Lack of effect of methimazole on thyrocyte cell-surface antigen expression.

The nature of the immunosuppressive effect of antithyroid drugs has been a subject of controversy. It has been claimed that these agents exert a direct effect on the immune system, although we and others have suggested that the drugs affect the thyroid cells primarily with consequent reduced thyrocyte-immunocyte signalling. This may occur from reduced thyroid hormone production and/or reduced antigen presentation by the thyrocytes to local T lymphocytes. Using a cytotoxicity assay system, with chromium-51 labelling, monoclonal antibodies against thyroperoxidase (TPO) and HLA-DR, and complement, we have measured the expression of TPO and HLA-DR on cultured normal human thyroid cells; we have also measured thyroglobulin (Tg) release by radioimmunoassay into the medium of the cultured cells. The thyroid cells were stimulated with TSH or thyrotropin binding inhibitory immunoglobulin (TBII) for 48 hours before measuring for TPO induction, and with interferon gamma (IFN-gamma) (with or without TSH or TBII) for thyrocyte HLA-DR expression. A dosage of 1.6 milliunits per ml of TSH resulted in a significant increase in TPO expression on thyrocytes when compared with control unstimulated thyroid cells (p less than 0.001). The concentrations of Tg released into the medium with TSH or TBII were also significantly higher than those of the control thyrocytes. IFN-gamma at 200 units per ml induced HLA-DR expression, but did not induce thyrocyte TPO expression, or Tg release. Addition of the antithyroid drug, methimazole (MMI), at different concentrations, in addition to the other stimulators, IFN-gamma, TSH, or TBII, did not result in any inhibition of TPO, Tg release, or HLA-DR expression on the thyroid cells. It would thus appear that the pathways for stimulation for the expression of TPO and HLA-DR appear to be different. Finally, MMI does not cause its immunosuppressive effect by any reduction of thyroid antigen expression or release.

Nontoxic nodular goiter and papillary thyroid carcinoma are not associated with peripheral blood lymphocyte sensitization to thyroid cells.

We tested the claim that uni- and multinodular goiter (UNG and MNG) and papillary carcinoma (PC) of the thyroid are autoimmune thyroid diseases (AITD) similar to Graves' disease (GD) and Hashimoto's thyroiditis (HT). The expression of HLA-DR on cultured thyroid epithelial cells (thyrocytes) from UNG, MNG, and PC after coculture with autologous peripheral blood mononuclear cells (PBMC) was compared with that on GD and HT cells. The thyrocytes also were cultured with interferon-gamma (IFN gamma) alone. A cytotoxicity assay involving 51Cr-labeled thyrocytes, anti-HLA-DR, and complement was used to determine HLA-DR expression. Stimulation of thyrocytes with 200 U/mL IFN gamma induced HLA-DR (expressed as a cytotoxicity index) equally well on all thyrocytes [AITD (n = 6): IFN gamma, 23.8 +/- 7.7 (+/- SD); unstimulated, 3.6 +/- 2.0; UNG (n = 6), MNG (n = 9), and PC (n = 5): IFN gamma, 22.5 +/- 4.7; unstimulated, 4.0 +/- 3.0]. When cocultured with autologous PBMC, the values were: AITD, 24.9 +/- 10.1; UNG, MNG, and PC, 3.8 +/- 3.7 (P less than 0.001). The supernatants from the AITD cocultures had higher IFN gamma concentrations (by RIA) than those from the other cocultures. We conclude that in UNG, MNG, and PC, the peripheral blood helper T-lymphocytes are not sensitized to thyrocyte membrane antigen(s); consequently, little if any IFN gamma is produced in cocultures, and hence, there is no increase in thyrocyte HLA-DR expression, unlike the situation in AITD (GD and HT). Thus, UNG, MNG, and PC are not primarily autoimmune in nature, as defined by a lack of sensitization of the PBMC of such patients to thyroid antigen(s).

Production of antigen specific procoagulant activity; effect of various culture supernatants.

