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Immunization with purified myelin-associated glycoprotein does not evoke myelination-inhibiting or demyelinating antibodies
470
B]]ain Research, 209 (1981) 470-475 © Elsevier/North-Holland Biomedical Press
Immunization with purified myelin-associated glycoprotein does not evoke myelination-inhibiting or demyelinating antibodies
FREDRICK J. SEIL*, RICHARD H. QUARLES, DAVID JOHNSON and ROSCOE O. BRADY Neurology Service, Portland Veterans Administration Medical Center and Department of Neurology, University of Oregon Health Sciences Center, Portland, Oreg. 97201 and Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Md. 20014 (U.S.A.)
(Accepted November 6th, 1980) Key words: myelin-associated glycoprotein - - demyelination - - myelination-inhibiting antibody - -
tissue culture
Antisera produced in rabbits against purified myelin-associated glycoprotein did not inhibit myelin formation in cerebellar tissue cultures or demyelinate centrally or peripherally myelinated fibers in spinal cord-dorsal root ganglia cultures. These results suggest that antibodies directed against other myelin components are responsible for the myelination-inhibition and demyelination produced by anti-central nervous system (CNS) and anti-myelin antisera in vitro. Antisera directed against whole central nervous system (CNS) tissue, purified myelin or a lipid component of myelin, galactocerebroside, have been shown to demyelinate CNS tissue cultures and to inhibit myelin formation in unmyelinated explants 2-6,9,1s. On the other hand, antibodies to the CNS myelin proteins, proteolipid protein and myelin basic protein, generally have no antimyelin activity in vitroS,9,15,16,1s. In spite of the failure of the major CNS myelin proteins to evoke demyelinating or myelination-inhibiting antibodies, any myelin protein is a potential antigen that may induce or contribute to the induction of antimyelin factors, particularly if some portion of that protein may possibly be localized on the surface of the myelin sheathlL Since membrane glycoproteins are surface components of many cells 7, this study was designed to evaluate antisera directed against CNS myelinassociated glycoprotein ( M A G ) for demyelinating and myelination-inhibiting activity in vitro. A preliminary report of this work was presented recently 17. A n t i - M A G antisera were prepared in 3 rabbits as described in detail elsewhere ~3, 19. Two rabbits were immunized with M A G purified by selective extraction with lithium diiodosalicylate (LIS) and phenol followed by preparative SDS gel electrophoresis. The antisera from these rabbits are designated MAG-1 and MAG-2 in Table I. The third rabbit was immunized with a preparation of M A G obtained by [,IS-phenol * To whom correspondence should be addressed at : Neurology Research (151N), Veterans Administration Medical Center, 3710 S.W.U.S. Veterans Hospital Road, Portland, Oreg, 97201 U.S.A.
Code
A1 B5 B8 A4 B1
A2 B3
* ** *** §
2 3 4 5
1
2/4 * * 11/14'** 5/7 6/9 6/7
0/5 0/7
4/6 7/7 5/6 5/6 5/7
Cultures myelinated (fraction)
÷ ÷
d
m
m
M1 (10% serum)
0/5 0/8"** 0/4 0/5 0/4
5/5 5/5
0/3 0/5 0/5 0/4 0/4
Cultures demyelinated* (fraction)
------
q+
------
Demyelination (23% serum)
anti-MAG-1 anti-MAG- 1 anti-MAG-1 § anti-MAG-2 anti-MAG-3
anti-myelin anti-myelin
prebleed prebleed prebleed P C A gel control P C A gel control
Serum
Figures given refer to CNS myelin. N o peripherally myelinated fibers were demyelinated in any cultures. Three o f four cultures myelinated when tested at a 23 ~o serum concentration. Results are cumulative from tests o f two aliquots o f the same serum. Boosted 1 year after original immunization.
