Switzerland. Dubois-Dalcq M., Niedieck B., and Buyse M. (1970) Action of anticerebroside sera on myelinated nervous tissue culture. Pathol. Eur. 5, 331-347. Fry J. M., Weissbarth S., Lehrer G. M., and Bornstein M. B. (1974) Cerebrosideantibody inhibits sulfatide synthesis and myelination and demyelinates in cord tissue cultures. Science 183,540-542. Glynn P. and Linington C. (1989) Cellular and molecular mechanisms of autoimmune demyelination in the central nervous system. Crit. Rev. Neurobiol. 4, 367-385. Honegger P. (1985) Biochemical differentiation in serum-free aggregating brain cell cultures, in Cell Culture in the Neurosciences (Bottenstein J. E. and Sato G., eds), pp. 223-243. Plenum Press, New York. Honegger P. and Matthieu J.-M. (1980) Myelination of aggregating fetal rat brain cell cultures grown in a chemically defined me-
J. Neurochrm.. Vol. 55, Nu. 2, 1990
dium, in Neurological Mutations Affecting Myelination (Baumann N., ed), pp. 48 1-488. Elsevierflorth Holland, Amsterdam. Honegger P., du Pasquier P., and Tenot M. (1986) Cholinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters. Dev. Brain Res. 29, 2 17-223. Lassmann H. and Linington C. (1987) The role of antibodies against myelin surface antigens in demyelination in chronic EAE, in A Multidisciplinary Approach to Myelin Diseases (Crescenzi G. S., ed), pp. 219-225. Plenum Press, New York. Lassmann H., Brunner C., Bradl M., and Linington C. (1988) Experimental allergic encephalomyelitis: the balance between encephalitogenic T lymphocytes and demyelinating antibodies determines size and structure of demyelinated lesions. Acta Neuropathol. (Berl.) 75, 566-576. Lebar R., Boutry J.-M., Vincent C., Robineaux R., and Voisin G. A. (1976) Studies on autoimmune encephalomyelitis in the guinea pig. 11. An in vitro investigation on the nature, properties, and specificity of the serum-demyelinating factor. J. Immunol. 116, 1439-1446. Lebar R., Vincent C., and Fischer-Le Boubennec E. (1979) Studies on autoimmune encephalomyelitis in the guinea pig. 111. A comparative study of two autoantigens of central nervous myelin. J. Neurochem. 32, 1451-1460. Lebar R., Lubetzki C., Vincent C., Lombrail P., and Boutry J.-M. (1986) The M2 autoantigen of central nervous myelin, a glycoprotein present in oligodendrocyte membrane. Clin.Exp. Immunol. 66,423-443. Linington C. and Lassmann H. (1987) Antibody responses in chronic relapsing experimental allergic encephalomyelitis correlation of serum demyelinating activity with antibody titre to the myelin/ oligodendrocyteglycoprotein (MOG). J. Neuroimmunol. 17,6169. Linington C., Webb M., and Woodhams P. L. (1984) A novel myelinassociated glycoprotein defined by a mouse monoclonal antibody. J. Neuroimmunol. 6, 387-396. Linington C., Bradl M., Lassmann H., Brunner C., and Vass K. (1988) Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein. Am. J. Pathol. 130,443-454. Liu W. T., Vangun P., and Shin M. L. (1983) Studies on demyelination in vitro: the requirement of membrane attack components of complement system. J. Zmmunol. 