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Differing responses of inbred rat strains in experimental autoimmune thyroiditis
CELLULAR
IMMUNOLOGY
Differing
18,
360-364 (1975)
Responses of Inbred Autoimmune NOEL
MRC
Cellular Iwamunology Oxford
Rat Strains
in Experimental
Thyroiditis R. ROSE1
Unit, Sir William Dunn School Universtiy, England
Received
February
of Pathology,
14,1975
Four inbred strains differed greatly in their response to a standard inoculation of rat thyroid extract with complete Freund’s adjuvant and pertussis vaccine. Strains HO and AU, both Ag-B5, developed only minimal lesions, although AU rats had a high titer of circulating antibodies to thyroid antigens. A0 rats (Ag-B2) and DA rats (Ag-B4) showed more severe thyroiditis. A0 rats had high titers of antibody while the DA strain had only moderate titers.
Experimental autoimmune thyroiditis can be produced in rats by injection of rat thyroid extract or purified rat thyroglobulin given with complete Freund’s adjuvant (l-3). It is characterized by lymphocytic and monocytic infiltration of the thyroid gland and production of thyroid-specific autoantibodies. Extensive studies have been carried out on factors influencing production of the disease and its transfer by viable lymph node cells (3, 4). McMaster et al. (6) have recently reported that Strain 2 guinea pigs do not develop thyroiditis as readily as the Hartley strain regardless of the dose of guinea pig thyroid extract, or of mycobacterium in the adjuvant, or the strain of origin of the antigen. In the mouse, experimental autoimmune thyroiditis is clearly under genetic control, with a major influence being attributed to a gene located at the Ir-1A region of the major histocompatibility (H-2) complex (7, 8). Other studies have shown that another experimental autoimmune disease, allergic encephalomyelitis, is genetically controlled in the rat (8). It was of interest, therefore, to compare four strains of inbred rats for their susceptibility to experimental autoimmune thyroiditis. MATERIAL
AND METHODS
Aniwtals. Four inbred strains of rats were used in this investigation : hooded (HO) Ag-B5 ; Albino, Oxford (AO) Ag-B2 ; dark Agouti (DA) Ag-B4 ; and August (AU) Ag-B5. They were raised under specific pathogen-free conditions in our own animal unit. Antigen. A mixed saline extract was prepared by mincing thyroids obtained from 1 Present address: Wayne State University, School of Medicine, Department of Immunology and Microbiology, 540 East Canfield Ave., Detroit, Michigan 48201. 360 Copyright All rights
c 1975 by Academic Press, Inc. o? reproduction in any form reserved.
DIFFERING
RESPONSES
TABLE
OF RATS
361
TO EAT
1
RESPONSE OF RAT STRAINS TO RAT THYROID CKUDE EXTIUCT
Rat
& type
Strain
Ag B5
HO HO
1
2 3
HO
10 11 12
2a 4 2
21 Day
Pathology
<2 <2 2
4 2 2
0 0 +
128 64
64 256 256
512 512 512
1024 1024 2048
++ ++ ++
8 8
8 <2
<2 <2 64
++ + ++
128 128 128
0 + +
Ag B4
DA DA DA
16
8
<2 <2 32
AU AU AU
32 32 64
32 32 32
32 32 16
Ag B5
28 Day
4 4 2
A0 A0 A0
7 8 9
14 Day
Ag B2
4
5 6
7 Day
16
a Figures indicate the titers obtained in tanned cell hemagglutination thyroid crude extract. All preinjection samples were negative (<2).
