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Demyelination in vitro
Journal of the Neurological Sciences, 1981, 52:117-131 Elsevier/North-Holland Biomedical Press
117
DEMYELINATION IN VITRO Absorption Studies Demonstrate that Galactocerebroside is a Major Target
CEDRIC S. RAINE, ANNE B. JOHNSON, DONALD M. MARCUS*, AKEMI SUZUKI** and MURRAY B. BORNSTEIN
Departments of Pathology (Neuropathology), Neuroscience, Neurology, Molecular Biology and Immunology and the Rose F. Kennedy Centerfor Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, 1300 Morris Park Avenue, The Bronx, N Y 10461 (U.S.A.) (Received 17 March, 1981) (Accepted 30 March, 1981)
SUMMARY
Myelinated cultures of mouse spinal cord have been exposed to sera raised in rabbits against whole white matter (anti-WM), myelin basic protein (anti-MBP) and galactocerebroside (anti-GC), the major glycolipid of CNS myelin, to determine which factor in central nervous system (CNS) tissue in vitro is the target of serum demyelinating and myelin swelling antibodies. The sera were tested by radioimmunoassay for activity against MBP and against GC and were also specifically absorbed with MBP, GC and control antigens. Studies were also performed with and without active complement. The findings show that demyelination and myelin swelling in vitro are caused by antibodies against GC and not against MBP. Ultrastructurally, the effects of anti-WM and anti-GC sera with and without complement were indistinguishable. This study demonstrates that GC is a major target in antibody-mediated demyelination.
INTRODUCTION
Anti-white matter serum (anti-WM) from animals sensitised for experimental allergic encephalomyelitis (EAE) possesses factors, now recognised as immunoSupported in part by USPHS grants NS-08952, NS-11920 and NS-07098; and by National Multiple Sclerosis Society grant RG 1001-C-3. * Present address: Department of Internal Medicine, Baylor College of Medicine, Texas Medical Center, Houston, TX 77030, U.S.A. ** Present address: Department of Biochemistry, Faculty of Medicine, University of Tokyo, Bunkyoku, Tokyo, Japan. 0022-510X/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
118 globulins, which selectively damage myelin in myelinated cultures of central nervous system (CNS) tissue. These include complement-dependent demyelinating antibodies (Bornstein and Appel 1961 ; Appel and Bornstein 1964; Raine and Bornstein 1970; Johnson and Bornstein 1978; Grundke-Iqbal et al. 1981), and complementindependent antibodies which cause myelin to swell and oligodendroglia to produce copious amounts of aberrant myelin (Bornstein and Raine 1976; Raine et al. 1978; Johnson et al. 1979; Grundke-Iqbal et al. 1981). These antibodies are reactive in vitro from very early stages of development onwards (Bornstein and Raine 1970; Bonnaud-Toulze et al. in press). To date, the specific myelin components against which these immunoglobulins are directed have not been identified, but indirect evidence suggests that myelin basic protein (MBP), the encephalitogenic component of central myelin (Kibler and Shapiro 1968; Eylar et al. 1969; Martenson et al. 1970), is not the target since anti-MBP serum does not cause demyelination in vitro (Seil et al. 1968). However, galactocerebroside (GC), the major glycolipid in CNS myelin, produces serum factors which are myelinotoxic in vitro (Dubois-Dalcq et al. 1970; Fry et al. 1974; Dorfman et al. 1978; Saida et al. 1979a). Since anti-GC serum factors are recognised as immunoglobulins (Johnson and Bornstein 1978; Raft et al. 1978; Johnson et al. 1980; Mirsky et al. 1980), it is possible that lipids may play a significant role in humoral immunity during demyelination. The present study describes tests performed in parallel to investigate the comparative effects upon myelinated CNS cultures of both intact and complementinactivated antisera raised against WM, MBP and GC, and before and after specific absorptions. The findings confirm that MBP is not the antigen responsible for demyelination in vitro, and that the lipid hapten, GC, does indeed play a major role in this phenomenon. MATERIALS A N D M E T H O D S
Tissue cultures
Myelinated organotypic cultures of embryonic mouse spinal cord were maintained in Maximow slide assemblies for 3-4 weeks by techniques standard in these laboratories (Bornstein 1973). Only well-myelinated cultures were used, and, when myelin was abundant, these were exposed to control and experimental sera for up to 4 days. Some cultures were exposed to complement-inactivated sera (see below) while others were exposed to intact serum (i.e. serum containing complement). In the latter case, the test serum was supplemented by the addition of 10~ normal guinea pig serum containing complement. Cultures were exposed to nutrient medium containing a 25~o concentration of the serum under test. Several different sera were compared simultaneously on sister cultures and each serum was used with 4-6 explants. In most cases, sera were tested on 2 separate occasions under code and the code was broken only after the cultures were assessed for demyelination.
119 Sera Control serum
In all experiments, control sera were examined. These consisted either of (a) normal New Zealand albino rabbit serum, usually a preimmune serum, or (b) serum from a rabbit inoculated with complete Freund's adjuvant (CFA) 14 days prior to sampling. The latter was used either without treatment or following passage through a control or a GC affinity column (see below) and in the presence or absence of active complement. Anti- W M serum
Adult, male New Zealand albino rabbits were inoculated subcutaneously in both hind foot-pads with 0.4 ml (0.2 ml each side) of an emulsion of fresh bovine white matter in saline and CFA in the ratio of 1 : 1 : 2. Prior to inoculation, preimmune serum was taken. Within the first 24 h of clinical EAE, 12-14 days postinoculation, animals were bled and the serum distributed into 1 ml aliquots and stored at - 90 °C. Anti-WM serum was used as follows: (a) Intact anti-WM serum containing complement. Before use, the MBP activity of the anti-WM serum was tested by radioimmunoassay (RIA) by Dr. S. R. Cohen, Johns Hopkins University, Baltimore and was found to contain significant specific binding to MBP at dilutions greater than 1 : 100; (b) Intact anti-WM serum absorbed against MBP. For this, 1 mg bovine MBP was added to 0.3 ml of serum and the serum centrifuged at 100,000 x g for 30 min to remove complexes. After absorption, MBP activity was not detectable by RIA; (c) Intact anti-WM serum absorbed against GC. The serum was passed through a G C affinity column (Kundu and Roy 1979) and then reconstituted to its original volume so that its concentration was similar to that of whole serum. To restore sterility, the serum was passed through a millipore filter prior to use in vitro; (d) Intact anti-WM serum absorbed against both MBP and GC. First the serum was collected from a GC affinity column as in (c) and then also absorbed with MBP as in (a), centrifuged and passed through a millipore filter; (e) Complement inactivated anti-WM serum. An aliquot of the above antiWM serum was heated at 56 °C for 30 min to inactivate complement. The MBP activity of this heated, unabsorbed anti-WM serum, measured by RIA, was found to have remained comparable to that of intact anti-WM serum; (f) Complement-inactivated anti-WM serum similarly absorbed against MBP and ultra-centrifuged; and (g) Complement-inactivated anti-WM serum absorbed with G C and centrifuged. A n t i - M B P serum
This was raised in a New Zealand male rabbit given one subcutaneous injection in the flank of 5 mg bovine MBP (obtained from Dr. E . H . Eylar, Toronto) emulsified in 0.5 ml saline and 0.5 ml CFA. This was followed at biweekly intervals by injections containing 1 mg MBP in saline and incomplete Freund's adjuvant (IFA). The animal did not develop signs of EAE. Two weeks
120
after the fifth booster injection, the animal was bled and the sterile serum stored as 3-5 ml aliquots at - 9 0 ° C . Prior to use, the serum was tested by RIA and found to bind MBP specifically, although at a low level. Immunofluorescence on brain sections using this serum followed by FITC-conjugated anti-rabbit IgG showed specific binding to white matter. Anti-GC s e r u m This was raised in two adult, male New Zealand albino rabbits. The GC preparation was obtained commercially (Supelco, Bellefonte, PA) and was of bovine brain origin. Prior to use, it was tested for purity by thin layer chromatography and was found to run as two spots only, corresponding to the two forms of galactosylceramide. The initial inoculation was subcutaneous in a rear flank and consisted of I ml containing 5 mg GC in 0.5 ml of micelles made up from lecithin (bovine heart - Supelco, Bellefonte, PA) and bovine serum albumin (Supelco) and 0.5 ml CFA. At biweekly intervals, 0.5 mg GC in micelles and IFA was similarly injected. Prior to each booster injection, about 50 ml of blood was drawn from the middle ear artery. Sterile serum was stored in 3-5 ml aliquots at - 9 0 °C. The anti-GC serum was tested for specific antibody to GC by RIA with a modification of the solid phase absorption technique of Holmgren et al. (1980). This technique involved coating the wells of microtitre plates with 0.1 /~g GC, incubation with diluted antiserum and then detection of bound lgG with radioiodinated protein A at a dilution of 1:400. The method does not detect IgM
TABLE 1 R A D I O I M M U N O A S S A Y OF A N T I - G A L A C T O C E R E B R O S I D E S E R U M IgG antibodies detected by RIA using a microtitre plate coated with GC and radioiodinated Protein A. Values are the average of 2 readings.
