concanavalin-a and Graves-Disease

Topics: Adult; Antigen-Antibody Complex; Autoantibodies; Autoimmune Diseases; Child; Concanavalin A; Graves Disease; Humans; T-Lymphocytes, Regulatory; Thyroglobulin; Thyroid Gland; Thyroid Neoplasms

A thyrotropin (TSH) binding inhibiting protein (TBIP) that inhibits TSH binding to the TSH receptor, as determined by the TSH receptor assay, was purified from human and porcine thyroid. The soluble fraction (100,000 x g supernatant of Graves' thyroid homogenate) was precipitated with ammonium sulfate between 1.75 to 2.5 mol/L. TBIP was eluted by 0.5 mol/L sodium chloride (NaCl) containing 20 mmol/L Tris buffer, pH 7.5 from a Q-sepharose column. The unbound fraction from concanavalin A (Con A) and blue-sepharose was gel-filtered using sephadex G-100, and finally purified by Resource Q column chromatography. Purified TBIP was confirmed as a single protein band of 17 kDa. The TBI activity in the purified TBIP was significantly decreased by either etnylene glycol tetraacetate (EGTA) (1 mmol/L) or antibody to calmodulin (CaM) in the TSH receptor assay. The TBIP was confirmed immunologically as CaM by the Ouchterlony method using antibody for CaM. These findings demonstrated that the TBIP purified from human and porcine thyroids was, in fact, CaM. We examined the effects of TBIP purified from human thyroid on bovine TSH (bTSH) or thyroid stimulating antibody (TSAb)-stimulated cyclic adenosine monophosphate (cAMP) production in porcine thyroid cells (PTC). TBIP itself did not increase basal levels of cAMP production, but inhibited bTSH (100 mU/L)-stimulated cAMP production. However, TBIP did not inhibit cAMP production stimulated by TSAb-IgG and various thyroid stimulators (GTPgammaS, forskolin and pituitary adenylate cyclase-activating polypeptide [PACAP, 27 and 38 amino acids]). Authentic CaM purified from bovine brain behaved in a manner similar to that of TBIP. These data showed that CaM differentially affects thyroid stimulation by TSH and TSAb in intact thyroid cell experiments.

Topics: Animals; Antibodies; Calmodulin; Cattle; Chromatography; Chromatography, Gel; Concanavalin A; Cyclic AMP; Dextrans; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Graves Disease; Humans; Immunodiffusion; Iodine Radioisotopes; Protein Binding; Receptors, Thyrotropin; Sepharose; Sodium Dodecyl Sulfate; Swine; Thyroid Gland; Thyrotropin

A number of immunological parameters have been monitored for up to 6 weeks following 131I treatment for hyperthyroidism in Graves' disease. The aim was to examine whether this isotope treatment normalizes or further accentuates some immunological abnormalities which may be a manifestation of autoimmune reactions in these patients. It was confirmed that both the cellular composition and immunological reactivities of the patients' blood lymphocytes were abnormal before treatment. After 131I administration a slight lymphopenia occurred and the ratio between CD4 and CD8 positive T lymphocytes (helper-inducer/suppressor-cytotoxic), which was increased before treatment, increased further. Moreover, PWM-triggered IgM secretion in vitro was reduced by 50%. No other immunological parameters studied, such as secretion of other Ig classes, mitogenic responses of lymphocytes, and distribution of other lymphocyte subsets, changed to any detectable extent. It remains speculative whether the 131I-induced changes of the immune system may further accelerate the underlying autoimmune disease processes.

Topics: Adult; Autoimmunity; B-Lymphocytes; Concanavalin A; Female; Graves Disease; Humans; Immunoglobulin M; Iodine Radioisotopes; Lymphocyte Activation; Lymphocytes; Male; Middle Aged; Phytohemagglutinins; T-Lymphocytes; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Thyroxine