131,778-782. Lowry 0.H., Rosebrough N. J., Farr A. L., and Randall R. J. (195 1) Protein measurement with the Folin phenol reagent. J. Efiol. Chem. 193,265-275. Matthieu J.-M., Honegger P., Favrod P., Gautier E., and Dolivo M. (1979) Biochemical characterization of a myelin fraction isolated from rat brain aggregating cell cultures. J. Neurochem. 32,869881. Patel A. J., Hunt A., Gordon R. D., and Balazs R. (1982) The activities in different neuronal cell types of certain enzymes associated with the metabolite compartmentation glutamate. Dev. Brain Res. 4, 3- 11. Pishak M. R. and Phillips A. T. (1979) A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts. Anal. Biochem. 94, 82-88. Raine C. S. ( 1 984) Biology of disease. Analysis of autoimmune demyelination: its impact upon multiple sclerosis. Lab. Invest. 50, 608-635. Raine C. S., Johnson A. B., Marcus D. M., Suzuki A., and Bornstein M. B. (1981) Demyelination in vitro. Absorption studies demonstrate that galactocerebroside is a major target. J. Neurol. Sci. 52, 117-131. Schluesener H. J., Sobel R. A,, Linington C., and Weiner H. L. (1987) A monoclonal antibody against a myelin oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease. J. Immunol. 139,4016-402 I . Schwerer B., Kitz K., Lassmann H., and Bernheimer H. (1984) Serum antibodies against glycosphingolipids in chronic relapsing ex-
ANTI-MOG-INDUCED DEMYELINA TION IN CELL CULTURE penmental allergic encephalomyelitis. Demonstration by ELlSA and relation to serum in vivo dcmyelinating activity. J . Neuroimmunol. 7, 107-1 19. Seil F. J. and Agrawal H. C. (1980) Myelin proteolipid protein does not induce demyelinating or myelination inhibiting antibodies. Bruin Res. 194, 273-277. Seil F. J. and Agrawal H. C . (1984) Serum antimyelin factors in experimental allergic encephalomyelitis and multiple sclerosis, in Experimental Allergic Encephalomyelitis:A Useful Mode1,for Multiple Sclerosis (Alvord E. C., Kies M. W., and Suckling A. J., eds), pp. 199-206. Alan R. Liss, New York. Seil F. J., Falk G. A., Kies M. W., and Alvord E. C. (1 968) The in vitro demyelinating activity of sera from guinea pigs sensitized with whole CNS and purified encephalitogen. Exp. Neurol. 22, 545-55s. Seil F. J., Quarks R. H., Johnson D., and Brady R. 0. (1981) Im-
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munization with purified myelin associated glycoprotein does not evoke myelination inhibiting or demyelinating antibodies. Brain Res. 209, 470-475. Silverman B. A., Carney D. F., Johnston C. A., Vanguri P., and Shin M. L. (1984) Isolation of membrane attack complex of complement from myelin membranes treated with serum complement. J Neurochem 42, 1024-1029. Sogin D. C. (1976) 2',3'-Cyclic NADP as a substrate for 2',3'-cyclic nucleotide 3'-phosphohydrolase. J. Neurochem. 27, 1333- 1337. Vanguri P. and Shin M. L. (1986) Activation of complement by myelin: identification of C1-binding proteins of human myelin from central nervous tissue. J. Neurochem. 46, 1535-1541. Walsh M. J. and Tourtellotte W. W. (1983) The cerebrospinal fluid in multiple sclerosis,in Mulliple Sclerosis (Hallpike J. F., Adams C . W. M., and Tourtellotte W. W., eds), pp. 275-358. Chapman and Hall, London.
