Antibodies Against Nicotinic Acetylcholine Receptor and Skeletal Muscle in Human and Experimental Myasthenia Gravis J. A. AARLI, C. MATTSSON & E. HEILBRONN Department of Neurology, School of Medicine, University of Bergen, Norway, and Division of Biochemistry, Department 4 (Foa 4 ) , National Defence Research Institute, Sundbyberg, Sweden
Aarli, J. A,, Mattsson, C. & Heilbronn, E. Antibodies Against Nicotinic Acetylcholine Receptor and Skeletal Muscle in Human and Experimental Myasthenia Gravis. Scand. J. Immunol. 4, 849-852, 1975. Using rocket immunoelectrophoresis and indirect haemagglutination tests, antibodies to nicotinic acetylcholine receptor (n-AChR) from Torpedo mawnorata were detected in sera from rabbits with experimental myasthenia but not in sera from patients with myasthenia gravis or in rabbit antisera to a partly purified skeletal muscle antigen. Antibodies to this muscle antigen were demonstrated in sera from patients with myasthenia gravis but not in sera from rabbits with experimental myasthenia. The results indicate that there is no immunological cross-reaction between the muscle antigen and n-AChR from Torpedo marrorata, and little or no cross-reactivity between these n-AChR antibodies and possible muscle n-AChR antibodies in myasthenia gravis patients.
J. A. Aarli, M.D., Department of Neurology, 5016 Haukeland sykehus, Bergen, Norway
Rabbits immunized with purified nicotinic acetylcholine receptor (n-AChR) from electric organs of Torpedo or Electroplax develop a neostigmine-sensitive muscular weakness (7, 8, 1 2 ) that has been accepted as an experimental model for myasthenia gravis. The appearance of clinical symptoms is preceded by the OCcurrence in serum of antibodies to n-AChR (11). Since sera from some patients with myasthenia gravis contain antibodies to protein components of the muscle cell membrane ( I ) , the present study was performed to investigate whether there is an immunological cross-reactivity between the muscle antibodies detected in the human disease and the antibodies to n-AChR present in animals with experimental myasthenia.
6
MATERIALS AND METHODS
Tissue p reparatio n.r Nicotinic acetylcholine receptor and CA antigen of human skeletal muscle were prepared as described previously (1, 7).
Sera Sera from 25 patients with myasthenia gravis (MG) were collected at the Department of Neurology. Rabbit antisera to n-AChR were produced by immunization of rabbits with nAChR in Freund’s adjuvant ( 7 ) . Rabbit antisera to sarcolemmic/subsarcolemmic antigen(s) were obtained by immunization of rabbits with their own erythrocytes, treated with tannic acid and coated with citric acid extract of skeletal
850
J. A . Aarli, C.Maitsson & E. Heilbronn
Table I. Results of indirect haemagglutination test with various sera Sera from
‘Experimental myasthenia’ Rabbits immunized with muscle antigen Myasthenia gravis
* - =
Titres* using erythrocytes sensitized with AChR protein
antigen
8,192
-
-
8,192 128,000
-
no agglutination; AChR tylcholine receptor.
nicotinic ace-
muscle (CA antigen) ( 2 ) . All sera were stored at -20OC until used. For inactivation of complement, the sera were heated at 56°C for 40 min. Indirpct haemagglutination ( I H A ) text In the IHA test for antibodies to n-AChR, a 5% suspension of rabbit erythrocytes in isotonic, phosphate-buffered saline, pH 7.2 (PBS), was mixed with an equal volume of tannic acid diluted 1:60,000 (wtlvol) in PBS and incubated at room temperature for 15 min. The suspension was centrifuged at 1,000 g for 5 min and the cells washed once in 10 ml of PBS. Equal amounts of a 2.5% suspension of the tanned erythrocytes and n-AChR diluted to a protein concentration of 0.02 mg/ml in PBS were mixed and incubated for 30 min at 37°C. After three washings, the cells were made up to 1.25% in PBS containing 1% normal rabbit serum (NRS). The sera were diluted 1:s in PBS and absorbed with y5 voIume of packed, tanned erythrocytes. Twofold dilutions of absorbed sera were prepared in 0.1-ml volumes of NRS. To each tube was added 0.1 ml of sensitized erythrocytes. Controls were (a) sera incubated with tanned erythrocytes but not exposed to n-AChR and (b) NRS incubated with sensitized erythrocytes. The racks were shaken and left at room temperature for 2 hr. Agglutination was recorded by observing the pattern formed by the eryth-
rocytes. A control reading after incubation overnight at 4°C was included. The reciprocal of the highest serum dilution giving agglutination was recorded as the titre. The IHA test for antibodies to the CA antigen was performed as described earlier ( I ) . Haernagglutination inhibition tejts Inhibition tests, both with n-AChR and with CA antigen, were performed as described earlier (1). Rocket immuizoelectvophoresjJ Rocket immunoelectrophoresis was performed in 2% agarose with lo-,.J antigen samples. Electrophoresis was run for 2 hr at a constant voltage of 10 V/cm. The gels were then treated as described by Weeke (15).