Date serum sampled a
cpm at 1 : 10 dilution
cpm at 1:50 dilution
cpm at 1:250 dilution
cpm at 1:1250 dilution
Animal No. 620
1/5/79 (preimmune) 1/19/79 2/5/79 2/21/79 3/7/79 3/26/79
110 3025 3059 3257 3548 3580
80 2278 2815 3336 3363 3320
66 1421 1947 1816 2264 2041
60 387 716 738 715 769
Animal No. 621
1/5/79 (preimmune) 1/19/79 2/5/79 2/21/79 3/7/79 3/26/79 5/2/79
85 2485 3346 3017 280 285 3532
75 1459 1378 1491 227 193 1433
68 542 531 490 104 119 468
69 259 180 215 72 87 201
a Blood drawn immediately prior to inoculation.
121 antibodies. The first animal developed transient signs of a peripheral neuropathy at 2 weeks post inoculation and had consistently high levels of anti-GC activity 3000 cpm at a 1 : 50 dilution (see Table 1). Two weeks prior to killing, the animal had a relapse and the signs persisted until the end of the experiment. The second rabbit developed no overt clinical signs of disease. Its serum showed a rapid rise in specific IgG to GC by the second booster injection which then dropped and rose again after the fourth booster injection. For the present experiments, anti-GC serum was used as: (a) Intact anti-GC serum (with complement); (b) Intact anti-GC serum passed over a control aminopropyl silica gel column containing Forssmann antigen as described by Kundu and Roy (1979); (c) Intact anti-GC serum passed over a GC affinity column (Kundu and Roy 1979). The anti-GC serum was passed twice over the column, reconcentrated prior to use and then passed through a millipore filter to restore sterility. The solid phase RIA showed that absorption removed about 60~ of the anti-GC IgG antibodies. (d) Complement-inactivated anti-GC serum; (e) Complement-inactivated anti-GC serum passed over a control column; and (f) Complement-inactivated anti-GC serum passed over a GC column.
lmmunocytochemistry Cultures treated with anti-WM, anti-GC and control sera were examined by immunocytochemistry. For this, living cultures were exposed to complement-inactivated sera for 1-4 h and then fixed and reacted with peroxidase labelled sheep Fab fragments against rabbit IgG. After washing, the samples were treated with diaminobenzidine and processed for light microscopy (LM) as whole mounts (Johnson and Bornstein 1978).
Morphology For each experimental serum, between 4 and 16 samples were studied by electron microscopy (EM). The cultures were taken between 12 to 96 h postexposure to test sera. While still on coverslips, they were fixed in 2.5~ glutaraldehyde, PO4-buffered to pH 7.4. The cultures were then processed and flat embedded in Epon for EM by techniques described in full elsewhere (Raine 1973). Thin sections for EM were double-stained with lead and uranyl salts, carboncoated and scanned in a Siemens 101. RESULTS A summary of the effects of the sera tested is given in Table 2. These were measured in living explants after 24 and 48 h of exposure. Preimmune and CFA serum (with and without specific absorptions) had no effect upon myelin in culture and no specific binding was observed by immunocytochemistry. Anti-WM serum with complement was exceedingly potent and by 48 h, had
122 TABLE 2 DEMYELINATING ACTIVITY OF SERA WITH AND WITHOUT ABSORPTION Serum
Demyelination a at 24 h
Demyelination a at 48 h
Anti-WM + C'b plain MBP-absorbed GC-absorbed pC- and MBP-absorbed
4.5 4.5 1.75 2.5
5.0 5.0 1.25 2.75
Anti-WM, no C'b plain MBP-absorbed GC-absorbed
1.5 1.5 0.5
1.75 1.5 0.5
Anti-MBP + C"b plain
1.0
1.5
Anti-GC + C 'b plain control absorption GC-absorbed
4.0 3.25 1.9
5.0 5.0 3.25
Anti-GC, no C 'b plain control absorption GC-absorbed
1.3 1.1 1.0
2.4 2.0 2.0
a Average of 4-6 sister cultures tested; 1.0 = minor swelling of myelin; 2.0 = up to 25~ demyelination; 3.0 = up to 50~ demyelination; 4.0 = up to 75~ demyelination; 5.0 = up to 100~ demyelination. b + C' = with added complement; no C' = complement inactivated by heating.