Culture supernatants (CS) of peripheral blood mononuclear cells (PBM) from normal subjects and patients with autoimmune thyroid disease (AITD) have been tested for procoagulant inducing activity. CS of PBM from patients with AITD stimulated with solubilized thyroid antigen induced significantly greater procoagulant activity (PCA) in a human monocyte-like cell line, U937, than CS of PBM from normal subjects similarly tested, suggesting that PBM from patients released a procoagulant inducing factor(s) into ambient fluid in response to thyroid antigen stimulation. There was a significant correlation between PCA inducing activity in PBM and that in CS from those same PBM cultures. CS from normal T lymphocytes stimulated with large amounts of thyroid antigen (putative suppressor factor) had no suppressive effect on thyroid antigen-induced PCA production in PBM from patients with AITD. These observations suggest that the helper activity for antigen-induced PCA production appears to be mediated, at least in part, by soluble factor(s); conversely, these helper cells were not apparently subject to suppressor influences under these circumstances.

In vitro induction of anti-thyroid microsomal antibody-secreting cells in peripheral blood mononuclear cells from normal subjects.

Secretion of immunoglobulin G (IgG) and IgM antithyroid microsomal antibodies (AMA) was induced in vitro by coculturing non-T cells (B lymphocytes) and autologous CD4 (helper/inducer) cells from normal subjects stimulated with pokeweed mitogen (PWM) or a combination of human thyroid microsomal antigen (McAg) and Staphylococcus aureus Cowan I (SAC) strain. With PWM stimulation, AMA production was induced in more IgM-secreting cells (AMA-M) than IgG-secreting cells (AMA-G). However, McAg plus SAC stimulation resulted in similar numbers of AMA-G- and AMA-M-secreting cells. PWM induced a significantly greater number of both AMA-M (and generalized IgM)-secreting cells than did McAg plus SAC, while the number of AMA-G-secreting cells induced by the two stimuli were similar. There were no significant differences between autologous or allogeneic CD4 cells from normal subjects or patients with autoimmune thyroid disease (AITD) when cocultured with B cells from normal subjects in terms of helper activity in the induction of AMA-M- or IgM-secreting cells with PWM stimulation. However, with McAg plus SAC, CD4 cells from patients with AITD induced a significantly greater number of AMA-M-secreting cells than did autologous or allogeneic CD4 cells from normal subjects. There was no difference in helper activity between autologous and allogeneic normal CD4 cells in the induction of generalized IgM-secreting cells regardless of the stimulus used. Normal autologous or allogeneic CD8 (suppressor/cytotoxic) cells cocultured with normal B cells and autologous CD4 cells suppressed the induction of AMA-M-secreting cells by PWM stimulation. On the other hand, CD8 cells from patients with AITD suppressed the induction of AMA-M-secreting cells significantly less effectively. All CD8 cells suppressed the induction of IgM-secreting cells equally well. We conclude that 1) B lymphocytes from normal subjects are capable of producing autoantibodies in vitro in the presence of CD4 cells; 2) the helper activity of CD4 cells from patients with AITD to induce AMA-M secreting cells is greater than that of normal CD4 cells with thyroid antigen stimulation; and 3) this helper activity may be due to relatively impaired suppressor activity in thyroid antigen-specific CD8 cells from patients with AITD, whereas the immunoregulatory function of CD8 cells from normal subjects appears to play an important role in the maintenance of self-tolerance.

Comparison of measurements of in vitro production of antithyroid microsomal antibody versus antithyroid peroxidase antibody.

In vitro production of antithyroid microsomal antibody (AMA) and antithyroid peroxidase antibody (APA) by peripheral blood lymphocytes from patients with autoimmune thyroid disease (AITD) has been studied and compared, in view of the evidence for identity of the two differently measured antibodies. Peripheral non-T cells (2 x 10(5)) and autologous CD4 (helper/inducer) cells (2 x 10(5)) from patients with positive serum AMA were cultured for 7 days with pokeweed mitogen (PWM). B cells secreting AMA or APA were detected by the enzyme-linked immunosorbent assay (ELISA) spot assay. AMA or APA in the culture supernatants of these cells was also measured by ELISA. There was a significant correlation between the number of AMA- (IgG class) secreting cells and APA- (IgG class) secreting cells (r = 0.89 p less than 0.001). There was also a significant correlation between AMA- and APA-ELISA indices (r = 0.86, p less than 0.001). Furthermore, the number of AMA- or APA-secreting cells significantly correlated with AMA or APA secreted in the culture supernatants (r = 0.91, r = 0.92), respectively. These data show that peripheral blood lymphocytes from patients with AITD were able to produce antibodies against thyroid peroxidase (TPO) in vitro, as well as antibodies against thyroid microsomal antigen, after PWM stimulation. The significant correlation between in vitro AMA versus APA production, or the number of AMA- versus APA-secreting cells, accords with the evidence that TPO is identical to, or at least the major antigenic protein component of, thyroid microsomal antigen.

