Immunology 1976 30 63
Autoimmume murine thyroiditis VIII. ROLE OF DIFFERENT THYROID ANTIGENS IN THE INDUCTION OF EXPERIMENTAL AUTOIMMUNE THYROIDITIS
VESNA TOMAZIC* & N. R. ROSE Department of Immunology and Microbiology, Wayne State University, School of Medicine, Detroit, Michigan, U.S.A.
Received 28 May 1975; acceptedfor publication 9 July 1975
Summary. Mice of the C57Br strain, which are susceptible to the induction of autoimmune thyroiditis with mouse thyroglobulin, and C57BI mice, which are resistant, were immunized with human and rabbit thyroglobulins in Freund's complete adjuvant. Susceptible strain C57Br developed higher degrees of thyroid infiltration than the resistant strain. The results indicate that the responses to xenogeneic (foreign) thyroglobulins parallel allogeneic and syngeneic (mouse) thyroglobulin. BSVS mice, which are highly susceptible to thyroiditis, were immunized with mouse thyroid extract from five different mouse strains including syngeneic antigen. Recipients of C57BI and DBA thyroid extracts showed lower indices of pathology than recipients of similar extracts from C3H, BSVS and non-inbred CF-1 mice. The results suggest that there is a difference in the immunogenicity of mouse thyroid extracts from different strains. Purified thyroglobulin was prepared from congenic strains B1O.D2 (H-2d, resistant) and B1OBr (H-2k, susceptible). H-2k thyroglobulin gave a greater response in both H-2 k and H-2d mice than H-2d thyroglobulin. * On leave of absence from 'Rudjer Boskovic' Institute
INTRODUCTION Extensive studies of experimental autoimmune thyroiditis in different animal species strongly suggested that thyroglobulin is the major autoantigen in thyroid tissue (Shulman, 1971). The circumstances under which thyroglobulin becomes immunogenic are still unclear. Autoimmune thyroiditis can be induced with an aqueous solution of xenogeneic thyroglobulin or with syngeneic thyroglobulin when incorporated into Freund's complete adjuvant, but an aqueous solution of syngeneic thyroglobulin is not effective. It may be assumed that thyroglobulin must possess some foreign antigenic determinants in its structure in order to become autoimmunogenic unless there is some abnormality of the immunological recognition process. In this paper we compare the ability of thyroglobulin from different sources to induce autoimmune thyroiditis in the mouse. The effectiveness of xenogeneic thyroglobulins and of allogeneic and syngeneic thyroglobulins were compared. A possible genetic polymorphism reflected in the antigenic determinants on the thyroglobulin molecule of mice is described.
Zagreb, Yugoslavia.
Correspondence: Prof. N. R. Rose, Department of Immunology and Microbiology, 7374 Gordon Scott Hall, Wayne State University School of Medicine, 540 East Clanfield Avenue, Detroit, Michigan 48201, U.S.A.
MATERIALS AND METHODS Animals Eight to 10-week-old female mice were used for 63
64
Vesna Tomazic & N. R. Rose
immunization in all experiments. The donors of the thyroid tissue were 45-50-week-old females. All strains of mice were supplied by Jackson Laboratories, Bar Harbor, Maine, except non-inbred line CF-1 obtained from Carworth Farms, New City, New York and BSVS mice raised in our own animal unit. Antigen
The preparation of thyroid extract from human, rabbit and mouse tissues was described previously (Rose, Twarog and Crowle, 1971). Extracts were prepared separately from each strain of mice. Thyroglobulin was purified utilizing a column of Sephadex G-200 (Pharmacia, Uppsala, Sweden) equilibrated with saline. In the mixture of soluble proteins from thyroid tissue, thyroglobulin appeared in the first peak. Purity of the fractions was tested by immunoelectrophoresis using antisera to mouse thyroid crude extract and mouse serum. A single precipitin band was found in the a-globulin zone using antiserum to thyroid extract, and no reaction occurred with antiserum to mouse serum. Immunization Mice in all experiments were immunized with two subcutaneous injections of thyroid extract or purified thyroglobulin incorporated into Freund's complete adjuvant (FCA) (Tomazic, Rose and Shreffler, 1974). Two injections containing 30 ,ug of antigen each were given 1 week apart.
