[Emphysematous cystitis and neuropathy; a report of the case with diabetes mellitus].

A study was made of a 55 years old male, who suffered from emphysematous cystitis with diabetes mellitus. He had multiple complications due to diabetic neuropathy such as foot ulceration, oculomotor nerve palsy, peroneal nerve palsy and a neurogenic bladder. Klebsiella pneumoniae and Pseudomonous aeruginosa were cultured from urine specimens. There have been only 19 reported cases of emphysematous cystitis since 1962. Fourteen of these cases had diabetes mellitus.

cDNA sequence analysis and characterization of a cytokine-inducing monoclonal antibody derived from autoimmune MRL/MP-lpr/lpr mouse.

We have previously reported that a monoclonal antibody 1D11 derived from an autoimmune MRL/Mp-lpr/lpr (MRL/lpr) mouse induced synthesis or increased production of IL-3, IL-6, and TNF-alpha in the IL-3-dependent bone marrow-derived cell line FDC-P2/185-4. In this report, we analyzed a sequence of cDNA encoding the V region of 1D11, and found that VH and VL segments of 1D11 belonged to the J558 and V kappa 21 family, respectively. The nucleotide sequence of 1D11 in the VH segment was highly homologous to that of AM9, a monoclonal RF derived from MRL/lpr mouse, and the only difference was the replacement of 3 nucleotides in the framework region 1 (FR1). However, the deduced amino acid sequence of 1D11 was identical to that of AM9. In contrast with the VH segment, the sequences of the VL regions of these two antibodies were quite different from each other; 1D11 showed a 3 base deletion in the FR2 and a 24 base insertion in the FR3 compared with AM9. At present, the mechanisms of such insertions or deletions in the FRs of autoantibodies are almost unknown. It is generally accepted that the differences in specificity and affinity of these autoantibodies depend on differences of CDR sequence. However, it is possible that not only CDRs but also FRs of autoantibodies play a critical role in pathogenicity and/or specificity in autoimmune diseases.

Activation of T lymphocyte subsets by synthetic TSH receptor peptides and recombinant glutamate decarboxylase in autoimmune thyroid disease and insulin-dependent diabetes.

We have postulated that a defect in specific antigenic induction of suppressor T lymphocytes may account for the immunoregulatory disorder in autoimmune thyroid disease. In this context, we have measured the proliferative responses of peripheral blood mononuclear cells (PBMC) to the synthetic peptides corresponding to the extracellular domain of the TSH receptor (TSHR) and recombinant glutamate decarboxylase (GAD65) by means of 3H thymidine incorporation. We have also studied the antigenic activation of CD4+ and CD8+ T lymphocytes by measuring human leukocyte antigen-DR (HLA-DR) expression on the cell surface by flow cytometric analysis. PBMC obtained from 47 patients with Graves' disease (GD) [including 19 hyperthyroid GD (hyper GD)], 18 with Hashimoto's thyroiditis (HT), 7 with nontoxic nodular goiter (NG), 18 with insulin-dependent diabetes (IDDM), and 20 normal controls (N), were cultured for 7 days in the presence or absence of the pool peptides representing 3 different segments of TSHR or GAD65 at final concentration of 30 micrograms/mL or 10 micrograms/mL. The proportion of subjects whose PBMC gave a positive proliferative response with a stimulation index (SI) of over 2.3 (i.e. above the mean +2 SD for N) to TSHR peptides was significantly higher in the hyper GD group than among euthyroid GD (eu GD), HT, IDDM, and N group. The corresponding differences in mean SI provided analogous results, showing significant responses above normal in only hyper GD. The CD4+ T lymphocytes from hyper GD group were significantly more activated by TSHR peptides compared to eu GD, HT, IDDM, and N, and this induction correlated to their thyroid hormone levels. Quite differently, the activation of CD8+ T lymphocytes from both hyper GD and eu GD group in response to TSHR peptides was impaired compared to HT, IDDM, and the N group; in contrast to the findings with CD4+ T lymphocytes, this was independent of thyroid hormone levels. On the other hand, while the CD8+ T lymphocytes from GD and N groups were activated equally by GAD65, the activation of CD8+ T lymphocytes from the IDDM group by GAD65 was impaired compared to the GD and N groups. In conclusion, the activation of CD8+ T lymphocytes from GD and IDDM by relevant antigens (i.e. TSHR peptides for GD and GAD65 for IDDM) was impaired, but not by irrelevant antigens (i.e. GAD65 for GD and TSHR peptides for IDDM). There was also a modest stimulation of CD8+ T cells from all groups by tetanus toxoid and cardiac myosin light chain peptide, both irrelevant antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of removing human Graves' thyroid xenografts after eight weeks in nude mice and rexenografting them into SCID mice.

