lmmunochemistry, 1977,Vol. 14. pp. 775 779. PergamonPress. Printedin Great Britain
I M M U N O G E N I C I T Y OF SULFATIDE* B. Z A L C t , C. JACQUE:~, N. S. R A D I N a n d P. D U P O U E Y Laboratoire de Neurochimie I.N.S.E.R.M.U.134, H6pital de la Satp~tri6re 75634 Paris Cddex 13. France; Mental Health Research Institute, University of Michigan, Ann Arbor, MI 48109, U.S.A.; and Laboratoire de Biochimie des Antig6nes, Institut Pasteur, 75015 Paris. France. (First received 18 March 1977; in revisedJbrm 3 June 1977) Abstract--The present study deals with the immunogenicity of sulfatide. Despite its acidic character, we succeeded in raising high titer antibodies. The most effective immunization procedure involved repeated intravenous injections of a mixture of sulfatid~lecithin~cholesterol-methylated bovine serum albumin (1:4: 10: 10). The respective role of the protein carrier and of the auxiliary lipids in inducing the antigencity was studied. The antibodies against sulfatide were purified by an immunoadsorption method and were characterized. They fixed complement and agglutinated. Nitrocatechol sulfate could inhibit the antibody reaction completely at high concentrations, while galactose and methyl galactoside had only a weak effect.
INTRODUCTION Sulfatide (3-sulfogalactopyranosyl ceramide) is located mainly in brain and, in h u m a n s accounts for a b o u t 5% of white matter lipids (Norton et al., 1966). It has been implicated in sodium transport and ATPase activity (Karlsson et al., 1974) in morphinebinding sites in the brain (Cho et al., 1976), in myelinogenesis (Fry et al., 1974) a n d in the h u m a n genetic disorder, metachromatic leukodystrophy (Austin, 1957). A specific antibody against sulfatide should find use in studying i m p o r t a n t m e m b r a n e problems. Only two attempts have been made to produce such antibodies: Niedieck (1967) a n d H a k o m o r i (1974). In both attempts, rabbits were injected with a mixture of sulfatide and BSA~ in CFA. Niedieck could not detect any antibodies while Hakomori, in a brief communication, reported a titer of 50 as assayed by CF. We present here a study of parameters involved in the antigenicity of sulfatide in rabbits. The conditions of producing a high titer, a purification procedure for the antibodies a n d specificity characterization of the antibodies are reported. MATERIALS AND METHODS
Lipid preparations Galactosylceramide and sulfatide were isolated from an alkaline hydrolysate of a brain lipid extract, by liquid
chromatography on a column of silica gel 60 (Merck) and again on Unisil (Hajra & Radin, 1963). Glucosylceramide was a generous gift of J. Polonovski (H6pital SaintAntoine, Paris). Lecithin was prepared from egg-yolk (Faure, 1950). The purity of these products was controlled by thin-layer chromatography on silica gel plates (Merck PF 254). Immune sera Immunization by i.v. injection. The antigenic suspensions were prepared following the procedure of Coulon-Morelec et al. (1970). Ethanolic solutions of cholesterol and lecithin (when used) and sulfatide were added in that order to distilled water under strong stirring. Most of the ethanol was removed by partial evaporation under vacuum. The protein carrier (BSA or methylated BSA) dissolved in distilled water was "then added. After one night at room temperature, the Suspension was centrifuged. The pellet, resuspended in distilled water, was distributed in l-ml portions in sealed ampules and kept at 4°C. Each rabbit received the contents of one ampule intravenously three times a week for three weeks and a tenth one after one week's rest. Antibody formation was followed by weekly trial bleedings starting seven days after the first injection. A larger bleeding was made when the antibody titer was highest, that is, on the 21st day. All sera were sterilized by Millipore filtration and divided in 0.5-ml or l-ml portions and kept at 4°C in sealed ampules. Four groups of five rabbits each were immunized. Control animals were injected with the same mixtures lacking the hapten. Immunization hy i.d. injection. Sulfatide (2.3 mg) was dispersed by sonication in 0.5 ml of PBS containing 2.3 mg of methylated BSA (Sigma). The mixture was then emulsified in 0.5 ml of FA containing 2.3 mg of heat-killed M. butyricum (Difco). The emulsion was injected by the procedure of Vaitukaitis et al. (1971) which involves 40~50 i.d. injections into the back, and 100pl in the hind feet. Crude Bordetella pertussis vaccine (0.5 ml) (generous gift of Dr. R. J. Hosley, Eli Lilly & Co.) was also injected subcutaneously on the same day. The four rabbits thus injected were given a booster injection 45 days later in the same way, except that the FA was incomplete and the pertussis vaccine was omitted. Another group of five rabbits was treated similarly, but the carrier protein and the pertussis vaccine were omitted. All of the emulsion was injected into the four foot pads. No booster injection was given. Sera of animals injected with CFA without the hapten were used as controls.