c a u s e d t o t a l d i s r u p t i o n o f all myelin sheaths. M o r p h o l o g i c a l l y , this was r e p r e s e n t e d b y the d i s i n t e g r a t i o n o f myelin into o v o i d s a n d the c o n c o m i t a n t d e a t h o f oligod e n d r o g l i a . D e t a i l s o f these p h e n o m e n a have been d e s c r i b e d p r e v i o u s l y ( R a i n e a n d B o r n s t e i n 1970). P r e v i o u s i m m u n o p e r o x i d a s e s t u d y o f this p h e n o m e n o n has shown it to be a s s o c i a t e d with specific b i n d i n g o f i m m u n o g l o b u l i n to myelin a n d o l i g o d e n d r o g l i a ( J o h n s o n a n d Bornstein 1978). A b s o r p t i o n o f a n t i - W M s e r u m with M B P h a d no m e a s u r a b l e effect u p o n the d e m y e l i n a t i n g a b i l i t y o f the serum, a l t h o u g h R I A s h o w e d t h a t the a b s o r b e d s e r u m h a d lost all activity a g a i n s t M B P . H o w e v e r , a b s o r p t i o n o f a n t i - W M s e r u m with G C h a d a m a r k e d effect o n the d e m y e l i n a t i o n a n d r e d u c e d the efficacy o f the s e r u m at 48 h f r o m 5.0 (before a b s o r p t i o n ) to less t h a n 2 (after a b s o r p t i o n ) (see T a b l e 2 for e x p l a n a t i o n o f scale). C o m b i n e d a b s o r p t i o n o f a n t i - W M s e r u m with G C a n d M B P also c a u s e d a r e d u c t i o n in the o b s e r v e d d e m y e l i n a t i o n , a l b e i t less m a r k e d t h a n after G C a b s o r p t i o n alone. T h e higher d e m y e l i n a t i n g scores with the c o m b i n e d a b s o r p t i o n were p r o b a b l y due to e x p e r i m e n t a l v a r i a t i o n since the dif-
123 ference was less at 24 h and since the 48 h reading was still significantly lower than that seen without absorption. If MBP absorption had had an additive effect with GC, a greater reduction in the amount of demyelination might have been expected after double absorption. Complement-inactivated anti-WM serum caused relatively few myelin abnormalities as perceived by LM. These consisted of some myelin irregularities, myelin swelling and ballooning but no frank demyelination. Immunocytochemistry showed extensive binding of immunoglobulin to myelin and oligodendrocytes, as described previously (Johnson et al. 1979). EM confirmed that the morphologic changes, described in detail elsewhere (Bornstein and Raine 1976; Raine et al. 1978), were related to the formation of aberrant myelin sheaths with an additional pair of leaflets at the intraperiod line and oligodendroglia which had proliferated an abundance of the same aberrant myelin within and around the cell somata (Figs. 1 and 2). Absorption of heated anti-WM serum with MBP did not significantly reduce the degree of myelin swelling (Fig. 3). However, absorption of complementinactivated anti-WM serum with GC reduced the myelin changes to almost imperceptible levels. Immunostaining was also markedly reduced. Anti-MBP serum had no effect upon myelin in vitro over a 48-h period (Fig. 4). Since it was ineffective in the presence of complement, anti-MBP was not tested without complement, nor was it deemed necessary to absorb the serum against MBP or GC. Anti-GC serum with complement, whether used plain or after absorption on a control column, proved to be a potent demyelinating agent (Table 2). The demyelinating ability of anti-GC serum was markedly reduced after passing the serum over a GC affinity column which was shown to decrease the GC IgG antibodies by about 60~. Without specific absorption, intact anti-GC serum was seen by EM to cause myelin to break down and dissociate from the axon. The dissociated myelin was phagocytosed by investing astrocytes which not infrequently contained an overabundance of glycogen (Figs. 5 and 6). Ultrastructurally, anti-GC serum-induced demyelination was generally similar to that with intact anti-WM serum, but with anti-GC serum it tended to be less severe in that oligodendroglia often survived and the process was slower. The demyelination induced by intact anti-GC serum could sometimes be seen to involve the myelin swelling which is typically associated with complement-inactivated anti-WM serum. After complement-inactivation, anti-GC serum caused relatively few myelin changes visible in the living state but there was pronounced immunostaining of myelin and oligodendroglia. At the EM level, abundant myelin swelling was apparent (Fig. 7). With GC-absorbed, heated anti-GC serum, there was a dramatic decrease in the amount of myelin swelling seen at the ultrastructural level (Fig. 8) and an associated decrease in peroxidase reaction product on myelin and oligodendrocytes.
Fig. 1. Anti-WM serum, complement-inactivated; 48 h exposure. This electron micrograph shows ata oligodendrocyte with proliferated aberrant, wide-spaced myelin within and around itself. Arrow indicates area depicted in Fig. 2. x 12,250. Fig. 2. Detail from Fig. 1. Note the wide-spaced myelin with a 22-nm periodicity. Between each pair of major dense lines, 4 instead of the usual 2 leaflets are associated with the intraperiod line. x 125,000.
125
Fig. 3. Anti-WM serum, complement-inactivated, absorbed against MBP; 48 h exposure. Note that myelin swelling still persists in the absence of antibody against MBP. × 30,000. Fig. 4. Anti-MBP serum, with complement; 48 h exposure. Note that this serum had no apparent effect upon myelin in vitro. Same magnification as Fig. 3 for comparison. × 30,000.
Fig. 5. Anti-GC serum, with complement ; 24 h exposure. A myelinated nerve has almost been completely divested of its myelin sheath which is in the process of being engulfed by encircling astroglial cell processes. Note the glycogen granules within the astroglial cytoplasm. Arrow indicates area enlarged in inset. × 22,500. Inset." Detail of the disintegrating myelin in Fig. 5. Note that some lamellae are widespaced (22 nm large arrow) while others are compacted (6 nm) and fragmented (small arrow). x 125,000. Fig. 6. A n t i - G C serum, with complement; 48 h exposure. Naked axons (arrows) and myelin debris are evident in this area from a culture showing total demyelination, x 15,000.
Fig. 7. Anti-GC serum, complement-inactivated; 72 h exposure. A group of myelinated axons displays a moderate degree o f myelin swelling, barely visible at this magnification. Arrow indicates area shown in inset, x 15,000. Inset: Detail of the swollen myelin sheaths from Fig. 7. The quadrilamellar intraperiod line is apparent in places (arrows). × 125,000. Fig. 8. Anti-GC serum, complement-inactivated, absorbed on a GC column to remove GC activity and to test the effect of specific absorption against GC upon myelin swelling; 72 h exposure. Note that the ability of G C serum to cause myelin swelling has been removed. × 15,000.