Human thyroid xenografts from four patients with Graves' disease (GD) and two normal persons were initially xenografted into nude mice. Eight weeks after xenografting, the thyroid tissue appeared normal; indeed, thyroid infiltrating lymphocytes in the GD xenograft could no longer be identified when analyzed histologically. Thus, human immunoglobulin G (IgG), thyroperoxidase (TPO)-antibodies (Abs), thyroglobulin (Tg)-Abs, thyroid-stimulating antibodies (TSAb), and thyrocyte histocompatibility leucocyte antigen (HLA)-DR expression were undetectable. These same tissues were retrieved from the nude mouse and rexenografted into severe combined immunodeficient (SCID) mice (with no prior xenograft); autologous peripheral blood mononuclear cells (PBMC) or CD8-depleted PBMC (non-CD8 cells) were simultaneously injected into some of these SCID mice. Engraftment of a GD thyroid rexenograft (TH) alone did not cause IgG, TSAb, TPO-Ab, or Tg-Ab production, thyrocyte HLA-DR expression, or lymphocytic infiltration in thyroid grafts. Engraftment of GD PBMC or non-CD8 cells alone (i.e. without a thyroid xenograft) caused human IgG to rise, but only minimal titers of thyroid antibodies appeared. When TSAb, TPO-Ab, and Tg-Ab were quantified, GD TH plus PBMC-engrafted SCID mice showed significantly higher production of each antibody than that of GD PBMC alone, and this phenomenon was further enhanced by the removal of CD8+ cells. GD thyrocytes showed marked HLA-DR expression at human surgery; however, after 8 weeks' sojourn in nude mice, DR expression disappeared. After a further 8 weeks following rexenografting into SCID mice, TH plus PBMC resulted in a reappearance of DR expression only in GD but not in grafts from normal persons, and this was enhanced by the depletion of CD8 cells. These results were also in parallel with histological findings inasmuch as the normal tissue remained normal with no thyroid antibodies appearing with PBMC or CD8-depleted cells. In experiments from two GD patients, autologous skeletal muscle as well as thyroid tissue were xenografted into nude mice. Eight weeks after xenografting, these were rexenografted into SCID mice that contained prior autologous primary GD thyroid xenografts. Histological findings showed new lymphocytic infiltration in rexenografted thyroid tissues in the SCID mice but not in autologous skeletal muscle. This signifies that the immune assault in GD is specifically targeted to the thyroid tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

An autoimmune MRL/Mp-Ipr/Ipr mouse-derived monoclonal IgG antibody stimulates cytokine production in bone-marrow-derived cell line by cross-linking of a cell surface antigen and Fc receptor.

An IgG1 mAb 1G10 derived from an autoimmune MRL/Mp-Ipr/Ipr (MRL/Ipr) mouse has previously been shown to induce IL-3, TNF-alpha and IL-6 production, and autocrine growth in an IL-3-dependent myeloid cell line, FDC-P2/185-4. In the present study, we have attempted to further define the molecular mechanism responsible for the 1G10-induced activation of FDC-P2/185-4 cells. We have shown that 1G10 lacked anti-IgG1 rheumatoid factor activity, failing to generate self-associated immune complexes. Since 1G10 stimulated cells in an Fc gamma R-dependent manner, it seems likely that cross-linking of a cell surface antigen and Fc gamma R by 1G10 antibody is responsible for the stimulation of FDC-P2/185-4 cells. Among several mAb specific to surface antigens expressed on FDC-P2/185-4 cells (MHC class I, LFA-1, and Fc gamma R), only a mAb specific to the alpha chain of LFA-1 alpha was able to induce the IL-3 and Fc gamma R-dependent proliferation of FDC-P2/185-4 cells, similar to that induced by 1G10. Immunoprecipitation analysis revealed that 1G10 recognized a polypeptide with a molecular mass of 140 kilodaltons (p140), which differed from Fc gamma R and from LFA-1 alpha chain. These results suggest that cross-linking of not general but particular cell surface antigens and Fc gamma R stimulates FDC-P2/185-4 cells to produce cytokines resulting in their proliferation.

