Altered balance of immunoregulatory T lymphocyte subsets in autoimmune thyroid diseases

Three monoclonal antibodies recognizing cell surface antigens of total peripheral (OKT3), helper/inducer (OKT4) and suppressor/cytotoxic (OKT8) T lymphocytes were used by an indirect immunofluorescence technique to enumerate peripheral T lymphocytes in 25 patients with Graves' disease (including 4 euthyroid Graves' patients), 16 patients with Hashimoto's thyroiditis and 22 normal controls. Total lymphocyte count and percentages of overall T and helper/inducer T cells among peripheral lymphocytes in these conditions showed no significant difference from those of the controls. Percentage of suppressor/cytotoxic T cells, however, was decreased in Graves' disease patients with or without hyperthyroidism. The ratio of helper/inducer T cells to suppressor/cytotoxic T cells was increased in Graves' disease population and slightly increased in hypothyroid Hashimoto's thyroiditis patients. The ratio correlated with the mitogenic response of peripheral mononuclear cells to phytohaemagglutinin, but not with the serum levels of thyroid hormones nor with the titres of thyroid autoantibodies. These findings are in accordance with the results of previous functional studies and indicate possible defects in suppressor T lymphocytes in autoimmune thyroid disease.     

BINDING OF IMMUNOGLOBULIN-G FROM PATIENTS WITH THYROID AUTOIMMUNE DISEASE TO NORMAL T LYMPHOCYTES

Sera from patients with Graves' disease and Hashimoto's thyroiditis were reacted with normal T lymphocyte preparations in an attempt to detect binding of immunoglobulin G (IgG) to T cells. Sera from normal subjects and patients with toxic adenomas served as controls. Each serum was reacted with at least three different preparations of normal T cells. Bound IgG was identified using a fluoresceinated second antibody, antihuman IgG. Positive cells were enumerated by means of epifluorescent microscopy. IgG from 57.8% of toxic Graves' patients, 30.7% of Graves' patients who were euthyroid after treatment, and 41.6% of Hashimoto's patients bound to normal T cells more than did IgG from normal controls. Reactivity of toxic adenoma sera was similar to that of normal sera. When the positive sera were reacted with helper or suppressor/cytotoxic T cell preparations (separated by negative selection technique), the binding was shown to be directed against suppressor/cytotoxic T cells but not against helper cells. These data indicate that a significant proportion of patients with autoimmune thyroid disease have IgG in their serum which react with a subset of normal T suppressor/cytotoxic cells. This phenomenon could be the expression of anti-lymphocyte antibodies, which may relate to previously recognized reductions in number and function of suppressor T cells in autoimmune thyroid disease.     

Circulating T cell subsets in euthyroid Graves' disease

T cell subpopulations recognized by surfaces markers of different functional meaning have been evaluated in 12 female patients with euthyroid Graves' disease and in 2 patients with ophthalmopathy and Hashimoto's thyroiditis. We have used the following markers: i) receptors for Fc fragments of IgG; ii) antigens recognized by the monoclonal antibodies MLR4, 5/9, BT 2/9 (anti-DR). In the 12 patients with euthyroid Graves' disease a marked decrease of TG cells (which proved to exert suppressor function in several in vitro systems) was observed, as previously reported in hyperthyroid Graves' disease. The 2 Hashimoto's patients with eye changes had normal or high TG. 5/9+ T cells (which contain cells with helper activity in vitro), as well as MLR4+ and BT 2/9+ cells (activated T cells) were normal in the majority of patients, but elevated in the 2 Hashimoto's thyroiditis. The observed abnormality of TG cells in euthyroid Graves' disease might be consistent with the hypothesized autoimmune pathogenesis of endocrine ophthalmopathy.     

Circulating activated T cell subsets in autoimmune thyroid diseases: differences between untreated and treated patients.