128 DISCUSSION The effects of anti-WM serum on organotypic cultures of CNS tissue have been known for some time but the antigen(s) involved has never been established. By specific absorptions and appropriate controls, this study with anti°WM, antiMBP, and anti-GC sera has shown that serum-induced demyelination, myelin swelling and positive immunostaining by anti-WM serum in vitro appear to be GC-related phenomena and are not dependent upon the presence of antibody against MBP. These conclusions emanate from groups of experiments in vitro carried out in parallel with well-characterised antisera and the protocols permitted simultaneous comparison of the effects of the various sera. It has previously been reported that anti-MBP serum is not myelinotoxic in vitro (Seil et al. 1968) and that antiGC serum has demyelinating activity (Dubois-Dalcq et al. 1970; Fry et al. 1974; Saida et al. 1979a,b). However, anti-MBP, anti-GC and anti-WM sera, with the various controls and specific absorptions, with and without active complement, have never been compared or tested in the same experimental series. The use of RIA systems and affinity columns provided valuable monitors in the analysis of the results. It should be emphasised that the tests were invariably carried out under code, providing added significance to the conclusions drawn from these experiments. A major goal of the present experiments was to determine whether demyelination in vitro and myelin swelling (in the absence of intact complement) could be produced, as well as removed, by the same or separate factors. It has recently been demonstrated that while demyelination in vitro is associated with IgG and non-IgG serum fractions, myelin swelling is linked only to IgG (Grundke-Iqbal et al. 1981). From the present studies, anti-GC serum was found to produce myelin swelling and demyelinating patterns similar to those found with anti-WM serum. In addition, it has been shown that GC absorption of anti-WM and anti-GC sera dramatically reduced the level of both phenomena in vitro. GC absorption also reduced the immunostaining with both these sera. Thus, galactocerebroside can at least be claimed to be a major component of myelin responsible for CNS myelin changes in vitro. The relative roles of other myelin lipids (e.g. sulfatides, gangliosides) remain to be determined. A second goal was to compare the efficacy of MBP and GC as demyelinating and absorption factors. Clearly, it has been demonstrated that MBP had little or no role in this humoral response tested in vitro and that GC appeared to be a major determinant. The importance of GC in experimental demyelination was also noted recently in a series of in vivo experiments comparing the effects of sensitisation with emulsions containing WM, MBP or GC (Raine et al., in press). It was found that emulsions containing GC alone induced no CNS signs or pathology, whereas MBP alone caused mild signs of EAE and produced inflammatory lesions which lacked demyelination. However, emulsions containing a combination of MBP and GC induced signs typical of EAE and led to the formation of CNS lesions displaying both inflammation and demyelination, a pattern which approximated that produced by sensitisation against whole white matter. From these experiments, it was sug-
129 gested that MBP provided the cell-mediated component of the immune response and that anti-GC serum factors were necessary to cause demyelination. The mechanism of demyelination in vivo might be through myelin opsonisation and attack by macrophages, through complement-mediated lysis because of bound antibody or through antibody-dependent cell-mediated demyelination (Brosnan et al. 1977). Perhaps what has been witnessed in the present study with cultures is the activity of anti-myelin antibodies which in vivo require lymphocyte activation to become effective, but in vitro are in themselves myelinotoxic. Possibly the cell-mediated immune response is required in vivo to alter the blood-brain barrier and local environment so that humoral factors gain access to CNS loci. Some evidence from in vivo experiments involving local infusion of anti-GC or anti-WM serum into the peripheral nerve suggests that the demyelination observed is purely humoral and that lymphocytes are not involved (Saida et al. 1978; Saida et al. 1979b). The relevance of the above studies to human demyelinating diseases, in particular multiple sclerosis (MS), remains somewhat obscure since current data suggest that most MS serum-induced demyelination is not related to immunoglobulin and MS serum does not give positive immunostaining in vitro (Johnson and Bornstein 1978; Grundke-Iqbal and Bornstein 1980). With anti-WM serum, demyelination has been known for some time to be related to antibody (Appel and Bornstein 1964; Johnson and Bornstein 1978). The question still remains, however, whether MS serum-induced demyelination is indeed MS-specific and whether it fluctuates with relapses and remissions. It is entirely possible, for example, that specific demyelinating antibody might appear in relationship to active disease and this may be distinguishable from non-specific demyelinating epiphenomena occurring at other stages of MS and in non-MS conditions. Also in MS, the use of organotypic cultures of CNS tissue to explore questions concerning the relative roles of different myelin components in the abnormal serum response or the interactions between possible cellular and humoral events using organotypic CNS cultures remain largely unexplored. With the current availability of a chronic relapsing EAE model (Raine et al. 1974) and its application to the MS problem (Raine et al. 1980a,b), it may be possible to approach in vivo several of the above problems and the implications of the present work. ACKNOWLEDGEMENTS The authors thank Michael Diaz, Vincent Spada, Everett Swanson, Howard Finch, Miriam Pakingan, Marie Vitale and Muhammad Farooq for invaluable technical expertise; and Mary Palumbo and Agnes Geoghan for expert secretarial assistance. The authors are indebted to Dr. William T. Norton for compilation of inocula; Dr. Steven R. Cohen, Dept. of Neurology, Johns Hopkins University, Baltimore, MD, who performed the RIA of the antisera for the determination of anti-MBP activity; to Dr. Samar Kundu, Dept. of Internal Medicine, Baylor College
130 of Medicine, Houston,
TX, who performed
and GC-specific columns;
a n d to D r .
the absorption
Edwin
of antisera on control
H. E y l a r , D e p t . o f B i o c h e m i s t r y ,
University of Toronto, Toronto, Ont. who supplied the bovine myelin basic protein. REFERENCES Appel, S. H. and M.B. Bornstein (1964) The application of tissue culture to the study of experimental "'allergic" encephalomyelitis, Part 2 (Serum factors responsible for demyelination), J. exp. Med., 119: 303-312. Bonnaud-Toulze, E.N., A.B. Johnson, M.B. Bornstein and C.S. Raine (19813 A marker for oligodendrocytes and its relation to myelinogenesis - - An immunocytochemical study with experimental allergic encephalomyelitis serum and CNS cultures, J. Neurocytol., In press. Bornstein, M. B. (1973) Organotypic mammalian central and peripheral nerve tissue. In : P. F. Kruse, Jr. and M.K. Patterson, Jr. (Eds.), Tissue Culture Methods and Applications, Academic Press, New York, NY, pp. 86-92. Bornstein, M. B. and S. H. Appel (19613 The application of tissue culture to the study of experimental "allergic" encephalomyelitis, Part 1 (Patterns of demyelination), J. Neuropath. exp. Neurol., 20: 141--157. Bornstein, M. B. and C. S. Raine (1970) Experimental allergic encephalomyelitis - - Antiserum inhibition of myelination in vitro, Lab. Invest., 23 : 536-542. Bornstein, M. B. and C. S. Raine (1976) The initial structural lesion in serum-induced demyelination in vitro, Lab. Invest., 35: 391-401. Brosnan, C.F., G.L. Stoner, B.R. Bloom and H. M. Wisniewski (1977) Studies on demyelination by activated lymphocytes in the rabbit eye, Part 2 (Antibody-dependent cell-mediated demyelination), J. lmmunol., 118:2103 2110. Dorfman, S. H., J. M. Fry, D. H. Silberberg, C. Grose and M.C. Manning (19783 Cerebroside antibody titers in antisera capable of myelination inhibition and demyelination, Brain Res., 147:410-415. Dubois-Dalcq, M., B. Niedieck and M. Buyse (1970) Action of anticerebroside sera on myelinated nervous tissue cultures - - Demyelination of cerebellum cultures, Path. Europ., 5 : 331-347. Eylar, E. H., J. Salk, G. C. Beveridge and L. V. Brown (1969) Experimental allergic encephalomyelitis An encephalitogenic basic protein from bovine myelin, Arch. Biochem. Biophys., 132: 34-48. Fry, J.M., S. Weissbarth, G.M. Lehrer and M.B. Bornstein (1974) Cerebroside antibody inhibits sulfatide synthesis and myelination and demyelinates in cord tissue cultures, Science, 183:540 542. Grundke-lqbal, I. and M.B. Bornstein (1980) Multiple sclerosis Serum gammaglobulin and demyelination in organ cultures, Neurology (Minneap.), 30: 749-754. Grundke-lqbal, I., C. S. Raine, A. B. Johnson, C. F. Brosnan and M. B. Bornstein (1981) Experimental allergic encephalomyelitis Characterization of serum factors causing demyelination and swelling of myelin, J. neurol. Sci., 50 : 63-79. Holmgren, J., H. Elwing, P. Fredman and L. Svennerholm (1980) lmmunoassays based on plasticabsorbed gangliosides. In: L. Svennerholm, P. Mandel, H. Dreyfus and F. F. Urban (Eds.), Structure and Function o f Gangliosides (Advances in Experimental Medicine and Biology~ Vol. 125), Plenum Press, New York, pp. 339-348. Johnson, A. B. and M.B. Bornstein (1978) Myelin-binding antibodies in vitro lmmunoperoxidase studies with experimental allergic encephalomyelitis, antigalactocerebroside and multiple sclerosis sera, Brain Res., 159:173 182. Johnson, A. B., C. S. Raine and M. B. Bornstein (1979) Experimental allergic encephalomyelitis: Serum immunoglobulin binds to myelin and oligodendrocytes in cultured tissue Ultrastructuralimmunoperoxidase observations, Lab. Invest., 40: 568-575. Johnson, A.B., M.B. Bornstein and C.S. Raine (1980) Effects of anti-galactocerebroside serum on organotypic CNS cultures, (Abstract), J. Neuropath. exp. Neurol., 39: 364. Kibler, R. F. and R. Shapiro (1968) Isolation and properties of an encephalitogenic protein from bovine, rabbit and human central nervous system tissue, J. biol. Chem., 243: 281-286. Kundu, S.R. and S.W. Roy (1979) Aminopropyl silica gel as a solid support for preparation of glycolipid immunoabsorbent and purification of antibodies, J. Lipid Res., 20: 825-833. Martenson, R. E., G. E. Deibler and M.W. Kies (1970) Myelin basic proteins of the rat central nervous system Purification, encephalitogenic properties, and amino acid compositions, Biochim. Biophys. Acta, 200: 353-362.