tests with mixed rat
(A0 x HO) F1, (A0 x DA) F 1, and (HO x DA) F1 hybrids, using equal parts (w/v) of tissue and isotonic buffered saline solution (pH 7.2). After overnight infusion, a clear supernatant was obtained by centrifuging in the cold at 69,000g for 45 min. The protein content was determined by absorption at 280 nm. Extracts of thyroids from each inbred strain were prepared separately using the same method. Female rats 8 wk of age were used for immunization. After a preliminary sample of serum was taken, every rat was injected with a emulsion of 6 mg of clarified thyroid extract emulsified with an equal volume of complete Freund’s adjuvant (Difco) and injected in 0.1 ml volumes in each of the four footpads. At the same time 0.5 ml concentrated pertussis vaccine (Parke, Davis & Co.) was given in the dorsum of each footpad. (2, 9). Assays. The tanned cell hemagglutination test for antibodies to thyroid was described previously as was the method of grading thyroiditis (2, 4). RESULTS The results of the weekly serological tests and the final histological examination are given in Table 1. It is obvious that the four inbred strains of rats differ markedly in their response to the standard stimulus of rat thyroid crude extract. HO rats responded very poorly, with low titers of antibody and with minimal pathological change apparent in only one of the three animals studied. A0 rats showed a vigorous response in terms of antibody titer and pathology. The antibody response of DA rats was generally poor, but thyroid lesions were pronounced, while AU rats produced antibody well but showed only minimal pathology. One possible explanation for the difference in response among the four rat strains tested is the presence of antigenic differences between thyroid antigens of the strains. Tanned red cells were, therefore, coated with each thyroid extract individually (using a 200 rg/ml solution) and tested with the final sample of serum from each rat. The results are given in Table 2.
362
NOELR.ROSE
TABLE
2
TANNED CELL HEMAGGLUTINATION WITH INDIVIDUAL RAT THYROID EXTRACTS Tanned
Antiserum Rat
Strain
HO
1 2 3
HO HO HO
<2 <2 <2
4 5 6
A0 A0 A0
7 8 9
DA DA DA
<2 <2 <2
10 11 12
AU AU AU
<2 4 32
RBC coated with thyroid
64 64 64
extract
from
DA
AU
<2 <2 <2
<2 <2 <2
<2
<2 <2 <2
512 512 2.56
256 256 256
<2 <2 128
8 <2 64
<2 <2 32
512 2048 2048
512 2048 1024
64 256 256
A0
<2 <2
There is a consistent tendency for antisera to react more strongly with allogeneic thyroid extract than the syngeneic extract. It is especially apparent in tests with the A0 antisera, which failed to react with thyroid extract of the same strain while reacting with all other rat thyroid extracts. These results suggested that there were distinct determinants on the thyroid antigens of each of the strains. Therefore inhibition of hemagglutination tests were performed. A thyroid extract of each strain was able to inhibit completely the reaction with every other strain. Ouchterlony tests were also carried out with each antiserum against all thyroid extracts. Positive reactions were found with antisera 4, 5, 6, 9, 10, 11, 12. When individual preparations of thyroid antigens were compared, all antisera produced lines of complete identity. In immunoelectrophoresis, all antisera tested produced a single arc of precipitation in the alpha globulin region typical of thyroglobulin. DISCUSSION The novel finding in this investigation is the great difference in responsiveness of various inbred rat strains to rat thyroid extract. Furthermore, there was a dissociation of antibody production from the severity of the thyroid lesions. Although antibody levels were followed for only 4 wk, experience has shown that animals that do not have antibody at that time remain negative. These differences in antibody formation are highly significant. In another serological test, precipitation, confirmatory results were obtained, since only antisera with hemagglutination titers of 64 or above were positive. The histological observations were performed after 4 wk. Although thyroiditis produced in rats by injection of rat thyroid extract in Freund’s adjuvant has been reported to regress, the use of pertussis vaccine as coadjuvant leads to progressive disease without any evidence of regression (2).