J Nrurochem.. Vol. 55. No. 2, 1990
Demyelination Induced in Aggregating Brain Cell Cultures by a Monoclonal Antibody Against Myelin/Oligodendrocyte Glycoprotein Nicole Kerlero de Rosbo, *Paul Honegger, ?Hans Lassmann, and Jean-Marie Matthieu Laboratoire de Neurochimie, Service de Pbdiatrie, Centre Hospitalier Universitaire Vaudois; *Institut de Physiologie de l'liniversite' de Lausanne, Latisunne, Switzerland; ?Institute for Bruin Research, Austrian Academy of Sciences: and ?Neurological Institute, University of Vienna, Vienna, Austria
Abstract: A monoclonal antibody (8- 18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures. With increasing doses of anti-MOG antibody in the presence of complement, myelin basic protein (MBP) concentrations decreased in a dose-related manner. A similar, albeit less pronounced, effect was observed on specific activity of 2',3'-cyclic nucleotide 3'phosphohydrolase. In the absence of Complement, anti-MOG antibody did not induce detectable demyelination.In contrast to the effect of anti-MOG antibody and as expected, antiMBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement. These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side ofthe myelin membrdne, is a good target antigen for antibodyinduced demyelination. Indeed, they show that a purified
anti-MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures. Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin-associated glycoprotein. These observations may have important implications regarding the possible role of antiMOG antibodies in demyelinating diseases. Key Words: Myelin/oligodendrocyte glycoprotein-Demyelination-Aggregating brain cell culture-Myelin basic protein-2',3'Cyclic nucleotide 3'-phosphohydrolase.Kerlero de Rosbo N. et al. Demyelination induced in aggregating brain cell cultures by a monoclonal antibody against myelin/oligodendrocyte glycoprotein. J. Neurochem. 55, 583-587 (1990).
Autoimmune responses to myelin and oligodendroglial components have been demonstrated in multiple sclerosis, a primary demyelinating disease (for review, see Walsh and Tourtellotte, 1983), and demyeh a t i n g activity has been linked to immunoglobulins in sera and CSF of multiple sclerosis patients (Walsh and Tourtellotte, 1983; Raine, 1984). However, the antigens responsible for induction of antibody-mediated demyelination in multiple sclerosis have not yet been identified. Sera from animals with the demyelinating form of experimental autoimmune encephalomyelitis (EAE), which are known to contain antibodies directed against myelin components such as myelin
basic protein (MBP), proteolipid protein (PLP), and galactocerebroside, are myelinotoxic in CNS cultures (Lassmann and Linington, 1987). Although polyclonal antisera raised in rabbits against purified MBP, PLP, or myelin-associated glycoprotein had no deleterious effect in such cultures (Seil et al., 1968, 1981; Seil and Agrawal, 1980), antibodies directed against myelin glycolipids initiated demyelination (Dubois-Dalcq et al., 1970; Fry et al., 1974; Raine et al., 1981; Seil and Agrawal, 1984). However, both in vitro (Lebar et al., 1976) and in vivo (Schwerer et al., 1984) studies have shown that the demyelinating activity in EAE sera is not attributable solely to detectable antiglycolipid an-
Received August 15, 1989; revised manuscript received December 10, 1989; accepted January 3, 1990. Address correspondence and reprint requests to Dr. J.-M. Matthieu at Laboratoire de Neurochimie, Service de PMiatrie, Centre Hospitalier Universitaire Vaudois, CH- I0 1 1 Lausanne, Switzerland. The present address of Dr. N. Kerlero de Rosbo is Neuroimmunology Laboratory, La Trobe University, Bundoora 3083, Victoria, Australia.
Abbreviaions used:CNP, 2',3'-cyclic nucleotide 3'-phosphohydrolase; EAE, experimental autoimmune encephalomyelitis; GS, glutamine synthetase; MBP, myelin basic protein; MOG, rnyelin/oligodendrocyte glycoprotein; PBS, phosphate-buffered saline; PLP, proteolipid protein.