A3 A5, B6 B2 B4 B7
C. Anti-MAG antisera
2
1
B. Positive controls
2 3 4 5
1
A. Negative controls
Group
27 100 64 85 55
4 2
0 0 0 0 0
Relative antibody levels to MAG
Abbreviations: M A G , myelin-associated glycoprotein; PCA, polyacrylamide; CNS, central nervous system; MI, myelination-inhibition.
Rabbit sera evaluatedfor myelination-inhibiting and demyelinating activity in vitro
TABLE I
"....I
472 extraction alone without preparative gel electrophoresis. The antiserum from this rabbit, immunized with MAG that had not been exposed to SDS, is designated MAG3. A control rabbit was injected on the same time schedule as MAG rabbit 1 with extracts from polyacrylamide preparative gels to which no glycoprotein sample was applied. Each rabbit was injected intradermally at multiple sites on its back on 3 occasions separated by about 3 weeks. The first two series of injections were done in complete Freund's adjuvant and the final series in incomplete Freund's adjuvant. In addition to the polyacrylamide gel control rabbit, other control sera were collected from rabbits prior to any inoculations (prebleed sera). Two rabbits were sensitized to CNS myelin by injection on a single occasion with 25 mg dry weight of purified rat brain myelin emulsified in complete Freunds' adjuvant, and sera collected from these animals served as positive controls for antimyelin activity in vitro. The properties of various antisera and a double antibody immunoassay used to measure serum levels of antibody to MAG are described elsewhere13,1.% The antiserum with the highest level of antibodies to MAG bound 360 #g of MAG per ml under the conditions of the assay, and the levels ofanti-MAG antibodies in other sera are presented relative to that value taken as 100. Cerebellar cultures derived from newborn Swiss-Webster mice were cultivated on collagen-coated coverslips in Maxinow chambers by established methods1,1~. Such cultures were exposed to nutrient media incorporating coded test rabbit sera at the time of explantation and continuously thereafter, and were observed for 15-16 days for myelin formation. Myelination in such preparations usually takes place between 9 and 12 days in vitro 14. A serum was considered positive for myelination-inhibition if less than half of the cultures exposed to it myelinated (usually 80'~'~,/, of cerebellar cultures in normal medium myelinate) s. Demyelination was evaluated in well myelinated 19-26 days in vitro spinal cord-dorsal root ganglia (DRG) explants also cultivated in Maximow chambers by standard methods1,1°. Such cultures contained both central and peripheral myelin. After application of coded test rabbit sera incorporated into nutrient medium, the cultures were observed for four days for signs of demyelination, as described previously TM. Two groups of rabbit sera were tested. The first group consisted of a series coded with numbers A l-A5 (Table I). This group, which was the subject of the preliminary report 17, included two sera collected at different intervals from a MAG inoculated rabbit (MAG-1), plus negative and positive control sera. These sera were tested for myelination-inhibition in both 10 °~tland 23 o concentrations in the nutrient medium, along with 10 ~ pooled normal guinea pig sera as a source of complement. Difficulties encountered with the interpretation of results of testing rabbit sera for myelinationinhibition at excessively high concentrations were discussed in the preliminary report 17, and only the results obtained with 10 % test sera for myelination-inhibition are reported here. Demyelinating activity was evaluated at a 23!~',/; concentration in nutrient medium, plus 10 ~/opooled normal guinea pig serum. The second group of sera was coded BI-B8 (Table I), and this group included sera from the original MAGinoculated animal (MAG-I) before and after another booster injection of MAG, and sera from two additional rabbits injected with MAG (MAG-2 and MAG-3). Negative