DIFFERING
RESPONSES
OF RATS
TO EAT
363
The findings can be accounted for in two ways, which are not mutually exclusive. The first is that there are strain-related differences between the thyroid antigens. The data presented in Table 2 suggest that this is the case. Thyroglobulin is the only demonstrable autoantigen of thyroid in the rat. All sera that could be tested showed only a single precipitation line with mobility in immunoelectrophoresis of thyroglobulin. Thyroid extract of any strain could neutralize completely the reaction of any other strain, suggesting that the antigenic determinants are all present, although they may differ in quantitative expression. At first it might seem that the HO rats responded only to the allogeneic determinants of the thyroglobulin molecule. However, the fact that the animals all had pathological changes in their thyroids indicated that autologous determinants were also involved. Perhaps the syngeneic determinants are involved more in cellular than humoral responses, suggesting that genetic control of T cell effecters may differ from B cell responses. Finally, the four strains show great differences in immunological responses to thyroid antigen. It is tempting to postulate that it is due to the inheritance of one or more immune response genes for particular determinants of thyroglobulin. It is interesting that immune responsiveness in terms of antibody formation does not appear to be linked to serologically demonstrable Ag-B antigens while the capacity to develop thyroid lesions may be. The AU and HO rats, which are both Ag-B5, show minimal thyroid pathology despite the fact that HO is an antibody nonresponder and AU is a good responder. A0 and DA also show no relation between antibody responses and severity of lesions. We considered the further possibility that lymphocyte-determined histocompatibility differences might distinguish HO from AU strains of rats, and performed local graft vs host assays by the lymph node enlargement method (10). Even 50 X 10G HO spleen cells failed to produce significant lymph node enlargement in (AU x HO) F1 recipient mice. Gasser ef al. (11) found that the genetic locus controlling susceptibility to experimental allergic encephalomyelitis in rat strains is closely associated with or linked to Ag-B but that the specific gene in question was not Ag-B4. Based on the data shown in Table 1, Ag-B4, in addition to Ag-B2, may be linked to the allele favoring susceptibility to experimental autoimmune thyroiditis. Thus, a contrasting relationship may be present with respect to genetic factors influencing development of experimental allergic encephalomyelitis as opposed to development of experimental autoimmune thyroiditis. An analogous situation has been described in the guinea pig, i.e., strain 2 animals are much more susceptible to induction of experimental allergic thyroiditis than are strain 13 guinea pigs, as recently reported by BraleyMulle, Sharp, and Kyriakos (12), whereas just the reverse situation has been described in studies of the comparative susceptibility of these two inbred strains of guinea pigs to experimental allergic encephalomyelitis (13). ACKNOWLEDGMENTS The author wishes to acknowledge the hospitality and encouragement of Prof. James L. Gowans and the technical assistance of Mr. Steven Simmonds. He received support from the Buswell Foundation and NIH Research Grant CA02357 from the National Cancer Institute.
REFERENCES 1. Jones, H. E. H., and Roitt, I. M., Brit. J. Exp. Pathol. 42, 546, 1961. 2. Twarog, F. J., and Rose, N. R., Proc. Sot. Exp. Biol. Med. 130, 435, 1969. 3. Twarog, F. J., Kenney, E., and Rose, N. R., Prof. Sot. Exp. Biol. Med. 133, 185, 1970.
364 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
NOELR.ROSE
Twarog, F. J., and Rose, N. R., J. Immzmol. 104, 1467, 1970. Rose, N. R., Molotchnikoff, M.-F., and Twarog, F. J., Immunology 24, 859, 1973. McMaster, P. R. B., Owens, J. D., and Kyriakos, W., Cell. Zmmunol. 14, 39, 1974. Vladutiu, A. O., and Rose, N. R., Science 174, 1137, 1971. Tomazic, B., Rose, N. R., and Shreffler, D. C., J. Immwnol. 112, 965, 1974. Paterson, P. Y., and Drobish, D. G., J. Immunol. 101, 1098, 1968. Ford, W. L., Burr, W., and Simensen, M., Trulzsphntation 10, 258, 1970. Gasser, D. L., Newlin, C. M., Palm, J., and Gonatas, N. K., Sc&ence 181, 872, 1973. Braley-Mullen, H., Sharp, G. C., and Kyriakos, M., J. Immunol. 114, 371, 1975. Stone, S. H., Lerner, E. M., and Goode, J. H., Proc. Sot. Exp. Biol. Med. 132, 341, 1969.