Serological analysis Sera were obtained from immunized mice 4 weeks after the first injection. The titration of antibodies to thyroglobulin was performed separately for each individual animal. Titrations were performed by a micro-modification of the passive haemagglutination test using tanned human erythrocytes coated with soluble thyroid extract or with purified thyroglobulin from different strains of mice (Tomazic et al., 1974). Histological examination Immediately after bleeding, mice were killed and thyroids were removed for histological examination Sections were made at six different levels throughout the organ. The scoring of the pathology was described previously (Vladutiu and Rose, 1971 b).
RESULTS
Properties of xenogeneic and mouse antigens In the first group of experiments, two different strains of mice were used for immunization. The C57Br/cd strain was shown to be susceptible to the induction of autoimmune thyroiditis while C57BI/10 mice were resistant (Vladutiu and Rose, 1971b). Three groups of C57BI/10 mice, twenty-four in each group, were immunized with mouse, human and rabbit thyroglobulin, respectively. Another three groups of C57Brfcd mice with seventeen to eighteen mice in each received similar immunization. All animals were given two injections of antigen in FCA and were killed 4 weeks after the first injection. Antibody titres of the sera were determined and the thyroid glands were examined for pathological changes. Strain-dependent differences in the immune response to thyroglobulin was evident in the cases of all three antigens used. Resistant strain C57B1/10 developed minimal thyroiditis when immunized with any of the three antigens, while C57Br/cd mice developed consistently higher degrees of infiltration (Table 1). In C57Br/cd mice, human and rabbit thyroglobulins were less effective than mouse thyroglobulin. It appears that there is a greater response of the good responder strains when xenogeneic as well as mouse antigens are used. In the next experiment we attempted to evaluate possible differences in autoimmune thyroiditis induced by thyroid extracts obtained from several different strains of mice. The use of crude extracts obviated any possible alterations induced by fractionation. Recipients in this experiment were BSVS mice, which had been found to be highly susceptible to immunization with mouse thyroid extract from CF-i donors (Rose, Vladutiu, David and Shreffler, 1973). Five groups of twelve BSVS mice were immunized with the same amount of mouse thyroid extract (MTE) from five different sources including the syngeneic antigen. The allogeneic donor strains were DBA/2 and C57BI/ 10, which were shown to be resistant to autoimmune thyroiditis; C3H/He mice, which are susceptible; and non-inbred CF-1 mice. All groups of mice received the same amount of antigen and were killed 4 weeks after the first injection. Results are shown in Table 2. Some degree of thyroiditis was found in all BSVS mice. However, when recipients were immunized with MTE from the poor responder strains, C57Bl/10 and DBA/2, the
Different thyroid antigens in autoimmune thyroiditis
65
Table 1. Immune response of mice injected with xenogeneic or mouse thyroglobulin
Antibody titrest
Antigen used for Recipient strains immunization MTg
C57B1/10 (H-2b)
HTg RTg MTg
C57Br/cd (H-2k)
HTg RTg
Pathology index*
MTgt
HTg
RTg
0-20+01
7T5+O07
3-2+03
4-9+0-3
025+02 0.10+0.1 300+0-8
90+10 7-6+0-7 12-1+0-9
16-3+1-1 11 1+10 7-3+0-6
6-4+0-5 12-4+0-9
1-61+07 1-O1+0-5
9-2+0 7 8 4+0 5
18-0+07 12-3+0-9
8-4+0-7 14-6+1-1
9-6+0-7
* Mean degree of thyroid infiltration ± s.d. t MTg= mouse thyroglobulin; HTg= human thyroglobulin; RTg= rabbit thyroglobulin. I Geometric mean of haemagglutination titre (log2 + s.d.). Table 2. Immune response of BSVS mice to mouse thyroid extracts from different strains of mice
Strains used as source of antigen (H-2 type) BSVS (H-2th) CF-1 (not inbred) C3H/He (H-2k)
C57B1/10 (H-2b) DBA/2 (H-2d)
Pathology
Antibody
index
Titre
3-10+0-4 2 35+05 2-70+0-6 1-75+0-2 1-50+0-3
12-3+ 1.0 12-3+ 1-2 11-4+0-5 11-0+ 0-8 11-1+0-7
pathology indices were lower than with MTE from good responder strains (P < 0-05; t-test). No marked difference in the antibody titres of the groups immunized with different antigens was observed. From these experiments it seems likely that there is a difference in the immunogenicity of mouse thyroid extracts from different strains.