To investigate the relationships between lymphocyte subsets and thyroid function, peripheral blood lymphocytes were analysed with cell surface antigens of activated (HLA-DR+) T, helper T (CD4+ 2H4-, CD4+ 4B4+) and suppressor-inducer T (CD4+ 2H4+, CD4+ 4B4-) cells subsets in 56 patients with Graves' disease, 16 patients with Hashimoto's thyroiditis, 7 patients with typical subacute thyroiditis and 2 patients with the thyrotoxic phase of autoimmune thyroiditis. Both patients with Graves' disease and Hashimoto's thyroiditis had increased percentages of HLA-DR+ T (Ia+ CD3+) cells as well as HLA-DR+ helper-inducer T (Ia+ CD4+) cells, which seemed to be independent of treatments. The percentage of HLA-DR+ suppressor-cytotoxic T (Ia+ CD8+) cells was increased in euthyroid or hypothyroid patients with Graves' disease following treatment, but was normal in hyperthyroid patients. The percentages of Ia+ CD4+ cells and Ia+ CD8+ were also increased in patients with thyroiditis, whereas these abnormal values normalized in the remission phase. These findings suggest that an increase in Ia+ CD4+ cells characteristically occurs during immune system activation in patients with hyperthyroid Graves' disease, Hashimoto's thyroiditis and the thyrotoxic phase of subacute thyroiditis, whereas the activated CD8+ cells in Graves' disease are induced by antithyroidal therapy.     

Studies on suppressor cell function in thyroid diseases.

Suppressor cell function of peripheral mononuclear cells has been examined in patients with Graves' disease, Hashimoto's thyroiditis, and thyroid cancer, as well as in healthy subjects. Suppressor cell function was assessed through two methods: 1) measurement of enhanced blastogenesis after 24-h preculture and 2) concanavalin A-inducible suppressor activity. The results from the two tests were coincident and indicate that suppressor cell function was significantly decreased in the Graves' disease population but not changed in either the Hashimoto's thyroiditis or the thyroid cancer groups compared to healthy controls. The impairment of suppressor cell function in the Graves' disease population was still observed when patients became euthyroid by treatment with antithyroid drugs, although the treated patients had improved suppressor cell function compared to untreated patients (P = NS). Low activity of suppressor cell function in the Graves' disease population might be a constitutional character based on an inherited abnormality specific for the disease population.     

Studies on the suppressor T cell function in patients with autoimmune thyroid diseases

Suppressor T cell function induced by concanavalin A (con A) was evaluated in patients with Graves' disease and Hashimoto's thyroiditis. Patients with Graves' disease were divided into the following two groups: (1) untreated, and (2) euthyroid during antithyroid drug (methylmercaptoimidazole) therapy. T cells (2 X 10(5)), activated by con A for 48 hours, were added to preincubated responder cells (2 X 10(5)) and re-incubated for 7 days in the presence of pokeweed mitogen (PWM). IgG produced in the culture medium was measured by radioimmunoassay and then % suppressions (IgG) were calculated. Thyroid stimulating activity (TSA) in serum was measured by McKenzie's method by means of normal human thyroid slices, and % suppressions (c-AMP) were calculated. IgG produced in lymphocyte culture medium was suppressed by added con A-activated cells in untreated and euthyroid groups of Graves' disease, Hashimoto's thyroiditis and normal controls. The value of % suppression (IgG) was reduced in each group of Graves' disease compared to normal controls. No significant relation was observed between TSA in serum and % suppression (IgG), but three cases with high serum TSA showed low % suppressions (IgG). In 12 cases of Graves' disease, % suppression (IgG) had a positive relation with % suppression (c-AMP) in same medium. The amount of c-AMP produced in thyroid slices incubated in medium, in which responder cells (8 X 10(5)), was elevated in all 7 untreated cases of Graves' disease, while not elevated in 7 euthyroid cases. The value of % suppression (c-AMP) in euthyroid cases with Graves' disease was significantly higher than that in untreated cases. The value of % suppression (IgG) was reduced and had a significant negative relation with logarithm of serum antimicrosomal antibody titer in patients with Hashimoto's thyroiditis. These results indicate that low activity of suppressor T cell had a role on antibody production, including thyroid stimulating antibody, and pathogenesis of autoimmune thyroid diseases.     