RESULTS Antibodies to n - AC h R Sera from 3 rabbits immunized with nAChR, 3 rabbits immunized with CA antigen, and 25 patients with MG were tested for antibodies to n-AChR by the IHA test. All three sera from rabbits with experimental myasthenia agglutinated the cells to high titres (2,048 to 8,192), whereas no agglutination occurred with the other sera (Table I). In rocket immunoelectrophoresis, precipitates were obtained only with the three sera from rabbits immunized with n-AChR. Serum was collected at intervals during immunization of a rabbit with n-AChR. The samples were tested for antibodies to n-AChR by IHA and rocket immunoelectrophoresis. The results showed good correlation between the two techniques (Fig. 1). Antibodies to nAChR were not detected in MG sera by rocket immunoelectrophoresis. Antibodies to C A antigen The same sera were examined for antibodies to CA antigen. The three sera from rabbits immunized with CA antigen gave agglutination
Nicotinic Aceiylcholine Receptor AntibodieJ ANTIBOW TITRE nAChR
OF n A C h R IMMUNIZED
RABBITS
I H A TlTRE
mg/rn
6 4
2
J I
10
20
0
IHA-TEST
0
ROCKET IE
o;
days a t t e r flrSt injection
Fig. 1. Antibodies to nicotinic acetylcholine receptor (n-AChR) in serum from a rabbit with experimental myasthenia. Comparison between titres in rocket immunoelectrophoresis ( I E ) and indirect haemagglutination (IHA) in relation to time after immunization.
of the sensitized cells to titres of 1,02416,384. With the MG sera, a positive reaction was recorded with five specimens. The sera from the three rabbits with experimental myasthenia did not agglutinate the cells (Table I).
Haemdgglutination inhibition te.rtS Erythrocytes sensitized with n-AChR were prepared. Agglutination of these cells by antiserum to n-AChR was inhibited by n-AChR but not by CA. On the other hand, agglutination of CA-sensitized erythrocytes was not inhibited by n-AChR but was inhibited by CA antigen. Accordingly, the results of the haemagglutination inhibition tests do not indicate that the two preparations share antigenic determinant (s) .
DISCUSSION The present results clearly indicate a serological difference between sera from animals with experimental myasthenia and sera from MG patients. Thus, tanned erythrocytes, sensitized with n-AChR from Torpedo marmorata, were
85 1
agglutinated by rabbit antisera but not by human sera or normal rabbit controls. The antibodies were demonstrated even with high serum dilutions, illustrating the high sensitivity of this method. Nevertheless, when the same technique was applied to human M G sera, no agglutination occurred. Nor did any reaction occur when rocket immunoelectrophoresis was used. Furthermore, in an investigation of sera from M G patients at Sodersjukhuset, Stockholm, antibodies to Torpedo n-AChR were not detected, and blocking of [3H)acetyl-~-neurotoxin binding to Torpedo n-AChR was not demonstrated ( 6 ) . A few reports indicate, the presence in some M G sera of a factor which blocks a-bungarotoxin binding to n-AChR of the neuromuscular junction ( 4 , 5 , 14). According to Bender et al. ( 5 ) , the factor also blocks the binding of a-bungarotoxin to human sarcolemmic AChR of disease-denervated muscle fibres. Since anti-muscle antibodies were present in all sera containing the serum blocking factor, the question was raised whether this factor is identical with the muscle antibodies occurring in human myasthenia. W e have not detected muscle antibodies in sera from rabbits immunized with n-AChR. Nor could we demonstrate antibodies to Torpedo n-AChR in sera from rabbits immunized with CA antigen of skeletal muscle. In haemagglutination inhibition experiments, CA antigen did not inhibit agglutination