* Preliminary results have been presented before the 10th Meeting of the IUB, Hamburg 1976 (Zalc et al., 1976). t "Stagiaire de Recherche 5. I'I.N.S.E.R.M." to whom proofs should be sent. Part of this investigation was carried out while he was a postdoctoral fellow of the Minist6re des Affaires Etrang6res in the Mental Health Research Institute, University of Michigan. Present address: Laboratoire de Neurochimie I.N.S.E.R.M.U.134, H6pital de la Salp~tri6re, 75634 Paris, C6dex 13, France. "Charg6 de Recherche 5. I'I.N.S.E.R.M." §Abbreviations used: BSA, bovine serum albumin; CFA, Freund's complete adjuvant; FA, Freund's adjuvant; PBS, phosphate-buffered saline, pH 7.4; CF, complement fixation; Agg, passive agglutination; i.d., intradermal; i.v., intravenous. 775
776
B. ZALC, C. JACQUE, N. S. RADIN and P. DUPOUEY
Preparation of pure antibodies Coulon-Morelec's procedure (1972) was used for the isolation of anti-sulfatide antibodies starting from immune sera. The immunoadsorbent was prepared by adsorption of 2 mg of the hapten on 70 mg of cholesterol particles dispersed in a final volume of 30 ml of saline. The immunoadsorbent was centrifuged and the pellet resuspended in 2.5 ml of the antiserum with stirring for 30 min at room temperature. After centrifugation the supernatant (SI) was kept as a test for antibody absorption and the immunoadsorbent was washed three times with 2.5 ml of saline. The washings were pooled (SII) and kept together with (SI) at -20°C. The specific antibodies were then released from the immunoadsorbent by stirring 15 rain at 4°C in 2.5 ml of a NaC1-BaC12 solution (2.5 and 0.01 M, respectively). The immunoadsorbent was separated from the pure antibodies by dissolving the former in ether containing 0.5°,~, lecithin. The antibodies in the aqueous phase were then dialyzed against saline at 4"C for 48 hr.
Serological techniques The CF technique has been detailed previously (Dupouey et al., 1972). The fixation is carried out in alveolated Plexiglass plates, using 6 H 50 units of complement at 4°C, overnight. The formula of hapten auxiliary lipids chosen is sulfatide:lecithin: cholesterol--0.5 : 0.5 : 9 (mg/ml of ethanol). In preliminary experiments, dilutions varying from 1/25 to 1/800 of this alcoholic solution in Mayer's buffer for CF (Mayer, 1971) have shown that the best specificity is observed for 1/100 or 1/200. The latter has been used throughout this work. This preparation was sometimes found to be anti-complementary. That effect was avoided by adding 230 of normal rabbit serum in the dilution buffer. Control experiments using lecithin:cholesterol 1:9 (w/w) at the same dilution were also run. Bovine myelin for the CF test was prepared as described previously (Dupouey et al., 1976). In order to double-check our antisera, we have also used the passive agglutination of cholesterol microcrystals sensitized with the hapten (Kline reaction). The antigenic suspension was prepared by dropwise addition of 0.9 ml of a l°i, alcoholic solution of cholesterol to 0.85 ml of distilled water. A solution of stilfatide (0.2 rag) and lecithin (1.5 rag) in 0.1 ml of ethanol was added to the mixture with vigorous swirling. The final volume was brought to 3.4 ml with 0.15 M NaCI. The final micellar suspension was then gently homogenized and allowed to remain 20min
at room temperature. The reaction was performed on glass plates by adding 16 #1 of the suspension to 50 #1 of increasing dilutions of the antisera. The plates were then submitted to a horizontal rotating movement for 4 rain. The extent of agglutination was estimated under a microscope on a scale ranging from + to 4 +. The test was used for the inhibition experiments by pre-incubating 34 #1 of the diluted antisera for 4 min with 16 ill of compounds related to sulfatide. When the test was used to check specificity of the antibodies, cerebrosides were substituted for sulfatide in preparing the antigenic suspension. RESULTS
Role of auxiliary substances in inducin9 the immuno,qenicity of sulfatide The best results were obtained with animals injected i.v. with methylated BSA, lecithin a n d cholesterol as the auxiliary substances (group c). Both assay procedures gave good titers, with passive agglutination showing less sensitivity (Table 1). An identical group of rabbits, for whom methylated BSA was replaced by BSA, yielded no detectable antibodies. The appearance of antibody in sera of animals immunized with mixture (c) can be followed on Fig. 1. The peak of antibody production was reached after nine injections. All animals injected i.v. with methylated BSA, cholesterol a n d sulfatide (group d) raised low titer antibodies. As with the lecithin-containing mixture, loss of antigenicity was produced by substituting BSA for methylated BSA. In the intradermal series of injections, three rabbits of group (a) and two of group (b) responded positively. Here too the use of methylated BSA resulted in an increased tiler. Pre-immunization sera were negative in all the tests, as were sera obtained from rabbits injected with the same immunization mixtures lacking the hapten.
Cross-reactivity test results N o n e of the sera reacted with glucocerebroside under the test conditions. In the case of group (a),
Table 1. Tilers of antisera obtained by different immunization procedures with sulfatide Sulfatide injected under following conditions Auxiliary Substances
Series
Route
(a)
i.d.
CFA alone
(b)
i.d.
(c)
i.v.
Methylated BSA + CFA Methylated BSA + Lecithin + Cholesterol Methylated BSA + Cholesterol
(d)
i.v.
Rabbit number (in each series) l
2
3
16 0 64 0
8 0 16 0
0 0 0 0
128 128 16 64 8 4 0 0
4
5
Test used for titration
0 0 0 0
8 4
CF Agg CF Agg
64 256 32 128 16 8 4 0
8 2 2 0
CF Agg CF Agg
(a) Hapten in CFA (2 mg and 2 mg mycobacteria). (b) Hapten: methylated BSA in CFA (2.3 mg, 2.3 mg and 2.3 mg mycobacteria) only four animals immunized. (c) Hapten:cholesterol:lecithin:methylated BSA (1:10:4:10). (d) Hapten : cholesterol :methylated BSA (2:10: 40).