Reduction in the suppressor-inducer T cell subset and increase in the helper T cell subset in thyroid tissue from patients with Graves' disease

The expression of surface markers associated with activation and characterization was compared among T cells in thyroid glands and peripheral blood of 10 patients with Graves' hyperthyroidism receiving chronic antithyroid drug therapy, in peripheral blood of 15 patients with untreated hyperthyroid Graves' disease, and in peripheral blood of 21 normal subjects using two-color flow cytometry. In the chronically treated Graves' disease patients, the percentage of activated T cells (HLA-DR+ T cells) among total T cells was significantly higher in thyroid tissue than in peripheral blood, and the increase in percent activated T cells was also significant among both helper/inducer T cell (CD4+ cell) and suppressor/cytotoxic T cell (CD8+ cell) subsets. The percentage of activated T cells in peripheral blood was not significantly different between chronically treated hyperthyroid Graves' patients and normal subjects, whereas the percentage of activated T cells in the peripheral blood from untreated hyperthyroid Graves' disease patients was significantly higher than that in normal subjects or chronically treated hyperthyroid Graves' patients. The percentages of CD4+ cells and CD8+ cells among total T cells were not different between thyroid tissues and peripheral blood in patients with chronically treated hyperthyroid Graves' disease. When CD4+ were further divided into helper T cells (CD4+2H4- cells) and suppressor-inducer T cells (CD4+2H4+ cells) using two-color flow cytometry, the percentage of helper T cells among CD4+ cells was significantly higher in thyroid tissue than in peripheral blood, resulting in an increased ratio of CD4+2H4- cells to CD4+2H4+ cells. The percentage of CD4+2H4+ cells in peripheral blood, however, was not significantly different among untreated and chronically treated Graves' disease patients and normal subjects. From the findings of abnormalities in intrathyroidal T cell subsets, we suggest that the decrease in the function of suppressor T cells within the thyroids of Graves' disease patients may be due to a decrease in CD4+2H4+ cells within thyroid tissue.     

Changes of peripheral mononuclear cell subpopulations in autoimmune thyroid diseases

To estimate abnormalities in humoral or cellular immunity that relate to the etiologies of Graves' disease (GD) and Hashimoto's thyroiditis (HT), peripheral mononuclear cell subpopulations were enumerated by an immunofluorescence technique using monoclonal antibodies. Also antithyrotropin receptor antibodies (thyrotropin-binding inhibitor immunoglobulin, TBII) were measured by the radioreceptor assay according to Smith's method; and antithyroglobulin antibodies (TGHA) and antithyroid microsomal antibodies (MCHA), by the tanned red cell hemagglutination technique. The data obtained were analyzed for the count of peripheral total white cells, lymphocytes and granulocytes, and to the level of serum free thyroxine (T4), free T4 index (FT4I) and free triiodothyronine index (FT3I). Peripheral white cells tended to be decreased in some cases of GD and HT. The absolute count of lymphocytes was slightly increased in GD but did not change in HT. On granulocytes (neutrophils mainly), the absolute count was considerably decreased in hyperthyroid GD. In this disease, FT3I showed significantly positive correlations to the percentage and absolute count of peripheral lymphocytes, whereas FT4I revealed significantly negative correlations to the percentage and count of granulocytes. These facts indicate that lymphocytosis and granulocytopenia in GD might be ascribed partially to the direct effects of thyroid hormones. The percentage of peripheral OKT3-positive cells (common T lymphocytes) was significantly decreased in untreated cases of GD, showing negative correlations to FT4, FT4I and FT3I. The absolute count of OKT4-positive cells (inducer/helper T lymphocytes) was increased in untreated cases of GD and hyperthyroid and euthyroid cases receiving antithyroid drugs (ATD) for GD. But peripheral OKT8-positive cells (suppressor/killer T lymphocytes) was significantly decreased in HT. The percentage of peripheral OKT8-positive cells in GD was also decreased in untreated and remitted cases, but slightly more increased in ATD-medicated cases than in nonmedicated cases. The ratios of OKT4-positive cells to OKT8-positive cells (OKT4+/OKT8+ ratios) were significantly increased in untreated, ATD-medicated euthyroid and remitted cases of GD, and in HT. It seems that ATD may exert direct effects on OKT8-positive cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Soluble Fas is increased in hyperthyroidism independent of the underlying thyroid disease

