0021-972X/90/7101-0040$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 1 Printed in U.S.A.
Incidence of Antibodies Blocking Thyrotropin Effect In Vitro in Patients with Euthyroid or Hypothyroid Autoimmune Thyroiditis* LUCA CHIOVATO, PAOLO VITTI, FERRUCCIO SANTINI, GUADALUPE LOPEZ, CLAUDIA MAMMOLI, PABLITO BASSI, LIA GIUSTI, MASSIMO TONACCHERA, GIANFRANCO FENZI, AND ALDO PINCHERA Cattedra di Endocrinologia e Medicina Costituzionale, University of Pisa, Pisa, Italy
32 (9.4%) with HT-SH, and in 20 of 55 (36%) with HT-H. The prevalence of TSH-BAb was higher in AT us. HT-H ( P < 0.001), HT-SH (P < 0.001), or HT-E (P < 0.001), and in HT-H vs. HTSH (P < 0.001) or HT-E (P < 0.001). Mean TSH-BAb levels in AT were higher than those in HT-H (P < 0.005) and HT-SH (P < 0.025); the difference was not significant between HT-H and HT-SH. An inverse correlation was found between TSHBAb levels and estimated goiter weight (P < 0.005). The results of the present study indicate that 1) in autoimmune thyroiditis TSH-BAb are detectable almost exclusively in hypothyroid patients, their prevalence being higher in overt hypothyroidism than in subclinical thyroid failure; 2) the prevalence of TSHBAb and their mean levels are higher in hypothyroid patients with AT than in those with HT; and 3) therefore, the presence of circulating TSH-BAb appears to be related to the development of hypothyroidism and thyroid atrophy. (J Clin Endocrinol Metab 7 1 : 40-45, 1990)
ABSTRACT. Autoantibodies blocking the TSH-dependent production of cAMP in thyroid cells (TSH-BAb) have been described in atrophic thyroiditis (AT; idiopathic myxedema) and in neonates with transient hypothyroidism, but their incidence in autoimmune thyroiditis in relation to thyroid status remains to be completely established. To this purpose TSH-BAb were evaluated in a group of 140 consecutive patients with autoimmune thyroiditis, which included 26 cases of AT and 114 subjects with goitrous Hashimoto's thyroiditis (HT); among the goitrous group 27 were euthyroid (HT-E), 32 had subclinical hypothyroidism (HT-SH), and 55 had clinical hypothyroidism (HT-H). TSH-BAb were measured in immunoglobulin G prepared by DEAE-Sephadex A-50 by determining their ability to inhibit TSH-dependent cAMP production in a differentiated strain of cultured rat thyroid cells (FRTL-5). Using this sensitive and reproducible method, TSH-BAb were detected in 12 of 26 (46%) patients with AT, in 1 of 27 (3.7%) subjects with HT-E, in 3 of
M
Early studies (2, 3,10) indicated that antibodies blocking the TSH-induced stimulation of thyroid adenylate cyclase (TSH-BAb) are present almost exclusively in AT and contribute to the pathogenesis of hypothyroidism in this condition, but not in HT. However, subsequent reports showed that a variable percentage of patients with HT also have circulating TSH-BAb (6,15,16). The latter observation suggests that TSH-BAb could play a substantial role in the pathogenesis of hypothyroidism independently of the presence or absence of goiter. To test this hypothesis we devised an epidemiological study of TSH-BAb in a large group of consecutive patients with AT or HT whose thyroid function ranged from complete euthyroidism to subclinical or overt hypothyroidism. A differentiated strain of rat thyroid cells (FRTL-5) was used to detect TSH-BAb (7).