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tibodies. It has been suggested that the CNS-specific myelin/oligodendrocyteglycoprotein (MOG)would be a good target for .antibody-induced demyelination (Lassmann and Linington, 1987), as it is located preferentially at the external surface of myelin sheaths and oligodendrocytes (Brunner et al., 1989). The potential role of anti-MOG antibodiesin demyelination has been tested in vivo: In acute EAE, where little or no demyelination is usually seen (Raine, 1984), extensive CNS demyelination can be induced by intravenous injection of a monoclonal anti-MOG antibody, clone 818C5 (Linington et al., 1984), at the time when the blood-brain bamer is breached (Schluesener et al., 1987). In addition, the possible involvement of MOG as a target antigen in demyelinating diseases is shown by the presence of anti-MOG antibodies in chronic relapsing EAE (Linington and Lassmann, 1987). In aggregating rat brain cell cultures, established and maintained in serum-free, chemically defined medium, cell development and differentiationproceed as in normal brain tissue (Honegger, 1985). Furthermore, the chemical composition of the cultured myelin membrane closely resembles that found in normal brain tissue (Matthieu et al., 1979). Thus, such a system is ideally suited to investigate the potentially neurotoxic effects of antibodies recognizing myelin and/or oligodendroglial components, avoiding the complex systemic interactions and possible artifacts inherent to in vivo demyelination assays. By combining such a culture system with the use of the purified immunoglobulin fraction of anti-MOG antibody, we show that the demyelinating effect of anti-MOG antibody is specific and dose related in the presence of complement.
PBS) in the presence or absence of complement (guinea pig serum; 25 pl/ml of culture medium). In control cultures, the same volume of PBS with or without complement (total volume, 500 pl) was added. On day 26, the aggregates of each culture flask were split into two equal parts; the volume of each flask was then made up to 8 ml by addition of 4 ml of fresh medium with or without guinea pig complement as above. Media were replenished by exchange of 5 ml of medium per flask on day 27, and the cultures were harvested on day 29.
MATERIALS AND METHODS
In aggregating fetal rat brain cell cultures, myelination starts at around culture day 16 and reaches a maximum -8 days later (Honegger and Matthieu, 1980). Thus, the potentially demyelinating effect of antimyelin antibodies on brain cell aggregates was investigated by the application of antibody preparations on culture day 25, in the presence or absence of complement (guinea pig serum). Three parameters were examined on treatment completion. The amount of myelin was assessed by measuring the content of MBP in the cultures. The effect of the treatments on dial cells was estimated by measuring changes of two specific markers: CNP for oligodendrocytes and GS for astrocytes. In the absence of complement, no demyelination was seen with the antimyelin antibodies at all concentrations used (data not shown). Complement alone had no effect on MBP content in the cultures (Table 1). However, in the presence of complement, a statistically highly significant loss of MBP was observed with anti-MOG IgG at a concentration of 62.5 pg/ml of culture (Table 1). In contrast, anti-MBP IgG at the same (Table 1) or a higher ( 125 pg/ml of culture; data not shown) concentration had no such effect. In the presence of complement, there was a significant de-
Aggregating brain cell cultures Rotation-mediated, serum-free aggregating cell cultures were prepared from mechanically dissociated fetal ( 15- 16 days of gestation) rat telencephalon (Wistar strain; Madorin AG, Fiillinsdorf, Switzerland), as previously described (Honegger, 1985; Honegger et al., 1986).
Antibodies Monoclonal antibody against rat MOG was derived from clone 8-18C5 (Linington et al., 1984); the IgG was purified from ascites fluid produced in mice by affinity chromatography using the Bio-Rad Econo-Pac protein A kit (Bio-Rad, Richmond, CA, U.S.A.). Polyclonal rabbit anti-MBP antibody was a gift from Dr. c. Bernard ( L a Trobe University, Bundoora, Victoria, Australia). It had been raised against a synthetic peptide comprising the sequence of amino acid residues 160- 167 of the I 8.5-kDa human MBP. The IgG fraction was affinity-purified from the antiserum on a protein ASepharose CL4B column as described previously (Bernard et al., 1981).