In Hashimoto's thyroiditis, Fas-induced apoptosis is one of the mechanisms leading to cell destruction, whereas thyroid tissue in Graves' disease is prevented from it. The soluble form of the Fas molecule produced by alternative splicing prevents from apoptosis. We measured soluble Fas in the sera of 112 patients with Graves' disease, 21 patients with toxic goiter, and 24 patients with subclinical hyperthyroidism due to suppressive therapy with levothyroxine after near-total resection of the thyroid gland for nodular goiter. Soluble Fas was increased in thyrotoxic patients, toxic goiter, and patients with subclinical hyperthyroidism. Decreased levels of soluble Fas were found in euthyroid patients with Graves' disease after surgery, whereas soluble Fas was normal in euthyroid patients with Graves' disease receiving antithyroid drug treatment and in patients in stable remission. There was a good correlation between soluble Fas with free T(3) (r = 0.6) and free T(4) (r = 0.5). Our results show that soluble Fas is increased in hyperthyroidism independent of the underlying thyroid disease.     

Relation of CD30 molecules on T-cell subsets to the severity of autoimmune thyroid disease.

The prognosis of patients with autoimmune thyroid disease (AITD) varies. To clarify the immunologic differences among patients with various severities of AITD, we examined two types of molecules on peripheral T lymphocytes: CD195 (CCR5), which express dominantly on CD4(+) type 1 helper T (T(H)1) cells, and CD30, which is known as a marker of CD4(+) type 2 helper T (T(H)2) cells and a regulatory molecule of CD8(+) autoreactive cytotoxic T cells. We found presence of patients with high proportion (> 9%) of CD30 expression in CD4(+) cells in a group of patients with Graves' disease (GD) in remission compared to the patients with intractable GD and a decrease in the intensity of CD30 expression on CD8(+) cells from patients with severe Hashimoto's disease (HD) treated for hypothyroidism compared to patients with untreated and euthyroid HD. There was no difference in CD195 expression between these patients with GD or HD with different severities, but there was a decreased intensity of CD195(+) cells in thyrotoxic patients with GD. These results indicate that CD30 molecules on CD4(+) and CD8(+) cells may be related to the severities of GD and HD, respectively.     

T lymphocyte subsets in autoimmune thyroid diseases and subacute thyroiditis detected with monoclonal antibodies

Peripheral T lymphocyte subsets were analysed with monoclonal antibodies, by highly standardized fluorescence-activated cell sorter analysis instead of manual counting by the indirect immunofluorescence method, in autoimmune thyroid diseases and subacute thyroiditis. Total lymphocyte counts were increased in patients with thyrotoxic Graves' disease and subacute thyroiditis. The percentage of total T (Leu 1) cells was significantly lower in patients with thyrotoxic Graves' disease and Hashimoto's disease with destructive thyrotoxicosis than in normal subjects. No significant changes were observed in the percentages of suppressor-cytotoxic T (Leu 2a) cells or helper-inducer T (Leu 3a) cells or in the Leu 3a-Leu 2a ratio in different groups of patients. There were no correlations between the percentages of E rosette-forming cells and Leu 1 cells and between the percentages of T gamma cells and Leu 2a cells in normal subjects and patients. The peak position of fluorescence intensity of Leu 2a cells showed a significant sex difference even in normal controls. The most important finding was a significant decrease in the peak position of Leu 2a cells in patients with thyrotoxic Graves' disease and with hypothyroid or thyrotoxic Hashimoto's disease. These findings indicate the significant association of qualitative, but not quantitative, abnormality of suppressor-cytotoxic T (Leu 2a) cells with thyroid dysfunction in autoimmune thyroid diseases.     

Peripheral blood T-lymphocyte subsets in autoimmune thyroid disease.