of cells sensitized with n-AChR and vice vefJa. Antibodies to the CA antigen, but not to n-AChR, were detected in some M G sera. Accordingly, the results of the present investigation strongly indicate that there is no immunological crossreaction between this muscle antigen and nAChR from Torpedo marmorata. The implication is either that M G sera d o not contain antibodies to n-AChR in general or that there are variations between n-AChR of various species. I n favour of the first theory are results reported by Keesey et al. ( 9 ) , who found no significant difference in a-bungarotoxin-binding between normal mouse diaphragms incubated in M G sera and those incubated in control sera. A recent study reports complement
352
J . A . Aarli, C. Maftsson & E. Heilbronn
fixation with MG sera using AChR from Torpedo culif ornicu as antigen ( 3 ) . However, data suggesting differences between n-AChR from various species have been reported ( i 3 ) . Lindstrom & Lennon have described the presence in MG sera of antibodies to human skeletal muscle n-AChR with minimal cross-reaction with n-AChR of eel origin (lo). Investigations of possible cross-reactivity between antibodies to human n-AChR and to human muscle antigens are obviously needed.
REFERENCES 1. Aarli, J. A. Myasthenia gravis. Antibodies to an acid-soluble antigen in striated muscle. Clin. exp. Immunol. 10, 453, 1972. 2. Aarli, J. A,, Tsnder, 0. & Closs, 0. Specificity of muscle antibodies in sera from patients with myasthenia gravis and from immunized rabbits. J. NeuroE. Neurosurg. PJychiat. 37, 991, 1974. 3. Aharonov, A,, Abramsky, O., Tarrab-Hazdai, R. & Fuchs, S. Humoral antibodies to acetylcholine receptor in patients with myasthenia gravis. Lancet 2, 340, 1975. 4. Almon, R. R., Andrew, C. G. & Appel, S. H. Serum globulin in myasthenia gravis: inhibition of a-bungarotoxin binding to acetylcholine receptor. Science 186, 55, 1974. 5. Bender, A. N., King Engel, W., Ringel, S. P., Daniels, M. P. & Vogel, Z. Myasthenia gravis: a serum factor blocking acetylcholine receptors of the human neuromuscular junction. Lancet I , 607, 1975.
Received 16 October 1975
6. Fischer, C. Unpublished data. 7. Heilbronn, E. & Mattsson, C. The nicotinic cholinergic receptorprotein: improved purification method, preliminary amino acid composition and observed autoimmuno response. J . Neworhem. 22, 315, 1974. 8. Heilbronn, E., Mattsson, C., StHlberg, E. & Hilton-Brown, P. Neurophysiological signs of Myasthenia in rabbits after receptor antibody development. J . neurol. Sci. 24, 59, 1975. 9. Keesey, J., Shaikh, I., Wolfgram, F. & Chao, L. P. Studies on the ability of acetylcholine receptors to bind alpha-bungarotoxin after exposure to myasthenic serum. Ann. N.Y. Acad. Sci. In press, 1975. 10. Lindstrom, J. & Lennon, V. Experimental autoimmune myasthenia and myasthenia gravis: biochemical and immunochemical aspects. Ann. N.Y . Acad. Sci. In press, 1975. 11. Mattsson, C. & Heilbronn, E. Immunization of rabbits with purified nicotinic acetylcholine receptor. Croat. chem. Arta. In press. 12. Patrick, J. & Lindstrom, J. Auto-immune response to acetylcholine receptor. Science 180, 871, 1973. 13. Penn, A. S., Chang, H. W., Lovelace, R. E., Niemi, W. & Miranda, A. Antibodies to acetylcholine receptor in rabbits. Ann. N . Y . Acad. SCI. In press, 1975. 14. Ringel, S. P., Bender, A. N., King Engel, W. & Smith, H. J. Myas'thenia gravis: relationship between serum factor blocking acetylcholine receptors and anti-striated-muscle antibody. Lancet 1, 1388, 1975. 15. Weeke, B. Rocket immunoelectrophoresis. pp. 37-46 in Axelsen, N . H., Krsll, J. & Weeke, B. (eds.) A Manual of Quaiititative Immunoelectrophoresis. Universitetsforlaget, Oslo, 1973.