131 Mirsky, R., J. Winter, E. R. Abney, R.H. Pruss, J. Gaurilovic and M.C. Raft (1980) Myelin-specific protein and glycolipids in rat Schwann cells and oligodendrocytes in culture, J. Cell Biol., 84: 483~,94. Raft, M.C., R. Mirsky, K.L. Fields, R.P. Lisak, S.H. Dorfman, D.H. Silberberg, N.A. Gregson, S. Leibowitz and M.C. Kennedy (1978) Galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture, Nature (Lond.), 274: 813-816. Raine, C.S. (1973) Ultrastructural applications of cultured nervous system tissue to neuropathology. In: H.M. Zimmerman (Ed.), Progress in Neuropathology, Fol. 2, Grune and Stratton, New York, NY, pp. 27-68. Raine, C. S. and M.B. Bornstein (1970) Experimental allergic encephalomyelitis - - An ultrastructural study of experirnental demyelination in vitro, J. Neuropath. exp. Neurol., 29: 177-191. Raine, C. S., D. H. Snyder, M. P. Valsamis and S. H. Stone (1974) Chronic experimental allergic encephalomyelitis in inbred guinea pigs - - An ultrastructural study, Lab. Invest., 31 : 369-380. Raine, C. S., U. Traugott and S. H. Stone (1980) Lymphocyte studies in acute and chronic relapsing EAE during suppression. In : A. N. Davison and M. L. Cuzner (Eds.), The Suppression of Experimental Allergic Encephalomyelitis and Multiple Sclerosis, Academic Press, New York, NY, pp. 119-140. Raine, C. S., U. Traugott and S. H. Stone (1980) Applications of chronic relapsing experimental allergic encephalomyelitis to multiple sclerosis. In: H. Bauer, S. Poser and G. Ritter (Eds.), Progress in Multiple Sclerosis Research, Springer-Verlag, Berlin, pp. 3 10. Raine, C.S., U. Traugott, M. Farooq, M.B. Bornstein and W.T. Norton (1981) Augmentation of immune-mediated demyelination by lipid haptens, Lab. Invest., In press. Saida, T., K. Saida, D.H. Silberberg and M.J. Brown (1978) Transfer of demyelination by intraneural injection of experimental allergic neuritis serum, Nature (Lond.), 272 : 639-641. Saida, T., K. Saida and D.H. Silberberg (1979a) Demyelination produced by experimental allergic neuritis serum and anti-galactocerebroside antiserum in CNS cultures, Acta neuropath. (Berlin), 48: 19-25. Saida, T., K. Saida, M.J. Brown and D. H. Silberberg (1979b) Peripheral nerve demyelination induced by intraneural injection of experimental allergic encephalomyelitis serum, J. Neuropath. exp. Neurol., 38: 498-518. Seil, F.J., G.A. Falk, M.W. Kies and E. C. Alvord (1968) The in vitro demyelinating activity of sera from guinea pigs sensitized with whole CNS and with purified encephalitogen, Exp. Neurol., 22: 545-555.
117
DEMYELINATION IN VITRO Absorption Studies Demonstrate that Galactocerebroside is a Major Target
CEDRIC S. RAINE, ANNE B. JOHNSON, DONALD M. MARCUS*, AKEMI SUZUKI** and MURRAY B. BORNSTEIN
Departments of Pathology (Neuropathology), Neuroscience, Neurology, Molecular Biology and Immunology and the Rose F. Kennedy Centerfor Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, 1300 Morris Park Avenue, The Bronx, N Y 10461 (U.S.A.) (Received 17 March, 1981) (Accepted 30 March, 1981)
SUMMARY
Myelinated cultures of mouse spinal cord have been exposed to sera raised in rabbits against whole white matter (anti-WM), myelin basic protein (anti-MBP) and galactocerebroside (anti-GC), the major glycolipid of CNS myelin, to determine which factor in central nervous system (CNS) tissue in vitro is the target of serum demyelinating and myelin swelling antibodies. The sera were tested by radioimmunoassay for activity against MBP and against GC and were also specifically absorbed with MBP, GC and control antigens. Studies were also performed with and without active complement. The findings show that demyelination and myelin swelling in vitro are caused by antibodies against GC and not against MBP. Ultrastructurally, the effects of anti-WM and anti-GC sera with and without complement were indistinguishable. This study demonstrates that GC is a major target in antibody-mediated demyelination.
INTRODUCTION
Anti-white matter serum (anti-WM) from animals sensitised for experimental allergic encephalomyelitis (EAE) possesses factors, now recognised as immunoSupported in part by USPHS grants NS-08952, NS-11920 and NS-07098; and by National Multiple Sclerosis Society grant RG 1001-C-3. * Present address: Department of Internal Medicine, Baylor College of Medicine, Texas Medical Center, Houston, TX 77030, U.S.A. ** Present address: Department of Biochemistry, Faculty of Medicine, University of Tokyo, Bunkyoku, Tokyo, Japan. 0022-510X/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
118 globulins, which selectively damage myelin in myelinated cultures of central nervous system (CNS) tissue. These include complement-dependent demyelinating antibodies (Bornstein and Appel 1961 ; Appel and Bornstein 1964; Raine and Bornstein 1970; Johnson and Bornstein 1978; Grundke-Iqbal et al. 1981), and complementindependent antibodies which cause myelin to swell and oligodendroglia to produce copious amounts of aberrant myelin (Bornstein and Raine 1976; Raine et al. 1978; Johnson et al. 1979; Grundke-Iqbal et al. 1981). These antibodies are reactive in vitro from very early stages of development onwards (Bornstein and Raine 1970; Bonnaud-Toulze et al. in press). To date, the specific myelin components against which these immunoglobulins are directed have not been identified, but indirect evidence suggests that myelin basic protein (MBP), the encephalitogenic component of central myelin (Kibler and Shapiro 1968; Eylar et al. 1969; Martenson et al. 1970), is not the target since anti-MBP serum does not cause demyelination in vitro (Seil et al. 1968). However, galactocerebroside (GC), the major glycolipid in CNS myelin, produces serum factors which are myelinotoxic in vitro (Dubois-Dalcq et al. 1970; Fry et al. 1974; Dorfman et al. 1978; Saida et al. 1979a). Since anti-GC serum factors are recognised as immunoglobulins (Johnson and Bornstein 1978; Raft et al. 1978; Johnson et al. 1980; Mirsky et al. 1980), it is possible that lipids may play a significant role in humoral immunity during demyelination. The present study describes tests performed in parallel to investigate the comparative effects upon myelinated CNS cultures of both intact and complementinactivated antisera raised against WM, MBP and GC, and before and after specific absorptions. The findings confirm that MBP is not the antigen responsible for demyelination in vitro, and that the lipid hapten, GC, does indeed play a major role in this phenomenon. MATERIALS A N D M E T H O D S
Tissue cultures
Myelinated organotypic cultures of embryonic mouse spinal cord were maintained in Maximow slide assemblies for 3-4 weeks by techniques standard in these laboratories (Bornstein 1973). Only well-myelinated cultures were used, and, when myelin was abundant, these were exposed to control and experimental sera for up to 4 days. Some cultures were exposed to complement-inactivated sera (see below) while others were exposed to intact serum (i.e. serum containing complement). In the latter case, the test serum was supplemented by the addition of 10~ normal guinea pig serum containing complement. Cultures were exposed to nutrient medium containing a 25~o concentration of the serum under test. Several different sera were compared simultaneously on sister cultures and each serum was used with 4-6 explants. In most cases, sera were tested on 2 separate occasions under code and the code was broken only after the cultures were assessed for demyelination.