Effect of HLA-DR positive thyrocytes on in vitro thyroid autoantibody production.

The function of HLA-DR positive thyrocytes on thyroid autoantibody production has been examined to test the hypothesis that such HLA-DR positive thyrocytes may initiate or aggravate autoimmune thyroid disease. Thyrocytes were cultured (precultured) with leucoagglutinin (which stimulated thyrocyte expression of HLA-DR, beta 2-microglobulin (beta 2-m) and thyroid microsomal antigens) and then cocultured with peripheral blood mononuclear cells. Thyroid antibody production by the latter was then measured. There was no evidence of induction or enhancement of thyroid-microsomal and thyroglobulin autoantibody production in supernatants from the cocultures of autologous peripheral blood mononuclear cells and HLA-DR positive thyrocytes from normal controls and patients with Graves' disease. Furthermore, stimulation of B lymphocytes from patients with autoimmune thyroid disease with a combination of Staphylococcus aureus Cowan I plus supernatants from autologous cocultures of peripheral blood mononuclear cells and HLA-DR positive thyrocytes from normal controls and Graves' disease, produced significantly less microsomal antibody and thyroglobulin antibody than similar cocultures with HLA-DR negative thyrocytes, although total immunoglobulin G (IgG) was similar in both groups. The effect of supernatants from allogeneic cocultures on microsomal antibody thyroglobulin antibody and total IgG production was no different between HLA-DR positive and HLA-DR negative thyrocytes. These data suggest that HLA-DR positive thyrocytes may have a protective role against thyroid autoimmunity rather than a pathogenic role for it.

Studies of HLA-DR expression on cultured human thyrocytes: effect of antithyroid drugs and other agents on interferon-gamma-induced HLA-DR expression.

There have been conflicting reports on whether antithyroid drugs (ATD) act as immunosuppressive agents in patients with autoimmune thyroid disease. While some have claimed that methimazole (MMI) affects the immune system directly, we and others have suggested that its apparent immunosuppressive activity is due to its ability to inhibit thyrocyte, rather than immunocyte, activity. To further address the question, we studied the action of ATD on interferon-gamma (IFN gamma)-induced HLA-DR expression on thyrocytes in tissue culture. We used a cytotoxicity assay, using chromium-51-labeled Graves' disease (GD) thyrocytes and normal thyrocytes incubated sequentially with a monoclonal antibody against HLA-DR and complement, with a cytotoxicity index as the measure of thyrocyte HLA-DR expression. MMI and propylthiouracil (PTU) were added along with 200 U/mL IFN gamma to thyrocytes cultured for 10-14 days. IFN gamma or supernatants from leukoagglutinin-stimulated peripheral blood mononuclear cells (PBMC) stimulated thyrocyte HLA-DR expression; however, the addition of MMI or PTU to either the PBMC or thyrocytes caused no inhibition of the IFN gamma or PBMC IFN gamma stimulation of thyrocyte HLA-DR expression, using either normal or GD thyrocytes. Potassium perchlorate and sodium iodide also had no effect on IFN gamma-induced thyrocyte HLA-DR expression. TSH (either bovine or human) did not induce HLA-DR expression on thyrocytes by itself, but did enhance IFN gamma-induced HLA-DR expression in normal, but not GD, thyrocytes; once again, the further addition of MMI or PTU did not inhibit the enhancing effect of TSH on thyrocyte HLA-DR expression. Low concentrations of TSH binding inhibitory immunoglobulin (TBII; 100 micrograms/mL) did not alter the cytotoxicity index, but at 400 micrograms/mL or more it enhanced HLA-DR expression on normal, but not GD, thyrocytes in a manner similar to TSH; like TSH, it did not induce thyrocyte HLA-DR expression by itself. Moreover, addition of MMI to the combination of IFN gamma and TBII did not inhibit the response of thyrocytes in terms of HLA-DR expression. We conclude that ATD do not alter thyrocyte HLA-DR expression in vitro; however, the ATD may still cause immune effects in vivo secondary to their influence on thyroid hormone formation or synthesis or by inhibition of thyroid antigen presentation which indirectly may result in an immunomodulatory effect. While TSH and TBII similarly enhanced the IFN gamma-induced expression of HLA-DR on normal thyrocytes, they did not do so in GD thyrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of the effect of suppressor T lymphocytes on the induction of antithyroid microsomal antibody-secreting cells in autoimmune thyroid disease.

