Immunology, 1975, 28, 213.
Role of Antibodies to Galactocerebroside in Experimental Allergic Encephalomyelitis R. A. C. HUGHES* AND S. LEIBOWITZ Department of Pathology, Guy's Hospital Medical School, London
(Received 13th April 1974; acceptedfor publication 4th July 1974) Summary. The possible role of antibodies to galactocerebroside was investigated in AS rats with experimental allergic encephalomyelitis (EAE) induced by guineapig spinal cord. Passive immunization with rat or rabbit antiserum to galactocerebroside did not protect rats from EAE. Active immunization with galactocerebroside before the encephalitogenic challenge produced antibody to galactocerebroside but also failed to protect rats from EAE. These experiments did not support previous suggestions that antibodies to galactocerebroside are protective.
INTRODUCTION The ability of rat antiserum to guinea-pig spinal cord to protect rats from EAE was demonstrated by Paterson and Harwin (1963) and confirmed by Hughes (1974). Paterson (1971) has suggested that the antibodies responsible for this protection are directed against galactocerebroside. Niedieck and Kuck (1967) provided some evidence in favour of this hypothesis by showing that pre-immunization with galactocerebroside would protect rabbits from developing EAE in response to a later injection of rabbit spinal cord. The present experiments were designed to see whether the protective properties of antiserum to guinea-pig spinal cord were shared by antiserum to galactocerebroside. MATERIALS AND METHODS Antiserum to galactocerebroside was raised in twenty-five outbred Wistar rats (ASH/W from Scientific Products Farm Ltd, Manston Road, Margate, Kent). Galactocerebroside was dissolved in absolute ethanol (1 volume) and diluted with saline (2 volumes) containing bovine albumin: the resultant solution was emulsified with an equal volume of complete adjuvant made up from incomplete adjuvant (Difco) and Mycobacterium tuberculosis H37RA (Difco). Each animal received two 0-05-ml injections into the pads of each hind foot, containing 10 mg each of galactocerebroside, bovine albumin and M. tuberculosis. Twenty-five control rats were immunized with a similar emulsion without galactocerebroside. Fourteen days later the sera were harvested by aortic puncture under ether anaesthesia and pooled aseptically. Inbred AS rats were treated with ten 2*0-ml intraperitoneal injections of this antiserum starting 4 hours before immunization, continuing on alternate days and finishing on day 18. The encephalitogenic emulsion consisted of 1 mg of guinea-pig spinal cord in complete adjuvant containing 5 mg M. tuberculosis, * Present address and correspondence: Maida Vale Hospital, London W9.
213
R. A. C. Hughes and S. Leibowitz 214 prepared as described by Hughes and Stedronska (1973). The animals were killed on day 19 and scored by a blind observer for clinical and histological disease as described by Hughes (1974). Rabbit antiserum to galactocerebroside was prepared by injecting rabbits with a similar emulsion of adjuvant containing 2 5 mg of galactocerebroside, 2-5 mg of bovine albumin and 0-625 mg of M. butyricum per animal (0 1 ml into each hind foot and three 0d1-ml injections into the back and the neck). The rabbits were bled from their ear veins between 7 and 21 days after immunization. The control sera consisted either of normal rabbit serum, or of antiserum to an emulsion of bovine albumin in complete adjuvant without galactocerebroside. In experiments 1 and 2 (Table 2) seven 2-0-ml intraperitoneal injections were given on alternate days from days 0-12; in experiment 3 (Table 2) six injections were given on alternate days from days 0-10. In all three experiments the encephalitogenic emulsion consisted of 15 mg of guinea-pig spinal cord and 5 0 mg of M. tuberculosis. In experiments 4 and 5 (Table 3) inbred AS rats were pre-immunized with 1-0 mg each of galactocerebroside, bovine albumin and M. tuberculosis as described above. Controls were pre-immunized with a similar emulsion without galactocerebroside. Fourteen days later at the time of the peak of the response of the complement-fixing antibodies to galactocerebroside, both groups were immunized with guinea-pig spinal cord (15 mg in experiment 4 and 115 mg in experiment 5) together with 5 mg of M. tuberculosis. Complement-fixation tests were performed with galactocerebroside as antigen and cholesterol and lecithin as auxiliary lipids as described by Hughes and Stedronska (1973). TABLE 1 EFFECT OF RAT ANTISERUM TO GALACTOCEREBROSIDE ON EAE
Number of rats with each grade of histological disease
Number of rats with each grade of clinical disease
Treatment Antiserum to
galactocerebroside
Antiserum to bovine albumin No treatment
0
+
++
+++
0
+
0
0
2
3*
0
2 0
0 2
2 1
1 2
1 0
Total
++
+++
1*
0
4
5
1 0
0 0
3 5
5 5
* Including one animal which died following typical signs of EAE.
RESULTS Prolonged treatment with rat antiserum to galactocerebroside produced no striking change in the severity of EAE by comparison with two control groups (Table 1). Treatment with rabbit antiserum to galactocerebroside did not produce any consistent change in the severity of EAE (Table 2). The apparent specific protection against clinical disease conferred by this antiserum compared with normal rabbit serum in experiment 1 was not confirmed in experiments 2 and 3 in which the control sera consisted of antisera to bovine albumin. The complement-fixation titre to galactocerebroside
215
Galactocerebroside Antibodies in EAE
8
8192
I
4096
0
2048
0
1024
*
0
512
0
0
co
256
@
0
00
*C
28
9
64
.-
332
o 0O *
16_
o
8 KX 0~~~~~~~~~ 4 aC
Ea,
E o
2 _
Role of Antibodies to Galactocerebroside in Experimental Allergic Encephalomyelitis R. A. C. HUGHES* AND S. LEIBOWITZ Department of Pathology, Guy's Hospital Medical School, London
(Received 13th April 1974; acceptedfor publication 4th July 1974) Summary. The possible role of antibodies to galactocerebroside was investigated in AS rats with experimental allergic encephalomyelitis (EAE) induced by guineapig spinal cord. Passive immunization with rat or rabbit antiserum to galactocerebroside did not protect rats from EAE. Active immunization with galactocerebroside before the encephalitogenic challenge produced antibody to galactocerebroside but also failed to protect rats from EAE. These experiments did not support previous suggestions that antibodies to galactocerebroside are protective.