777
Immunogenicity of Sulfatide
,I, ,I, ,I, 2.4. 2.1 L. I.B "1.5 ""6 ~1.2 0.9 0.6 0.3
L
~J"
0
5
i
I0
x
X
x
X
I
I
I
l
15 20 Days
25
30
35
I
Fig. 1. Changes in the titer of anti-sulfatide antibodies in 5 rabbits injected i.v. with the antigenic preparation: sulfatide-cholesterol-lecithin-methylated BSA (1:10:4:10). Rabbit number 1 ( x x ), 2 (On-O), 3 ( A - - A ) , 4 (~------e) and 5 (x x); days of injection are indicated by arrows. Sera were assayed by the CF test. methyl-fl-glucopyranoside) had any effect on the absorption of anti-sulfatide antibodies, fl-D-galactose, lactose and 1-o-methyl-fl-D-galactopyranoside were found to be slightly more efficient, especially the methyl glycoside when used at the highest concentration tested. Only p-nitrocatechol sulfate, an artificial substrate of arylsulfatases A and B, could bind up to 100% of the antibodies at a concentration of 100 mM. In control experiments it was demonstrated that nitrocatechol sulfate had no inhibitory effect on anti-galactosylceramide antisera in the same test.
none of the sera reacted with galactocerebroside either, except for rabbit 5a. With this animal, the titer in both tests was the same as the titer obtained with sulfatide. The two rabbits of group (b) reacting with sulfatide did not react with galactocerebroside, but they did react slightly with myelin. The animals injected i.v. yielded positive reactions with galactosylceramide in the C F test. In group (d) rabbits, the titer was equal to that obtained with sulfatide. Inclusion of lecithin in the immunizing mixture (group c) reduced considerably the cross-reactivity with galactocerebroside. Purification of the serum from rabbit 4c by immunoadsorption (Table 2) resulted in a yield of 50% of the original titratable sulfatide antibodies. Part or all of the missing antibodies were found in the original supernatant liquid (SI) and in the wash liquids (SII). The pure antibody solution did not react anymore with methylated BSA but still fixed complement with galactosylceramide and myelin. The control mixture, lecithin-cholesterol (1:9) did not react with any of the serum preparations.
DISCUSSION The study of glycolipid immunology has been hampered by difficulties in producing antibodies and in detecting antibodies in vitro. These problems have been discussed in detail in the excellent review by Rapport several years ago (Rapport & Graf, 1969). Glycolipids are haptens. In order to be immunogenic, they need to be complexed first to a carrier and then either to an adjuvant or to auxiliary lipids. As glycolipids are hydrophobic, auxiliary lipids are also needed to run sensitive and specific in vitro reactions with the corresponding antibodies. In the present study, we have found that when sulfatide, emulsified in CFA, was injected intradermally without protein carrier (series a), our rabbits yield
Specific inhibition of the passive agglutination test We tested eight potential inhibitors at various concentrations (Fig. 2). Xylose was chosen as reference for having no inhibitory power. None of the three glucose derivatives tested (fl-D-glucose, cellobiose, I-o-
Table 2. Comparison of the reactivity of anti-sulfatide antiserum and pure antibodies Titers observed with the following antigens (in the CF test) Fractions tested Antisulfatide antiserum SP SIIb Pure antisulfatide antibodies
Sulfatide
Methylated GalactosylBSA ceramide
Glucosylceramide
Myelin
256
64
32
0
32
8 4 128
32 8 0
2 2 16
0 0 0
0 0 16
a SI is the first supernatant after absorption of antibodies on the immunoadsorbent. b SII is the fraction obtained by pooling the three washings before elution.
778
B. ZALC, C. JACQUE, N. S. RADIN and P. DUPOUEY
1.8
1.5
.....
:::.: p y r ' o f l o | i Oe
1.2
"6 0.9 0
0.6
0.3 A ~
50
I00
L
L
150
200
Concentrotion of inhibitors odded in mM
Fig. 2. Inhibition of anti-sulfatide antibodies. The stippled area represents the inhibitory range of all sugars tested: the upper line corresponds to the non-inhibitory compounds; the lower line reflects the inhibition power of methyl-/~-D-galactopyranoside. The effect of galactose (×) and lactose (©) is in between. The effect of p-nitrocatechol sulfate is represented by (~ ~). no antibody. Thus it behaves differently from galactosylceramide. Czlonkowska reported that galactocerebroside injected following the same procedure can induce strong antibody formation (titer 2560) in rats (Czlonkowska & Leibowitz, 1974). We have confirmed this result in rabbits (Dupouey, unpublished data). Czlonkowska explained this phenomenon by a combination of galactocerebroside with the mycobacteria, which then function as the carrier. The binding of glactosylceramide to the mycobacteria must occur by hydrophobic forces. Sulfatide, being much more polar than galactosylceramide, may bind much more poorly to the bacteria. In addition, the sulfatide (which has detergent-like properties) might be attracted to the interface between the mineral oil of FA and the water in the immunizing emulsion. The addition of methylated BSA to the emulsion of sulfatide in CFA rendered the mixture immunogenic as shown by group (b). Our results with this immunogenic preparation are very close to those found by Hakomori (1974) with a similar mixture, with respect to titer and non-reactivity with galactocerebroside. The other groups of animals were immunized intravenously with a mixture of hapten-prorein carrier and auxiliary lipids. Thus we were able to study two types of factors: firstly the nature of the carrier, secondly the influence of the auxiliary lipids, both playing a role in the conversion of a lipidic hapten into an immunogenic antigen. Methylated BSA was far superior to BSA and lecithin proved markedly helpful. Methylated BSA has already been used as carrier with other acidic lipids, ganglioside (Naiki et al., 1974) cardiolipin and phosphatidyl inositol (Kataoka & Nogima, 1970). The superiority of methylated BSA is due to the conjunction of two of its properties: its basicity and its hydrophobicity. Both characteristics increase its binding