The suppressor function in CD8 (suppressor/cytotoxic) T lymphocytes from patients with autoimmune thyroid disease and normal subjects has been studied. CD8 and CD4 (helper/inducer) cells were separated by the panning method. Patient's non-T cells and autologous CD4 cells were cultured with or without autologous or allogeneic CD8 cells in the presence of either pokeweed mitogen or Staphylococcus aureus strain Cowan 1 plus human thyroid microsomal antigen. Antithyroid microsomal antibody (AMA) and total immunoglobulin G (IgG)-secreting non-T cells were measured by enzyme-linked immunosorbent spot assay. With pokeweed mitogen stimulation, the suppressor effect of CD8 cells from patients with serum AMA on the induction of AMA (of IgG type)-secreting cells was significantly less than that of CD8 cells from normal subjects. CD8 cells from patients with no serum AMA suppressed the induction of AMA-secreting cells as much as did normal CD8 cells. CD8 cells from both patients and normal subjects suppressed the induction of IgG-secreting cells equally well. On the other hand, with the combination of S. aureus strain Cowan 1 and human thyroid microsomal antigen (1 mg/L) stimulation, CD8 cells from both normal subjects and patients only slightly suppressed the induction of IgG-secreting cells. However, under these circumstances, once again, CD8 cells from both normal subjects and patients with no serum AMA suppressed the induction of AMA-secreting cells, whereas CD8 cells from patients with serum AMA suppressed the induction of the AMA-secreting cells significantly less. Higher TMc concentrations enhanced the suppressor effect of CD8 cells from patients with serum AMA on the induction of AMA-secreting cells. Furthermore, Concanavalin A, when added to the stimuli described above, further inhibited the induction of both AMA- and IgG-secreting cells by CD8 cells from patients with serum AMA. There thus appears to be a relative defect of antigen-specific suppressor T lymphocyte function in CD8 cells from patients with autoimmune thyroid disease, which may result in the presence of autoantibody-secreting cells in those patients.

Induction of thyroid autoantibody production: synergistic effect of B cell mitogen combined with T cell mitogen.

In vitro production of thyroglobulin autoantibodies (TgAb) and thyroid microsomal autoantibodies (McAb) by peripheral blood mononuclear cells (PBMC) stimulated with the B cell mitogen Staphylococcus aureus Cowan I (SAC) and the T cell mitogen pokeweed mitogen (PWM) was examined in 35 normal subjects (NC) and 64 patients with autoimmune thyroid disease (AITD) using an enzyme-linked immunosorbent assay technique. Low concentrations of SAC plus PWM resulted in a synergistic effect on thyroid autoantibody production as well as nonspecific immunoglobulin G production. With such maximal stimulation, TgAb production was detected in all PBMC preparations from serum TgAb-positive patients with AITD; TgAb production was also detected in some NC (46%) and serum TgAb-negative patients with AITD (39%), but the levels of TgAb production were low. Similarly, McAb production was marked in PBMC preparations from serum TgAb-negative but McAb-positive patients. TgAb-secreting cells were also detected in NC by the plaque-forming cell (PFC) assay. The response patterns of PBMC to mitogen (Nil, PWM, and SAC plus PWM) in terms of TgAb production varied among serum TgAb-positive patients with AITD, but not among NC and serum TgAb-negative patients with AITD. Serum TgAb titers were significantly correlated with the in vitro production of TgAb by PBMC with no stimulation (r = 0.64; n = 99; P less than 0.001), with stimulation by PWM (r = 0.75), and with stimulation by SAC plus PWM (r = 0.87); the correlation coefficient increased with the efficiency of stimulation of B cell differentiation. Similar results were found for McAb production. These data suggest that 1) optimal in vitro thyroid autoantibody production occurs with B cell mitogen (SAC) acting synergistically with T cell mitogen (PWM); 2) sufficient numbers of resting B lymphocytes specific for Tg or microsomal antigens are present in some NC PBMC; 3) stages of thyroid-specific B cell differentiation in PBMC vary among serum thyroid autoantibody-positive patients with AITD; and 4) the potential of PBMC to produce thyroid autoantibodies may correlate with the capacity of thyroid-derived lymphocytes. Thus, the circulating lymphocytes may provide a useful vehicle by which sequential changes occurring at the tissue level may be examined.