OST cases of spontaneous hypothyroidism in adults are due to a chronic autoimmune thyroiditis that may result in either thyroid enlargement [goitrous Hashimoto's thyroiditis (HT)] or atrophy [atrophic thyroiditis (AT)]. The inability of the thyroid to respond with a compensatory hormone production and tissue regrowth to the excess TSH in AT led to the discovery of blocking antibodies that compete with TSH for its receptor (1) and/or inhibit the biological effects of TSH on cAMP production (2-10), iodine uptake and organification (4,5,11), T 3 secretion (12), or thyroid cell growth (6,13, 14). Received August 4, 1989. Address all correspondence and requests for reprints to: Dr. Luca Chiovato, Cattedra di Endocrinologia e Medicina Costituzionale, Istituto di Endocrinologia, Metodologia Clinica e Medicina del Lavoro, University of Pisa, viale del Tirreno 64, 56018, Tirrenia-Pisa, Italy. * This work was supported by grants from the Consiglio Nazionale delle Ricerche, Progetti Finalizzati: Medicina Preventiva e Riabilitativa, Sottoprogetto 2 (Meccanismi di Invecchiamento; Grant 84.02464.56); Biotecnologie e Biostrumentazione, Sottoprogetto 6 (Biofarmaci: Biotecnologie e Meccanismo D'azione dei Farmaci e Fattori di Crescita); and the Ministero Pubblica Istruzione, Rome, Italy.
Materials and Methods Patients One hundred and forty consecutive patients with autoimmune thyroiditis were included in the study. One hundred and 40
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 18:57 For personal use only. No other uses without permission. . All rights reserved.
AUTOANTIBODIES BLOCKING TSH-DEPENDENT cAMP PRODUCTION fourteen had a palpable goiter (HT); among them 27 were euthyroid (HT-E), 32 had subclinical hypothyroidism (HTSH), and 55 had overt hypothyroidism (HT-H). The remaining 26 patients had AT with the clinical picture of idiopathic myxedema. Mean ages were 34.5 ± 12 yr (mean ± SD) in HTE, 38.0 ± 13 yr in HT-SH, 49.4 ± 13 yr in HT-H, and 52 ± 11 yr in AT. There were 103 female patients with HT, and 21 females with AT. Forty normal subjects were specifically selected as a control group, which shared with the group of patients the same male/female ratio (1/8) and age range (1076 yr). The diagnosis of HT was based on the evidence of a typical goiter associated with high titers of circulating microsomal (MAb; >l:1.600) and/or thyroglobulin (Tg-Ab; >l:400) antibodies. Cytological evidence of lymphocytic infiltration of the gland was obtained in 36 patients (mainly euthyroid), in whom fine needle aspiration was performed to rule out thyroid cancer. AT was diagnosed in patients with primary hypothyroidism associated with a nonpalpable gland and circulating M-Ab and/or Tg-Ab. Euthyroidism was diagnosed on the basis of normal serum total T4 and T 3 associated with TSH in the normal range by an ultrasensitive immunoradiometric assay (ELSA-2 TSH, CIS International, France). Subclinical hypothyroidism was defined by evidence of elevated serum TSH levels with normal total T4 and T 3 and no clinical sign of hypothyroidism. In clinical or overt hypothyroidism T4 and T 3 were low, and TSH was high. Goiter weight was estimated by palpation and scintiscan; thyroid ultrasonography was performed in all patients with AT and in 55 of those with HT. It is relevant to note that in our experience there is a good correlation between thyroid weight estimated by palpation and gland volume measured by ultrasonography (17), and that in normal adult subjects from our area the mean size of the thyroid is equivalent to 20 g. Thyroid radioiodine uptake at 24 h (normal range in our area, 30-50%) was determined in 84 patients. Sera were obtained before any treatment in all patients with HT-E and HT-SH, while 14 subjects with HT-H and 7 with AT had been receiving L-T4 for 4-6 months when sample was drawn. Immunoglobulin G (IgG) was prepared by double separation on DEAE-Sephadex A-50 and subsequent precipitation by ammonium sulfate (7). The protein concentration was determined by optical density at 280 nm using an extinction coefficient of 1.46. Tg-Ab, M-Ab, and thyroid peroxidase antibodies (TPO-Ab) Tg-Ab and M-Ab were assessed in sera by passive hemagglutination (Thyroid Test Kit and Microsome Test Kit, Fujizoki, Tokyo, Japan) and by immunoradiometric methods (18, 19). TPO-Ab were measured by an immunoradiometric method (20).