Treatment of brain cell aggregates On culture day 25, brain cell aggregates were treated with varying concentrations of purified anti-MOG or anti-MBP IgG in phosphate-buffered saline (PBS; 1 mg of IgG/ml of
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Analytical procedures For biochemical analyses, the aggregates of each flask were washed twice with 5 ml of ice-cold PBS and homogenized in 0.5 ml of 2 mM potassium phosphate containing 1 mM EDTA (pH 6.8) using 2-ml glass-glass homogenizers (Bellco, Vineland, NJ, U.S.A.). The homogenates were sonicated briefly and stored at -80°C as aliquots for the different assays. MBP content was measured by radioimmunoassay (Burgisser, 1983). 2’,3’-Cyclic nucleotide 3‘-phosphohydrolase (CNP; EC 3.1.4.37) activity was determined by the method of Sogin (1976). Glutamine synthetase (GS;EC 6.3.1.2) activity was assayed by a modification of the method of Pishak and Phillips (1 979), using L-[ l-’4C]glutamicacid (New England Nuclear-DuPont, Boston, MA, U.S.A.) as the precursor and phosphoenolpyruvate/pyruvate kinase as the ATP-regenerating system (Pate1 et al., 1982). Protein concentrations were measured by the Folin phenol method (Lowry et al., 1951) using bovine serum albumin as the standard.
Statistics Statistical significance was assessed using Student’s t test. Differences with p < 0.05 were considered as significant.
RESULTS
ANTI-MOG-IIVDUCED DEMYELINA TION IN CELL CUL TUKE TABLE 1. Complement (C)-mediated efect oJantibodies to MBP and MOG on oligodendrocyte and myelin markers
Treatment
MBP (pg/mg of total protein)
CNP (rmol of 2'-NADP/min/ mg of total protein)
None C Anti-MBP C Anti-MOG C
1.18 f 0.39 1.01 k 0 . 1 4 1.25 f 0.17 0.25 ? O.lOb
1.81 f 0.33 3.09 f 0.57"
+ +
3.23 f 0.45" 1.88 & 0.45
Guinea pig serum and purified IgG were added to aggregating cell cultures at a concentration of 25 p1 and 62.5 pgjml, respectively. Data are mean f SD values for four culture flasks. Significant differences by Student's t test from untreated cultures were indicated: " p < 0.0 I , ' p < 0.001.
crease in CNP activity in cultures treated with antiMOG IgG (62.5 pg/ml of culture; Table I). Anti-MBP antibody had no deleterious effect on CNP activity in the presence of complement; in fact, the effect was quite the opposite, showing a twofold increase as compared with control cultures. This stimulation appears to be imputable to a trophic effect of guinea pig serum, because the same effect was observed in cultures treated with guinea pig serum alone (Table 1). A similar increase was observed on GS activity, an astrocytic marker, with complement and in the presence or absence of anti-myelin IgG (data not shown). Increasing concentrations of anti-MOG antibody in the cultures led to increasing losses of MBP (Fig. 1j. At the highest concentration tested (62.5 pg of IgG/ ml), the levels of MBP detected were -25% of those measured in control cultures (Fig. 1 and Table 1j. Similar observations were made for CNP activity, except that the decrease was less drastic than that observed for MBP. Indeed, a maximal decrease of CNP specific activity of 40% was already reached with antiMOG antibody concentrations of 12.5 pg of IgG/ml. However, it is possible that further loss in CNP specific activity was masked by the trophic effect observed in cultures treated with guinea pig serum alone (Table 1). DISCUSSION
One can postulate that epitopes relevant for demyelination should be exposed on the external surface of myelin and/or oligodendrocytes. They may not be the ones recognized by antisera raised against purified myelin proteins. In contrast, antisera raised against myelin membranes or CNS homogenates are more likely to recognize epitopes externally exposed (Seil and Agrawal, 1980; Seil et al., 1981). Although the MOG epitope recognized by the monoclonal 8- 18C5 antibody is still unknown, this antibody was not obtained from immunization with myelin membranes or CNS homogenates but with a glycoprotein fraction isolated from cerebellum (Linington et al., 1984). It should