Immune response of mice to purified mouse thyroglobulin from congenic donors In order to clarify the possible existence of different antigenic determinants on the molecules of thyroglobulin from different strains of mice the following experiments were designed. Two congenic strains of mice, B1O.D2/n and BlO.Br/SgSn, were used as donors. Except for the H-2 complex, these two strains are identical. The BlO.Br strain, bearing H-2k, was determined to be a good responder to CF-1 thyroglobulin while BlO.D2 (H-2d) was a poor responder.
Purified thyroglobulins from the extract of each strain were used as immunogen. Using thyroglobulin from BlO.D2/n mice in FCA we immunized fifteen B1O.D2 mice, fifteen mice of DBA/2 strain, which is also H-2d, and fifteen congenic BlO.Br mice. The same procedure was repeated using thyroglobulin from BlO.BrSg donors. Again there were fifteen mice in each immunized group. Recipients were the BlO.Br strain; another H-2k bearing strain, C57Br/cd; and the congenic H-2d mice, B1O.D2. All animals received two injections of antigen and were killed 4 weeks after the first injection. Results are summarized in Table 3. A difference in the degree of thyroiditis was observed in members of the same strain immunized with two different antigens. BMO.D2 and DBA/2 strains developed minimal thyroiditis when immunized with thyroglobulin from the BMO.D2 strain, but a higher degree of disease occurred in BlO.D2 mice when immunized with BlO.Br antigen (P
Autoimmume murine thyroiditis VIII. ROLE OF DIFFERENT THYROID ANTIGENS IN THE INDUCTION OF EXPERIMENTAL AUTOIMMUNE THYROIDITIS
VESNA TOMAZIC* & N. R. ROSE Department of Immunology and Microbiology, Wayne State University, School of Medicine, Detroit, Michigan, U.S.A.
Received 28 May 1975; acceptedfor publication 9 July 1975
Summary. Mice of the C57Br strain, which are susceptible to the induction of autoimmune thyroiditis with mouse thyroglobulin, and C57BI mice, which are resistant, were immunized with human and rabbit thyroglobulins in Freund's complete adjuvant. Susceptible strain C57Br developed higher degrees of thyroid infiltration than the resistant strain. The results indicate that the responses to xenogeneic (foreign) thyroglobulins parallel allogeneic and syngeneic (mouse) thyroglobulin. BSVS mice, which are highly susceptible to thyroiditis, were immunized with mouse thyroid extract from five different mouse strains including syngeneic antigen. Recipients of C57BI and DBA thyroid extracts showed lower indices of pathology than recipients of similar extracts from C3H, BSVS and non-inbred CF-1 mice. The results suggest that there is a difference in the immunogenicity of mouse thyroid extracts from different strains. Purified thyroglobulin was prepared from congenic strains B1O.D2 (H-2d, resistant) and B1OBr (H-2k, susceptible). H-2k thyroglobulin gave a greater response in both H-2 k and H-2d mice than H-2d thyroglobulin. * On leave of absence from 'Rudjer Boskovic' Institute
INTRODUCTION Extensive studies of experimental autoimmune thyroiditis in different animal species strongly suggested that thyroglobulin is the major autoantigen in thyroid tissue (Shulman, 1971). The circumstances under which thyroglobulin becomes immunogenic are still unclear. Autoimmune thyroiditis can be induced with an aqueous solution of xenogeneic thyroglobulin or with syngeneic thyroglobulin when incorporated into Freund's complete adjuvant, but an aqueous solution of syngeneic thyroglobulin is not effective. It may be assumed that thyroglobulin must possess some foreign antigenic determinants in its structure in order to become autoimmunogenic unless there is some abnormality of the immunological recognition process. In this paper we compare the ability of thyroglobulin from different sources to induce autoimmune thyroiditis in the mouse. The effectiveness of xenogeneic thyroglobulins and of allogeneic and syngeneic thyroglobulins were compared. A possible genetic polymorphism reflected in the antigenic determinants on the thyroglobulin molecule of mice is described.