with acidic lipid haptens and thus enhance its carrier function. In addition the methylated BSA may protect sulfatide from attack by the widely prevalent arylsulfatase A, for which sulfatide is the natural substrate (Stinshoff & Jatzkewitz, 1975). Perhaps more interesting is the role played by lecithin. The poor immunogenicity of the mixture sulfatide-cholesterol carrier can be explained by an incorrect hapten-carrier ratio due to the acidic character of the hapten, since in the case of galactosylceramide this formula is immunogenic (Dupouey, 1976). The addition of lecithin has the following consequences: it decreases the binding of the protein to the cholesterol particles by creating a hydrated layer around the cholesterol; and it enhances the haptenprotein association (M. J. Coulon-Morelec personal communication). The proposed intravenous immunization procedure is advantageous not only because of the intense antibody response, but also because it provides a means to obtain antibodies using a mixture where, besides the hapten, the only antigenic component is well defined (i.e. methylated BSA). Cholesterol and lecithin are indeed not immunogenic while administered following this intravenous procedure, as shown by our controls. There is an additional advantage of making CFA unnecessary: an acylated trehalose 2-sulfate has been desqribed as constituent of mycobacteria membranes (Goren, 1972). If this lipid is antigenic, the antibodies thus produced might contaminate our pure anti-sulfatide antibodies. The finding that even pure antibodies still slightly cross-reacted with galactosylceramide is not in favor of a partial hydrolysis of sulfatide, with formation of galactosylceramide. But this cross-reactivity, as well as the partial inhibitory power of methyl fl-Dgalactopyranosyl, suggests the intervention of the gly-
Immunogenicity of Sulfatide cosidic bond in the antigenic determinant of sulfatide. This is not as potent as in the case of galactosylceramide as ascertained by Niedieck (1975) and by ourselves (Zalc, Coulon-Morelec and Dupouey, in preparation). Of much greater interest seems to be the hydroxyl in the C4 position. Its importance is obvious by the absence of the slightest cross-reaction with glucosylceramide and by the absence of inhibition observed with glucose, cellobiose or even methyl fl-Dglucopyranoside. The total inhibition caused by p-nitrocatechol sulfate, although at a high concentration, even emphasizes the role of this C4 hydroxyl group and brings to evidence the importance of the neighbour sulfate group in the C3 position. The important role played by the location of this sulfate group has already been suggested by Hakomori's data when comparing the cross-reactivity of native sulfatide with a synthetic C6-sulfate cerebroside (Hakomori, 1974). It is possible that other lipid sulfates, such as sterol sulfate or seminolipid, could react, at least in vitro, with anti-sulfatide antibodies.
Acknowledoements--The author wish to thank Ms. D. Gomez for excellent technical help and Ms. J. Marechal for preparing the immunization mixtures. They are also greatly indebted to Dr. M. J. Coulon-Morelec for her invaluable advice. This work was supported by grants from D.R.M.E., conv. 76.34.041 and the U.S. Public Health Service, No. NS 03192. REFERENCES
Austin J. H. (1957) Neurology 7, 716.
779
Cho T. M., Cho J. S. & Loh H. H. (1976) LiJe Sci. 18, 231. Coulon-Morelec M. J., Faure M. & Marechal J. (1970) Ann. Inst. Pasteur, Paris 119, 17. Coulon-Morelec M. J. (1972) Ann. Inst. Pasteur, Paris 123, 619. Czlonkowska A. & Leibowitz L. (1974) Immunology 27, 1117. Dupouey P. (1972) J. lmmun. 109, 146. Dupouey P., Billecocq A. & Lefroit M. (1976) lmmunochemistry 13, 289. Faure M. (1950) Bull. Soc. Chim. Biol. 32, 503. Fry J. M., Lehrer G. M. & Bornstein M. B. (1974) J. Neurochem. 22, 459. Goren M. B. (1972) Bacteriol. Rev. 36, 33. Hajra A. K. and Radin N. S. (1963) J. Lipid Res. 4, 448. Hakomori S. I. (1974) J. Immun. 112, 424. Karlsson K. A., Samuelsson B. E. & Steen G. O. (1974) Eur. J. Biochem. 46, 243. Kataoka I. and Nogima S. (1970) J. lmmun. 105, 502. Mayer M. M. (1971) Experimental lmmunochemistry, 2nd edn. (Edited by Kabat E. A. & Mayer M. M.) p. 149. Thomas, Springfield, IL. Naiki M., Marcus D. M. & Ledeen R. (1974) J. Immun. 113, 84. Niedieck B. (1967) Z. Immun. Forsch. exp. Ther. 132, 139. Niedieck B. (1975) Prog. Allergy 18, 353. Norton W. I., Poduslo S. & Suzuki K. (1966) J. Neuropathol, exp. Neurol. 25, 582. Rapport M. M. & Graf L. (1969) Pro9. Allergy 13, 273. Stinshoff K. & Jatzkewitz H. (1975)Biochim. biophys. Acta 377, 126. Vaitukaitis J., Robbins J. B., Nieschlag E. & Ross G. I. (1971) J. clin. Endocr. 33, 988. Zalc B., Coulon-Morelec M. J. and Dupouey P. (in preparation). Zalc B., Jacque C., Radin N. S. & Dupouey P. (1976) Abstr. lOth Int. Cong. Biochem. Hamburg, 566.