Aarli, J. A,, Mattsson, C. & Heilbronn, E. Antibodies Against Nicotinic Acetylcholine Receptor and Skeletal Muscle in Human and Experimental Myasthenia Gravis. Scand. J. Immunol. 4, 849-852, 1975. Using rocket immunoelectrophoresis and indirect haemagglutination tests, antibodies to nicotinic acetylcholine receptor (n-AChR) from Torpedo mawnorata were detected in sera from rabbits with experimental myasthenia but not in sera from patients with myasthenia gravis or in rabbit antisera to a partly purified skeletal muscle antigen. Antibodies to this muscle antigen were demonstrated in sera from patients with myasthenia gravis but not in sera from rabbits with experimental myasthenia. The results indicate that there is no immunological cross-reaction between the muscle antigen and n-AChR from Torpedo marrorata, and little or no cross-reactivity between these n-AChR antibodies and possible muscle n-AChR antibodies in myasthenia gravis patients.
J. A. Aarli, M.D., Department of Neurology, 5016 Haukeland sykehus, Bergen, Norway
Rabbits immunized with purified nicotinic acetylcholine receptor (n-AChR) from electric organs of Torpedo or Electroplax develop a neostigmine-sensitive muscular weakness (7, 8, 1 2 ) that has been accepted as an experimental model for myasthenia gravis. The appearance of clinical symptoms is preceded by the OCcurrence in serum of antibodies to n-AChR (11). Since sera from some patients with myasthenia gravis contain antibodies to protein components of the muscle cell membrane ( I ) , the present study was performed to investigate whether there is an immunological cross-reactivity between the muscle antibodies detected in the human disease and the antibodies to n-AChR present in animals with experimental myasthenia.
6
MATERIALS AND METHODS
Tissue p reparatio n.r Nicotinic acetylcholine receptor and CA antigen of human skeletal muscle were prepared as described previously (1, 7).
Sera Sera from 25 patients with myasthenia gravis (MG) were collected at the Department of Neurology. Rabbit antisera to n-AChR were produced by immunization of rabbits with nAChR in Freund’s adjuvant ( 7 ) . Rabbit antisera to sarcolemmic/subsarcolemmic antigen(s) were obtained by immunization of rabbits with their own erythrocytes, treated with tannic acid and coated with citric acid extract of skeletal
850
J. A . Aarli, C.Maitsson & E. Heilbronn
Table I. Results of indirect haemagglutination test with various sera Sera from
‘Experimental myasthenia’ Rabbits immunized with muscle antigen Myasthenia gravis
* - =
Titres* using erythrocytes sensitized with AChR protein
antigen
8,192
-
-
8,192 128,000
-
no agglutination; AChR tylcholine receptor.
nicotinic ace-
muscle (CA antigen) ( 2 ) . All sera were stored at -20OC until used. For inactivation of complement, the sera were heated at 56°C for 40 min. Indirpct haemagglutination ( I H A ) text In the IHA test for antibodies to n-AChR, a 5% suspension of rabbit erythrocytes in isotonic, phosphate-buffered saline, pH 7.2 (PBS), was mixed with an equal volume of tannic acid diluted 1:60,000 (wtlvol) in PBS and incubated at room temperature for 15 min. The suspension was centrifuged at 1,000 g for 5 min and the cells washed once in 10 ml of PBS. Equal amounts of a 2.5% suspension of the tanned erythrocytes and n-AChR diluted to a protein concentration of 0.02 mg/ml in PBS were mixed and incubated for 30 min at 37°C. After three washings, the cells were made up to 1.25% in PBS containing 1% normal rabbit serum (NRS). The sera were diluted 1:s in PBS and absorbed with y5 voIume of packed, tanned erythrocytes. Twofold dilutions of absorbed sera were prepared in 0.1-ml volumes of NRS. To each tube was added 0.1 ml of sensitized erythrocytes. Controls were (a) sera incubated with tanned erythrocytes but not exposed to n-AChR and (b) NRS incubated with sensitized erythrocytes. The racks were shaken and left at room temperature for 2 hr. Agglutination was recorded by observing the pattern formed by the eryth-
rocytes. A control reading after incubation overnight at 4°C was included. The reciprocal of the highest serum dilution giving agglutination was recorded as the titre. The IHA test for antibodies to the CA antigen was performed as described earlier ( I ) . Haernagglutination inhibition tejts Inhibition tests, both with n-AChR and with CA antigen, were performed as described earlier (1). Rocket immuizoelectvophoresjJ Rocket immunoelectrophoresis was performed in 2% agarose with lo-,.J antigen samples. Electrophoresis was run for 2 hr at a constant voltage of 10 V/cm. The gels were then treated as described by Weeke (15).