be noted that EAE sera devoid of reactivity against MBP, PLP, and cerebrosides but containing antibodies against the CNS myelin glycoprotein M2 have been shown to demyelinate CNS cultures (Lebar et al., 1976, 1979). Additional studies have shown that M2 seems to be the same antigen as MOG (Lebar et al., 1986; Glynn and Linington, 1989). Induction of demyelination by anti-MOG antibody in acute EAE (Schluesener et al., 1987), aggravation of the disease in T cell-induced EAE (Lassmann et al., 1988; Linington et al., 1988), and in vivo demyelination in normal animals following intrathecal injection of anti-MOG antibody (Lassmann and Linington, 1987) suggest that anti-MOG-induced demyelination may occur in primary demyelinating diseases. The role of complement in antibody-mediated demyelination in vitro appears to be linked to the generation of complement membrane attack complexes (Liu et al., 1983j, following complement fixation and activation by myelin-specific antibodies. Complement alone had no demyelinating action in the aggregating brain cell cultures; similar findings have been reported in CNS cultures (Liu et al., 1983).The possibility exists that myelin isolation procedures cause unmasking of
1.3
1
2 1.0
en i
As previously described in other CNS culture systems
(Raine, 1984), anti-MBP antibodies did not cause demyelination in brain cell aggregates. In contrast to polyclonal antisera raised against purified MBP, PLP, or myelin-associated glycoprotein (Seil et al., 1968, 1981; Seil and Agrawal, 1980), the monoclonal antiMOG antibody had a strong demyelinating effect. This effect, observed with a purified anti-MOG antibody preparation, was dose related and required the presence of complement. These results bring further support to the suggestion that anti-MOG antibodies have a potentially demyelinating role (Lassmann and Linington, 1987).
585
0.8
0
I
0.6 \ m
3
0.4
n
5"
0.2 0
l
0
I
I
I
(
I
I
I
10 20 30 LO 50 60 70 anti-MOG IgG l u g / m l i
FIG. 1. Demyelination response to increasing concentrations of anti-MOG antibody in aggregating brain cell cultures in the presence of complement. Aggregating brain cell cultures were treated from day 25 with increasing concentrations of anti-MOG antibody, and levels of MBP and CNP activity were measured as indicated in Materials and Methods.
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N . KERLERO DE ROSBO ET AL.
586
binding sites for complement on myelin (Vangun and Shin, 1986), whereas such binding sites are only made available on binding by myelin-specific antibodies in cultures. The highly increased specific activity of the enzymes CNP and GS observed in cultures treated with guinea pig serum can be explained by the presence of several hormones and trophic factors in guinea pig serum. Indeed, we have shown that addition of normal serum to culture medium increases oligodendrocyte differentiation and myelin production (Honegger and Matthieu, 1980). The decrease in CNP specific activity on treatment of the cultures with anti-MOG antibody did not parallel the dose-related loss of MBP content in the same cultures. Such a differentiated effect of the anti-MOG antibody on MBP, a myelin constituent, and CNP, which is mostly localized in oligodendrocytes, suggests that myelin can be degraded yet oligodendrocytes could survive. Whichever process operates, discontinuation of exposure to anti-MOG antibody should allow remyelination. This hypothesis is currently being tested in our laboratory. Acknowledgment: We would like to express o u r gratitude to Dr. Christopher Linington and Dr. Walter Fierz for providing us with the 8-18C5 clone and monoclonal antibody used i n this study and to Dr. Claude Bernard for purified anti-MBP antibody. T h e technical help of Mr. V a n T h a n h Nguyen is gratefully acknowledged. We thank Dr. M. Gardinier for critically reading the manuscript. This work was supported by grants from t h e Multiple Sclerosis Society of Switzerland and the Swiss National Science Foundation (grants 3.601.87 a n d 3 1-9400.88).