I M M U N O G E N I C I T Y OF SULFATIDE* B. Z A L C t , C. JACQUE:~, N. S. R A D I N a n d P. D U P O U E Y Laboratoire de Neurochimie I.N.S.E.R.M.U.134, H6pital de la Satp~tri6re 75634 Paris Cddex 13. France; Mental Health Research Institute, University of Michigan, Ann Arbor, MI 48109, U.S.A.; and Laboratoire de Biochimie des Antig6nes, Institut Pasteur, 75015 Paris. France. (First received 18 March 1977; in revisedJbrm 3 June 1977) Abstract--The present study deals with the immunogenicity of sulfatide. Despite its acidic character, we succeeded in raising high titer antibodies. The most effective immunization procedure involved repeated intravenous injections of a mixture of sulfatid~lecithin~cholesterol-methylated bovine serum albumin (1:4: 10: 10). The respective role of the protein carrier and of the auxiliary lipids in inducing the antigencity was studied. The antibodies against sulfatide were purified by an immunoadsorption method and were characterized. They fixed complement and agglutinated. Nitrocatechol sulfate could inhibit the antibody reaction completely at high concentrations, while galactose and methyl galactoside had only a weak effect.
INTRODUCTION Sulfatide (3-sulfogalactopyranosyl ceramide) is located mainly in brain and, in h u m a n s accounts for a b o u t 5% of white matter lipids (Norton et al., 1966). It has been implicated in sodium transport and ATPase activity (Karlsson et al., 1974) in morphinebinding sites in the brain (Cho et al., 1976), in myelinogenesis (Fry et al., 1974) a n d in the h u m a n genetic disorder, metachromatic leukodystrophy (Austin, 1957). A specific antibody against sulfatide should find use in studying i m p o r t a n t m e m b r a n e problems. Only two attempts have been made to produce such antibodies: Niedieck (1967) a n d H a k o m o r i (1974). In both attempts, rabbits were injected with a mixture of sulfatide and BSA~ in CFA. Niedieck could not detect any antibodies while Hakomori, in a brief communication, reported a titer of 50 as assayed by CF. We present here a study of parameters involved in the antigenicity of sulfatide in rabbits. The conditions of producing a high titer, a purification procedure for the antibodies a n d specificity characterization of the antibodies are reported. MATERIALS AND METHODS
Lipid preparations Galactosylceramide and sulfatide were isolated from an alkaline hydrolysate of a brain lipid extract, by liquid
chromatography on a column of silica gel 60 (Merck) and again on Unisil (Hajra & Radin, 1963). Glucosylceramide was a generous gift of J. Polonovski (H6pital SaintAntoine, Paris). Lecithin was prepared from egg-yolk (Faure, 1950). The purity of these products was controlled by thin-layer chromatography on silica gel plates (Merck PF 254). Immune sera Immunization by i.v. injection. The antigenic suspensions were prepared following the procedure of Coulon-Morelec et al. (1970). Ethanolic solutions of cholesterol and lecithin (when used) and sulfatide were added in that order to distilled water under strong stirring. Most of the ethanol was removed by partial evaporation under vacuum. The protein carrier (BSA or methylated BSA) dissolved in distilled water was "then added. After one night at room temperature, the Suspension was centrifuged. The pellet, resuspended in distilled water, was distributed in l-ml portions in sealed ampules and kept at 4°C. Each rabbit received the contents of one ampule intravenously three times a week for three weeks and a tenth one after one week's rest. Antibody formation was followed by weekly trial bleedings starting seven days after the first injection. A larger bleeding was made when the antibody titer was highest, that is, on the 21st day. All sera were sterilized by Millipore filtration and divided in 0.5-ml or l-ml portions and kept at 4°C in sealed ampules. Four groups of five rabbits each were immunized. Control animals were injected with the same mixtures lacking the hapten. Immunization hy i.d. injection. Sulfatide (2.3 mg) was dispersed by sonication in 0.5 ml of PBS containing 2.3 mg of methylated BSA (Sigma). The mixture was then emulsified in 0.5 ml of FA containing 2.3 mg of heat-killed M. butyricum (Difco). The emulsion was injected by the procedure of Vaitukaitis et al. (1971) which involves 40~50 i.d. injections into the back, and 100pl in the hind feet. Crude Bordetella pertussis vaccine (0.5 ml) (generous gift of Dr. R. J. Hosley, Eli Lilly & Co.) was also injected subcutaneously on the same day. The four rabbits thus injected were given a booster injection 45 days later in the same way, except that the FA was incomplete and the pertussis vaccine was omitted. Another group of five rabbits was treated similarly, but the carrier protein and the pertussis vaccine were omitted. All of the emulsion was injected into the four foot pads. No booster injection was given. Sera of animals injected with CFA without the hapten were used as controls.