Interest in T-lymphocyte subsets has arisen because of their involvement in the autoimmune process. Contradictory results have been published in the literature about the number of peripheral blood lymphocyte subsets in autoimmune diseases. In order to investigate the number and distribution of peripheral blood lymphocyte subsets in autoimmune thyroid disease, the levels of total T-lymphocytes (CD3), T-helper (CD4) and T-suppressor/cytotoxic (CD8) lymphocytes were determined in 44 patients with Graves' disease (1), multinodular goiter (2) and Hashimoto's thyroiditis (3). All patients had high levels of antithyroglobulin and thyroid antiperoxidase (antimicrosomal) antibodies. The T subset levels were related to the functional thyroid status, measured as serum free thyroxine (FT4) and thyrotropin (TSH). Our data show the existence of a strong influence of functional status on CD3, CD4 and CD8 levels, as reflected in the significant correlations obtained with FT4 (negative) and TSH (positive). A significant decrease in all populations was observed in Graves' disease hyperthyroid patients. A decrease in the CD4/CD8 ratio in Hashimoto's thyroiditis hypothyroid patients was observed, in contrast to an increase in the ratio in autoimmune hyperthyroid patients. This points to the CD4/CD8 ratio as a differential characteristic between the two autoimmune (hypothyroid and hyperthyroid) entities, independent of free thyroxine levels. No significant correlation was found between antithyroid antibody levels and peripheral blood T-lymphocyte subsets or serum levels of FT4 and TSH.     

Thyroid function and thyroid autoimmunity independently modulate serum concentration of soluble interleukin 2 (IL-2) receptor (sIL-2R) in thyroid diseases

OBJECTIVE: The serum concentration of soluble interleukin-2 receptor (sIL-2R) is a marker of T-lymphocyte activation. Increased circulating sIL-2R has been reported in untreated Graves' disease. This finding has been interpreted as the consequence of the autoimmune activation, but recent data suggest that sIL-2R is directly correlated to thyroid state. The aim of this study was to elucidate the respective roles of autoimmunity and thyroid function in modulating serum sIL-2R. DESIGN AND PATIENTS: sIL-2R was evaluated in 20 normal euthyroid subjects and in a large series of patients with autoimmune and non-autoimmune thyroid disorders in different functional state. MEASUREMENTS: sIL-2R was assayed by a solid-phase monoclonal antibody assisted ELISA method. RESULTS: Serum sIL-2R in normals was 461 +/- 186 U/ml (mean +/- SD). Increased sIL-2R was found in 61 hyperthyroid patients with Graves' disease (1610 +/- 962 U/ml, P < 0.0001) and in 23 with toxic adenoma (1121 +/- 598 U/ml, P < 0.0001). Restoration of euthyroidism lowered to normal sIL-2R in both groups. Serum sIL-2R was higher in euthyroid Graves' disease patients with active than in those with non-active ophthalmopathy. Decreased serum sIL-2R (228 +/- 93 U/ml, P < 0.0001) was found in 30 patients hypothyroid after total thyroidectomy. Highly variable circulating sIL-2R (range 100-1456 U/ml, mean +/- SD: 379 +/- 301 U/ml) was found in 49 patients with hypothyroid Hashimoto's thyroiditis (P = NS vs normals; P < 0.02 vs post-thyroidectomy hypothyroid patients). Treatment with L-thyroxine increased sIL-2R in all thyroidectomized and in the majority of Hashimoto's thyroiditis patients. In individual Hashimoto's thyroiditis patients (mostly with increased serum sIL-2R), L-thyroxine caused a decrease of circulating sIL-2R, sIL-2R was normal in 29 patients with euthyroid Hashimoto's thyroiditis. Both in Graves' disease and in Hashimoto's thyroiditis, no correlation was found between sIL-2R and anti-thyroglobulin, anti-thyroid peroxidase and anti-thyrotrophin-receptor autoantibodies. Highly significant positive correlation between serum thyroid hormones and sIL-2R was found in all study groups. CONCLUSIONS: In thyroid disorders thyroid hormones are the main regulator of serum sIL-2R concentration. The contribution of autoimmune activation may be detected only in some patients with autoimmune hypothyroidism, while in Graves' disease the role of the immune system is masked by the hyperthyroid state.     