RESULTS Antibodies to n - AC h R Sera from 3 rabbits immunized with nAChR, 3 rabbits immunized with CA antigen, and 25 patients with MG were tested for antibodies to n-AChR by the IHA test. All three sera from rabbits with experimental myasthenia agglutinated the cells to high titres (2,048 to 8,192), whereas no agglutination occurred with the other sera (Table I). In rocket immunoelectrophoresis, precipitates were obtained only with the three sera from rabbits immunized with n-AChR. Serum was collected at intervals during immunization of a rabbit with n-AChR. The samples were tested for antibodies to n-AChR by IHA and rocket immunoelectrophoresis. The results showed good correlation between the two techniques (Fig. 1). Antibodies to nAChR were not detected in MG sera by rocket immunoelectrophoresis. Antibodies to C A antigen The same sera were examined for antibodies to CA antigen. The three sera from rabbits immunized with CA antigen gave agglutination
Nicotinic Aceiylcholine Receptor AntibodieJ ANTIBOW TITRE nAChR
OF n A C h R IMMUNIZED
RABBITS
I H A TlTRE
mg/rn
6 4
2
J I
10
20
0
IHA-TEST
0
ROCKET IE
o;
days a t t e r flrSt injection
Fig. 1. Antibodies to nicotinic acetylcholine receptor (n-AChR) in serum from a rabbit with experimental myasthenia. Comparison between titres in rocket immunoelectrophoresis ( I E ) and indirect haemagglutination (IHA) in relation to time after immunization.
of the sensitized cells to titres of 1,02416,384. With the MG sera, a positive reaction was recorded with five specimens. The sera from the three rabbits with experimental myasthenia did not agglutinate the cells (Table I).
Haemdgglutination inhibition te.rtS Erythrocytes sensitized with n-AChR were prepared. Agglutination of these cells by antiserum to n-AChR was inhibited by n-AChR but not by CA. On the other hand, agglutination of CA-sensitized erythrocytes was not inhibited by n-AChR but was inhibited by CA antigen. Accordingly, the results of the haemagglutination inhibition tests do not indicate that the two preparations share antigenic determinant (s) .
DISCUSSION The present results clearly indicate a serological difference between sera from animals with experimental myasthenia and sera from MG patients. Thus, tanned erythrocytes, sensitized with n-AChR from Torpedo marmorata, were
85 1
agglutinated by rabbit antisera but not by human sera or normal rabbit controls. The antibodies were demonstrated even with high serum dilutions, illustrating the high sensitivity of this method. Nevertheless, when the same technique was applied to human M G sera, no agglutination occurred. Nor did any reaction occur when rocket immunoelectrophoresis was used. Furthermore, in an investigation of sera from M G patients at Sodersjukhuset, Stockholm, antibodies to Torpedo n-AChR were not detected, and blocking of [3H)acetyl-~-neurotoxin binding to Torpedo n-AChR was not demonstrated ( 6 ) . A few reports indicate, the presence in some M G sera of a factor which blocks a-bungarotoxin binding to n-AChR of the neuromuscular junction ( 4 , 5 , 14). According to Bender et al. ( 5 ) , the factor also blocks the binding of a-bungarotoxin to human sarcolemmic AChR of disease-denervated muscle fibres. Since anti-muscle antibodies were present in all sera containing the serum blocking factor, the question was raised whether this factor is identical with the muscle antibodies occurring in human myasthenia. W e have not detected muscle antibodies in sera from rabbits immunized with n-AChR. Nor could we demonstrate antibodies to Torpedo n-AChR in sera from rabbits immunized with CA antigen of skeletal muscle. In haemagglutination inhibition experiments, CA antigen did not inhibit agglutination of cells sensitized with n-AChR and vice vefJa. Antibodies to the CA antigen, but not to n-AChR, were detected in some M G sera. Accordingly, the results of the present investigation strongly indicate that there is no immunological crossreaction between this muscle antigen and nAChR from Torpedo marmorata. The implication is either that M G sera d o not contain antibodies to n-AChR in general or that there are variations between n-AChR of various species. I n favour of the first theory are results reported by Keesey et al. ( 9 ) , who found no significant difference in a-bungarotoxin-binding between normal mouse diaphragms incubated in M G sera and those incubated in control sera. A recent study reports complement
352
J . A . Aarli, C. Maftsson & E. Heilbronn
fixation with MG sera using AChR from Torpedo culif ornicu as antigen ( 3 ) . However, data suggesting differences between n-AChR from various species have been reported ( i 3 ) . Lindstrom & Lennon have described the presence in MG sera of antibodies to human skeletal muscle n-AChR with minimal cross-reaction with n-AChR of eel origin (lo). Investigations of possible cross-reactivity between antibodies to human n-AChR and to human muscle antigens are obviously needed.