Immunomodulatory effect of the treatment of Graves' disease on antigen-specific monocyte procoagulant activity production.

The monocyte procoagulant activity (PCA) production assay has been shown to be a good parameter of cell-mediated immunity. We have studied antigen-specific PCA production in peripheral blood mononuclear cells from patients with Graves' disease to determine the effect of the treatment on the cell-mediated immune response. Peripheral blood mononuclear cells from patients with untreated or relapsed Graves' disease produced significantly greater PCA with thyroid antigen stimulation than those from normal subjects. Patients both on antithyroid drugs in the hyperthyroid state and within 3 months post-131I therapy also produced significantly larger amount of PCA than normal subjects. However, there was no significant difference in PCA production with thyroid antigen stimulation between normal subjects and patients on anti-thyroid drugs in the euthyroid state, or patients over 3 months post-131I therapy. The ratio of positive to negative PCA production in patients on anti-thyroid drugs in the euthyroid state or over 3 months post-131I therapy was significantly lower than in untreated or relapsed Graves' disease patients. Mononuclear cells from patients on propylthiouracil responded to propylthiouracil in vitro by production of PCA. Cells from normal subjects, untreated Graves' disease patients, or patients with Hashimoto's thyroiditis did not produce PCA with propylthiouracil stimulation. Mononuclear cells from patients who were on propylthiouracil for more than 3 months produced greater PCA than those on the drug for less than 3 months, suggesting sensitization of lymphocytes to propylthiouracil during the course of treatment. However, after 131I therapy, they gradually became unresponsive to propylthiouracil. This study has shown that the activity of the antigen-specific response assessed by PCA production in mononuclear cells from Graves' disease patients declined after treatment, suggesting that the treatment exerted immunomodulatory effects.

Induction of monocyte procoagulant activity with OKT3 antibody.

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.

The decrease in non-specific suppressor T lymphocytes in female hyperthyroid Graves' disease is secondary to the hyperthyroidism.

There has recently been considerable interest generated in the significance of changes in the peripheral blood T lymphocyte subsets in patients with autoimmune thyroid disease. Previously, monoclonal antibodies that recognized T cells (Leu 1+ cells), T helper/inducer cells (Leu 3a+ cells), and T suppressor/cytotoxic cells (Leu 2a+ cells), have been used to enumerate these subsets. Using 2 new monoclonal antibodies (anti-Leu-8 and anti-Leu-15), in addition to the above 3 antibodies, and 2-colour flow cytometry, we have enumerated the total T, T helper/inducer, T suppressor/cytotoxic, T helper (Leu 3a+8-), T inducer (Leu 3a+8+), T suppressor (Leu 2a+15+), and T cytotoxic (Leu 2a+15-) cells in 22 patients with hyperthyroid Graves' disease, 38 patients with 131I-treated Graves' disease and 10 patients with Hashimoto's thyroiditis. All patients and controls were female. We found that hyperthyroid patients with Graves' disease had significantly lower T suppressor/cytotoxic cells (p less than 0.05) than did controls, and that this was mainly due to a decrease in T suppressor cells (p less than 0.01). Furthermore, patients with severe hyperthyroidism had a more significant decrease in T suppressor/cytotoxic (p less than 0.001) and T suppressor (p less than 0.001) cells, and an increase in the T helper/inducer:T suppressor/cytotoxic (p less than 0.01) and T helper:T suppressor (p less than 0.01) cell ratios. Patients who were euthyroid more than 1 year after 131I therapy for Graves' disease had normal T cell subsets and ratios, whether or not TSH receptor antibody or other thyroid auto-antibody titres were elevated. Ten females with Hashimoto's thyroiditis also had normal T cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)