Zagreb, Yugoslavia.
Correspondence: Prof. N. R. Rose, Department of Immunology and Microbiology, 7374 Gordon Scott Hall, Wayne State University School of Medicine, 540 East Clanfield Avenue, Detroit, Michigan 48201, U.S.A.
MATERIALS AND METHODS Animals Eight to 10-week-old female mice were used for 63
64
Vesna Tomazic & N. R. Rose
immunization in all experiments. The donors of the thyroid tissue were 45-50-week-old females. All strains of mice were supplied by Jackson Laboratories, Bar Harbor, Maine, except non-inbred line CF-1 obtained from Carworth Farms, New City, New York and BSVS mice raised in our own animal unit. Antigen
The preparation of thyroid extract from human, rabbit and mouse tissues was described previously (Rose, Twarog and Crowle, 1971). Extracts were prepared separately from each strain of mice. Thyroglobulin was purified utilizing a column of Sephadex G-200 (Pharmacia, Uppsala, Sweden) equilibrated with saline. In the mixture of soluble proteins from thyroid tissue, thyroglobulin appeared in the first peak. Purity of the fractions was tested by immunoelectrophoresis using antisera to mouse thyroid crude extract and mouse serum. A single precipitin band was found in the a-globulin zone using antiserum to thyroid extract, and no reaction occurred with antiserum to mouse serum. Immunization Mice in all experiments were immunized with two subcutaneous injections of thyroid extract or purified thyroglobulin incorporated into Freund's complete adjuvant (FCA) (Tomazic, Rose and Shreffler, 1974). Two injections containing 30 ,ug of antigen each were given 1 week apart.
Serological analysis Sera were obtained from immunized mice 4 weeks after the first injection. The titration of antibodies to thyroglobulin was performed separately for each individual animal. Titrations were performed by a micro-modification of the passive haemagglutination test using tanned human erythrocytes coated with soluble thyroid extract or with purified thyroglobulin from different strains of mice (Tomazic et al., 1974). Histological examination Immediately after bleeding, mice were killed and thyroids were removed for histological examination Sections were made at six different levels throughout the organ. The scoring of the pathology was described previously (Vladutiu and Rose, 1971 b).
RESULTS
Properties of xenogeneic and mouse antigens In the first group of experiments, two different strains of mice were used for immunization. The C57Br/cd strain was shown to be susceptible to the induction of autoimmune thyroiditis while C57BI/10 mice were resistant (Vladutiu and Rose, 1971b). Three groups of C57BI/10 mice, twenty-four in each group, were immunized with mouse, human and rabbit thyroglobulin, respectively. Another three groups of C57Brfcd mice with seventeen to eighteen mice in each received similar immunization. All animals were given two injections of antigen in FCA and were killed 4 weeks after the first injection. Antibody titres of the sera were determined and the thyroid glands were examined for pathological changes. Strain-dependent differences in the immune response to thyroglobulin was evident in the cases of all three antigens used. Resistant strain C57B1/10 developed minimal thyroiditis when immunized with any of the three antigens, while C57Br/cd mice developed consistently higher degrees of infiltration (Table 1). In C57Br/cd mice, human and rabbit thyroglobulins were less effective than mouse thyroglobulin. It appears that there is a greater response of the good responder strains when xenogeneic as well as mouse antigens are used. In the next experiment we attempted to evaluate possible differences in autoimmune thyroiditis induced by thyroid extracts obtained from several different