473 and positive control sera were again included. Sera from this group were tested for myelination-inhibition only in a 10~ concentration in nutrient medium, plus 10~o pooled normal guinea pig serum. Demyelination was again evaluated at a 23 concentration of test serum in nutrient medium, along with 10~ pooled normal guinea pig serum. The results of this study are summarized in Table I. All of the prebleed sera and sera from the polyacrylamide extract injected animal were negative for myelinationinhibition and for demyelination. Sera from both of the rabbits sensitized with purified CNS myelin inhibited myelination in cerebellar cultures and demyelinated centrally myelinated fibers in spinal cord-DRG cultures. Peripherally myelinated axons were spared, confirming previous observations of the specificity of anti-CNS myelin antisera for activity against central myelin2,1~. None of the 5 anti-MAG antisera derived from 3 rabbits and containing a range of titers of antibody to MAG inhibited myelin formation in cerebellar explants or demyelinated either centrally or peripherally myelinated fibers in mature spinal cord-DRG cultures. These results indicate that rabbit antisera directed against MAG do not demyelinate centrally or peripherally myelinated axons in vitro, nor do they inhibit myelin formation in cerebellar cultures. Thus purified MAG, like myelin basic protein and proteolipid protein, does not induce demyelinating or myelination-inhibiting antibodies. It follows that such antibodies are evoked in response to some other myelin antigen(s). As antisera with high levels of anti-MAG antibody were inactive against myelin in tissue cultures, possible reasons as to why an antibody directed against a presumably surface-exposed myelin protein did not disrupt myelin in vitro must be considered. Among such possible reasons are: (1) the antibody may bind to the protein without causing myelin destruction; and (2) the protein, or its antigenetically active site, may not be exposed at the surface of the myelin sheath. While no studies have been directed at the former possibility, there is evidence to suggest that MAG may not be as exposed at the surface of the myelin sheath as early surface probe studies 11 with galactose oxidase and sodium [aH]borohydride had indicated. More recent immunocytochemical studies have shown that most of the MAG is localized periaxonally on the inside of myelin sheaths 19, a location that is not readily accessible to extracellular macromolecules. Moreover, anti-MAG antisera cannot be absorbed by whole brain homogenate or purified myelin13. Rather, the MAG must be extracted from the membranes or the membranes disrupted with detergent before MAG can react with the antibodies. This suggests that the antigenic sites are buried in the lipid bilayer or in some other way blocked by the molecular structure in the intact membranes. Thus the MAG or its antigenically active site may not be accessible for formation of antigen-antibody complexes when myelin is exposed to anti-MAG antisera in tissue cultures. Table I shows that the positive control anti-myelin antisera did contain antibodies to MAG, but the levels were much lower than those in the rabbits that had been hyperimmunized with purified MAG la. It is of interest that over half of these anti-MAG antibodies in the anti-myelin serum could be absorbed by whole brain
474 h o m o g e n a t e , indicating that they are directed against a different a n d m o r e accessible site on the M A G molecule than the antibodies in the rabbits t h a t had been h y p e r i m m u n i z e d with purified M A G . This low level o f a n t i - M A G a n t i b o d i e s reacting with a m o r e accessible p o r t i o n o f the molecule could help mediate or potentiate the myelination-inhibiting or d e m y e l i n a t i n g activity o f anti-myelin a n t i s e r u m by acting in concert with antibodies to other myelin components. However, in general, the results o f this a n d previous studies suggest that the principal antibodies responsible for myelination-inhibition or d e m y e l i n a t i o n in vitro are directed against one o r m o r e other myelin c o m p o n e n t s , such as galactocerebroside, a n d not against M A G o r the other myelin proteins studied to date. S u p p o r t e d by the Veterans A d m i n i s t r a t i o n (F.J.S.). The technical assistance o f Benson F o n g , James Jetton, Michele Mass, Jean Quigley, C a r o l Pasnak, G a r y B a r b a r a s h a n d K a t h r y n Winchell is gratefully acknowledged. Carol P a s n a k was s u p p o r t e d by a grant from the N a t i o n a l Multiple Sclerosis Society. 