Microsomal antigen-reactive lymphocyte lines and clones derived from thyroid tissue of patients with Graves' disease

Thyroid mononuclear cells (TMC) were maintained in long term cocultures with thyroid fibroblasts and thyroid epithelial cells from patients with Graves' disease, using medium supplemented with thyroid microsomal antigen (McAg) and IL-2. The TMC consisted predominantly of T4+ (CD4+, helper) and, to a lesser extent, T8+ (CD8+, cytotoxic/suppressor) lymphocytes, with a small number of macrophages and natural killer cells. The average T4+ to T8+ ratio was 3.2. From these cultures we obtained thyroid T cell lines and clones reactive to thyroid antigens. T Cell lines were tested in a microproliferation assay using thyroglobulin (Tg), McAg, tetanus toxoid, and IL-2. Of 14 lines from 6 patients, 2 proliferated in response to McAg when TMC plus thyroid fibroblasts were used as antigen-presenting cells. Clones of thyroid lymphocytes were obtained by culturing cells at limiting dilution with IL-2, McAg, and different types of autologous accessory cells. Peripheral blood mononuclear cells plus skin fibroblasts provided the best source of accessory cells, allowing near 100% cloning efficiency. Of 26 clones tested, 6 recognized McAg, 2 were Tg reactive, and 3 were autoreactive. All phenotyped clones were of the T4+ phenotype. Our method results in production of thyroid T cell lines and clones. The fibroblasts probably provided growth factors and/or collaborated with peripheral blood mononuclear cells as antigen-presenting cells. These lines and clones from patients with Graves' disease were predominantly helper T cells, in contrast to the previously demonstrated cytotoxic/suppressor cell predominance in cells from patients with Hashimoto's thyroiditis. This difference in cell function may help explain the differing clinical courses of these two closely related autoimmune thyroid diseases. The availability of long term microsomal antigen-specific T cell clones should allow careful analysis of the role these cells play in thyroid autoimmunity.     

Normal immunoregulation of in vitro antibody secretion in autoimmune thyroid disease

Defective suppressor cell function may be a causative factor in autoimmune disease in animals and man. In autoimmune thyroid disease, decreased suppressor cell activity could, under appropriate conditions, account for excess production of thyroid autoantibodies. We evaluated suppressor cell function in patients with Graves' disease and Hashimoto's thyroiditis and in normal controls. The method used is based on the principle that immunoglobulin synthesis by pokeweed mitogen (PWM)-stimulated lymphocytes is inhibited by Concanavalin A (Con A) stimulation of suppressor T cells. We studied suppressor cell control of polyclonal immunoglobulin G (IgG) and the thyroid-specific autoantibody, antimicrosomal antibody. PWM-stimulated IgG secretion (mean +/- SD) by lymphocytes from patients with Graves' disease (2797 +/- 718 ng/ml) and Hashimoto's thyroiditis (2201 +/- 423 ng/ml) did not differ from normal subjects (2431 +/- 485 ng/ml). The addition of Con A to PWM-stimulated lymphocytes suppressed IgG production in all three groups: Graves', 475 +/- 137 ng/ml; Hashimoto's, 507 +/- 74 ng/ml; and normal subjects, 460 +/- 156 ng/ml. The degree of suppression by the disease groups did not differ from the normal controls. Antimicrosomal antibody was detected in the concentrated, PWM-stimulated culture media of two of four Hashimoto's lymphocytes, three of five Graves' lymphocytes, and none of nine normal controls. Con A induced marked suppression of this organ-specific antibody in all cases. We conclude that Con A-stimulated lymphocytes from patients with Hashimoto's thyroiditis and Graves' disease can suppress antimicrosomal antibody and polyclonal IgG synthesis. These findings do not support the postulate of a generalized defect of suppressor cell function in these thyroid disorders.     

In situ immunologic characterization of cellular constituents in lymph nodes and spleens involved by Hodgkin's disease

The cellular constituents in lymph nodes and spleens of patients with Hodgkin's disease were studied with a series of monoclonal antibodies directed against human thymocyte, peripheral T-cell, and la antigens. Utilizing both an immunoperoxidase technique on frozen tissue sections and indirect immunofluorescence on cell suspensions, wer found that a majority of lymphocytes were T cells, since they stained with anti-T1 and anti-T3 antibodies, which react with all peripheral T cells. In addition, most of these cells were reactive with anti-T4 antibody, which defines the helper/inducer T-cell population, whereas only a minority of cells stained with anti-T5 and anti-T8 antibodies, which are reactive with suppressor/cytotoxic T cells. Moreover, a large proportion of T cells expressed T10 antigen, which is found on activated T cells. A minority of the T cells also expressed la antigen(s), again suggesting that some of the T cells are activated. In contrast, the Reed-sternberg cells did not react with any of these anti- T-cell antibodies or with anti-IgM antiserum, but displayed strong membrane and cytoplasmic staining with anti-la antibody. Taken together, these findings suggest that Reed-Sternberg cells are not of T- cell lineage but may be derived from antigen-presenting reticulum cells in the thymus-dependent areas of lymphoid tissues; these cells are normally associated with T4+ cells.     