New animal models for human autoimmune thyroid disease. Xenografts of human thyroid tissue in severe combined immunodeficient (SCID) and nude mice.

We have been employing two mouse models for the study of human thyroid xenografts from patients with autoimmune thyroid disease (AITD). The first mouse strain is that of the athymic "nude" mouse which accepts human thyroid xenografts, but the passenger lymphocytes are lysed in this model over several weeks; in the second model, the severe combined immunodeficient (SCID) mouse, both the xenograft and its lymphocytes survive. The AITD thyroid xenograft returns to normal function and morphology in the nude mouse over several weeks whereas the same AITD thyroid xenografts undergo aggravation of their lesions in the SCID mouse. Normalized ("cleansed") thyroid tissue in the nude mouse, now bereft of the passenger lymphocytes, can be removed and then re-xenografted into SCID mice. There it will remain normal unless autologous peripheral blood mononuclear cells are added, whereupon the AITD lesion will be reproduced. Autologous "irrelevant" muscle tissue having undergone the same process will not show such lesions. There is thus no evidence for a primary thyroid cell disturbance in AITD, the abnormality appearing to be only in the immune system. Moreover peripheral blood mononuclear cells appear to contain sufficient memory cells to be able to mount an immune assault on the autologous normalized thyroid tissue (to which they had been previously sensitized) but not on irrelevant autologous (muscle) tissue.

In vitro effects of cytokines and human thyroglobulin on the induction of antibody-secreting cells in patients with auto-immune thyroid disease.

We have investigated the in vitro effects of interleukin-1-beta (IL-1-beta), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) on the induction of anti-thyroperoxidase (anti-TPO), anti-thyroglobulin (anti-Tg), and IgG-secreting cells (SC) by cultures of pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC) obtained from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD), and controls (C). The effect of human thyroglobulin (Tg) together with cytokines was also studied. No differences in the numbers of PWM-induced IgG-SC were found. However, greater numbers of PWM-induced anti-TPO-SC were observed in the HT preparations compared to C, either with or without the addition of Tg (p < 0.05). Inhibition of numbers of IgG-SC with certain cytokines in all 3 groups of preparations was observed (0.01 < p < 0.05). Decreased numbers of anti-TPO-SC were noted in HT preparations after exposing cells to IL-2 and TNF-alpha. Induction of anti-TPO-SC after addition of Tg plus cytokines was significantly lower in HT than in the other 2 groups. In contrast, induction of anti-TPO-SC in C was markedly increased by all cytokines, which was enhanced by Tg. These data suggest that cytokines play a role in auto-antibody regulation in auto-immune thyroid disease (AITD).

Studies of thyroid xenografts from Graves' disease in severe combined immunodeficient mice.

Thyroid tissues from normal (paranodular) subjects and patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT) were xenografted to severe combined immunodeficiency (SCID) mice, and the same tissues were engrafted into nude mice; in addition, peripheral blood mononuclear cells were engrafted to separate SCID mice (SCID-PB). Thyroglobulin (TG) and microsomal antibodies (Abs) became detectable with high titers by hemagglutination assays in SCID mice xenografted with thyroid tissues (SCID-TH) from GD and HT patients; moreover, TG Ab was detectable even in SCID-TH from TG Ab-negative GD and HT donors. On the other hand, only 2 of 10 SCID-PB had detectable Abs with low titers. TSH receptor (TSH-R) Ab was detectable in all sets of SCID-TH from GD. After peaking (3-7 weeks), their levels decreased despite the fact that immunoglobulin G levels increased. In addition, in 3 of 4 sets of SCID-PB from GD patients, TSH-R Ab was also detectable. SCID-TH from GD and HT patients showed transient hyperthyroxinemia, peaking at 2 weeks; these values were significantly higher [free T4, 6.48 +/- 0.90 and 5.50 +/- 0.77 pmol/L (mean +/- SE), respectively; P < 0.05] than SCID-TH from normal controls (2.5 +/- 0.24). Histologically, intrathyroidal infiltrating lymphocytes (ITL) survived in SCID mice, but not in nude mice after 8 weeks. The follicles of GD tissue in SCID mice were virtually destroyed with ITL, and their appearance was similar to that in HT. In conclusion, TSH-R Ab was clearly produced from ITL, and some peripheral blood mononuclear cells grafts could also produce TSH-R Ab. In spite of the presence of TSH-R Ab, SCID-TH from GD patients did not show persistent hyperthyroxinemia, presumably because destructive thyroiditis may be occurring in the grafted tissue, with decreasing levels of TSH-R Ab.