REFERENCES Bernard C. C. A., Randell V. B., Horvath L. B., Carnegie P. R., and Mackay 1. R. ( I 98 I ) Antibody to myelin basic protein in extracts of multiple sclerosis brain. Immiinology 43, 447-457. Brunner C., Lassmann H., Waehneldt T. V., Matthieu J.-M., and Linington C. ( 1989) Differential ultrastructural localization of myelin basic protein, myelin/oligodendroglial glycoprotein, and 2’,3%yclic nucleotide 3’-phosphodiesterase in the CNS of adult rats. J. Neurochem. 52, 296-304. Biirgisser P. (1983) Mise au point d’un dosage radioimmunologique de la prottine basiquc de la my&linedans le liquide ckphalorachidien et dans le tissu nerveux [M.D. thesis]. Universitk de Lausanne, Lausanne, Switzerland. Dubois-Dalcq M., Niedieck B., and Buyse M. (1970) Action of anticerebroside sera on myelinated nervous tissue culture. Pathol. Eur. 5, 331-347. Fry J. M., Weissbarth S., Lehrer G. M., and Bornstein M. B. (1974) Cerebrosideantibody inhibits sulfatide synthesis and myelination and demyelinates in cord tissue cultures. Science 183,540-542. Glynn P. and Linington C. (1989) Cellular and molecular mechanisms of autoimmune demyelination in the central nervous system. Crit. Rev. Neurobiol. 4, 367-385. Honegger P. (1985) Biochemical differentiation in serum-free aggregating brain cell cultures, in Cell Culture in the Neurosciences (Bottenstein J. E. and Sato G., eds), pp. 223-243. Plenum Press, New York. Honegger P. and Matthieu J.-M. (1980) Myelination of aggregating fetal rat brain cell cultures grown in a chemically defined me-
J. Neurochrm.. Vol. 55, Nu. 2, 1990
dium, in Neurological Mutations Affecting Myelination (Baumann N., ed), pp. 48 1-488. Elsevierflorth Holland, Amsterdam. Honegger P., du Pasquier P., and Tenot M. (1986) Cholinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters. Dev. Brain Res. 29, 2 17-223. Lassmann H. and Linington C. (1987) The role of antibodies against myelin surface antigens in demyelination in chronic EAE, in A Multidisciplinary Approach to Myelin Diseases (Crescenzi G. S., ed), pp. 219-225. Plenum Press, New York. Lassmann H., Brunner C., Bradl M., and Linington C. (1988) Experimental allergic encephalomyelitis: the balance between encephalitogenic T lymphocytes and demyelinating antibodies determines size and structure of demyelinated lesions. Acta Neuropathol. (Berl.) 75, 566-576. Lebar R., Boutry J.-M., Vincent C., Robineaux R., and Voisin G. A. (1976) Studies on autoimmune encephalomyelitis in the guinea pig. 11. An in vitro investigation on the nature, properties, and specificity of the serum-demyelinating factor. J. Immunol. 116, 1439-1446. Lebar R., Vincent C., and Fischer-Le Boubennec E. (1979) Studies on autoimmune encephalomyelitis in the guinea pig. 111. A comparative study of two autoantigens of central nervous myelin. J. Neurochem. 32, 1451-1460. Lebar R., Lubetzki C., Vincent C., Lombrail P., and Boutry J.-M. (1986) The M2 autoantigen of central nervous myelin, a glycoprotein present in oligodendrocyte membrane. Clin.Exp. Immunol. 66,423-443. Linington C. and Lassmann H. (1987) Antibody responses in chronic relapsing experimental allergic encephalomyelitis correlation of serum demyelinating activity with antibody titre to the myelin/ oligodendrocyteglycoprotein (MOG). J. Neuroimmunol. 17,6169. Linington C., Webb M., and Woodhams P. L. (1984) A novel myelinassociated glycoprotein defined by a mouse monoclonal antibody. J. Neuroimmunol. 6, 387-396. Linington C., Bradl M., Lassmann H., Brunner C., and Vass K. (1988) Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein. Am. J. Pathol. 