We have developed a sensitive, reproducible and specific radioimmunoassay for human interleukin-2. Using 125I-labeled interleukin-2 and polyclonal rabbit antisera raised against recombinant human interleukin-2, a competitive inhibition assay was described which could detect 1 U/ml of human interleukin-2. Substances such as interleukin-1 alpha, interferon beta, nerve growth factor, tissue necrotizing factor, various hormones, peptides and lectins did not affect the assay. Interleukin-2 was measured in supernatants of culture media of stimulated human blood mononuclear cells. Kinetics of interleukin-2 production in seven normal lymphocytes revealed that in both PHA- and Con A-stimulations, the peak levels of interleukin-2 were seen at the end of 72 hours (113.9 +/- 54.4 U/ml, 111.6 +/- 37.3 U/ml, respectively) and then declined. Interleukin-2 levels in PHA- and Con A-stimulations of untreated Graves' disease were significantly lower (14.5 +/- 15.5 U/ml, 12.3 +/- 12.7 U/ml, respectively) than normal controls. However, the improvement of decreased interleukin-2 production in methimazole-treated patients with Graves' disease was observed (38.2 +/- 28.1 U/ml, 48.0 +/- 35.6 U/ml, respectively). The present study demonstrates the usefulness of quantitating human interleukin-2 produced by human blood mononuclear cells and that there exists an impaired production of interleukin-2 in Graves' disease.

Topics: Antibody Specificity; Chromatography, Ion Exchange; Concanavalin A; Graves Disease; Humans; Interleukin-1; Interleukin-2; Iodine Radioisotopes; Kinetics; Lymphocytes; Monocytes; Phytohemagglutinins; Radioimmunoassay

The expression of phenotypic markers and Concanavalin-A-induced suppressor activity was compared among mononuclear cells isolated from thyroid glands and peripheral blood of thionamide-treated patients with hyperthyroid Graves' disease and peripheral blood from normal subjects. Intrathyroidal lymphocytes were obtained by two different methods (TG-1 and TG-2 cells), gradient centrifugation of supernatants of minced thyroid tissue and overnight culture of thyroid debris after mechanical disaggregation and enzymatic digestion, respectively. The percentages of CD3+ cells (all mature T cells) among peripheral blood and TG-1 and TG-2 cells from Graves' patients were similar, but the percentages of B1+ cells (pan B cells) among the TG-1 and TG-2 cells were markedly increased compared to that in peripheral blood. The percentages of CD4+ cells among the TG-1 and TG-2 cells were significantly less than that in peripheral blood. The percentages of CD4+2H4+ cells among CD4+ cells in TG-1 and TG-2 cells also were significantly less than that in peripheral blood. The percentage of CD4+4B4+ cells among CD4+ cells in thyroid glands was markedly higher than that in peripheral blood. The percentages of CD8+ cells and CD8+CD11b- cells (cytotoxic T cells) in thyroid glands were significantly higher than those in peripheral blood from Graves' patients and peripheral blood from normal subjects. The CD8+CD11b+ cells were subdivided into two subpopulations on the basis of CD8 antigen density. The percentage of dull CD8+CD11b+ cells (natural killer cells) among TG-2 cells was lower than that in peripheral blood, but there was no significant difference in bright CD8+CD11b+ cells (suppressor-effector T cells) between thyroid glands and peripheral blood. The percent suppression induced by Concanavalin-A in both TG-1 and TG-2 cells was significantly decreased compared with that in peripheral blood. These results suggest that impairment of suppressor cell activity and an increased number of B cells exist in thyroid glands of patients with Graves' disease compared to those in peripheral blood. It, thus, appears likely that both B cell hyperactivity and suppressor T cell dysfunction may induce excess production of autoantibodies in the thyroid glands of such patients.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Concanavalin A; Female; Flow Cytometry; Graves Disease; Humans; Male; Phenotype; T-Lymphocytes, Regulatory; Thyroid Gland

Following cross-linking with disuccinimidyl suberate and analysis by SDS-PAGE and autoradiography, both the high- and low-affinity TSH binding components exhibited two similar 125I-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves' disease inhibited 125I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the high- and low-affinity components share similar subunit composition and antigenic determinants.

Topics: Animals; Antibody Specificity; Autoantibodies; Concanavalin A; Graves Disease; Humans; In Vitro Techniques; Macromolecular Substances; Molecular Weight; Receptors, Cell Surface; Receptors, Thyrotropin; Structure-Activity Relationship; Swine; Thyrotropin

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.