1 Bornstein, M. B., Organotypic mammalian central and peripheral nervous tissue. In P. F. Kruse, Jr, and M. K. Patterson, Jr. (Eds.), Tissue Culture Methods and Applications, Academic Press, New York, N.Y., 1973, pp. 86-92. 2 Bornstein, M. B. and Appel, S. H., The application of tissue culture to the study of experimental 'allergic' encephalomyelitis: I. Patterns of demyelination, J. Neuropath. exp. NeuroL, 20 (1961) 141-157. 3 Bornstein, M. B. and Raine, C. S., Experimental allergic encephalomyelitis: antiserum inhibition of myelination in vitro, Lab. lnvest., 23 (1970) 536-542. 4 Dubois-Dalcq, M., Niedieck, B. and Buyse, M., Action of anti-cerebroside sera on myelinated tissue cultures, Path. Europ., 5 (1970) 331-347. 5 Fry, J. M., Weissbarth, S., Lehrer, G. M. and Bornstein, M. B., Cerebroside antibody inhibits sulfatide synthesis and myelination and demyelinates in cord tissue cultures, Science, 183 (1974) 540-542. 6 Hruby, S., Alvord, E. C., Jr. and Sell, F. J., Synthetic galactocerebrosides evoke myelinationinhibiting antibodies, Science, 195 (1977) 173-175. 7 Hughes, R. C., Membrane Glycoproteins: ,4 Review of Structure and Function, Butterworth, Woburn, Mass., 1976. 8 Kies, M. W., Driscoll, B. F., Seil, F. J. and Alvord, E. C., Jr., Myelination inhibition factor: dissociation from induction of experimental allergic encephalomyelitis, Science, 179 (1973) 689690. 9 Lebar, R., Boutry, J. M., Vincent, C. H., Robineaux, R. and Voisin, G. A., Studies on autoimmune encephalomyelitis in the guinea pig: II. An in vitro investigation of the nature, properties and specificity of the serum demyelinating factor, J. lmmunol., 116 (1976) 1439-1446. 10 Peterson, E. R., Crain, S. M. and Murray, M. R., Differentiation and prolonged maintenance of bioelectricallyactive spinal cord cultures (rat, chick and human), Z. Zellforsch., 66 (1965) 130154. 11 Poduslo, J. F., Quarles, R. H. and Brady, R. O., External labeling of galactose in surface membrane glycoproteins of the intact myelin sheath, J. biol. Chem., 251 (1976) 153-158. 12 Quarles, R. H., Glycoproteins in myelin and myelin related membranes. In R. U. Margolis and R. K. Margolis (Eds.), Complex Carbohydrates of the Nervous System, Plenum Press, New York, N.Y., 1979, pp. 209-233. 13 Quarles, R. H., Johnson, D., Brady, R. O. and Sternberger, N. H., Preparation and characterization of antisera to the myelin-associated glycoprotein, Neurochem. Res., in press. 14 Seil, F. J., Cerebellum in tissue culture. In D. M. Schneider (Ed.), Reviews of Neuroscience, Vol. 4, Raven Press, New York, N.Y., 1979, pp. 105-177. 15 Seil, F. J. and Agrawal, H. C., Myelin-proteolipid protein does not induce demyelinating or myelination-inhibiting antibodies, Brain Research, 194 (1980) 273-277.
475 16 Seil, F. J., Falk, G. A., Kies, M. W. and Alvord, E. C., Jr., The in vitro demyelinatingactivity of sera from guinea pigs sensitized with whole CNS and with purified encephalitogen, Exp. Neurol., 22 (1968) 545-555. 17 Seil, F. J., Kies, M. W., Agrawal, H. C., Quarles, R. H. and Brady, R. O., Myelin proteins dissociated from induction of antimyelin antibodies. In E. Giacobini, A. Vernadakis and A. Shahar (Eds.), Tissue Culture in Neurobiology, Raven Press, New York, N.Y., 1980, pp. 477--488. 18 Seil, F. J., Smith, M. E , Leiman, A. L. and Kelly, J. M., Myelination inhibiting and neuroelectric blocking factors in experimental allergic encephalomyelitis, Science, 187 (1975) 951-953. 19 Sternberger, N. H., Quarles, R. H., Itoyama, Y. and Webster, H. F., Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 1510-1514.