The use of the severe combined immunodeficient mouse and the athymic "nude" mouse as models for the study of human autoimmune thyroid disease.

The athymic "nude" mouse and the severe combined immunodeficient (SCID) mouse have differing immunological properties which permit complementary studies of autoimmune thyroid disease (AITD). The nude mouse accepts human thyroid xenografts, but lyses the passenger lymphocytes, whereas in the SCID mouse both the xenograft and its lymphocytes survive. Human AITD thyroid xenografts manifest a worsening of the pathological picture in the SCID mouse, but show a return to normal morphology and function in the nude mouse at the time of sacrifice 6-8 weeks after engraftment. Such tissue which has been grafted into the nude mouse can be retrieved and then can be re-xenografted into the SCID mouse. Normalized AITD thyroid tissue (from the nude mouse xenograft) will remain normal in the SCID mouse, but if xenografted into a SCID mouse which already has a primary autologous AITD thyroid xenograft, or if autologous PBMC are added, the AITD lesion will be reproduced. This demonstrates the primacy of the immune system in AITD and constitutes a useful model for the study of this human disorder.

Sensitization of T lymphocytes to thyroglobulin and thyroperoxidase in autoimmune thyroid diseases.

To investigate T-cell sensitization to thyroid autoantigens in autoimmune thyroid disease (AITD), purified soluble human thyroglobulin (Tg) and thyroid peroxidase (TPO) were used. Peripheral blood mononuclear cells (PBMC) as well as CD8-depleted, CD4-enriched PBMC ("selected" PBMC) from 9 patients with Graves' disease (GD), 13 Hashimoto's thyroiditis (HT) and 10 healthy subjects, were cultured for 6 days with or without varying concentrations (0.1, 1.0 and 5.0 micrograms/ml, respectively) of Tg or TPO and their responses were evaluated using the 3H-thymidine incorporation assay. Total PBMC as well as selected PBMC from GD and HT responded to both TPO and Tg, but normal PBMC did not. This induction was more marked in "selected" PBMC; on the other hand, CD8 depletion did not permit normal PBMC to respond to either antigen. However, reactivity of selected AITD PBMC to Tg differed from that of TPO. Two way analysis of variance showed that the proliferative response was significantly greater with Tg than with TPO, (again particularly notable with the "selected" PBMC) in both GD and HT. There was no difference between control and AITD preparations when an irrelevant (renal microsomal) antigen was employed. Taken together with our previous report that CD4 cells were induced by TPO even when cultured with CD8 cells, it is evident that suppressor CD8 cells do play a role in CD4 cells from proliferating against Tg and TPO; however their function alone or in combination with suppressor-inducer CD4 cells is partially disturbed, so that T cell sensitization to Tg and TPO can be identified in the AITD PBMC.

Reconstitution of severe combined immunodeficient mice with intrathyroidal lymphocytes of thyroid xenografts from patients with Hashimoto's thyroiditis.