Separate induction of MHC and thyroid microsomal antigen (McAg) expression on thyroid cell monolayers: enhancement of lectin-induced McAg expression by interferon-gamma.

Interferon-gamma (IFN gamma) induced the expression of the MHC class II antigens HLA-DR and -DQ on 1- to 2-week-old thyrocytes from normal thyroid tissue and thyroid tissue from patients with autoimmune thyroid disease; it also enhanced the expression of B2-microglobulin, which is associated with MHC class I molecules. However, the expression of thyroglobulin and thyroid microsomal antigen (McAg) was not detected after IFN gamma stimulation. Autologous and allogeneic peripheral blood mononuclear cells had the same ability as IFN gamma to induce antigen expression when cocultured with thyrocytes. In contrast, leucoagglutinin (LAG) induced McAg as well as HLA-DR and B2-microglobulin expression on thyrocytes, but not thyroglobulin expression. Concanavalin A and pokeweed mitogen also induced McAg expression. The time course of LAG induction of McAg was not always correlated with that of HLA-DR. Anti-IFN gamma, antiinterleukin-2 receptor, and anti-HLA-DR monoclonal antibodies inhibited LAG or peripheral blood mononuclear cell induction of HLA-DR expression, but not LAG induction of McAg expression. Anti-HLA-DR reduced the IFN gamma induction of HLA-DR. INF gamma enhanced thyrocyte McAg expression induced by LAG, especially when thyrocytes were incubated with IFN gamma for 24 h before LAG stimulation. In contrast, in the absence of LAG stimulation, IFN gamma suppressed already present spontaneous McAg expression. TSH did not induce McAg and HLA-DR expression on DR-negative thyrocytes, but enhanced weak DR expression induced by other stimulants, e.g. IFN gamma or lectins. These data suggest that in vitro induction mechanisms of MHC class I and II antigens and McAg are different; MHC antigens are induced by IFN gamma, whereas McAg is induced by lectin, probably acting on thyrocytes directly; and IFN gamma has an enhancing effect on LAG-induced thyrocyte McAg expression.

Sensitization of T lymphocytes to thyroid antigen in autoimmune thyroid disease as demonstrated by the monocyte procoagulant activity test.

Monocyte procoagulant activity (PCA) production has been reported to have a close relation to cell-mediated immunity (CMI), and the collaboration of T lymphocytes is necessary to induce PCA. Antigen-specific sensitization of lymphocytes in patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT) has been demonstrated by means of the production of cell-bound PCA by monocytes following antigen stimulation of whole peripheral blood mononuclear cells (PBM). These cells, obtained from both normal subjects and patients with autoimmune thyroid diseases, produced significant amounts of PCA with non-specific lectin, concanavalin A (Con A) stimulation; however, there was no significant difference between the two groups, suggesting that Con A stimulated T cells induced monocyte PCA nonspecifically. Peripheral mononuclear cells from patients with autoimmune thyroid diseases produced significantly greater amounts of PCA than PBM from normal subjects when stimulated with solubilized and IgG-free thyroid antigen. On the other hand, liver antigen did not induce significant amounts of PCA production in PBM from either normal subjects or patients. Significantly larger amounts of PCA were produced by PBM from patients following thyroid antigen stimulation than with liver antigen stimulation. Although monocytes were the major source of PCA, T cells were necessary to induce PCA in monocytes with thyroid antigen and Con A stimulation. Elimination of lymphocyte subsets in PBM from patients by negative selection (using monoclonal antibodies and complement) suggested that the collaboration of T lymphocytes, especially helper/inducer (T4+) T cells, was necessary to produce PCA with thyroid antigen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro regulation of antithyroglobulin synthesis by lymphocytes of patients with Hashimoto's disease by an antigen-specific "suppressor factor" derived from cultured normal human T-lymphocytes.