B]]ain Research, 209 (1981) 470-475 © Elsevier/North-Holland Biomedical Press
Immunization with purified myelin-associated glycoprotein does not evoke myelination-inhibiting or demyelinating antibodies
FREDRICK J. SEIL*, RICHARD H. QUARLES, DAVID JOHNSON and ROSCOE O. BRADY Neurology Service, Portland Veterans Administration Medical Center and Department of Neurology, University of Oregon Health Sciences Center, Portland, Oreg. 97201 and Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Md. 20014 (U.S.A.)
(Accepted November 6th, 1980) Key words: myelin-associated glycoprotein - - demyelination - - myelination-inhibiting antibody - -
tissue culture
Antisera produced in rabbits against purified myelin-associated glycoprotein did not inhibit myelin formation in cerebellar tissue cultures or demyelinate centrally or peripherally myelinated fibers in spinal cord-dorsal root ganglia cultures. These results suggest that antibodies directed against other myelin components are responsible for the myelination-inhibition and demyelination produced by anti-central nervous system (CNS) and anti-myelin antisera in vitro. Antisera directed against whole central nervous system (CNS) tissue, purified myelin or a lipid component of myelin, galactocerebroside, have been shown to demyelinate CNS tissue cultures and to inhibit myelin formation in unmyelinated explants 2-6,9,1s. On the other hand, antibodies to the CNS myelin proteins, proteolipid protein and myelin basic protein, generally have no antimyelin activity in vitroS,9,15,16,1s. In spite of the failure of the major CNS myelin proteins to evoke demyelinating or myelination-inhibiting antibodies, any myelin protein is a potential antigen that may induce or contribute to the induction of antimyelin factors, particularly if some portion of that protein may possibly be localized on the surface of the myelin sheathlL Since membrane glycoproteins are surface components of many cells 7, this study was designed to evaluate antisera directed against CNS myelinassociated glycoprotein ( M A G ) for demyelinating and myelination-inhibiting activity in vitro. A preliminary report of this work was presented recently 17. A n t i - M A G antisera were prepared in 3 rabbits as described in detail elsewhere ~3, 19. Two rabbits were immunized with M A G purified by selective extraction with lithium diiodosalicylate (LIS) and phenol followed by preparative SDS gel electrophoresis. The antisera from these rabbits are designated MAG-1 and MAG-2 in Table I. The third rabbit was immunized with a preparation of M A G obtained by [,IS-phenol * To whom correspondence should be addressed at : Neurology Research (151N), Veterans Administration Medical Center, 3710 S.W.U.S. Veterans Hospital Road, Portland, Oreg, 97201 U.S.A.
Code
A1 B5 B8 A4 B1
A2 B3
* ** *** §
2 3 4 5
1
2/4 * * 11/14'** 5/7 6/9 6/7
0/5 0/7
4/6 7/7 5/6 5/6 5/7
Cultures myelinated (fraction)
÷ ÷
d
m
m
M1 (10% serum)
0/5 0/8"** 0/4 0/5 0/4
5/5 5/5
0/3 0/5 0/5 0/4 0/4
Cultures demyelinated* (fraction)
------
q+
------
Demyelination (23% serum)
anti-MAG-1 anti-MAG- 1 anti-MAG-1 § anti-MAG-2 anti-MAG-3
anti-myelin anti-myelin
prebleed prebleed prebleed P C A gel control P C A gel control
Serum
Figures given refer to CNS myelin. N o peripherally myelinated fibers were demyelinated in any cultures. Three o f four cultures myelinated when tested at a 23 ~o serum concentration. Results are cumulative from tests o f two aliquots o f the same serum. Boosted 1 year after original immunization.
A3 A5, B6 B2 B4 B7
C. Anti-MAG antisera
2
1
B. Positive controls
2 3 4 5
1
A. Negative controls
Group
27 100 64 85 55
4 2
0 0 0 0 0
Relative antibody levels to MAG
Abbreviations: M A G , myelin-associated glycoprotein; PCA, polyacrylamide; CNS, central nervous system; MI, myelination-inhibition.
