J Endocrinol. Invest. 13. 871
~878.
1990
Effects of recombinant human interferon gamma on human thyroid· tissues from patients with Graves' disease and normal subjects transplanted into nude mice 1,2 y. Kasuga 3 , S. Matsubayashi 4 , Y. Sakatsume 4 , N. Miller**, C. Jamieson***, and R. Volpe* *Endocrinology Research Laboratory and Departments of Medicine, **Pathology, and ***Surgery, The Wellesley Hospital, University of Toronto, Toronto, Ontario, Canada
ABSTRACT. We have attempted to determine whether inferferon gamma (IFNI') would enhance, sustain or induce autoimmune thyroid disease (AITD) in xenotransplanted thyroid tissue fram patients with Graves' disease or normal persons (actually paranodular tissue) in nude athymic mice, in the absence of an intact immune system. A dosage of 4000 U/mouse of human IFNI'(hIFNI') was injected intraperitoneally daily for six consecutive weeks into the xenotransplanted mice. The parameters measured included the free T4 index, thyroid autoantibodies and TSH during the course of hlFNI' injections. Thyroid epithelial cell (TEC) HLA-DR expression was measured in the thyroid tissue before xenotransplantation and at sacrifice; in addition, light and electron microscopic studies were carried out at those times. There were no significant differences in thyroid function between the control results and those obtained with hlFNI'
in either graup of tissues. TEC HLA-DR expression was significantly increased by hlFNI' in the normal group, but insignificantly in the Graves' group. In both light and electron microscopic observations, Graves' tissue (whether or not treated with hIFNI') was indistinguishable at sacrifice from normal thyraid tissue. The appearance had markedly altered from the same Graves' tissue examined at the time of the initial human surgery, wh ich then showed the usual histological appearance of this disorder. We conclude that IFNI' induced HLA-DR expression alone is not sufficient to sustain the ongoing process of AITD in this model. Graves' TEC appear to be essentially normal when removed from their immune environment; it may be proposed that TEC may be mere passive captives to immunologie events in terms of the pathogenesis of AITD, without any intrinsic functional abnormality.
INTRODUCTION The role of interferon gamma (IFNI') in the development of autoimmune thyroid disease (AITD) has been the subject of considerable interest over the past few years (1 ~3). There is now considerable
evidenee that the expression of thyrocyte HLA-DR in AITD is a seeondary phenomenon indueed by the elaboration of IFNI' by al ready sensitized intrathyroidal T Iymphoeytes (4,5). There is evidenee that monoeytes are also necessary for these events to oceur (4), presumably aeting as the initial antigenpresenting cells, Interferon gamma has been shown to induee an inerease in eyelic AMP in target eel/s, suggesting an inerease in eellular activity as a result of this Iymphokine (6,7). However, there have also been reports that administered interferons have induced AITD and hypothyroidism in humans as weil as in experimental animals (8~ 14). The experimental studies of Kawakami et al. (9) were of eonsiderable interest in this respeet, since the authors noted that
Key-words: Nude mice. Interferon gamma. Graves' disease. xenografts. Correspondence' Prof Robert Volpe. Endocrinology Research Laboratory. The Wellesley Hospital. 160 Wellesley Street East. Toronto. Ontario M4Y IJ3 Canada Received March 1. 1990; accepted August 29, 1990 'This work was supported by a grant from the Medlcal Research Council of Canada (MT 859). 2Presented in part at the 64th Annual Meeting of the American Thyroid Associat,on, San Francisco, Californla, September 6-9, 1989.
3Fellow. Wellesley Hospital Research Foundation, 'Fellows, Angus Foundation, The Wellesley Hospital.