strains of mice. The use of crude extracts obviated any possible alterations induced by fractionation. Recipients in this experiment were BSVS mice, which had been found to be highly susceptible to immunization with mouse thyroid extract from CF-i donors (Rose, Vladutiu, David and Shreffler, 1973). Five groups of twelve BSVS mice were immunized with the same amount of mouse thyroid extract (MTE) from five different sources including the syngeneic antigen. The allogeneic donor strains were DBA/2 and C57BI/ 10, which were shown to be resistant to autoimmune thyroiditis; C3H/He mice, which are susceptible; and non-inbred CF-1 mice. All groups of mice received the same amount of antigen and were killed 4 weeks after the first injection. Results are shown in Table 2. Some degree of thyroiditis was found in all BSVS mice. However, when recipients were immunized with MTE from the poor responder strains, C57Bl/10 and DBA/2, the
Different thyroid antigens in autoimmune thyroiditis
65
Table 1. Immune response of mice injected with xenogeneic or mouse thyroglobulin
Antibody titrest
Antigen used for Recipient strains immunization MTg
C57B1/10 (H-2b)
HTg RTg MTg
C57Br/cd (H-2k)
HTg RTg
Pathology index*
MTgt
HTg
RTg
0-20+01
7T5+O07
3-2+03
4-9+0-3
025+02 0.10+0.1 300+0-8
90+10 7-6+0-7 12-1+0-9
16-3+1-1 11 1+10 7-3+0-6
6-4+0-5 12-4+0-9
1-61+07 1-O1+0-5
9-2+0 7 8 4+0 5
18-0+07 12-3+0-9
8-4+0-7 14-6+1-1
9-6+0-7
* Mean degree of thyroid infiltration ± s.d. t MTg= mouse thyroglobulin; HTg= human thyroglobulin; RTg= rabbit thyroglobulin. I Geometric mean of haemagglutination titre (log2 + s.d.). Table 2. Immune response of BSVS mice to mouse thyroid extracts from different strains of mice
Strains used as source of antigen (H-2 type) BSVS (H-2th) CF-1 (not inbred) C3H/He (H-2k)
C57B1/10 (H-2b) DBA/2 (H-2d)
Pathology
Antibody
index
Titre
3-10+0-4 2 35+05 2-70+0-6 1-75+0-2 1-50+0-3
12-3+ 1.0 12-3+ 1-2 11-4+0-5 11-0+ 0-8 11-1+0-7
pathology indices were lower than with MTE from good responder strains (P < 0-05; t-test). No marked difference in the antibody titres of the groups immunized with different antigens was observed. From these experiments it seems likely that there is a difference in the immunogenicity of mouse thyroid extracts from different strains.
Immune response of mice to purified mouse thyroglobulin from congenic donors In order to clarify the possible existence of different antigenic determinants on the molecules of thyroglobulin from different strains of mice the following experiments were designed. Two congenic strains of mice, B1O.D2/n and BlO.Br/SgSn, were used as donors. Except for the H-2 complex, these two strains are identical. The BlO.Br strain, bearing H-2k, was determined to be a good responder to CF-1 thyroglobulin while BlO.D2 (H-2d) was a poor responder.
Purified thyroglobulins from the extract of each strain were used as immunogen. Using thyroglobulin from BlO.D2/n mice in FCA we immunized fifteen B1O.D2 mice, fifteen mice of DBA/2 strain, which is also H-2d, and fifteen congenic BlO.Br mice. The same procedure was repeated using thyroglobulin from BlO.BrSg donors. Again there were fifteen mice in each immunized group. Recipients were the BlO.Br strain; another H-2k bearing strain, C57Br/cd; and the congenic H-2d mice, B1O.D2. All animals received two injections of antigen and were killed 4 weeks after the first injection. Results are summarized in Table 3. A difference in the degree of thyroiditis was observed in members of the same strain immunized with two different antigens. BMO.D2 and DBA/2 strains developed minimal thyroiditis when immunized with thyroglobulin from the BMO.D2 strain, but a higher degree of disease occurred in BlO.D2 mice when immunized with BlO.Br antigen (P