41
transferrin (62.5 nmol/L), 1-glycil-l-hystydil-l-lysine (25 nmol/L), somatostatin (6.25 nmol/L), and TSH (300 mU/L). For the assay of TSH-BAb, 30,000 cells were seeded in each well of a 96-well Costar plate (Costar, Cambridge, MA). After 3 days of culture in the presence of TSH, FRTL-5 cells were switched to a medium deprived of the hormone (5H medium) and maintained in this medium for 4 days before the assay for TSH-BAb. Assay for TSH-BAb in FRTL-5 cells Before the assay the culture medium was aspirated, and cells were washed with Hanks' Balanced Salt Solution. IgG were dialyzed in hypotonic buffer (22) and then diluted in the same buffer containing 4 g/L BSA and 0.5 mmol/L isobutylmethylxanthine (IBMX) to a concentration of 2 mg/mL. A purified preparation of bovine TSH (kindly provided by Dr. L. D. Kohn, NIDDK, Bethesda, MD) was also diluted in hypotonic buffer-BSA-IBMX to a concentration of 20 mU/L. Cell cultures were incubated with IgG alone, TSH alone, or IgG plus TSH in a total volume of 100 /zL hypotonic buffer-BSA-IBMX. The final concentrations of IgG and TSH in the incubation mixture were 1 mg/mL and 10 mU/L, respectively. Hypotonic bufferBSA-IBMX alone was added to some cultures in each experiment to measure basal cAMP production. After 1 h of incubation at 37 C in 5% CO2-95% air atmosphere, cAMP was measured in the extracellular medium by RIA, as previously described (7). All experiments were performed in triplicate, and the results (in picomoles per well) were expressed as the average of these. To evaluate the effect of IgG, an index of inhibition of TSH-dependent cAMP production was calculated with the following formula: 1
_ cAMP (TSH + sample IgG) - cAMP (sample IgG) ~ cAMP (TSH) - cAMP (control buffer)
J
x 100
Statistical analysis Comparison between means was performed by Student's t test; the prevalence of TSH-BAb in different groups of patients was evaluated by x2 analysis. Regression analysis was used to evaluate the relation between TSH-BAb and various clinical and laboratory parameters; the correlation coefficient (r), as a parameter of regression analysis, was used to determine the degree of association. Distribution of variables, analysis of residuals, and F test were checked before applying the regression model.
Results
Cell cultures
Assay for TSH-BAb
As previously described (21), FRTL-5 cells were grown in Coon's modified Ham's F-12 medium supplemented with 5% calf serum and a six-hormone mixture (6H medium) consisting of insulin (1.6 x 106 pmol/L), hydrocortisone (1 nmol/L),
Basal cAMP (0.5-1.0 pmol/well) was unaffected by incubation with normal IgG (1 mg/mL). TSH produced an increase in extracellular cAMP that was always significant at 1 mU/L and dose dependent up to 100 mU/
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 18:57 For personal use only. No other uses without permission. . All rights reserved.
CHIOVATO ET AL.
42
L. In the assay for TSH-BAb, TSH was used at a standard concentration of 10 mU/L, which elicited a 4to 6-fold increase in extracellular cAMP with respect to the basal value. This dose of TSH, while not producing a plateau stimulation, induced a reproducible increase in cAMP (intra- and interassay coefficients of variation, 20
X SH
AT
HT
TSH-BAb in patients with HT and AT In patients with AT 12 of 26 IgG (46%) produced inhibition of TSH-dependent cAMP production (Fig. 2). Among hypothyroid patients with HT 20 of 55 IgG (36%) were positive for TSH-BAb, while in the group with HTSH only 3 of 22 (9.4%) inhibited TSH action. With the exception of 1 patient, TSH-BAb were not detectable in euthyroid HT. Statistical analysis confirmed a higher prevalence of TSH-BAb in AT than in any group of patients with HT (Fig. 3). Among patients with goiter the prevalence of TSH-BAb was significantly higher in those with overt hypothyroidism than in those who were euthyroid or had
FIG. 2. Results obtained in the assay for TSH-BAb using IgG from patients with AT or HT. In the latter group patients were divided according to their thyroid status: euthyroidism (E), subclinical hypothyroidism (SH), and clinical hypothyroidism (H). The results obtained with normal IgG are also shown. The shaded area indicates +2 SD from the mean of normal IgG. HT(E) < HT(H) p
Vol. 71, No. 1 Printed in U.S.A.