Thyroid vascularity and blood flow are not dependent on serum thyroid hormone levels: studies in vivo by color flow doppler sonography

OBJECTIVE: Thyroid blood flow is greatly enhanced in untreated Graves' disease, but it is not known whether it is due to thyroid hormone excess or to thyroid hyperstimulation by TSH-receptor antibody. To address this issue in vivo patients with different thyroid disorders were submitted to color flow doppler sonography (CFDS). SUBJECTS AND METHODS: We investigated 24 normal subjects, and 78 patients with untreated hyperthyroidism (49 with Graves' hyperthyroidism, 24 with toxic adenoma, and 5 patients with TSH-secreting pituitary adenoma (TSHoma)), 19 patients with thyrotoxicosis (7 with thyrotoxicosis factitia, and 12 with subacute thyroiditis), 37 euthyroid patients with goitrous Hashimoto's thyroiditis, and 21 untreated hypothyroid patients with Hashimoto's thyroiditis. RESULTS: Normal subjects had CFDS pattern 0 (absent or minimal intraparenchimal spots) and mean intraparenchimal peak systolic velocity (PSV) of 4.8+/-1.2cm/s. Patients with spontaneous hyperthyroidism due to Graves' disease, TSHoma, and toxic adenoma had significantly increased PSV (P<0.0001, P=0.0004, P<0.0001 respectively vs controls) and CFDS pattern. Patients with Graves' disease had CFDS pattern II (mild increase of color flow doppler signal) in 10 (20%) and pattern III (marked increase) in 39 cases (80%). Mean PSV was 15+/-3cm/s. Patients with toxic adenoma had CFDS pattern I (presence of parenchymal blood flow with patchy uneven distribution) in 2 (8%), pattern II in 16 (70%) and pattern III in 5 (22%). Mean PSV was 11+/-2.4cm/s. Patients with TSHoma showed CFDS pattern I in one case (20%) and pattern II in 4 (80%). Mean PSV was 14.8+/-4.2cm/s. Patients with thyrotoxicosis had normal PSV (4.2+/-1. 1cm/s in subacute thyroiditis, 4+/-0.8cm/s in thyrotoxicosis factitia, P=not significant vs controls) and CFDS pattern 0. Untreated euthyroid patients with goitrous Hashimoto's thyroiditis had CFDS pattern 0, and mean PSV (4.3+/-0.9cm/s; P=not significant vs controls). Untreated hypothyroid patients with goitrous Hashimoto's thyroiditis had CFDS pattern I in 14 cases (67%), pattern II in 4 (19%) and pattern 0 in 3 (14%) and mean PSV (5.6+/-1. 4cm/s) was higher than that of controls (P=0.026). CONCLUSIONS: An increase in both intrathyroidal vascularity and blood velocity was observed in patients with spontaneous hyperthyroidism but not in thyrotoxicosis due to either ingestion of thyroid hormones or to a thyroidal destructive process. The slightly increased vascularity and blood velocity observed in patients with hypothyroid Hashimoto's thyroiditis suggests that thyroid stimulation by either TSH-receptor antibody or TSH is responsible for the increased thyroid blood flow.     