Thyroid tissues from patients with Hashimoto's thyroiditis (HT) have been xenografted to both severe combined immunodeficiency (SCID) mice and nude mice to study the intrathyroidal lymphocytes which were expected to migrate from the xenografts in the SCID mice. Peripheral blood mononuclear cells from HT, Graves' disease, and normal donors have also been separately engrafted. SCID mice, but not nude mice with HT thyroid grafts produce human immunoglobulins. More immunoglobulin G (IgG), but less IgM and IgA is produced in SCID mice with HT thyroid grafts (SCID-TH), compared to SCID mice injected with peripheral blood mononuclear cells from patients with HT or normal donors (SCID-PB), suggesting that different B cell subpopulations were active in the SCID-PB vs. SCID-TH. Production of IgG by SCID-PB and SCID-TH was maintained 6 weeks after engraftment, and decreased thereafter. SCID mice but not nude mice grafted with HT thyroid tissue produce antibodies to thyroglobulin and thyroperoxidase. Lymphocytes within intact HT thyroid grafts persist in SCID mice, and migrate to the spleen, whereas human lymphocytes do not survive in the thyroid grafts or other tissues of the nude mouse. In 6 weeks, the xenografts in nude mice became histologically normal. In contrast, xenografts from SCID mice showed more marked inflammatory changes than in the original human lesion, although the ratio of T/B cells is unchanged. This worsening of the lesion may relate to the increase in activation of the T-lymphocytes.

Effect of human thyroglobulin on the production of platelet activating factor from peripheral blood mononuclear cells from patients with autoimmune thyroid diseases.

Platelet activating factor (PAF), a phospholipid mediator, has been found to play a role in immune reactions, as well as in many pathophysiological alterations in certain disorders. To determine whether there might be a potential role of PAF in the development of autoimmune thyroid diseases (AITD) we have measured in vitro production of PAF by cultures of pokeweed mitogen-stimulated peripheral blood mononuclear cells (PBMC) obtained from 13 patients with Hashimoto's thyroiditis (HT) and 22 patients with Graves' disease (GD), as well as 18 normal control subjects. Similarly, the levels of PAF in cultures of PBMC after relevant [human thyroglobulin (Tg) and human thyroperoxidase (TPO)] antigenic stimulation in the same preparations were measured by a RIA kit. The basal values of PAF were significantly higher in the PBMC preparations from HT patients than in other two groups. In HT preparations, but not in controls, Tg antigen significantly increased the production of PAF. In GD preparations the response to Tg antigen was also present, but the release of PAF did not reach the levels in control group of preparations. Significantly lower values of PAF production were found in preparations from hyperthyroid GD when compared to the results of preparations from GD patients who were euthyroid and to the results of normal control preparations. The type of treatment and chronicity of disease may also have played some role in these findings, since those treated with radioactive iodine had lower values than those patients who became euthyroid using only antithyroid drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired generation of high-affinity interleukin-2 receptors in the autologous mixed lymphocyte reaction in rheumatoid arthritis.

We examined the expression of high-affinity interleukin (IL)-2 receptors (IL-2R) as well as Tac and HLA-DR antigens on peripheral blood (PB) T cells from 11 rheumatoid arthritis (RA) patients and 8 healthy controls induced in the autologous mixed lymphocyte reaction (AMLR). The proportion of HLA-DR- and Tac-bearing T cells and expression of these activation antigens were higher in patients relative to controls (P less than 0.01) in freshly isolated unstimulated PB mononuclear cells. AMLR stimulation of RA T cells failed to induce an increase in the proportion of HLA-DR and Tac-bearing T cells which was observed in health controls. After AMLR stimulation the number of high-affinity IL-2R were significantly lower in RA patients compared with controls (P less than 0.01). The number of high-affinity IL-2R on patient T cells correlated strongly with AMLR reactivity as measured by [3H]thymidine incorporation (r = 0.821, P = 0.002). The results suggest that the AMLR defect in RA may result from impaired generation of high-affinity IL-2R.

The effect of xenotransplantation of human thyroid tissue following radioactive iodine-induced thyroid ablation on thyroid function in the nude mouse.