INTRODUCTION The ability of rat antiserum to guinea-pig spinal cord to protect rats from EAE was demonstrated by Paterson and Harwin (1963) and confirmed by Hughes (1974). Paterson (1971) has suggested that the antibodies responsible for this protection are directed against galactocerebroside. Niedieck and Kuck (1967) provided some evidence in favour of this hypothesis by showing that pre-immunization with galactocerebroside would protect rabbits from developing EAE in response to a later injection of rabbit spinal cord. The present experiments were designed to see whether the protective properties of antiserum to guinea-pig spinal cord were shared by antiserum to galactocerebroside. MATERIALS AND METHODS Antiserum to galactocerebroside was raised in twenty-five outbred Wistar rats (ASH/W from Scientific Products Farm Ltd, Manston Road, Margate, Kent). Galactocerebroside was dissolved in absolute ethanol (1 volume) and diluted with saline (2 volumes) containing bovine albumin: the resultant solution was emulsified with an equal volume of complete adjuvant made up from incomplete adjuvant (Difco) and Mycobacterium tuberculosis H37RA (Difco). Each animal received two 0-05-ml injections into the pads of each hind foot, containing 10 mg each of galactocerebroside, bovine albumin and M. tuberculosis. Twenty-five control rats were immunized with a similar emulsion without galactocerebroside. Fourteen days later the sera were harvested by aortic puncture under ether anaesthesia and pooled aseptically. Inbred AS rats were treated with ten 2*0-ml intraperitoneal injections of this antiserum starting 4 hours before immunization, continuing on alternate days and finishing on day 18. The encephalitogenic emulsion consisted of 1 mg of guinea-pig spinal cord in complete adjuvant containing 5 mg M. tuberculosis, * Present address and correspondence: Maida Vale Hospital, London W9.
213
R. A. C. Hughes and S. Leibowitz 214 prepared as described by Hughes and Stedronska (1973). The animals were killed on day 19 and scored by a blind observer for clinical and histological disease as described by Hughes (1974). Rabbit antiserum to galactocerebroside was prepared by injecting rabbits with a similar emulsion of adjuvant containing 2 5 mg of galactocerebroside, 2-5 mg of bovine albumin and 0-625 mg of M. butyricum per animal (0 1 ml into each hind foot and three 0d1-ml injections into the back and the neck). The rabbits were bled from their ear veins between 7 and 21 days after immunization. The control sera consisted either of normal rabbit serum, or of antiserum to an emulsion of bovine albumin in complete adjuvant without galactocerebroside. In experiments 1 and 2 (Table 2) seven 2-0-ml intraperitoneal injections were given on alternate days from days 0-12; in experiment 3 (Table 2) six injections were given on alternate days from days 0-10. In all three experiments the encephalitogenic emulsion consisted of 15 mg of guinea-pig spinal cord and 5 0 mg of M. tuberculosis. In experiments 4 and 5 (Table 3) inbred AS rats were pre-immunized with 1-0 mg each of galactocerebroside, bovine albumin and M. tuberculosis as described above. Controls were pre-immunized with a similar emulsion without galactocerebroside. Fourteen days later at the time of the peak of the response of the complement-fixing antibodies to galactocerebroside, both groups were immunized with guinea-pig spinal cord (15 mg in experiment 4 and 115 mg in experiment 5) together with 5 mg of M. tuberculosis. Complement-fixation tests were performed with galactocerebroside as antigen and cholesterol and lecithin as auxiliary lipids as described by Hughes and Stedronska (1973). TABLE 1 EFFECT OF RAT ANTISERUM TO GALACTOCEREBROSIDE ON EAE
Number of rats with each grade of histological disease
Number of rats with each grade of clinical disease
Treatment Antiserum to
galactocerebroside
Antiserum to bovine albumin No treatment
0
+
++
+++
0
+
0
0
2
3*
0
2 0
0 2
2 1
1 2
1 0
Total
++
+++
1*
0
4
5
1 0
0 0
3 5
5 5
* Including one animal which died following typical signs of EAE.
RESULTS Prolonged treatment with rat antiserum to galactocerebroside produced no striking change in the severity of EAE by comparison with two control groups (Table 1). Treatment with rabbit antiserum to galactocerebroside did not produce any consistent change in the severity of EAE (Table 2). The apparent specific protection against clinical disease conferred by this antiserum compared with normal rabbit serum in experiment 1 was not confirmed in experiments 2 and 3 in which the control sera consisted of antisera to bovine albumin. The complement-fixation titre to galactocerebroside
215
Galactocerebroside Antibodies in EAE
8
8192
I
4096
0
2048
0
1024
*
0
512
0
0
co
256
@
0
00
*C
28
9
64
.-
332
o 0O *
16_
o
8 KX 0~~~~~~~~~ 4 aC
Ea,
E o
2 _