Incidence of Antibodies Blocking Thyrotropin Effect In Vitro in Patients with Euthyroid or Hypothyroid Autoimmune Thyroiditis* LUCA CHIOVATO, PAOLO VITTI, FERRUCCIO SANTINI, GUADALUPE LOPEZ, CLAUDIA MAMMOLI, PABLITO BASSI, LIA GIUSTI, MASSIMO TONACCHERA, GIANFRANCO FENZI, AND ALDO PINCHERA Cattedra di Endocrinologia e Medicina Costituzionale, University of Pisa, Pisa, Italy
32 (9.4%) with HT-SH, and in 20 of 55 (36%) with HT-H. The prevalence of TSH-BAb was higher in AT us. HT-H ( P < 0.001), HT-SH (P < 0.001), or HT-E (P < 0.001), and in HT-H vs. HTSH (P < 0.001) or HT-E (P < 0.001). Mean TSH-BAb levels in AT were higher than those in HT-H (P < 0.005) and HT-SH (P < 0.025); the difference was not significant between HT-H and HT-SH. An inverse correlation was found between TSHBAb levels and estimated goiter weight (P < 0.005). The results of the present study indicate that 1) in autoimmune thyroiditis TSH-BAb are detectable almost exclusively in hypothyroid patients, their prevalence being higher in overt hypothyroidism than in subclinical thyroid failure; 2) the prevalence of TSHBAb and their mean levels are higher in hypothyroid patients with AT than in those with HT; and 3) therefore, the presence of circulating TSH-BAb appears to be related to the development of hypothyroidism and thyroid atrophy. (J Clin Endocrinol Metab 7 1 : 40-45, 1990)
ABSTRACT. Autoantibodies blocking the TSH-dependent production of cAMP in thyroid cells (TSH-BAb) have been described in atrophic thyroiditis (AT; idiopathic myxedema) and in neonates with transient hypothyroidism, but their incidence in autoimmune thyroiditis in relation to thyroid status remains to be completely established. To this purpose TSH-BAb were evaluated in a group of 140 consecutive patients with autoimmune thyroiditis, which included 26 cases of AT and 114 subjects with goitrous Hashimoto's thyroiditis (HT); among the goitrous group 27 were euthyroid (HT-E), 32 had subclinical hypothyroidism (HT-SH), and 55 had clinical hypothyroidism (HT-H). TSH-BAb were measured in immunoglobulin G prepared by DEAE-Sephadex A-50 by determining their ability to inhibit TSH-dependent cAMP production in a differentiated strain of cultured rat thyroid cells (FRTL-5). Using this sensitive and reproducible method, TSH-BAb were detected in 12 of 26 (46%) patients with AT, in 1 of 27 (3.7%) subjects with HT-E, in 3 of
M
Early studies (2, 3,10) indicated that antibodies blocking the TSH-induced stimulation of thyroid adenylate cyclase (TSH-BAb) are present almost exclusively in AT and contribute to the pathogenesis of hypothyroidism in this condition, but not in HT. However, subsequent reports showed that a variable percentage of patients with HT also have circulating TSH-BAb (6,15,16). The latter observation suggests that TSH-BAb could play a substantial role in the pathogenesis of hypothyroidism independently of the presence or absence of goiter. To test this hypothesis we devised an epidemiological study of TSH-BAb in a large group of consecutive patients with AT or HT whose thyroid function ranged from complete euthyroidism to subclinical or overt hypothyroidism. A differentiated strain of rat thyroid cells (FRTL-5) was used to detect TSH-BAb (7).