119 Sera Control serum
In all experiments, control sera were examined. These consisted either of (a) normal New Zealand albino rabbit serum, usually a preimmune serum, or (b) serum from a rabbit inoculated with complete Freund's adjuvant (CFA) 14 days prior to sampling. The latter was used either without treatment or following passage through a control or a GC affinity column (see below) and in the presence or absence of active complement. Anti- W M serum
Adult, male New Zealand albino rabbits were inoculated subcutaneously in both hind foot-pads with 0.4 ml (0.2 ml each side) of an emulsion of fresh bovine white matter in saline and CFA in the ratio of 1 : 1 : 2. Prior to inoculation, preimmune serum was taken. Within the first 24 h of clinical EAE, 12-14 days postinoculation, animals were bled and the serum distributed into 1 ml aliquots and stored at - 90 °C. Anti-WM serum was used as follows: (a) Intact anti-WM serum containing complement. Before use, the MBP activity of the anti-WM serum was tested by radioimmunoassay (RIA) by Dr. S. R. Cohen, Johns Hopkins University, Baltimore and was found to contain significant specific binding to MBP at dilutions greater than 1 : 100; (b) Intact anti-WM serum absorbed against MBP. For this, 1 mg bovine MBP was added to 0.3 ml of serum and the serum centrifuged at 100,000 x g for 30 min to remove complexes. After absorption, MBP activity was not detectable by RIA; (c) Intact anti-WM serum absorbed against GC. The serum was passed through a G C affinity column (Kundu and Roy 1979) and then reconstituted to its original volume so that its concentration was similar to that of whole serum. To restore sterility, the serum was passed through a millipore filter prior to use in vitro; (d) Intact anti-WM serum absorbed against both MBP and GC. First the serum was collected from a GC affinity column as in (c) and then also absorbed with MBP as in (a), centrifuged and passed through a millipore filter; (e) Complement inactivated anti-WM serum. An aliquot of the above antiWM serum was heated at 56 °C for 30 min to inactivate complement. The MBP activity of this heated, unabsorbed anti-WM serum, measured by RIA, was found to have remained comparable to that of intact anti-WM serum; (f) Complement-inactivated anti-WM serum similarly absorbed against MBP and ultra-centrifuged; and (g) Complement-inactivated anti-WM serum absorbed with G C and centrifuged. A n t i - M B P serum
This was raised in a New Zealand male rabbit given one subcutaneous injection in the flank of 5 mg bovine MBP (obtained from Dr. E . H . Eylar, Toronto) emulsified in 0.5 ml saline and 0.5 ml CFA. This was followed at biweekly intervals by injections containing 1 mg MBP in saline and incomplete Freund's adjuvant (IFA). The animal did not develop signs of EAE. Two weeks
120
after the fifth booster injection, the animal was bled and the sterile serum stored as 3-5 ml aliquots at - 9 0 ° C . Prior to use, the serum was tested by RIA and found to bind MBP specifically, although at a low level. Immunofluorescence on brain sections using this serum followed by FITC-conjugated anti-rabbit IgG showed specific binding to white matter. Anti-GC s e r u m This was raised in two adult, male New Zealand albino rabbits. The GC preparation was obtained commercially (Supelco, Bellefonte, PA) and was of bovine brain origin. Prior to use, it was tested for purity by thin layer chromatography and was found to run as two spots only, corresponding to the two forms of galactosylceramide. The initial inoculation was subcutaneous in a rear flank and consisted of I ml containing 5 mg GC in 0.5 ml of micelles made up from lecithin (bovine heart - Supelco, Bellefonte, PA) and bovine serum albumin (Supelco) and 0.5 ml CFA. At biweekly intervals, 0.5 mg GC in micelles and IFA was similarly injected. Prior to each booster injection, about 50 ml of blood was drawn from the middle ear artery. Sterile serum was stored in 3-5 ml aliquots at - 9 0 °C. The anti-GC serum was tested for specific antibody to GC by RIA with a modification of the solid phase absorption technique of Holmgren et al. (1980). This technique involved coating the wells of microtitre plates with 0.1 /~g GC, incubation with diluted antiserum and then detection of bound lgG with radioiodinated protein A at a dilution of 1:400. The method does not detect IgM
TABLE 1 R A D I O I M M U N O A S S A Y OF A N T I - G A L A C T O C E R E B R O S I D E S E R U M IgG antibodies detected by RIA using a microtitre plate coated with GC and radioiodinated Protein A. Values are the average of 2 readings.
Date serum sampled a
cpm at 1 : 10 dilution
cpm at 1:50 dilution
cpm at 1:250 dilution
cpm at 1:1250 dilution
Animal No. 620
1/5/79 (preimmune) 1/19/79 2/5/79 2/21/79 3/7/79 3/26/79
110 3025 3059 3257 3548 3580
80 2278 2815 3336 3363 3320
66 1421 1947 1816 2264 2041
60 387 716 738 715 769
Animal No. 621
1/5/79 (preimmune) 1/19/79 2/5/79 2/21/79 3/7/79 3/26/79 5/2/79
85 2485 3346 3017 280 285 3532
75 1459 1378 1491 227 193 1433
68 542 531 490 104 119 468
69 259 180 215 72 87 201
a Blood drawn immediately prior to inoculation.
121 antibodies. The first animal developed transient signs of a peripheral neuropathy at 2 weeks post inoculation and had consistently high levels of anti-GC activity 3000 cpm at a 1 : 50 dilution (see Table 1). Two weeks prior to killing, the animal had a relapse and the signs persisted until the end of the experiment. The second rabbit developed no overt clinical signs of disease. Its serum showed a rapid rise in specific IgG to GC by the second booster injection which then dropped and rose again after the fourth booster injection. For the present experiments, anti-GC serum was used as: (a) Intact anti-GC serum (with complement); (b) Intact anti-GC serum passed over a control aminopropyl silica gel column containing Forssmann antigen as described by Kundu and Roy (1979); (c) Intact anti-GC serum passed over a GC affinity column (Kundu and Roy 1979). The anti-GC serum was passed twice over the column, reconcentrated prior to use and then passed through a millipore filter to restore sterility. The solid phase RIA showed that absorption removed about 60~ of the anti-GC IgG antibodies. (d) Complement-inactivated anti-GC serum; (e) Complement-inactivated anti-GC serum passed over a control column; and (f) Complement-inactivated anti-GC serum passed over a GC column.
lmmunocytochemistry Cultures treated with anti-WM, anti-GC and control sera were examined by immunocytochemistry. For this, living cultures were exposed to complement-inactivated sera for 1-4 h and then fixed and reacted with peroxidase labelled sheep Fab fragments against rabbit IgG. After washing, the samples were treated with diaminobenzidine and processed for light microscopy (LM) as whole mounts (Johnson and Bornstein 1978).