IMMUNOLOGY
Differing
18,
360-364 (1975)
Responses of Inbred Autoimmune NOEL
MRC
Cellular Iwamunology Oxford
Rat Strains
in Experimental
Thyroiditis R. ROSE1
Unit, Sir William Dunn School Universtiy, England
Received
February
of Pathology,
14,1975
Four inbred strains differed greatly in their response to a standard inoculation of rat thyroid extract with complete Freund’s adjuvant and pertussis vaccine. Strains HO and AU, both Ag-B5, developed only minimal lesions, although AU rats had a high titer of circulating antibodies to thyroid antigens. A0 rats (Ag-B2) and DA rats (Ag-B4) showed more severe thyroiditis. A0 rats had high titers of antibody while the DA strain had only moderate titers.
Experimental autoimmune thyroiditis can be produced in rats by injection of rat thyroid extract or purified rat thyroglobulin given with complete Freund’s adjuvant (l-3). It is characterized by lymphocytic and monocytic infiltration of the thyroid gland and production of thyroid-specific autoantibodies. Extensive studies have been carried out on factors influencing production of the disease and its transfer by viable lymph node cells (3, 4). McMaster et al. (6) have recently reported that Strain 2 guinea pigs do not develop thyroiditis as readily as the Hartley strain regardless of the dose of guinea pig thyroid extract, or of mycobacterium in the adjuvant, or the strain of origin of the antigen. In the mouse, experimental autoimmune thyroiditis is clearly under genetic control, with a major influence being attributed to a gene located at the Ir-1A region of the major histocompatibility (H-2) complex (7, 8). Other studies have shown that another experimental autoimmune disease, allergic encephalomyelitis, is genetically controlled in the rat (8). It was of interest, therefore, to compare four strains of inbred rats for their susceptibility to experimental autoimmune thyroiditis. MATERIAL
AND METHODS
Aniwtals. Four inbred strains of rats were used in this investigation : hooded (HO) Ag-B5 ; Albino, Oxford (AO) Ag-B2 ; dark Agouti (DA) Ag-B4 ; and August (AU) Ag-B5. They were raised under specific pathogen-free conditions in our own animal unit. Antigen. A mixed saline extract was prepared by mincing thyroids obtained from 1 Present address: Wayne State University, School of Medicine, Department of Immunology and Microbiology, 540 East Canfield Ave., Detroit, Michigan 48201. 360 Copyright All rights
c 1975 by Academic Press, Inc. o? reproduction in any form reserved.
DIFFERING
RESPONSES
TABLE
OF RATS
361
TO EAT
1
RESPONSE OF RAT STRAINS TO RAT THYROID CKUDE EXTIUCT
Rat
& type
Strain
Ag B5
HO HO
1
2 3
HO
10 11 12
2a 4 2
21 Day
Pathology
<2 <2 2
4 2 2
0 0 +
128 64
64 256 256
512 512 512
1024 1024 2048
++ ++ ++
8 8
8 <2
<2 <2 64
++ + ++
128 128 128
0 + +
Ag B4
DA DA DA
16
8
<2 <2 32
AU AU AU
32 32 64
32 32 32
32 32 16
Ag B5
28 Day
4 4 2
A0 A0 A0
7 8 9
14 Day
Ag B2
4
5 6
7 Day
16
a Figures indicate the titers obtained in tanned cell hemagglutination thyroid crude extract. All preinjection samples were negative (<2).