We have attempted to determine whether xenotransplanted human thyroid tissue into nude mice would act as a physiological substitute for the mouse thyroid gland after the mice had been rendered hypothyroid, using radioactive iodine (131I). The dosage of 0.2 millicuries of 131I was given to each mouse. The xenotransplantations of human thyroid tissue, i.e., normal, Graves' and nontoxic multinodular goitre, were carried out three weeks after radioactive ablation. The values of TSH in all mice rose to high levels (71 +/- 15.6 ng/ml, +/- SD) by three weeks after 131I administration. The TSH values in the mice declined rapidly and reached normal levels by 3-5 weeks after xenotransplantation. In addition, the serum T4 values were generally in the euthyroid range by 3-6 weeks after xenotransplantation. There were no marked differences in the changes of serum T4 and TSH when the three groups were compared. These results indicated that the xenografted human thyroid tissue permitted a return to a normal feedback system as reflected by normal serum TSH and T4 values in the animals. The Graves' thyroid tissue reverted to normal physiological function when removed from its human (abnormal) immune environment, signifying that Graves' thyrocytes are mere passive captives to immune events. This model should prove to be useful in the study of human thyroid physiology and pathophysiology.

Effects of recombinant human interleukin-2 and tumor necrosis factor-alpha with or without interferon-gamma on human thyroid tissues from patients with Graves' disease and from normal subjects xenografted into nude mice.

We have compared the effects of interleukin-2 (IL-2) or tumor necrosis factor-alpha (TNF alpha) administration with or without interferon-gamma (IFN gamma) on Graves' and normal thyroid tissue xenografts in the nude mouse (in the absence of an intact immune system) in terms of possible functional, immunological, or histological changes. The dosages of recombinant human IL-2, TNF alpha, and IFN gamma given to each mouse were 250, 800, and 4000 U, respectively; they were injected ip daily for 6 consecutive weeks. The parameters measured included the free T4 index, thyroid autoantibodies, and mouse TSH during the course of the study. Thyroid epithelial cell (TEC) HLA-DR expression was measured in thyroid tissue before xenotransplantation and at death; in addition, light microscopic studies were carried out at those times. There were no significant differences in thyroid function between the results in unstimulated (control) animals and those obtained with cytokine administration in either group of tissues, with the exception of the group receiving TNF alpha together with IFN gamma; in this latter group, the free T4 index declined significantly 4-6 weeks after commencement of treatment in the animals with normal thyroid tissue xenografts. The reduction of thyroid function induced by the combination of IFN gamma and TNF alpha observed in normal thyroid tissue may be due to inhibition of thyroperoxidase and thyroglobulin gene transcription. However, there was no such effect on the Graves' thyroid tissue xenografts, perhaps because of down-regulation of this tissue in response to cytokines, after having been released from long term in vivo immune stimulation. On the other hand, TNF alpha plus IFN gamma induced TEC HLA-DR expression on both types of thyroid xenografts at death, although IL-2 alone did not induce HLA-DR expression, and IFN gamma induced TEC significantly only on normal thyroid xenografts (but not on Graves' xenografts). In light microscopic examination, Graves' thyroid xenografts treated with IL-2 alone or TNF alpha plus IFN gamma appeared normal at death. In addition, normal thyroid xenografts treated with the same cytokines did not show discernible differences compared to those at human surgery or when the xenografts were untreated at death. We conclude that Graves' TEC did not differ from normal TEC in any significant fashion at the time of death, aside from a reduced responsiveness to the stimuli applied.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal IgGs from an autoimmune MRL/Mp-lpr/lpr mouse induce an interleukin-3-dependent myeloid cell line to produce tumor necrosis factor alpha and interleukin-6.

We previously reported that two IgG mAbs, 1D11 and 1G10, derived from an autoimmune MRL/Mp-lpr/lpr(MRL/l) mouse, induced IL-3 synthesis in the IL-3-dependent myeloid cell line, FDC-P2/185-4. In this study, we found that these mAbs induced TNF-alpha and IL-6 production in FDC-P2/185-4 cells. Both TNF-alpha and IL-6 were secreted rapidly within 1 hr after the addition of mAb to the cells. Increases of TNF-alpha and IL-6 mRNA were also observed in FDC-P2/185-4 cells stimulated with MRL/l-derived mAb. The anti-Fc gamma RII mAb 2.4G2 suppressed TNF-alpha and IL-6 production induced by these mAbs. Our results suggest that some IgGs of MRL/l mice may have the capacity to induce cytokine synthesis in Fc gamma R-bearing cells.

Studies of CD4+ (helper/inducer) T lymphocytes in autoimmune thyroid disease: demonstration of specific induction in response to thyroid peroxidase (TPO) in vitro and its relationship with thyroid status in vivo.