Morphology For each experimental serum, between 4 and 16 samples were studied by electron microscopy (EM). The cultures were taken between 12 to 96 h postexposure to test sera. While still on coverslips, they were fixed in 2.5~ glutaraldehyde, PO4-buffered to pH 7.4. The cultures were then processed and flat embedded in Epon for EM by techniques described in full elsewhere (Raine 1973). Thin sections for EM were double-stained with lead and uranyl salts, carboncoated and scanned in a Siemens 101. RESULTS A summary of the effects of the sera tested is given in Table 2. These were measured in living explants after 24 and 48 h of exposure. Preimmune and CFA serum (with and without specific absorptions) had no effect upon myelin in culture and no specific binding was observed by immunocytochemistry. Anti-WM serum with complement was exceedingly potent and by 48 h, had
122 TABLE 2 DEMYELINATING ACTIVITY OF SERA WITH AND WITHOUT ABSORPTION Serum
Demyelination a at 24 h
Demyelination a at 48 h
Anti-WM + C'b plain MBP-absorbed GC-absorbed pC- and MBP-absorbed
4.5 4.5 1.75 2.5
5.0 5.0 1.25 2.75
Anti-WM, no C'b plain MBP-absorbed GC-absorbed
1.5 1.5 0.5
1.75 1.5 0.5
Anti-MBP + C"b plain
1.0
1.5
Anti-GC + C 'b plain control absorption GC-absorbed
4.0 3.25 1.9
5.0 5.0 3.25
Anti-GC, no C 'b plain control absorption GC-absorbed
1.3 1.1 1.0
2.4 2.0 2.0
a Average of 4-6 sister cultures tested; 1.0 = minor swelling of myelin; 2.0 = up to 25~ demyelination; 3.0 = up to 50~ demyelination; 4.0 = up to 75~ demyelination; 5.0 = up to 100~ demyelination. b + C' = with added complement; no C' = complement inactivated by heating.
c a u s e d t o t a l d i s r u p t i o n o f all myelin sheaths. M o r p h o l o g i c a l l y , this was r e p r e s e n t e d b y the d i s i n t e g r a t i o n o f myelin into o v o i d s a n d the c o n c o m i t a n t d e a t h o f oligod e n d r o g l i a . D e t a i l s o f these p h e n o m e n a have been d e s c r i b e d p r e v i o u s l y ( R a i n e a n d B o r n s t e i n 1970). P r e v i o u s i m m u n o p e r o x i d a s e s t u d y o f this p h e n o m e n o n has shown it to be a s s o c i a t e d with specific b i n d i n g o f i m m u n o g l o b u l i n to myelin a n d o l i g o d e n d r o g l i a ( J o h n s o n a n d Bornstein 1978). A b s o r p t i o n o f a n t i - W M s e r u m with M B P h a d no m e a s u r a b l e effect u p o n the d e m y e l i n a t i n g a b i l i t y o f the serum, a l t h o u g h R I A s h o w e d t h a t the a b s o r b e d s e r u m h a d lost all activity a g a i n s t M B P . H o w e v e r , a b s o r p t i o n o f a n t i - W M s e r u m with G C h a d a m a r k e d effect o n the d e m y e l i n a t i o n a n d r e d u c e d the efficacy o f the s e r u m at 48 h f r o m 5.0 (before a b s o r p t i o n ) to less t h a n 2 (after a b s o r p t i o n ) (see T a b l e 2 for e x p l a n a t i o n o f scale). C o m b i n e d a b s o r p t i o n o f a n t i - W M s e r u m with G C a n d M B P also c a u s e d a r e d u c t i o n in the o b s e r v e d d e m y e l i n a t i o n , a l b e i t less m a r k e d t h a n after G C a b s o r p t i o n alone. T h e higher d e m y e l i n a t i n g scores with the c o m b i n e d a b s o r p t i o n were p r o b a b l y due to e x p e r i m e n t a l v a r i a t i o n since the dif-
123 ference was less at 24 h and since the 48 h reading was still significantly lower than that seen without absorption. If MBP absorption had had an additive effect with GC, a greater reduction in the amount of demyelination might have been expected after double absorption. Complement-inactivated anti-WM serum caused relatively few myelin abnormalities as perceived by LM. These consisted of some myelin irregularities, myelin swelling and ballooning but no frank demyelination. Immunocytochemistry showed extensive binding of immunoglobulin to myelin and oligodendrocytes, as described previously (Johnson et al. 1979). EM confirmed that the morphologic changes, described in detail elsewhere (Bornstein and Raine 1976; Raine et al. 1978), were related to the formation of aberrant myelin sheaths with an additional pair of leaflets at the intraperiod line and oligodendroglia which had proliferated an abundance of the same aberrant myelin within and around the cell somata (Figs. 1 and 2). Absorption of heated anti-WM serum with MBP did not significantly reduce the degree of myelin swelling (Fig. 3). However, absorption of complementinactivated anti-WM serum with GC reduced the myelin changes to almost imperceptible levels. Immunostaining was also markedly reduced. Anti-MBP serum had no effect upon myelin in vitro over a 48-h period (Fig. 4). Since it was ineffective in the presence of complement, anti-MBP was not tested without complement, nor was it deemed necessary to absorb the serum against MBP or GC. Anti-GC serum with complement, whether used plain or after absorption on a control column, proved to be a potent demyelinating agent (Table 2). The demyelinating ability of anti-GC serum was markedly reduced after passing the serum over a GC affinity column which was shown to decrease the GC IgG antibodies by about 60~. Without specific absorption, intact anti-GC serum was seen by EM to cause myelin to break down and dissociate from the axon. The dissociated myelin was phagocytosed by investing astrocytes which not infrequently contained an overabundance of glycogen (Figs. 5 and 6). Ultrastructurally, anti-GC serum-induced demyelination was generally similar to that with intact anti-WM serum, but with anti-GC serum it tended to be less severe in that oligodendroglia often survived and the process was slower. The demyelination induced by intact anti-GC serum could sometimes be seen to involve the myelin swelling which is typically associated with complement-inactivated anti-WM serum. After complement-inactivation, anti-GC serum caused relatively few myelin changes visible in the living state but there was pronounced immunostaining of myelin and oligodendroglia. At the EM level, abundant myelin swelling was apparent (Fig. 7). With GC-absorbed, heated anti-GC serum, there was a dramatic decrease in the amount of myelin swelling seen at the ultrastructural level (Fig. 8) and an associated decrease in peroxidase reaction product on myelin and oligodendrocytes.
Fig. 1. Anti-WM serum, complement-inactivated; 48 h exposure. This electron micrograph shows ata oligodendrocyte with proliferated aberrant, wide-spaced myelin within and around itself. Arrow indicates area depicted in Fig. 2. x 12,250. Fig. 2. Detail from Fig. 1. Note the wide-spaced myelin with a 22-nm periodicity. Between each pair of major dense lines, 4 instead of the usual 2 leaflets are associated with the intraperiod line. x 125,000.