130,443-454. Liu W. T., Vangun P., and Shin M. L. (1983) Studies on demyelination in vitro: the requirement of membrane attack components of complement system. J. Zmmunol. 131,778-782. Lowry 0.H., Rosebrough N. J., Farr A. L., and Randall R. J. (195 1) Protein measurement with the Folin phenol reagent. J. Efiol. Chem. 193,265-275. Matthieu J.-M., Honegger P., Favrod P., Gautier E., and Dolivo M. (1979) Biochemical characterization of a myelin fraction isolated from rat brain aggregating cell cultures. J. Neurochem. 32,869881. Patel A. J., Hunt A., Gordon R. D., and Balazs R. (1982) The activities in different neuronal cell types of certain enzymes associated with the metabolite compartmentation glutamate. Dev. Brain Res. 4, 3- 11. Pishak M. R. and Phillips A. T. (1979) A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts. Anal. Biochem. 94, 82-88. Raine C. S. ( 1 984) Biology of disease. Analysis of autoimmune demyelination: its impact upon multiple sclerosis. Lab. Invest. 50, 608-635. Raine C. S., Johnson A. B., Marcus D. M., Suzuki A., and Bornstein M. B. (1981) Demyelination in vitro. Absorption studies demonstrate that galactocerebroside is a major target. J. Neurol. Sci. 52, 117-131. Schluesener H. J., Sobel R. A,, Linington C., and Weiner H. L. (1987) A monoclonal antibody against a myelin oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease. J. Immunol. 139,4016-402 I . Schwerer B., Kitz K., Lassmann H., and Bernheimer H. (1984) Serum antibodies against glycosphingolipids in chronic relapsing ex-
ANTI-MOG-INDUCED DEMYELINA TION IN CELL CULTURE penmental allergic encephalomyelitis. Demonstration by ELlSA and relation to serum in vivo dcmyelinating activity. J . Neuroimmunol. 7, 107-1 19. Seil F. J. and Agrawal H. C. (1980) Myelin proteolipid protein does not induce demyelinating or myelination inhibiting antibodies. Bruin Res. 194, 273-277. Seil F. J. and Agrawal H. C . (1984) Serum antimyelin factors in experimental allergic encephalomyelitis and multiple sclerosis, in Experimental Allergic Encephalomyelitis:A Useful Mode1,for Multiple Sclerosis (Alvord E. C., Kies M. W., and Suckling A. J., eds), pp. 199-206. Alan R. Liss, New York. Seil F. J., Falk G. A., Kies M. W., and Alvord E. C. (1 968) The in vitro demyelinating activity of sera from guinea pigs sensitized with whole CNS and purified encephalitogen. Exp. Neurol. 22, 545-55s. Seil F. J., Quarks R. H., Johnson D., and Brady R. 0. (1981) Im-
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munization with purified myelin associated glycoprotein does not evoke myelination inhibiting or demyelinating antibodies. Brain Res. 209, 470-475. Silverman B. A., Carney D. F., Johnston C. A., Vanguri P., and Shin M. L. (1984) Isolation of membrane attack complex of complement from myelin membranes treated with serum complement. J Neurochem 42, 1024-1029. Sogin D. C. (1976) 2',3'-Cyclic NADP as a substrate for 2',3'-cyclic nucleotide 3'-phosphohydrolase. J. Neurochem. 27, 1333- 1337. Vanguri P. and Shin M. L. (1986) Activation of complement by myelin: identification of C1-binding proteins of human myelin from central nervous tissue. J. Neurochem. 46, 1535-1541. Walsh M. J. and Tourtellotte W. W. (1983) The cerebrospinal fluid in multiple sclerosis,in Mulliple Sclerosis (Hallpike J. F., Adams C . W. M., and Tourtellotte W. W., eds), pp. 275-358. Chapman and Hall, London.
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