871
Y. Kasuga, S. Matsubayashi, Y. Sakatsume, et al.
despite continuing injections of IFN')' to intact mice, the induction of thyroid autoantibodies and thyroiditis so induced ultimately declined. These observations cast into so me doubt a central role for IFN')' in the induction of AITD. The present study was initiated to determine whether IFN')' could enhance, sustain, or induce AITD in xenotransplanted thyroid tissue from patients with Graves' disease or normal persons (actually paranodular tissue) in a nude athymic mouse model, in which there is largely lacking a T Iymphocyte immune system (the mice do retain so me immune functions, such as B Iymphocytes, and some gamma-delta T Iymphocytes). They however cannot reject solid tissue xenografts, although xenogeneic Iymphocytes do not survive (unpublished data). We have therefore studied the functional, immunological and histological effects of administering this Iymphokine on the human thyroid tissue under these circumstances. It was our view that this model had so me advantages over in vitra thyrocyte cultures, because of the intact architectural structure of the tissue. MATERIALS AND METHODS Balb/C nude (athymic) mice of an outbred strain (Harlan Sprague Dawley Inc., Indianapolis, USA) were used in this study. They were 4-6 weeks old with an initial body weight of approximately 20 g. The thyroid gland of each mouse was completely ablated with 0.2 millicuries of 131 1(Merck Frost Canada, Kirkland Lake, Canada). The mice were shown to be hypothyroid by routine laboratory tests including low free thyroxine and high TSH values. Xenotransplantation was performed 3-4 weeks after the 131 1 ablation at a time when hypothyroidism was clearly demonstrable by the above laboratory tests. Xenotransplantation was carried out by the method of Smeds et al. (15) and Usadei et al. (16). Briefly, human thyroid tissues were obtained at surgery from two patients with Graves' disease and paranodular ("normal") tissue was obtained at the time of removal of thyroid nodules from two patients. The patients with Graves' disease had been treated with antithyroid drugs prior to surgery; however, both were still positive for thyroid stimulating antibody and microsomal antibody, and both displayed Iymphocytic infiltration in their thyroid histology. Surgical specimens selected from several areas of the resected tissue for xenograft were cut into fragments measuring about 2 x 2 x 2 mm. The initial
pathological examinations on the resected tissue were performed on sam pies taken adjacent to the above specimens. About five fragments were xenografted subcutaneously into each groin of the mouse within two hours after the human surgery. Survival of the xenograft was clearly indicated by the return of thyroid function to normal in the animals that were xenografted, whereas there was no return to normal in those that were left as controls. After approximately four weeks following the xenografts, recombinant human IFN')' (hIFN')') (Genzyme Company, Boston, USA) was injected intraperitoneally into the mice on a daily basis for six consecutive weeks. The dosage provided was 4,000 units of hIFN')', considered analogous to dosages employed for anticancer therapy (12-14). Blood from the tail vein of the mice was taken every two weeks until sacrifice. In this study, Graves' thyroid tissue was xenografted into nine mice, and normal thyroid tissue into seven mice. The animals survived the procedure weil. The study consisted of two sequential components: i.e., segments of one Graves' gland (into 5 mice) and one normal gland (into 4 mice) were xenografted at the same time, followed bya similar procedure with 4 and 3 animals respectively. The results did not differ significantly between these two procedures and the means of the various groups are thus considered together.
Measurements perfarmed The serum thyroxine (T 4 ) concentrations were measured by a commercial radioimmunoassay (Joldon Diagnostics, Scarborough, Ontario). A T3 resin uptake was also measured by a similar commercial kit and the free thyroxine index (fTI) was calculated as the serum thyroxine in nmol/L x T; resin uptake (17,18). The mouse TSH was measured by radioimmunoassay using rat TSH and anti-TSH antibody provided by a generous gift of Dr. Salvatore Raiti, National Institutes of Diabetes, Digestive and Kidney Disease, Bethesda, Maryland, and a mouse TSH standard generously provided by Dr. A.F. Parlow, UCLA, Los Angeles, and the National Hormone and Pituitary Program, University of Maryland School of Medicine. Thyroid autoantibodies (antithyroglobulin and antimicrosomal antibody) were measured by haemagglutination kits (Wellcome Diagnostics, Dartford, England). For measurements of thyrocyte HLA-DR expression, the thyroid tissue was cultured as previously de-
872
Effect of fFNy on Graves' thyroid grafts in nude mice