* Preliminary results have been presented before the 10th Meeting of the IUB, Hamburg 1976 (Zalc et al., 1976). t "Stagiaire de Recherche 5. I'I.N.S.E.R.M." to whom proofs should be sent. Part of this investigation was carried out while he was a postdoctoral fellow of the Minist6re des Affaires Etrang6res in the Mental Health Research Institute, University of Michigan. Present address: Laboratoire de Neurochimie I.N.S.E.R.M.U.134, H6pital de la Salp~tri6re, 75634 Paris, C6dex 13, France. "Charg6 de Recherche 5. I'I.N.S.E.R.M." §Abbreviations used: BSA, bovine serum albumin; CFA, Freund's complete adjuvant; FA, Freund's adjuvant; PBS, phosphate-buffered saline, pH 7.4; CF, complement fixation; Agg, passive agglutination; i.d., intradermal; i.v., intravenous. 775
776
B. ZALC, C. JACQUE, N. S. RADIN and P. DUPOUEY
Preparation of pure antibodies Coulon-Morelec's procedure (1972) was used for the isolation of anti-sulfatide antibodies starting from immune sera. The immunoadsorbent was prepared by adsorption of 2 mg of the hapten on 70 mg of cholesterol particles dispersed in a final volume of 30 ml of saline. The immunoadsorbent was centrifuged and the pellet resuspended in 2.5 ml of the antiserum with stirring for 30 min at room temperature. After centrifugation the supernatant (SI) was kept as a test for antibody absorption and the immunoadsorbent was washed three times with 2.5 ml of saline. The washings were pooled (SII) and kept together with (SI) at -20°C. The specific antibodies were then released from the immunoadsorbent by stirring 15 rain at 4°C in 2.5 ml of a NaC1-BaC12 solution (2.5 and 0.01 M, respectively). The immunoadsorbent was separated from the pure antibodies by dissolving the former in ether containing 0.5°,~, lecithin. The antibodies in the aqueous phase were then dialyzed against saline at 4"C for 48 hr.
Serological techniques The CF technique has been detailed previously (Dupouey et al., 1972). The fixation is carried out in alveolated Plexiglass plates, using 6 H 50 units of complement at 4°C, overnight. The formula of hapten auxiliary lipids chosen is sulfatide:lecithin: cholesterol--0.5 : 0.5 : 9 (mg/ml of ethanol). In preliminary experiments, dilutions varying from 1/25 to 1/800 of this alcoholic solution in Mayer's buffer for CF (Mayer, 1971) have shown that the best specificity is observed for 1/100 or 1/200. The latter has been used throughout this work. This preparation was sometimes found to be anti-complementary. That effect was avoided by adding 230 of normal rabbit serum in the dilution buffer. Control experiments using lecithin:cholesterol 1:9 (w/w) at the same dilution were also run. Bovine myelin for the CF test was prepared as described previously (Dupouey et al., 1976). In order to double-check our antisera, we have also used the passive agglutination of cholesterol microcrystals sensitized with the hapten (Kline reaction). The antigenic suspension was prepared by dropwise addition of 0.9 ml of a l°i, alcoholic solution of cholesterol to 0.85 ml of distilled water. A solution of stilfatide (0.2 rag) and lecithin (1.5 rag) in 0.1 ml of ethanol was added to the mixture with vigorous swirling. The final volume was brought to 3.4 ml with 0.15 M NaCI. The final micellar suspension was then gently homogenized and allowed to remain 20min
at room temperature. The reaction was performed on glass plates by adding 16 #1 of the suspension to 50 #1 of increasing dilutions of the antisera. The plates were then submitted to a horizontal rotating movement for 4 rain. The extent of agglutination was estimated under a microscope on a scale ranging from + to 4 +. The test was used for the inhibition experiments by pre-incubating 34 #1 of the diluted antisera for 4 min with 16 ill of compounds related to sulfatide. When the test was used to check specificity of the antibodies, cerebrosides were substituted for sulfatide in preparing the antigenic suspension. RESULTS
Role of auxiliary substances in inducin9 the immuno,qenicity of sulfatide The best results were obtained with animals injected i.v. with methylated BSA, lecithin a n d cholesterol as the auxiliary substances (group c). Both assay procedures gave good titers, with passive agglutination showing less sensitivity (Table 1). An identical group of rabbits, for whom methylated BSA was replaced by BSA, yielded no detectable antibodies. The appearance of antibody in sera of animals immunized with mixture (c) can be followed on Fig. 1. The peak of antibody production was reached after nine injections. All animals injected i.v. with methylated BSA, cholesterol a n d sulfatide (group d) raised low titer antibodies. As with the lecithin-containing mixture, loss of antigenicity was produced by substituting BSA for methylated BSA. In the intradermal series of injections, three rabbits of group (a) and two of group (b) responded positively. Here too the use of methylated BSA resulted in an increased tiler. Pre-immunization sera were negative in all the tests, as were sera obtained from rabbits injected with the same immunization mixtures lacking the hapten.
Cross-reactivity test results N o n e of the sera reacted with glucocerebroside under the test conditions. In the case of group (a),
Table 1. Tilers of antisera obtained by different immunization procedures with sulfatide Sulfatide injected under following conditions Auxiliary Substances
Series
Route
(a)
i.d.
CFA alone
(b)
i.d.
(c)
i.v.
Methylated BSA + CFA Methylated BSA + Lecithin + Cholesterol Methylated BSA + Cholesterol
(d)
i.v.
Rabbit number (in each series) l
2
3
16 0 64 0
8 0 16 0
0 0 0 0
128 128 16 64 8 4 0 0
4
5
Test used for titration
0 0 0 0
8 4
CF Agg CF Agg
64 256 32 128 16 8 4 0
8 2 2 0
CF Agg CF Agg
(a) Hapten in CFA (2 mg and 2 mg mycobacteria). (b) Hapten: methylated BSA in CFA (2.3 mg, 2.3 mg and 2.3 mg mycobacteria) only four animals immunized. (c) Hapten:cholesterol:lecithin:methylated BSA (1:10:4:10). (d) Hapten : cholesterol :methylated BSA (2:10: 40).