Rabbit sera evaluatedfor myelination-inhibiting and demyelinating activity in vitro
TABLE I
"....I
472 extraction alone without preparative gel electrophoresis. The antiserum from this rabbit, immunized with MAG that had not been exposed to SDS, is designated MAG3. A control rabbit was injected on the same time schedule as MAG rabbit 1 with extracts from polyacrylamide preparative gels to which no glycoprotein sample was applied. Each rabbit was injected intradermally at multiple sites on its back on 3 occasions separated by about 3 weeks. The first two series of injections were done in complete Freund's adjuvant and the final series in incomplete Freund's adjuvant. In addition to the polyacrylamide gel control rabbit, other control sera were collected from rabbits prior to any inoculations (prebleed sera). Two rabbits were sensitized to CNS myelin by injection on a single occasion with 25 mg dry weight of purified rat brain myelin emulsified in complete Freunds' adjuvant, and sera collected from these animals served as positive controls for antimyelin activity in vitro. The properties of various antisera and a double antibody immunoassay used to measure serum levels of antibody to MAG are described elsewhere13,1.% The antiserum with the highest level of antibodies to MAG bound 360 #g of MAG per ml under the conditions of the assay, and the levels ofanti-MAG antibodies in other sera are presented relative to that value taken as 100. Cerebellar cultures derived from newborn Swiss-Webster mice were cultivated on collagen-coated coverslips in Maxinow chambers by established methods1,1~. Such cultures were exposed to nutrient media incorporating coded test rabbit sera at the time of explantation and continuously thereafter, and were observed for 15-16 days for myelin formation. Myelination in such preparations usually takes place between 9 and 12 days in vitro 14. A serum was considered positive for myelination-inhibition if less than half of the cultures exposed to it myelinated (usually 80'~'~,/, of cerebellar cultures in normal medium myelinate) s. Demyelination was evaluated in well myelinated 19-26 days in vitro spinal cord-dorsal root ganglia (DRG) explants also cultivated in Maximow chambers by standard methods1,1°. Such cultures contained both central and peripheral myelin. After application of coded test rabbit sera incorporated into nutrient medium, the cultures were observed for four days for signs of demyelination, as described previously TM. Two groups of rabbit sera were tested. The first group consisted of a series coded with numbers A l-A5 (Table I). This group, which was the subject of the preliminary report 17, included two sera collected at different intervals from a MAG inoculated rabbit (MAG-1), plus negative and positive control sera. These sera were tested for myelination-inhibition in both 10 °~tland 23 o concentrations in the nutrient medium, along with 10 ~ pooled normal guinea pig sera as a source of complement. Difficulties encountered with the interpretation of results of testing rabbit sera for myelinationinhibition at excessively high concentrations were discussed in the preliminary report 17, and only the results obtained with 10 % test sera for myelination-inhibition are reported here. Demyelinating activity was evaluated at a 23!~',/; concentration in nutrient medium, plus 10 ~/opooled normal guinea pig serum. The second group of sera was coded BI-B8 (Table I), and this group included sera from the original MAGinoculated animal (MAG-I) before and after another booster injection of MAG, and sera from two additional rabbits injected with MAG (MAG-2 and MAG-3). Negative