tests with mixed rat
(A0 x HO) F1, (A0 x DA) F 1, and (HO x DA) F1 hybrids, using equal parts (w/v) of tissue and isotonic buffered saline solution (pH 7.2). After overnight infusion, a clear supernatant was obtained by centrifuging in the cold at 69,000g for 45 min. The protein content was determined by absorption at 280 nm. Extracts of thyroids from each inbred strain were prepared separately using the same method. Female rats 8 wk of age were used for immunization. After a preliminary sample of serum was taken, every rat was injected with a emulsion of 6 mg of clarified thyroid extract emulsified with an equal volume of complete Freund’s adjuvant (Difco) and injected in 0.1 ml volumes in each of the four footpads. At the same time 0.5 ml concentrated pertussis vaccine (Parke, Davis & Co.) was given in the dorsum of each footpad. (2, 9). Assays. The tanned cell hemagglutination test for antibodies to thyroid was described previously as was the method of grading thyroiditis (2, 4). RESULTS The results of the weekly serological tests and the final histological examination are given in Table 1. It is obvious that the four inbred strains of rats differ markedly in their response to the standard stimulus of rat thyroid crude extract. HO rats responded very poorly, with low titers of antibody and with minimal pathological change apparent in only one of the three animals studied. A0 rats showed a vigorous response in terms of antibody titer and pathology. The antibody response of DA rats was generally poor, but thyroid lesions were pronounced, while AU rats produced antibody well but showed only minimal pathology. One possible explanation for the difference in response among the four rat strains tested is the presence of antigenic differences between thyroid antigens of the strains. Tanned red cells were, therefore, coated with each thyroid extract individually (using a 200 rg/ml solution) and tested with the final sample of serum from each rat. The results are given in Table 2.
362
NOELR.ROSE
TABLE
2
TANNED CELL HEMAGGLUTINATION WITH INDIVIDUAL RAT THYROID EXTRACTS Tanned
Antiserum Rat
Strain
HO
1 2 3
HO HO HO
<2 <2 <2
4 5 6
A0 A0 A0
7 8 9
DA DA DA
<2 <2 <2
10 11 12
AU AU AU
<2 4 32
RBC coated with thyroid
64 64 64
extract
from
DA
AU
<2 <2 <2
<2 <2 <2
<2
<2 <2 <2
512 512 2.56
256 256 256
<2 <2 128
8 <2 64
<2 <2 32
512 2048 2048
512 2048 1024
64 256 256
A0
<2 <2
There is a consistent tendency for antisera to react more strongly with allogeneic thyroid extract than the syngeneic extract. It is especially apparent in tests with the A0 antisera, which failed to react with thyroid extract of the same strain while reacting with all other rat thyroid extracts. These results suggested that there were distinct determinants on the thyroid antigens of each of the strains. Therefore inhibition of hemagglutination tests were performed. A thyroid extract of each strain was able to inhibit completely the reaction with every other strain. Ouchterlony tests were also carried out with each antiserum against all thyroid extracts. Positive reactions were found with antisera 4, 5, 6, 9, 10, 11, 12. When individual preparations of thyroid antigens were compared, all antisera produced lines of complete identity. In immunoelectrophoresis, all antisera tested produced a single arc of precipitation in the alpha globulin region typical of thyroglobulin. DISCUSSION The novel finding in this investigation is the great difference in responsiveness of various inbred rat strains to rat thyroid extract. Furthermore, there was a dissociation of antibody production from the severity of the thyroid lesions. Although antibody levels were followed for only 4 wk, experience has shown that animals that do not have antibody at that time remain negative. These differences in antibody formation are highly significant. In another serological test, precipitation, confirmatory results were obtained, since only antisera with hemagglutination titers of 64 or above were positive. The histological observations were performed after 4 wk. Although thyroiditis produced in rats by injection of rat thyroid extract in Freund’s adjuvant has been reported to regress, the use of pertussis vaccine as coadjuvant leads to progressive disease without any evidence of regression (2).