Suppressor factors (SF) elaborated by peripheral blood T-lymphocytes in culture from normal subjects activated by thyroglobulin as well as those from patients with Hashimoto's thyroiditis (HT), were tested for their ability to modulate antithyroglobulin synthesis by cultured HT-lymphocytes. A micro ELISA system and a micro reverse haemolytic plaque assay system were employed to detect antithyroglobulin and antithyroglobulin secreting B-lymphocytes respectively. In the latter system, the SF from normal T-lymphocytes caused suppression of thyroglobulin-specific plaque formation by HT lymphocytes while the "SF" from HT T-lymphocytes did not cause any suppression in most of the cases studied. A "SF" obtained by using liver antigen as a non-specific antigen, did not cause suppression of plaques. These results were highly significant (p less than 0.001). Although differences were seen with and without SF in the ELISA system, they were not statistically significant, likely due to a lack of sensitivity of the ELISA system as compared with the plaque assay. These observations imply that the HT-lymphocytes were deficient in thyroid antigen specific T-suppressor cell function and/or numbers to begin with and that the added normal SF afforded the signal necessary to the helper T-cells to suppress antibody synthesis by the B-lymphocytes. It is thus inferred that there is a defect in antigen-specific suppressor T-lymphocyte function in Hashimoto's thyroiditis.

Thyrocyte HLA-DR expression and interferon-gamma production in autoimmune thyroid disease.

We examined the expression of HLA-DR antigen induced by mitogen, mitogen-free supernatants from mitogen-stimulated peripheral blood mononuclear cells (PBMC), or autologous and allogeneic PBMC on thyrocytes cultured for 1-2 weeks (precultured) before the addition of the stimulant. Leucoagglutinin (LAG) and concanavalin A, but not lipopolysaccharide induced HLA-DR expression on thyrocytes from normal subjects (NC) and patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). The degree of DR expression induced by LAG was significantly less in GD than in NC thyrocytes. This response was dependent on contaminating T cells, especially suppressor-cytotoxic T (Ts/c) cells, NK cells, and HLA-DR+ cells, but not helper-inducer T (Th/i) cells or B cells, in the thyrocyte cultures. OKT3 monoclonal antibody, which activates T cells specifically in the presence of monocytes, also induced thyrocyte HLA-DR expression. Furthermore, interferon-gamma (IFN-gamma) was detected in culture supernatants from LAG-stimulated thyrocytes. Anti-IFN-gamma monoclonal antibody eliminated the ability of LAG to induce HLA-DR. Mitogen-free supernatants from mitogen-stimulated PBMC also induced thyrocyte HLA-DR expression, which was inhibited by anti-IFN-gamma. The supernatants of concanavalin A- or LAG-stimulated PBMC from either untreated or recently treated patients with GD or hypothyroid HT induced less thyrocyte DR expression than NC PBMC. Indeed, the levels of IFN-gamma in supernatants from such patients were lower than those in NC, and the correlation between DR expression and IFN-gamma levels was significant. This IFN-gamma production by PBMC required Th/i cells, NK cells, and HLA-DR+ cells. Before the addition of autologous or allogeneic PBMC, only precultured HT thyrocytes expressed HLA-DR, whereas GD and NC thyrocytes did not. The induction or enhancement of DR expression on autologous thyrocytes by direct coculture with PBMC occurred within 8 days in GD and HT, but not in NC. There was a significant correlation between the serum titer of antithyroid microsomal antibodies and the degree of DR expression. Allogeneic normal PBMC also induced DR expression on NC and GD thyrocytes within 8 days, the effect on the latter being more pronounced than with autologous GD PBMC. Thyrocyte HLA-DR expression induced by autologous GD PBMC and allogeneic normal PBMC required monocytes. Th/i, and NK cells and was blocked by anti-IFN-gamma. However, the enhancement of thyrocyte DR expression by autologous HT PBMC did not require monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on the immunoregulation of thyroid autoantibody production in vitro.