OST cases of spontaneous hypothyroidism in adults are due to a chronic autoimmune thyroiditis that may result in either thyroid enlargement [goitrous Hashimoto's thyroiditis (HT)] or atrophy [atrophic thyroiditis (AT)]. The inability of the thyroid to respond with a compensatory hormone production and tissue regrowth to the excess TSH in AT led to the discovery of blocking antibodies that compete with TSH for its receptor (1) and/or inhibit the biological effects of TSH on cAMP production (2-10), iodine uptake and organification (4,5,11), T 3 secretion (12), or thyroid cell growth (6,13, 14). Received August 4, 1989. Address all correspondence and requests for reprints to: Dr. Luca Chiovato, Cattedra di Endocrinologia e Medicina Costituzionale, Istituto di Endocrinologia, Metodologia Clinica e Medicina del Lavoro, University of Pisa, viale del Tirreno 64, 56018, Tirrenia-Pisa, Italy. * This work was supported by grants from the Consiglio Nazionale delle Ricerche, Progetti Finalizzati: Medicina Preventiva e Riabilitativa, Sottoprogetto 2 (Meccanismi di Invecchiamento; Grant 84.02464.56); Biotecnologie e Biostrumentazione, Sottoprogetto 6 (Biofarmaci: Biotecnologie e Meccanismo D'azione dei Farmaci e Fattori di Crescita); and the Ministero Pubblica Istruzione, Rome, Italy.
Materials and Methods Patients One hundred and forty consecutive patients with autoimmune thyroiditis were included in the study. One hundred and 40
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 18:57 For personal use only. No other uses without permission. . All rights reserved.
AUTOANTIBODIES BLOCKING TSH-DEPENDENT cAMP PRODUCTION fourteen had a palpable goiter (HT); among them 27 were euthyroid (HT-E), 32 had subclinical hypothyroidism (HTSH), and 55 had overt hypothyroidism (HT-H). The remaining 26 patients had AT with the clinical picture of idiopathic myxedema. Mean ages were 34.5 ± 12 yr (mean ± SD) in HTE, 38.0 ± 13 yr in HT-SH, 49.4 ± 13 yr in HT-H, and 52 ± 11 yr in AT. There were 103 female patients with HT, and 21 females with AT. Forty normal subjects were specifically selected as a control group, which shared with the group of patients the same male/female ratio (1/8) and age range (1076 yr). The diagnosis of HT was based on the evidence of a typical goiter associated with high titers of circulating microsomal (MAb; >l:1.600) and/or thyroglobulin (Tg-Ab; >l:400) antibodies. Cytological evidence of lymphocytic infiltration of the gland was obtained in 36 patients (mainly euthyroid), in whom fine needle aspiration was performed to rule out thyroid cancer. AT was diagnosed in patients with primary hypothyroidism associated with a nonpalpable gland and circulating M-Ab and/or Tg-Ab. Euthyroidism was diagnosed on the basis of normal serum total T4 and T 3 associated with TSH in the normal range by an ultrasensitive immunoradiometric assay (ELSA-2 TSH, CIS International, France). Subclinical hypothyroidism was defined by evidence of elevated serum TSH levels with normal total T4 and T 3 and no clinical sign of hypothyroidism. In clinical or overt hypothyroidism T4 and T 3 were low, and TSH was high. Goiter weight was estimated by palpation and scintiscan; thyroid ultrasonography was performed in all patients with AT and in 55 of those with HT. It is relevant to note that in our experience there is a good correlation between thyroid weight estimated by palpation and gland volume measured by ultrasonography (17), and that in normal adult subjects from our area the mean size of the thyroid is equivalent to 20 g. Thyroid radioiodine uptake at 24 h (normal range in our area, 30-50%) was determined in 84 patients. Sera were obtained before any treatment in all patients with HT-E and HT-SH, while 14 subjects with HT-H and 7 with AT had been receiving L-T4 for 4-6 months when sample was drawn. Immunoglobulin G (IgG) was prepared by double separation on DEAE-Sephadex A-50 and subsequent precipitation by ammonium sulfate (7). The protein concentration was determined by optical density at 280 nm using an extinction coefficient of 1.46. Tg-Ab, M-Ab, and thyroid peroxidase antibodies (TPO-Ab) Tg-Ab and M-Ab were assessed in sera by passive hemagglutination (Thyroid Test Kit and Microsome Test Kit, Fujizoki, Tokyo, Japan) and by immunoradiometric methods (18, 19). TPO-Ab were measured by an immunoradiometric method (20).