scribed (4,19,20). Briefly, the finely minced tissue was digested with 2 mg/mi collagen ase (Type 11, Sigma, St. Louis, Missouri) at 37 C for sixty minutes. The pellet obtained after ten minutes of centrifugation at 600 x 9 was washed twice with phosphatebuffered saline (PBS) and once with complete medium [RPMI-1640 (Gibco, Burlington, Ontario)] containing 20% fetal bovine serum substitute (Sigma), 1% antibiotic-antimycotic solution and 2 nmol/I glutamine (Gibco), plated in Petri a dish and incubated at 37 C in a humidified atmosphere of 5% CO 2 . The confluent cells after one day of culture were trypsinized [0.5% trypsin in 0.02% EDTA (Gibco)], washed with PBS and medium and transferred into the wells (40,000 cells/well) of a Linbro 96-well tissue culture plate (Flow Laboratories Inc., Virginia). Cell viability by trypan blue exclusion was greater than 90%. The thyrocyte HLA-DR expression was measured by an enzyme linked immunosorbent assay (ELlSA) technique which has been published elsewhere (2123). This was performed on thyroid tissue before xenotransplantation (Le., immediately following the human surgery) and on the same tissues after sacrifice of the nude mice. Briefly, after two days' culture in the plates, the medium was removed and the cells washed twice in 0.1 % bovine serum albumin (BSA) in PBS; 50 JLI of anti-human HLA-DR mouse monoclonal antibody I1 :15 (Becton-Dickinson, Mississauga, Ontario)] was added to the thyroid epithelial cells (TEC) and incubated at 37 C for 30 minutes and then washed three times in 0.1 % BSA-PBS. Next, 100 JLI of 1:1 000 peroxidase-conjugated goat anti-mouse IgG (Sigma) was added in the weil and incubated for about 45 minutes at 37 C and washed twice in 0.1 % BSA-PBS. The volume of each weil was adjusted to 100 JLI with 0.1 % BSA-PBS. Then 100 JLI of substrate containing 0.04% o-phenylenediamine [OPO (Sigma)] and 0.01 % H20 2 in phosphate citrate buffer (pH 5) was added. The plates were left in the dark at room temperature for 10 minutes. The reaction was stopped by the addition of 100 JLI of 10N H2 S0 4 to each weil and read in a spectrophotometer at 490 nm. HLA-DR expression in the thyrocytes was calculated as foliows: Experimental optical density with anti-HLA-DR antibody - optical density of control without anti-HLA-DR antibody x 103 .
was submitted to the Department of Pathology for routine light and electron microscopic studies for the purpose of comparison with the subsequent appearance of the same tissues. Following sacrifice, the xenografted thyroid tissues were subjected to the same studies. These tissues were fixed in neutral formalin and embedded in paraffin for routine light microscopic examination. The staining was that of hematoxylin-eosin. The tissue for electron microscopic studies was post fixed in 2.5% glutaraldehyde and then dehydrated in graded se ries of ethanol and embedded in Epon. Ultrathin sections were then analyzed in the electron microscope. Statistical analysis Statistical analysais of data was performed by the student's t test. p
~878.
1990
Effects of recombinant human interferon gamma on human thyroid· tissues from patients with Graves' disease and normal subjects transplanted into nude mice 1,2 y. Kasuga 3 , S. Matsubayashi 4 , Y. Sakatsume 4 , N. Miller**, C. Jamieson***, and R. Volpe* *Endocrinology Research Laboratory and Departments of Medicine, **Pathology, and ***Surgery, The Wellesley Hospital, University of Toronto, Toronto, Ontario, Canada
ABSTRACT. We have attempted to determine whether inferferon gamma (IFNI') would enhance, sustain or induce autoimmune thyroid disease (AITD) in xenotransplanted thyroid tissue fram patients with Graves' disease or normal persons (actually paranodular tissue) in nude athymic mice, in the absence of an intact immune system. A dosage of 4000 U/mouse of human IFNI'(hIFNI') was injected intraperitoneally daily for six consecutive weeks into the xenotransplanted mice. The parameters measured included the free T4 index, thyroid autoantibodies and TSH during the course of hlFNI' injections. Thyroid epithelial cell (TEC) HLA-DR expression was measured in the thyroid tissue before xenotransplantation and at sacrifice; in addition, light and electron microscopic studies were carried out at those times. There were no significant differences in thyroid function between the control results and those obtained with hlFNI'
in either graup of tissues. TEC HLA-DR expression was significantly increased by hlFNI' in the normal group, but insignificantly in the Graves' group. In both light and electron microscopic observations, Graves' tissue (whether or not treated with hIFNI') was indistinguishable at sacrifice from normal thyraid tissue. The appearance had markedly altered from the same Graves' tissue examined at the time of the initial human surgery, wh ich then showed the usual histological appearance of this disorder. We conclude that IFNI' induced HLA-DR expression alone is not sufficient to sustain the ongoing process of AITD in this model. Graves' TEC appear to be essentially normal when removed from their immune environment; it may be proposed that TEC may be mere passive captives to immunologie events in terms of the pathogenesis of AITD, without any intrinsic functional abnormality.