REFERENCES 1. Aarli, J. A. Myasthenia gravis. Antibodies to an acid-soluble antigen in striated muscle. Clin. exp. Immunol. 10, 453, 1972. 2. Aarli, J. A,, Tsnder, 0. & Closs, 0. Specificity of muscle antibodies in sera from patients with myasthenia gravis and from immunized rabbits. J. NeuroE. Neurosurg. PJychiat. 37, 991, 1974. 3. Aharonov, A,, Abramsky, O., Tarrab-Hazdai, R. & Fuchs, S. Humoral antibodies to acetylcholine receptor in patients with myasthenia gravis. Lancet 2, 340, 1975. 4. Almon, R. R., Andrew, C. G. & Appel, S. H. Serum globulin in myasthenia gravis: inhibition of a-bungarotoxin binding to acetylcholine receptor. Science 186, 55, 1974. 5. Bender, A. N., King Engel, W., Ringel, S. P., Daniels, M. P. & Vogel, Z. Myasthenia gravis: a serum factor blocking acetylcholine receptors of the human neuromuscular junction. Lancet I , 607, 1975.
Received 16 October 1975
6. Fischer, C. Unpublished data. 7. Heilbronn, E. & Mattsson, C. The nicotinic cholinergic receptorprotein: improved purification method, preliminary amino acid composition and observed autoimmuno response. J . Neworhem. 22, 315, 1974. 8. Heilbronn, E., Mattsson, C., StHlberg, E. & Hilton-Brown, P. Neurophysiological signs of Myasthenia in rabbits after receptor antibody development. J . neurol. Sci. 24, 59, 1975. 9. Keesey, J., Shaikh, I., Wolfgram, F. & Chao, L. P. Studies on the ability of acetylcholine receptors to bind alpha-bungarotoxin after exposure to myasthenic serum. Ann. N.Y. Acad. Sci. In press, 1975. 10. Lindstrom, J. & Lennon, V. Experimental autoimmune myasthenia and myasthenia gravis: biochemical and immunochemical aspects. Ann. N.Y . Acad. Sci. In press, 1975. 11. Mattsson, C. & Heilbronn, E. Immunization of rabbits with purified nicotinic acetylcholine receptor. Croat. chem. Arta. In press. 12. Patrick, J. & Lindstrom, J. Auto-immune response to acetylcholine receptor. Science 180, 871, 1973. 13. Penn, A. S., Chang, H. W., Lovelace, R. E., Niemi, W. & Miranda, A. Antibodies to acetylcholine receptor in rabbits. Ann. N . Y . Acad. SCI. In press, 1975. 14. Ringel, S. P., Bender, A. N., King Engel, W. & Smith, H. J. Myas'thenia gravis: relationship between serum factor blocking acetylcholine receptors and anti-striated-muscle antibody. Lancet 1, 1388, 1975. 15. Weeke, B. Rocket immunoelectrophoresis. pp. 37-46 in Axelsen, N . H., Krsll, J. & Weeke, B. (eds.) A Manual of Quaiititative Immunoelectrophoresis. Universitetsforlaget, Oslo, 1973.