The spontaneous production (without mitogen or antigen) of antithyroglobulin and antimicrosomal antibodies by peripheral (PBL) and thyroid-derived lymphocytes from patients with Hashimoto's thyroiditis (HT) has been studied with particular emphasis on the regulation of this phenomenon. Based on studies of DNA and protein synthesis, kinetic studies and B/T reconstitution experiments, in most HT patients, spontaneous production by PBL is accounted for by secretion of preformed antithyroglobulin (termed Type 1 patients), whereas active production is observed in a small minority (termed Type 2). In none of 24 HT patients could active antimicrosomal antibody production by PBL be detected. Conversely, thyroid-derived lymphocytes produced both autoantibodies by an active process. Pokeweed mitogen (PWM) stimulation enhanced antibody production by PBL in the Type 1 group but not in Type 2 or thyroid-derived lymphocytes. T lymphocytes were required for antibody synthesis in both thyroid antigen-driven and peripheral PWM-driven cultures. By separating T lymphocytes into T4+ (helper) and T8+ (suppressor) subsets with monoclonal antibodies, T-cell modulation of autoantibody production in both systems was studied. In a PWM-induced system, both thyroid and peripheral T-cell subsets were capable of modulating peripheral antibody production. In the thyroid lymphocyte antigen-specific system, further addition of thyroid derived T8+ cells alone caused partial suppression of antibody production but not with peripheral T8+ cells. Of interest was the partial decrease of antibody production by the thyroid lymphocytes by added peripheral T4+ cells. The fact that the production of thyroid autoantibodies by thyroid-derived mononuclear cells (which included T suppressor, T helper and B lymphocytes) could be reduced by the addition of more suppressor T lymphocytes suggests that an antigen-specific defect in the T4+/T8+ thyroid cell balance may account for the in vivo production of these antibodies in patients with Hashimoto's thyroiditis.

A micro reverse haemolytic plaque technique for the detection of antithyroglobulin antibody producing-lymphocytes in Hashimoto's thyroiditis.

Employing a micro reverse haemolytic plaque (RHP) technique, antithyroglobulin antibody producing B-lymphocytes have been detected in thyroglobulin challenged peripheral blood lymphocyte cultures from patients with Hashimoto's thyroiditis. The method employs a monolayer of sheep red blood cells instead of a semi-solid matrix as a medium for the development of plaques. This technique which requires very small amounts of immunoreagents, is therefore much less expensive than the macro methods. In our studies of patients with Hashimoto's thyroiditis, peripheral lymphocytes of patients produced a significantly greater number of plaques than lymphocytes from normal subjects on challenging with thyroglobulin. There was a good correlation between the thyroid antibodies detected by the haemagglutination tests in the sera of the patients and the production of plaques by their lymphocytes. The RHP assay also appeared to be more sensitive than the haemagglutination test. The technique should lend itself for the study of the effect of various lymphokines on cultured lymphocytes.

Spontaneous secretion of thyroid autoantibodies by cultured peripheral blood lymphocytes from patients with Hashimoto's thyroiditis detected by micro-ELISA techniques.

The spontaneous in vitro production of anti-thyroglobulin (aTg) and anti-microsomal (aM) antibodies by mononuclear cells (MNC) from patients with Hashimoto's thyroiditis (HT) was analysed by an ELISA detection system. MNC from 35 HT patients spontaneously produced detectable levels of both autoantibodies in vitro (i.e., without mitogenic or antigenic stimulation). aTg was quantified using a reference aTg IgG standard and ranged from 55 to 9,000 ng aTg. Specificity of aTg by ELISA was assessed using heterologous Tg antigen (Ag). Microsomal Ag obtained by gel filtration was far less contaminated with Tg than the ultracentrifugation pellet. Specificity of aM ELISA was assessed using insulinoma membrane as unrelated Ag and by blocking aM detection only with microsomal Ag. aM levels in the 35 supernatants ranged from 0.1 to 1.12 OD. A direct correlation was found between aM serum titres detected by haemagglutination and in vitro aM spontaneous production, but not for aTg. This lack of correlation for aTg might have biological relevance. Tg restimulation in vitro enhanced aTg production in only four out of 18 cases, of which only one was significant. This system provides a tool for studies of the immunoregulation of thyroid autoantibody formation in vitro.