125
Fig. 3. Anti-WM serum, complement-inactivated, absorbed against MBP; 48 h exposure. Note that myelin swelling still persists in the absence of antibody against MBP. × 30,000. Fig. 4. Anti-MBP serum, with complement; 48 h exposure. Note that this serum had no apparent effect upon myelin in vitro. Same magnification as Fig. 3 for comparison. × 30,000.
Fig. 5. Anti-GC serum, with complement ; 24 h exposure. A myelinated nerve has almost been completely divested of its myelin sheath which is in the process of being engulfed by encircling astroglial cell processes. Note the glycogen granules within the astroglial cytoplasm. Arrow indicates area enlarged in inset. × 22,500. Inset." Detail of the disintegrating myelin in Fig. 5. Note that some lamellae are widespaced (22 nm large arrow) while others are compacted (6 nm) and fragmented (small arrow). x 125,000. Fig. 6. A n t i - G C serum, with complement; 48 h exposure. Naked axons (arrows) and myelin debris are evident in this area from a culture showing total demyelination, x 15,000.
Fig. 7. Anti-GC serum, complement-inactivated; 72 h exposure. A group of myelinated axons displays a moderate degree o f myelin swelling, barely visible at this magnification. Arrow indicates area shown in inset, x 15,000. Inset: Detail of the swollen myelin sheaths from Fig. 7. The quadrilamellar intraperiod line is apparent in places (arrows). × 125,000. Fig. 8. Anti-GC serum, complement-inactivated, absorbed on a GC column to remove GC activity and to test the effect of specific absorption against GC upon myelin swelling; 72 h exposure. Note that the ability of G C serum to cause myelin swelling has been removed. × 15,000.
128 DISCUSSION The effects of anti-WM serum on organotypic cultures of CNS tissue have been known for some time but the antigen(s) involved has never been established. By specific absorptions and appropriate controls, this study with anti°WM, antiMBP, and anti-GC sera has shown that serum-induced demyelination, myelin swelling and positive immunostaining by anti-WM serum in vitro appear to be GC-related phenomena and are not dependent upon the presence of antibody against MBP. These conclusions emanate from groups of experiments in vitro carried out in parallel with well-characterised antisera and the protocols permitted simultaneous comparison of the effects of the various sera. It has previously been reported that anti-MBP serum is not myelinotoxic in vitro (Seil et al. 1968) and that antiGC serum has demyelinating activity (Dubois-Dalcq et al. 1970; Fry et al. 1974; Saida et al. 1979a,b). However, anti-MBP, anti-GC and anti-WM sera, with the various controls and specific absorptions, with and without active complement, have never been compared or tested in the same experimental series. The use of RIA systems and affinity columns provided valuable monitors in the analysis of the results. It should be emphasised that the tests were invariably carried out under code, providing added significance to the conclusions drawn from these experiments. A major goal of the present experiments was to determine whether demyelination in vitro and myelin swelling (in the absence of intact complement) could be produced, as well as removed, by the same or separate factors. It has recently been demonstrated that while demyelination in vitro is associated with IgG and non-IgG serum fractions, myelin swelling is linked only to IgG (Grundke-Iqbal et al. 1981). From the present studies, anti-GC serum was found to produce myelin swelling and demyelinating patterns similar to those found with anti-WM serum. In addition, it has been shown that GC absorption of anti-WM and anti-GC sera dramatically reduced the level of both phenomena in vitro. GC absorption also reduced the immunostaining with both these sera. Thus, galactocerebroside can at least be claimed to be a major component of myelin responsible for CNS myelin changes in vitro. The relative roles of other myelin lipids (e.g. sulfatides, gangliosides) remain to be determined. A second goal was to compare the efficacy of MBP and GC as demyelinating and absorption factors. Clearly, it has been demonstrated that MBP had little or no role in this humoral response tested in vitro and that GC appeared to be a major determinant. The importance of GC in experimental demyelination was also noted recently in a series of in vivo experiments comparing the effects of sensitisation with emulsions containing WM, MBP or GC (Raine et al., in press). It was found that emulsions containing GC alone induced no CNS signs or pathology, whereas MBP alone caused mild signs of EAE and produced inflammatory lesions which lacked demyelination. However, emulsions containing a combination of MBP and GC induced signs typical of EAE and led to the formation of CNS lesions displaying both inflammation and demyelination, a pattern which approximated that produced by sensitisation against whole white matter. From these experiments, it was sug-
129 gested that MBP provided the cell-mediated component of the immune response and that anti-GC serum factors were necessary to cause demyelination. The mechanism of demyelination in vivo might be through myelin opsonisation and attack by macrophages, through complement-mediated lysis because of bound antibody or through antibody-dependent cell-mediated demyelination (Brosnan et al. 1977). Perhaps what has been witnessed in the present study with cultures is the activity of anti-myelin antibodies which in vivo require lymphocyte activation to become effective, but in vitro are in themselves myelinotoxic. Possibly the cell-mediated immune response is required in vivo to alter the blood-brain barrier and local environment so that humoral factors gain access to CNS loci. Some evidence from in vivo experiments involving local infusion of anti-GC or anti-WM serum into the peripheral nerve suggests that the demyelination observed is purely humoral and that lymphocytes are not involved (Saida et al. 1978; Saida et al. 1979b). The relevance of the above studies to human demyelinating diseases, in particular multiple sclerosis (MS), remains somewhat obscure since current data suggest that most MS serum-induced demyelination is not related to immunoglobulin and MS serum does not give positive immunostaining in vitro (Johnson and Bornstein 1978; Grundke-Iqbal and Bornstein 1980). With anti-WM serum, demyelination has been known for some time to be related to antibody (Appel and Bornstein 1964; Johnson and Bornstein 1978). The question still remains, however, whether MS serum-induced demyelination is indeed MS-specific and whether it fluctuates with relapses and remissions. It is entirely possible, for example, that specific demyelinating antibody might appear in relationship to active disease and this may be distinguishable from non-specific demyelinating epiphenomena occurring at other stages of MS and in non-MS conditions. Also in MS, the use of organotypic cultures of CNS tissue to explore questions concerning the relative roles of different myelin components in the abnormal serum response or the interactions between possible cellular and humoral events using organotypic CNS cultures remain largely unexplored. With the current availability of a chronic relapsing EAE model (Raine et al. 1974) and its application to the MS problem (Raine et al. 1980a,b), it may be possible to approach in vivo several of the above problems and the implications of the present work. ACKNOWLEDGEMENTS The authors thank Michael Diaz, Vincent Spada, Everett Swanson, Howard Finch, Miriam Pakingan, Marie Vitale and Muhammad Farooq for invaluable technical expertise; and Mary Palumbo and Agnes Geoghan for expert secretarial assistance. The authors are indebted to Dr. William T. Norton for compilation of inocula; Dr. Steven R. Cohen, Dept. of Neurology, Johns Hopkins University, Baltimore, MD, who performed the RIA of the antisera for the determination of anti-MBP activity; to Dr. Samar Kundu, Dept. of Internal Medicine, Baylor College
130 of Medicine, Houston,
TX, who performed
and GC-specific columns;
a n d to D r .
the absorption
Edwin
of antisera on control
H. E y l a r , D e p t . o f B i o c h e m i s t r y ,
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