777
Immunogenicity of Sulfatide
,I, ,I, ,I, 2.4. 2.1 L. I.B "1.5 ""6 ~1.2 0.9 0.6 0.3
L
~J"
0
5
i
I0
x
X
x
X
I
I
I
l
15 20 Days
25
30
35
I
Fig. 1. Changes in the titer of anti-sulfatide antibodies in 5 rabbits injected i.v. with the antigenic preparation: sulfatide-cholesterol-lecithin-methylated BSA (1:10:4:10). Rabbit number 1 ( x x ), 2 (On-O), 3 ( A - - A ) , 4 (~------e) and 5 (x x); days of injection are indicated by arrows. Sera were assayed by the CF test. methyl-fl-glucopyranoside) had any effect on the absorption of anti-sulfatide antibodies, fl-D-galactose, lactose and 1-o-methyl-fl-D-galactopyranoside were found to be slightly more efficient, especially the methyl glycoside when used at the highest concentration tested. Only p-nitrocatechol sulfate, an artificial substrate of arylsulfatases A and B, could bind up to 100% of the antibodies at a concentration of 100 mM. In control experiments it was demonstrated that nitrocatechol sulfate had no inhibitory effect on anti-galactosylceramide antisera in the same test.
none of the sera reacted with galactocerebroside either, except for rabbit 5a. With this animal, the titer in both tests was the same as the titer obtained with sulfatide. The two rabbits of group (b) reacting with sulfatide did not react with galactocerebroside, but they did react slightly with myelin. The animals injected i.v. yielded positive reactions with galactosylceramide in the C F test. In group (d) rabbits, the titer was equal to that obtained with sulfatide. Inclusion of lecithin in the immunizing mixture (group c) reduced considerably the cross-reactivity with galactocerebroside. Purification of the serum from rabbit 4c by immunoadsorption (Table 2) resulted in a yield of 50% of the original titratable sulfatide antibodies. Part or all of the missing antibodies were found in the original supernatant liquid (SI) and in the wash liquids (SII). The pure antibody solution did not react anymore with methylated BSA but still fixed complement with galactosylceramide and myelin. The control mixture, lecithin-cholesterol (1:9) did not react with any of the serum preparations.
DISCUSSION The study of glycolipid immunology has been hampered by difficulties in producing antibodies and in detecting antibodies in vitro. These problems have been discussed in detail in the excellent review by Rapport several years ago (Rapport & Graf, 1969). Glycolipids are haptens. In order to be immunogenic, they need to be complexed first to a carrier and then either to an adjuvant or to auxiliary lipids. As glycolipids are hydrophobic, auxiliary lipids are also needed to run sensitive and specific in vitro reactions with the corresponding antibodies. In the present study, we have found that when sulfatide, emulsified in CFA, was injected intradermally without protein carrier (series a), our rabbits yield
Specific inhibition of the passive agglutination test We tested eight potential inhibitors at various concentrations (Fig. 2). Xylose was chosen as reference for having no inhibitory power. None of the three glucose derivatives tested (fl-D-glucose, cellobiose, I-o-
Table 2. Comparison of the reactivity of anti-sulfatide antiserum and pure antibodies Titers observed with the following antigens (in the CF test) Fractions tested Antisulfatide antiserum SP SIIb Pure antisulfatide antibodies
Sulfatide
Methylated GalactosylBSA ceramide
Glucosylceramide
Myelin
256
64
32
0
32
8 4 128
32 8 0
2 2 16
0 0 0
0 0 16
a SI is the first supernatant after absorption of antibodies on the immunoadsorbent. b SII is the fraction obtained by pooling the three washings before elution.
778
B. ZALC, C. JACQUE, N. S. RADIN and P. DUPOUEY
1.8
1.5
.....
:::.: p y r ' o f l o | i Oe
1.2
"6 0.9 0
0.6
0.3 A ~
50
I00
L
L
150
200
Concentrotion of inhibitors odded in mM
Fig. 2. Inhibition of anti-sulfatide antibodies. The stippled area represents the inhibitory range of all sugars tested: the upper line corresponds to the non-inhibitory compounds; the lower line reflects the inhibition power of methyl-/~-D-galactopyranoside. The effect of galactose (×) and lactose (©) is in between. The effect of p-nitrocatechol sulfate is represented by (~ ~). no antibody. Thus it behaves differently from galactosylceramide. Czlonkowska reported that galactocerebroside injected following the same procedure can induce strong antibody formation (titer 2560) in rats (Czlonkowska & Leibowitz, 1974). We have confirmed this result in rabbits (Dupouey, unpublished data). Czlonkowska explained this phenomenon by a combination of galactocerebroside with the mycobacteria, which then function as the carrier. The binding of glactosylceramide to the mycobacteria must occur by hydrophobic forces. Sulfatide, being much more polar than galactosylceramide, may bind much more poorly to the bacteria. In addition, the sulfatide (which has detergent-like properties) might be attracted to the interface between the mineral oil of FA and the water in the immunizing emulsion. The addition of methylated BSA to the emulsion of sulfatide in CFA rendered the mixture immunogenic as shown by group (b). Our results with this immunogenic preparation are very close to those found by Hakomori (1974) with a similar mixture, with respect to titer and non-reactivity with galactocerebroside. The other groups of animals were immunized intravenously with a mixture of hapten-prorein carrier and auxiliary lipids. Thus we were able to study two types of factors: firstly the nature of the carrier, secondly the influence of the auxiliary lipids, both playing a role in the conversion of a lipidic hapten into an immunogenic antigen. Methylated BSA was far superior to BSA and lecithin proved markedly helpful. Methylated BSA has already been used as carrier with other acidic lipids, ganglioside (Naiki et al., 1974) cardiolipin and phosphatidyl inositol (Kataoka & Nogima, 1970). The superiority of methylated BSA is due to the conjunction of two of its properties: its basicity and its hydrophobicity. Both characteristics increase its binding