Intrathyroidal T cell clones from patients with autoimmune thyroid disease

We cloned activated T cells from thyroid tissue of patients with autoimmune thyroid disease. After separation on 40% Percoll gradients, T cells were cultured for 2-7 days with T cell growth factor (interleukin 2; 20 U/mL) and cloned by limiting dilution (0.3 cells/well) in the presence of irradiated autologous peripheral blood mononuclear cells (PMC; 10,000/well) as feeder cells. Fifty-seven clones were successfully expanded and tested for reactivity, cytotoxicity, helper/suppressor function, and phenotype. In the reactivity assays clones were tested for responses to autologous and allogeneic PMC, thyroid cells, human thyroglobulin (hTg), and microsomal antigen. Two distinct patterns of functional T cell clones emerged from these characterization studies. Seventy-five percent of T cell clones recovered from Graves' disease thyroid tissue (n = 21) were of helper-induced (CD4+/4B4+) phenotype, and most were effective immunoglobulin helper clones. Fifty percent of Graves' T cell clones responded to autologous PMC, and 33% had a proliferative response to autologous thyroid cells. No cytotoxic clones were derived from Graves' thyroid tissue. By contrast, intrathyroidal T cell clones from patients with autoimmune thyroiditis (n = 36) were 59% suppressor/cytotoxic (CD8+) phenotype, 17% suppressed immunoglobulin secretion, and 55% were cytotoxic to allogeneic blast cells. Fifty-five percent of clones also responded to autologous PMC, and one clone was nonspecifically autocytotoxic. In the thyroid antigen proliferation assays 11% of thyroiditis clones reacted to human thyroglobulin, but none responded to microsomal antigen. Two clones were cytotoxic to autologous but not allogeneic thyroid cells. These data demonstrate that the majority of intrathyroidal T cells in autoimmune thyroid disease are autoreactive. However, small numbers of thyroid-specific T cell clones are present within the thyroid of such patients; they are principally helper-inducer T cells in Graves' disease thyroid and cytotoxic T cells in autoimmune thyroiditis.     

Increased serum concentration of eosinophil-derived neurotoxin in patients with Graves' disease.

Eosinophil-derived neurotoxin (EDN) is released after activation and stimulation of eosinophils in allergic disease, which is a T(H)2-predominant condition. We previously reported that Graves' thyrotoxicosis develops or relapses after an attack of allergic rhinitis. In this study, to confirm the relation between Graves' disease and the allergic condition, we determined the serum level of EDN in 30 untreated patients with Graves' disease, 50 patients with Hashimoto's thyroiditis, and 39 normal controls. Compared to the serum level in normal subjects (30.1 +/- 15.6 ng/mL), EDN was increased in untreated patients with Graves' disease (52.4 +/- 27.6 ng/mL), but not in patients with Hashimoto's thyroiditis (thyrotoxic, 30.9 +/- 13.4 ng/mL; euthyroid, 30.0 +/- 11.9 ng/mL; hypothyroid, 23.4 +/- 10.2 ng/mL). A significant correlation was observed between the EDN level and the serum activity of thyrotropin (TSH) receptor antibody (r = 0.541, p < 0.0001). These data suggest that the allergic condition is closely related to Graves' disease and that a T(H)2-type immune response is crucial in the pathogenesis of Graves' disease.     

Phenotypic and functional analysis at the clonal level of infiltrating T lymphocytes in papillary carcinoma of the thyroid: prevalence of cytolytic T cells with natural killer-like or lymphokine-activated killer activity

We investigated the phenotype and function of thyroid tumor-, metastatic lymph node-, and peripheral blood-derived T lymphocytes of four patients with papillary thyroid cancer. Both phenotypic analysis of freshly isolated cells and clonal analysis, using a high efficiency cloning technique, were performed. For comparison, intrathyroid and peripheral blood T lymphocytes derived from patients with autoimmune thyroid diseases (Graves' disease and Hashimoto's thyroiditis) have been studied. In papillary cancer, the phenotype of thyroid and lymph node-derived T lymphocytes did not differ from that of peripheral blood lymphocytes of the same patients or lymphocytes from normal peripheral blood. At variance with respect to autoimmune thyroid disease, activation markers were poorly represented. The functional analysis of T cell clones showed similar proportions of interleukin-2-producing (helper-inducer) clones in thyroid, lymph node, and peripheral blood, slightly lower than in Hashimoto's thyroiditis and slightly higher than in Graves' disease. With regard to effector function, we found lower proportion of clones with cytolytic activity in a lectin-dependent assay compared to that in Hashimoto's thyroiditis. Interestingly, however, the proportions of cytolytic clones displaying cytolytic activity against the neoplastic cell line K562 (natural killer-like activity) or fresh unrelated tumor cells (lymphokine-activated killer activity) were relatively high in thyroid cancer infiltrates.