Topics: Adult; Autoimmune Diseases; Concanavalin A; Cyclic AMP; Female; Graves Disease; Humans; Immunoglobulin G; Male; Methimazole; T-Lymphocytes, Regulatory; Thyroiditis, Autoimmune; Triiodothyronine

Evidence concerning the role of hyperthyroidism per se in suppressor cell dysfunction in Graves' disease is conflicting. To investigate this issue, we studied the effect of triiodothyronine (T3) administration to ten healthy volunteers for 7 days on spontaneous (short-lived) and Concanavalin A (Con A)-induced suppressor cell activities. The short-lived suppressor cell index is a ratio of [3H] thymidine incorporation induced by Concanavalin A in lymphocytes preincubated for 24 h (to remove adherent cells) to that of non-incubated lymphocytes; the more active the adherent suppressor cells, the higher this index. The mean short-lived suppressor cell index rose from a mean of 2.46 +/- 0.15 (+/- S.D.) before T3 treatment to 3.29 +/- 0.46 (p less than 0.01) after 7 days of T3. Concanavalin A-induced suppressor cell activity, which measures the potential suppressive activity of lymphocytes triggered by Con A is expressed as the percentage decrease in [3H] thymidine incorporation by lymphocytes cultured with Con A for 72 h in relation to that by fresh lymphocytes. Con A-induced suppressor cell activity also improved from 55.8% +/- 2.5 before T3 treatment to 62.4% +/- 1.9 (p less than 0.05) at 7 days of this treatment. These studies show that within the time limitations of the study, T3 appears to enhance short-lived and Concanavalin A-induced suppressor cell activities.

Topics: Adult; Autoantibodies; Autoimmune Diseases; Cells, Cultured; Concanavalin A; Graves Disease; Humans; Lymphocyte Activation; Middle Aged; Receptors, Cell Surface; Receptors, Thyrotropin; T-Lymphocytes, Regulatory; Thyroglobulin; Triiodothyronine

Immunoregulatory defects have been suggested in autoimmune disorders including Graves' disease. The finding that Concanavalin A-induced suppressor T cell function was sub-optimal in Graves' disease has been disputed; a restricted defect in TSH-receptor antigen-specific suppressor cells has instead been proposed by Okita et al. (1980). To explore this further, we studied both specific and non-specific suppressor cell function in a pair of HLA identical twins, one of whom had Graves' disease. By contrast to the euthyroid healthy twin and 10 healthy controls (612 cpm/10(6) cells) the patient's mononuclear cells (MNCs) incorporated more (3H)-thymidine (7365 cpm/10(6) cells) in response to thyroid membrane antigen (TMA). Removal of glass-adherent cells before addition of antigen increased (3H)-uptake by cells from the healthy twin to 1808 cpm but reduced those from the Graves' twin to 3411 cpm. The influence of MNCs cultured with Con A or TMA for 24 h upon (3H)-thymidine uptake by 2 X 10(6) indicator cells triggered by Con A for 72 h or TMA for 96 h was taken as a measure of non-specific and specific suppressor cell function respectively. Both Con A and TMA induced suppressor cells were reduced, the latter to a more marked degree, in the patient compared to the healthy twin; mixing of MNCs from patient and healthy twin in a 1:1 ratio improved the patient suppressor cell function. When the patient's MNCs triggered for 24 h with Con A were mixed in a 1:1 ratio with her fresh MNCs and TMA, less blast transformation was found compared to an equal number of fresh cells (3H-thymidine uptake 3250 vs 7365 cpm/10(6). Similarly, preincubated cells from the healthy twin had greater suppressive effect (1820 cpm/10(6) cells). We conclude that (1) the HLA identical healthy twin has TMA autoreactive lymphocytes regulated by adherent regulatory cells; (2) the increased ratio of helper/suppressor cells in the adherent cell population in the patient leads to a decrease of (3H) incorporation upon their removal; (3) in the patient, the specific suppressor cell defect is more severe than the non-specific defect; (4) lack of specific TMA induced triggering may be the critical immunoregulatory defect in Graves' disease.

Topics: Adult; Antigens, Surface; Cell Division; Concanavalin A; Diseases in Twins; Female; Graves Disease; Humans; Lymphocyte Activation; Pregnancy; T-Lymphocytes, Regulatory; Thymidine; Thyroid Gland; Twins, Monozygotic

Topics: Adolescent; Adult; B-Lymphocytes; Cells, Cultured; Concanavalin A; Female; Graves Disease; Humans; Immunoglobulin G; Leukocyte Count; Male; Middle Aged; Pokeweed Mitogens; Rosette Formation; T-Lymphocytes; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory

Using peripheral blood lymphocytes from 8 healthy individuals and 5 patients with untreated Graves' disease, direct effects of methimazole (MMI) and propylthiouracil (PTU) on lectin-induced lymphocyte proliferative response were studied. Lymphocytes were cultured for 72 hr in the presence of lectins and antithyroid drugs. Lymphocyte DNA synthesis was counted by incorporation of 3H-thymidine. MMI at 1,000 microM enhanced lectin-induced lymphoproliferation of peripheral blood lymphocytes from both patients with Graves' disease and healthy individuals, at every point of culture time, while PTU showed a tendency toward suppression. These results suggest that this lympho-stimulation by MMI may be a causative factor related to insulin autoimmune syndrome, as deduced from the clinical reports that insulin autoimmune syndrome is, sometimes, found in patients with Graves' disease treated with MMI. This lympho-stimulation was evident regardless of the time of MMI addition, thus indicating that MMI is, by its action, a lymphoid stimulator and may lead to the insulin autoimmune syndrome in predisposed subjects with underlying Graves' disease.

Topics: Autoimmune Diseases; Concanavalin A; Graves Disease; Humans; Insulin Antibodies; Lymphocyte Activation; Methimazole; Phytohemagglutinins; Pokeweed Mitogens; Propylthiouracil; Syndrome

Suppressor lymphocyte function was evaluated in control subjects and in patients with autoimmune thyroid disease, utilizing an assay in which indomethacin was added to lymphocyte cultures to inhibit prostaglandin-producing suppressor cells. This assay is based on the observation that the addition of indomethacin, a potent prostaglandin synthesis inhibitor, to phytohemagglutinin-stimulated peripheral blood lymphocytes should cause an increase in the incorporation of iododeoxyuridine in control subjects and a smaller increase in diseases with reduced prostaglandin-producing suppressor cells. The addition of indomethacin, 1 microgram/ml, stimulated iododeoxyuridine incorporation in phytohemagglutinin-stimulated cultures in control subjects to an index value of 1.43 (i.e., the increment in iododeoxyuridine incorporation with both indomethacin and phytohemagglutinin was 43% greater than the incorporation with phytohemagglutinin alone). The stimulation index was significantly lower in patients with Graves' disease who were toxic and untreated (1.18 +/- 0.25, mean +/- SD; P less than 0.003). Patients who were toxic while receiving antithyroid drugs or after radioiodine therapy or patients euthyroid after treatment had a mean stimulation index in the normal range, although the spread of data was very large in these groups. Responses in patients with Hashimoto's thyroiditis were also quite variable. The average response was 1.74 +/- 0.72, with 40% of the patients showing a high stimulation index. This study supports our previous investigations in which we used different assay systems for measuring suppressor-cell function in patients with thyroid autoimmune diseases and indicates that a defect in suppressor lymphocyte function is measureable by another technique. The abnormality persists in some cases after metabolic control has been achieved, but usually returns toward normal over months or years.

Topics: Adolescent; Adult; Aged; Autoimmune Diseases; Cell Adhesion; Concanavalin A; Female; Graves Disease; Humans; In Vitro Techniques; Indomethacin; Lymphocyte Activation; Male; Middle Aged; Phytohemagglutinins; Prostaglandins; T-Lymphocytes, Regulatory; Thyroiditis, Autoimmune

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.

Topics: Adult; Autoantibodies; Autoimmune Diseases; Concanavalin A; Female; Graves Disease; Humans; Immunoglobulin G; Male; Microsomes; Middle Aged; Pokeweed Mitogens; T-Lymphocytes, Regulatory; Thyroiditis, Autoimmune

Function of short lived and concanavalin-A activated suppressor T cells of peripheral blood was studied in patients with thyrotoxicosis and normal individuals. It was found that the activity of short lived and concanavalin-A activated suppressort T cells decreased in untreated patients. Function of concanavalin-A activated suppressor cells relatively increased in euthyroid stage induced by methimazole treatment but failed to reach the normal level. Both the short lived and Concanavalin-A activated suppressor cell activity proved to be in inverse relationships with titres of anti-thyroglobulin antibodies and transformation indices to human thyroglobulin. These findings suggest a possible role of suppressor T cell function in the pathogenesis of Graves' disease.

Topics: Adolescent; Adult; Antibodies; Cell Survival; Concanavalin A; Female; Graves Disease; Humans; Male; Methimazole; Middle Aged; T-Lymphocytes, Regulatory; Thyroglobulin

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.

Topics: Adolescent; Adult; Cells, Cultured; Concanavalin A; DNA; Female; Graves Disease; Humans; Male; Propylthiouracil; T-Lymphocytes; Thyroid Neoplasms; Thyroiditis, Autoimmune; Time Factors