with acidic lipid haptens and thus enhance its carrier function. In addition the methylated BSA may protect sulfatide from attack by the widely prevalent arylsulfatase A, for which sulfatide is the natural substrate (Stinshoff & Jatzkewitz, 1975). Perhaps more interesting is the role played by lecithin. The poor immunogenicity of the mixture sulfatide-cholesterol carrier can be explained by an incorrect hapten-carrier ratio due to the acidic character of the hapten, since in the case of galactosylceramide this formula is immunogenic (Dupouey, 1976). The addition of lecithin has the following consequences: it decreases the binding of the protein to the cholesterol particles by creating a hydrated layer around the cholesterol; and it enhances the haptenprotein association (M. J. Coulon-Morelec personal communication). The proposed intravenous immunization procedure is advantageous not only because of the intense antibody response, but also because it provides a means to obtain antibodies using a mixture where, besides the hapten, the only antigenic component is well defined (i.e. methylated BSA). Cholesterol and lecithin are indeed not immunogenic while administered following this intravenous procedure, as shown by our controls. There is an additional advantage of making CFA unnecessary: an acylated trehalose 2-sulfate has been desqribed as constituent of mycobacteria membranes (Goren, 1972). If this lipid is antigenic, the antibodies thus produced might contaminate our pure anti-sulfatide antibodies. The finding that even pure antibodies still slightly cross-reacted with galactosylceramide is not in favor of a partial hydrolysis of sulfatide, with formation of galactosylceramide. But this cross-reactivity, as well as the partial inhibitory power of methyl fl-Dgalactopyranosyl, suggests the intervention of the gly-
Immunogenicity of Sulfatide cosidic bond in the antigenic determinant of sulfatide. This is not as potent as in the case of galactosylceramide as ascertained by Niedieck (1975) and by ourselves (Zalc, Coulon-Morelec and Dupouey, in preparation). Of much greater interest seems to be the hydroxyl in the C4 position. Its importance is obvious by the absence of the slightest cross-reaction with glucosylceramide and by the absence of inhibition observed with glucose, cellobiose or even methyl fl-Dglucopyranoside. The total inhibition caused by p-nitrocatechol sulfate, although at a high concentration, even emphasizes the role of this C4 hydroxyl group and brings to evidence the importance of the neighbour sulfate group in the C3 position. The important role played by the location of this sulfate group has already been suggested by Hakomori's data when comparing the cross-reactivity of native sulfatide with a synthetic C6-sulfate cerebroside (Hakomori, 1974). It is possible that other lipid sulfates, such as sterol sulfate or seminolipid, could react, at least in vitro, with anti-sulfatide antibodies.
Acknowledoements--The author wish to thank Ms. D. Gomez for excellent technical help and Ms. J. Marechal for preparing the immunization mixtures. They are also greatly indebted to Dr. M. J. Coulon-Morelec for her invaluable advice. This work was supported by grants from D.R.M.E., conv. 76.34.041 and the U.S. Public Health Service, No. NS 03192. REFERENCES
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779
Cho T. M., Cho J. S. & Loh H. H. (1976) LiJe Sci. 18, 231. Coulon-Morelec M. J., Faure M. & Marechal J. (1970) Ann. Inst. Pasteur, Paris 119, 17. Coulon-Morelec M. J. (1972) Ann. Inst. Pasteur, Paris 123, 619. Czlonkowska A. & Leibowitz L. (1974) Immunology 27, 1117. Dupouey P. (1972) J. lmmun. 109, 146. Dupouey P., Billecocq A. & Lefroit M. (1976) lmmunochemistry 13, 289. Faure M. (1950) Bull. Soc. Chim. Biol. 32, 503. Fry J. M., Lehrer G. M. & Bornstein M. B. (1974) J. Neurochem. 22, 459. Goren M. B. (1972) Bacteriol. Rev. 36, 33. Hajra A. K. and Radin N. S. (1963) J. Lipid Res. 4, 448. Hakomori S. I. (1974) J. Immun. 112, 424. Karlsson K. A., Samuelsson B. E. & Steen G. O. (1974) Eur. J. Biochem. 46, 243. Kataoka I. and Nogima S. (1970) J. lmmun. 105, 502. Mayer M. M. (1971) Experimental lmmunochemistry, 2nd edn. (Edited by Kabat E. A. & Mayer M. M.) p. 149. Thomas, Springfield, IL. Naiki M., Marcus D. M. & Ledeen R. (1974) J. Immun. 113, 84. Niedieck B. (1967) Z. Immun. Forsch. exp. Ther. 132, 139. Niedieck B. (1975) Prog. Allergy 18, 353. Norton W. I., Poduslo S. & Suzuki K. (1966) J. Neuropathol, exp. Neurol. 25, 582. Rapport M. M. & Graf L. (1969) Pro9. Allergy 13, 273. Stinshoff K. & Jatzkewitz H. (1975)Biochim. biophys. Acta 377, 126. Vaitukaitis J., Robbins J. B., Nieschlag E. & Ross G. I. (1971) J. clin. Endocr. 33, 988. Zalc B., Coulon-Morelec M. J. and Dupouey P. (in preparation). Zalc B., Jacque C., Radin N. S. & Dupouey P. (1976) Abstr. lOth Int. Cong. Biochem. Hamburg, 566.