473 and positive control sera were again included. Sera from this group were tested for myelination-inhibition only in a 10~ concentration in nutrient medium, plus 10~o pooled normal guinea pig serum. Demyelination was again evaluated at a 23 concentration of test serum in nutrient medium, along with 10~ pooled normal guinea pig serum. The results of this study are summarized in Table I. All of the prebleed sera and sera from the polyacrylamide extract injected animal were negative for myelinationinhibition and for demyelination. Sera from both of the rabbits sensitized with purified CNS myelin inhibited myelination in cerebellar cultures and demyelinated centrally myelinated fibers in spinal cord-DRG cultures. Peripherally myelinated axons were spared, confirming previous observations of the specificity of anti-CNS myelin antisera for activity against central myelin2,1~. None of the 5 anti-MAG antisera derived from 3 rabbits and containing a range of titers of antibody to MAG inhibited myelin formation in cerebellar explants or demyelinated either centrally or peripherally myelinated fibers in mature spinal cord-DRG cultures. These results indicate that rabbit antisera directed against MAG do not demyelinate centrally or peripherally myelinated axons in vitro, nor do they inhibit myelin formation in cerebellar cultures. Thus purified MAG, like myelin basic protein and proteolipid protein, does not induce demyelinating or myelination-inhibiting antibodies. It follows that such antibodies are evoked in response to some other myelin antigen(s). As antisera with high levels of anti-MAG antibody were inactive against myelin in tissue cultures, possible reasons as to why an antibody directed against a presumably surface-exposed myelin protein did not disrupt myelin in vitro must be considered. Among such possible reasons are: (1) the antibody may bind to the protein without causing myelin destruction; and (2) the protein, or its antigenetically active site, may not be exposed at the surface of the myelin sheath. While no studies have been directed at the former possibility, there is evidence to suggest that MAG may not be as exposed at the surface of the myelin sheath as early surface probe studies 11 with galactose oxidase and sodium [aH]borohydride had indicated. More recent immunocytochemical studies have shown that most of the MAG is localized periaxonally on the inside of myelin sheaths 19, a location that is not readily accessible to extracellular macromolecules. Moreover, anti-MAG antisera cannot be absorbed by whole brain homogenate or purified myelin13. Rather, the MAG must be extracted from the membranes or the membranes disrupted with detergent before MAG can react with the antibodies. This suggests that the antigenic sites are buried in the lipid bilayer or in some other way blocked by the molecular structure in the intact membranes. Thus the MAG or its antigenically active site may not be accessible for formation of antigen-antibody complexes when myelin is exposed to anti-MAG antisera in tissue cultures. Table I shows that the positive control anti-myelin antisera did contain antibodies to MAG, but the levels were much lower than those in the rabbits that had been hyperimmunized with purified MAG la. It is of interest that over half of these anti-MAG antibodies in the anti-myelin serum could be absorbed by whole brain
474 h o m o g e n a t e , indicating that they are directed against a different a n d m o r e accessible site on the M A G molecule than the antibodies in the rabbits t h a t had been h y p e r i m m u n i z e d with purified M A G . This low level o f a n t i - M A G a n t i b o d i e s reacting with a m o r e accessible p o r t i o n o f the molecule could help mediate or potentiate the myelination-inhibiting or d e m y e l i n a t i n g activity o f anti-myelin a n t i s e r u m by acting in concert with antibodies to other myelin components. However, in general, the results o f this a n d previous studies suggest that the principal antibodies responsible for myelination-inhibition or d e m y e l i n a t i o n in vitro are directed against one o r m o r e other myelin c o m p o n e n t s , such as galactocerebroside, a n d not against M A G o r the other myelin proteins studied to date. S u p p o r t e d by the Veterans A d m i n i s t r a t i o n (F.J.S.). The technical assistance o f Benson F o n g , James Jetton, Michele Mass, Jean Quigley, C a r o l Pasnak, G a r y B a r b a r a s h a n d K a t h r y n Winchell is gratefully acknowledged. Carol P a s n a k was s u p p o r t e d by a grant from the N a t i o n a l Multiple Sclerosis Society. 