41
transferrin (62.5 nmol/L), 1-glycil-l-hystydil-l-lysine (25 nmol/L), somatostatin (6.25 nmol/L), and TSH (300 mU/L). For the assay of TSH-BAb, 30,000 cells were seeded in each well of a 96-well Costar plate (Costar, Cambridge, MA). After 3 days of culture in the presence of TSH, FRTL-5 cells were switched to a medium deprived of the hormone (5H medium) and maintained in this medium for 4 days before the assay for TSH-BAb. Assay for TSH-BAb in FRTL-5 cells Before the assay the culture medium was aspirated, and cells were washed with Hanks' Balanced Salt Solution. IgG were dialyzed in hypotonic buffer (22) and then diluted in the same buffer containing 4 g/L BSA and 0.5 mmol/L isobutylmethylxanthine (IBMX) to a concentration of 2 mg/mL. A purified preparation of bovine TSH (kindly provided by Dr. L. D. Kohn, NIDDK, Bethesda, MD) was also diluted in hypotonic buffer-BSA-IBMX to a concentration of 20 mU/L. Cell cultures were incubated with IgG alone, TSH alone, or IgG plus TSH in a total volume of 100 /zL hypotonic buffer-BSA-IBMX. The final concentrations of IgG and TSH in the incubation mixture were 1 mg/mL and 10 mU/L, respectively. Hypotonic bufferBSA-IBMX alone was added to some cultures in each experiment to measure basal cAMP production. After 1 h of incubation at 37 C in 5% CO2-95% air atmosphere, cAMP was measured in the extracellular medium by RIA, as previously described (7). All experiments were performed in triplicate, and the results (in picomoles per well) were expressed as the average of these. To evaluate the effect of IgG, an index of inhibition of TSH-dependent cAMP production was calculated with the following formula: 1
_ cAMP (TSH + sample IgG) - cAMP (sample IgG) ~ cAMP (TSH) - cAMP (control buffer)
J
x 100
Statistical analysis Comparison between means was performed by Student's t test; the prevalence of TSH-BAb in different groups of patients was evaluated by x2 analysis. Regression analysis was used to evaluate the relation between TSH-BAb and various clinical and laboratory parameters; the correlation coefficient (r), as a parameter of regression analysis, was used to determine the degree of association. Distribution of variables, analysis of residuals, and F test were checked before applying the regression model.
Results
Cell cultures
Assay for TSH-BAb
As previously described (21), FRTL-5 cells were grown in Coon's modified Ham's F-12 medium supplemented with 5% calf serum and a six-hormone mixture (6H medium) consisting of insulin (1.6 x 106 pmol/L), hydrocortisone (1 nmol/L),
Basal cAMP (0.5-1.0 pmol/well) was unaffected by incubation with normal IgG (1 mg/mL). TSH produced an increase in extracellular cAMP that was always significant at 1 mU/L and dose dependent up to 100 mU/
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 18:57 For personal use only. No other uses without permission. . All rights reserved.
CHIOVATO ET AL.
42
L. In the assay for TSH-BAb, TSH was used at a standard concentration of 10 mU/L, which elicited a 4to 6-fold increase in extracellular cAMP with respect to the basal value. This dose of TSH, while not producing a plateau stimulation, induced a reproducible increase in cAMP (intra- and interassay coefficients of variation, 20
X SH
AT
HT
TSH-BAb in patients with HT and AT In patients with AT 12 of 26 IgG (46%) produced inhibition of TSH-dependent cAMP production (Fig. 2). Among hypothyroid patients with HT 20 of 55 IgG (36%) were positive for TSH-BAb, while in the group with HTSH only 3 of 22 (9.4%) inhibited TSH action. With the exception of 1 patient, TSH-BAb were not detectable in euthyroid HT. Statistical analysis confirmed a higher prevalence of TSH-BAb in AT than in any group of patients with HT (Fig. 3). Among patients with goiter the prevalence of TSH-BAb was significantly higher in those with overt hypothyroidism than in those who were euthyroid or had
FIG. 2. Results obtained in the assay for TSH-BAb using IgG from patients with AT or HT. In the latter group patients were divided according to their thyroid status: euthyroidism (E), subclinical hypothyroidism (SH), and clinical hypothyroidism (H). The results obtained with normal IgG are also shown. The shaded area indicates +2 SD from the mean of normal IgG. HT(E) < HT(H) p