INTRODUCTION The role of interferon gamma (IFNI') in the development of autoimmune thyroid disease (AITD) has been the subject of considerable interest over the past few years (1 ~3). There is now considerable
evidenee that the expression of thyrocyte HLA-DR in AITD is a seeondary phenomenon indueed by the elaboration of IFNI' by al ready sensitized intrathyroidal T Iymphoeytes (4,5). There is evidenee that monoeytes are also necessary for these events to oceur (4), presumably aeting as the initial antigenpresenting cells, Interferon gamma has been shown to induee an inerease in eyelic AMP in target eel/s, suggesting an inerease in eellular activity as a result of this Iymphokine (6,7). However, there have also been reports that administered interferons have induced AITD and hypothyroidism in humans as weil as in experimental animals (8~ 14). The experimental studies of Kawakami et al. (9) were of eonsiderable interest in this respeet, since the authors noted that
Key-words: Nude mice. Interferon gamma. Graves' disease. xenografts. Correspondence' Prof Robert Volpe. Endocrinology Research Laboratory. The Wellesley Hospital. 160 Wellesley Street East. Toronto. Ontario M4Y IJ3 Canada Received March 1. 1990; accepted August 29, 1990 'This work was supported by a grant from the Medlcal Research Council of Canada (MT 859). 2Presented in part at the 64th Annual Meeting of the American Thyroid Associat,on, San Francisco, Californla, September 6-9, 1989.
3Fellow. Wellesley Hospital Research Foundation, 'Fellows, Angus Foundation, The Wellesley Hospital.
871
Y. Kasuga, S. Matsubayashi, Y. Sakatsume, et al.
despite continuing injections of IFN')' to intact mice, the induction of thyroid autoantibodies and thyroiditis so induced ultimately declined. These observations cast into so me doubt a central role for IFN')' in the induction of AITD. The present study was initiated to determine whether IFN')' could enhance, sustain, or induce AITD in xenotransplanted thyroid tissue from patients with Graves' disease or normal persons (actually paranodular tissue) in a nude athymic mouse model, in which there is largely lacking a T Iymphocyte immune system (the mice do retain so me immune functions, such as B Iymphocytes, and some gamma-delta T Iymphocytes). They however cannot reject solid tissue xenografts, although xenogeneic Iymphocytes do not survive (unpublished data). We have therefore studied the functional, immunological and histological effects of administering this Iymphokine on the human thyroid tissue under these circumstances. It was our view that this model had so me advantages over in vitra thyrocyte cultures, because of the intact architectural structure of the tissue. MATERIALS AND METHODS Balb/C nude (athymic) mice of an outbred strain (Harlan Sprague Dawley Inc., Indianapolis, USA) were used in this study. They were 4-6 weeks old with an initial body weight of approximately 20 g. The thyroid gland of each mouse was completely ablated with 0.2 millicuries of 131 1(Merck Frost Canada, Kirkland Lake, Canada). The mice were shown to be hypothyroid by routine laboratory tests including low free thyroxine and high TSH values. Xenotransplantation was performed 3-4 weeks after the 131 1 ablation at a time when hypothyroidism was clearly demonstrable by the above laboratory tests. Xenotransplantation was carried out by the method of Smeds et al. (15) and Usadei et al. (16). Briefly, human thyroid tissues were obtained at surgery from two patients with Graves' disease and paranodular ("normal") tissue was obtained at the time of removal of thyroid nodules from two patients. The patients with Graves' disease had been treated with antithyroid drugs prior to surgery; however, both were still positive for thyroid stimulating antibody and microsomal antibody, and both displayed Iymphocytic infiltration in their thyroid histology. Surgical specimens selected from several areas of the resected tissue for xenograft were cut into fragments measuring about 2 x 2 x 2 mm. The initial
pathological examinations on the resected tissue were performed on sam pies taken adjacent to the above specimens. About five fragments were xenografted subcutaneously into each groin of the mouse within two hours after the human surgery. Survival of the xenograft was clearly indicated by the return of thyroid function to normal in the animals that were xenografted, whereas there was no return to normal in those that were left as controls. After approximately four weeks following the xenografts, recombinant human IFN')' (hIFN')') (Genzyme Company, Boston, USA) was injected intraperitoneally into the mice on a daily basis for six consecutive weeks. The dosage provided was 4,000 units of hIFN')', considered analogous to dosages employed for anticancer therapy (12-14). Blood from the tail vein of the mice was taken every two weeks until sacrifice. In this study, Graves' thyroid tissue was xenografted into nine mice, and normal thyroid tissue into seven mice. The animals survived the procedure weil. The study consisted of two sequential components: i.e., segments of one Graves' gland (into 5 mice) and one normal gland (into 4 mice) were xenografted at the same time, followed bya similar procedure with 4 and 3 animals respectively. The results did not differ significantly between these two procedures and the means of the various groups are thus considered together.