1 Bornstein, M. B., Organotypic mammalian central and peripheral nervous tissue. In P. F. Kruse, Jr, and M. K. Patterson, Jr. (Eds.), Tissue Culture Methods and Applications, Academic Press, New York, N.Y., 1973, pp. 86-92. 2 Bornstein, M. B. and Appel, S. H., The application of tissue culture to the study of experimental 'allergic' encephalomyelitis: I. Patterns of demyelination, J. Neuropath. exp. NeuroL, 20 (1961) 141-157. 3 Bornstein, M. B. and Raine, C. S., Experimental allergic encephalomyelitis: antiserum inhibition of myelination in vitro, Lab. lnvest., 23 (1970) 536-542. 4 Dubois-Dalcq, M., Niedieck, B. and Buyse, M., Action of anti-cerebroside sera on myelinated tissue cultures, Path. Europ., 5 (1970) 331-347. 5 Fry, J. M., Weissbarth, S., Lehrer, G. M. and Bornstein, M. B., Cerebroside antibody inhibits sulfatide synthesis and myelination and demyelinates in cord tissue cultures, Science, 183 (1974) 540-542. 6 Hruby, S., Alvord, E. C., Jr. and Sell, F. J., Synthetic galactocerebrosides evoke myelinationinhibiting antibodies, Science, 195 (1977) 173-175. 7 Hughes, R. C., Membrane Glycoproteins: ,4 Review of Structure and Function, Butterworth, Woburn, Mass., 1976. 8 Kies, M. W., Driscoll, B. F., Seil, F. J. and Alvord, E. C., Jr., Myelination inhibition factor: dissociation from induction of experimental allergic encephalomyelitis, Science, 179 (1973) 689690. 9 Lebar, R., Boutry, J. M., Vincent, C. H., Robineaux, R. and Voisin, G. A., Studies on autoimmune encephalomyelitis in the guinea pig: II. An in vitro investigation of the nature, properties and specificity of the serum demyelinating factor, J. lmmunol., 116 (1976) 1439-1446. 10 Peterson, E. R., Crain, S. M. and Murray, M. R., Differentiation and prolonged maintenance of bioelectricallyactive spinal cord cultures (rat, chick and human), Z. Zellforsch., 66 (1965) 130154. 11 Poduslo, J. F., Quarles, R. H. and Brady, R. O., External labeling of galactose in surface membrane glycoproteins of the intact myelin sheath, J. biol. Chem., 251 (1976) 153-158. 12 Quarles, R. H., Glycoproteins in myelin and myelin related membranes. In R. U. Margolis and R. K. Margolis (Eds.), Complex Carbohydrates of the Nervous System, Plenum Press, New York, N.Y., 1979, pp. 209-233. 13 Quarles, R. H., Johnson, D., Brady, R. O. and Sternberger, N. H., Preparation and characterization of antisera to the myelin-associated glycoprotein, Neurochem. Res., in press. 14 Seil, F. J., Cerebellum in tissue culture. In D. M. Schneider (Ed.), Reviews of Neuroscience, Vol. 4, Raven Press, New York, N.Y., 1979, pp. 105-177. 15 Seil, F. J. and Agrawal, H. C., Myelin-proteolipid protein does not induce demyelinating or myelination-inhibiting antibodies, Brain Research, 194 (1980) 273-277.
475 16 Seil, F. J., Falk, G. A., Kies, M. W. and Alvord, E. C., Jr., The in vitro demyelinatingactivity of sera from guinea pigs sensitized with whole CNS and with purified encephalitogen, Exp. Neurol., 22 (1968) 545-555. 17 Seil, F. J., Kies, M. W., Agrawal, H. C., Quarles, R. H. and Brady, R. O., Myelin proteins dissociated from induction of antimyelin antibodies. In E. Giacobini, A. Vernadakis and A. Shahar (Eds.), Tissue Culture in Neurobiology, Raven Press, New York, N.Y., 1980, pp. 477--488. 18 Seil, F. J., Smith, M. E , Leiman, A. L. and Kelly, J. M., Myelination inhibiting and neuroelectric blocking factors in experimental allergic encephalomyelitis, Science, 187 (1975) 951-953. 19 Sternberger, N. H., Quarles, R. H., Itoyama, Y. and Webster, H. F., Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 1510-1514.