DIFFERING
RESPONSES
OF RATS
TO EAT
363
The findings can be accounted for in two ways, which are not mutually exclusive. The first is that there are strain-related differences between the thyroid antigens. The data presented in Table 2 suggest that this is the case. Thyroglobulin is the only demonstrable autoantigen of thyroid in the rat. All sera that could be tested showed only a single precipitation line with mobility in immunoelectrophoresis of thyroglobulin. Thyroid extract of any strain could neutralize completely the reaction of any other strain, suggesting that the antigenic determinants are all present, although they may differ in quantitative expression. At first it might seem that the HO rats responded only to the allogeneic determinants of the thyroglobulin molecule. However, the fact that the animals all had pathological changes in their thyroids indicated that autologous determinants were also involved. Perhaps the syngeneic determinants are involved more in cellular than humoral responses, suggesting that genetic control of T cell effecters may differ from B cell responses. Finally, the four strains show great differences in immunological responses to thyroid antigen. It is tempting to postulate that it is due to the inheritance of one or more immune response genes for particular determinants of thyroglobulin. It is interesting that immune responsiveness in terms of antibody formation does not appear to be linked to serologically demonstrable Ag-B antigens while the capacity to develop thyroid lesions may be. The AU and HO rats, which are both Ag-B5, show minimal thyroid pathology despite the fact that HO is an antibody nonresponder and AU is a good responder. A0 and DA also show no relation between antibody responses and severity of lesions. We considered the further possibility that lymphocyte-determined histocompatibility differences might distinguish HO from AU strains of rats, and performed local graft vs host assays by the lymph node enlargement method (10). Even 50 X 10G HO spleen cells failed to produce significant lymph node enlargement in (AU x HO) F1 recipient mice. Gasser ef al. (11) found that the genetic locus controlling susceptibility to experimental allergic encephalomyelitis in rat strains is closely associated with or linked to Ag-B but that the specific gene in question was not Ag-B4. Based on the data shown in Table 1, Ag-B4, in addition to Ag-B2, may be linked to the allele favoring susceptibility to experimental autoimmune thyroiditis. Thus, a contrasting relationship may be present with respect to genetic factors influencing development of experimental allergic encephalomyelitis as opposed to development of experimental autoimmune thyroiditis. An analogous situation has been described in the guinea pig, i.e., strain 2 animals are much more susceptible to induction of experimental allergic thyroiditis than are strain 13 guinea pigs, as recently reported by BraleyMulle, Sharp, and Kyriakos (12), whereas just the reverse situation has been described in studies of the comparative susceptibility of these two inbred strains of guinea pigs to experimental allergic encephalomyelitis (13). ACKNOWLEDGMENTS The author wishes to acknowledge the hospitality and encouragement of Prof. James L. Gowans and the technical assistance of Mr. Steven Simmonds. He received support from the Buswell Foundation and NIH Research Grant CA02357 from the National Cancer Institute.
REFERENCES 1. Jones, H. E. H., and Roitt, I. M., Brit. J. Exp. Pathol. 42, 546, 1961. 2. Twarog, F. J., and Rose, N. R., Proc. Sot. Exp. Biol. Med. 130, 435, 1969. 3. Twarog, F. J., Kenney, E., and Rose, N. R., Prof. Sot. Exp. Biol. Med. 133, 185, 1970.
364 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
NOELR.ROSE
Twarog, F. J., and Rose, N. R., J. Immzmol. 104, 1467, 1970. Rose, N. R., Molotchnikoff, M.-F., and Twarog, F. J., Immunology 24, 859, 1973. McMaster, P. R. B., Owens, J. D., and Kyriakos, W., Cell. Zmmunol. 14, 39, 1974. Vladutiu, A. O., and Rose, N. R., Science 174, 1137, 1971. Tomazic, B., Rose, N. R., and Shreffler, D. C., J. Immwnol. 112, 965, 1974. Paterson, P. Y., and Drobish, D. G., J. Immunol. 101, 1098, 1968. Ford, W. L., Burr, W., and Simensen, M., Trulzsphntation 10, 258, 1970. Gasser, D. L., Newlin, C. M., Palm, J., and Gonatas, N. K., Sc&ence 181, 872, 1973. Braley-Mullen, H., Sharp, G. C., and Kyriakos, M., J. Immunol. 114, 371, 1975. Stone, S. H., Lerner, E. M., and Goode, J. H., Proc. Sot. Exp. Biol. Med. 132, 341, 1969.