Measurements perfarmed The serum thyroxine (T 4 ) concentrations were measured by a commercial radioimmunoassay (Joldon Diagnostics, Scarborough, Ontario). A T3 resin uptake was also measured by a similar commercial kit and the free thyroxine index (fTI) was calculated as the serum thyroxine in nmol/L x T; resin uptake (17,18). The mouse TSH was measured by radioimmunoassay using rat TSH and anti-TSH antibody provided by a generous gift of Dr. Salvatore Raiti, National Institutes of Diabetes, Digestive and Kidney Disease, Bethesda, Maryland, and a mouse TSH standard generously provided by Dr. A.F. Parlow, UCLA, Los Angeles, and the National Hormone and Pituitary Program, University of Maryland School of Medicine. Thyroid autoantibodies (antithyroglobulin and antimicrosomal antibody) were measured by haemagglutination kits (Wellcome Diagnostics, Dartford, England). For measurements of thyrocyte HLA-DR expression, the thyroid tissue was cultured as previously de-
872
Effect of fFNy on Graves' thyroid grafts in nude mice
scribed (4,19,20). Briefly, the finely minced tissue was digested with 2 mg/mi collagen ase (Type 11, Sigma, St. Louis, Missouri) at 37 C for sixty minutes. The pellet obtained after ten minutes of centrifugation at 600 x 9 was washed twice with phosphatebuffered saline (PBS) and once with complete medium [RPMI-1640 (Gibco, Burlington, Ontario)] containing 20% fetal bovine serum substitute (Sigma), 1% antibiotic-antimycotic solution and 2 nmol/I glutamine (Gibco), plated in Petri a dish and incubated at 37 C in a humidified atmosphere of 5% CO 2 . The confluent cells after one day of culture were trypsinized [0.5% trypsin in 0.02% EDTA (Gibco)], washed with PBS and medium and transferred into the wells (40,000 cells/well) of a Linbro 96-well tissue culture plate (Flow Laboratories Inc., Virginia). Cell viability by trypan blue exclusion was greater than 90%. The thyrocyte HLA-DR expression was measured by an enzyme linked immunosorbent assay (ELlSA) technique which has been published elsewhere (2123). This was performed on thyroid tissue before xenotransplantation (Le., immediately following the human surgery) and on the same tissues after sacrifice of the nude mice. Briefly, after two days' culture in the plates, the medium was removed and the cells washed twice in 0.1 % bovine serum albumin (BSA) in PBS; 50 JLI of anti-human HLA-DR mouse monoclonal antibody I1 :15 (Becton-Dickinson, Mississauga, Ontario)] was added to the thyroid epithelial cells (TEC) and incubated at 37 C for 30 minutes and then washed three times in 0.1 % BSA-PBS. Next, 100 JLI of 1:1 000 peroxidase-conjugated goat anti-mouse IgG (Sigma) was added in the weil and incubated for about 45 minutes at 37 C and washed twice in 0.1 % BSA-PBS. The volume of each weil was adjusted to 100 JLI with 0.1 % BSA-PBS. Then 100 JLI of substrate containing 0.04% o-phenylenediamine [OPO (Sigma)] and 0.01 % H20 2 in phosphate citrate buffer (pH 5) was added. The plates were left in the dark at room temperature for 10 minutes. The reaction was stopped by the addition of 100 JLI of 10N H2 S0 4 to each weil and read in a spectrophotometer at 490 nm. HLA-DR expression in the thyrocytes was calculated as foliows: Experimental optical density with anti-HLA-DR antibody - optical density of control without anti-HLA-DR antibody x 103 .
was submitted to the Department of Pathology for routine light and electron microscopic studies for the purpose of comparison with the subsequent appearance of the same tissues. Following sacrifice, the xenografted thyroid tissues were subjected to the same studies. These tissues were fixed in neutral formalin and embedded in paraffin for routine light microscopic examination. The staining was that of hematoxylin-eosin. The tissue for electron microscopic studies was post fixed in 2.5% glutaraldehyde and then dehydrated in graded se ries of ethanol and embedded in Epon. Ultrathin sections were then analyzed in the electron microscope. Statistical analysis Statistical analysais of data was performed by the student's t test. p