We have studied by flow cytometric analysis the antigen specific activation of CD4+ (helper/inducer) T lymphocytes by purified human thyroid peroxidase (TPO). Peripheral blood mononuclear cells were obtained from 26 patients with Graves' disease (GD), 16 with Hashimoto's thyroiditis (HT), 7 with nontoxic nodular goiter (NG), and 14 normal subjects (N). Cells were cultured for 7 days in the presence or absence of TPO at final concentrations of 3, 30, and 300 ng/mL. When harvested, cells were reacted with an FITC-conjugated anti-CD4 and a PE-conjugated anti-HLA-DR murine monoclonal antibodies. The percentage of HLA-DR+ CD4+ cells (activated CD4+ cells) was determined by a flow cytometer. In the absence of TPO, CD4+ cells had been activated without any specific stimulant. This is known as the autologous mixed lymphocyte reaction (AMLR). In the AMLR, CD4+ cells from GD and HT were less activated compared to those from NG and N. Results of TPO-specific activation were expressed as an incremental increase of activated CD4+ cells (II) (percentage of activated CD4+ cells cultured with TPO minus percentage of activated CD4+ cells cultured without TPO). II of N, GD, HT, and NG were 0.37 +/- 0.21, 2.20 +/- 0.45,** 2.0 +/- 0.66,* and 0.35 +/- 0.27 (mean +/- SEM), respectively (**p less than 0.01; *p less than 0.05 vs N). When patients were further subdivided, the highest mean II was found in patients with hyperthyroid GD (p less than 0.01), followed by euthyroid HT (p less than 0.05) and euthyroid GD (p less than 0.05), however there was no significant difference between hypothyroid HT and N. In conclusion (1) AMLR reactivity of CD4+ cells from GD and HT was impaired, (2) however, CD4+ cells from both GD and HT were significantly more induced by TPO compared to N, and (3) this induction depends, in part, on the in vivo thyroid status.

Interleukin 2-activated killer cells do not mediate autologous thyrocyte lysis in autoimmune thyroid disease in vitro.

Because of interest in IL-2, and IL-2-activated killer cell-induced hypothyroidism in humans, we attempted to study an in vitro system that might prove to illuminate this disorder. We have thus studied interleukin 2 (IL-2--0, 12.5, 25, or 50 U/mL) activated killer cell-mediated autologous thyrocyte lysis, as well as cytotoxic activity in IL-2-stimulated mononuclear cell supernatants in 7 patients with autoimmune thyroid disease (2 Graves' disease and 5 Hashimoto's thyroiditis) using the 51Cr release assay. Controls included 14 patients with nonautoimmune thyroid disease (3 nontoxic goiter, 8 follicular thyroid adenoma, 2 papillary thyroid carcinoma, and 1 medullary carcinoma of the thyroid). Soluble IL-2 receptor (sIL-2R) in supernatants of peripheral mononuclear cells stimulated by IL-2 from these patients also was measured. Whereas in the control preparations, IL-2-activated killer cell activity was increased in a dose-dependent fashion relative to the IL-2 concentration, as well as to the effector cell/target cell ratio, in preparations from patients with autoimmune thyroid disease, this activity was not elevated as the IL-2 concentration was increased. The susceptibility of thyrocytes to the lytic effect of IL-2-activated killer cells was higher in controls than that in autoimmune thyroid disease (at concentrations of IL-2 of 0, 12.5, 25, and 50 U/mL) (p less than 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral blood T lymphocyte sensitization to thyroid microsomal antigen from patients with Graves' disease negative for circulating anti-thyroid microsomal antibodies.

We have studied thyrocyte HLA-DR expression induced by supernatants of peripheral blood mononuclear cells (PBMC) stimulated by thyroid microsomal antigen (TMA), as an index of sensitization of the T lymphocyte in autoimmune thyroid diseases; we have studied PBMC from 11 normal control persons and 19 patients with Graves' disease (GD) in whom serum anti-thyroid microsomal antibodies (AMA) were either not detectable (9 patients) or were positive (10 patients). Thyrocyte HLA-DR induction in response to TMA-treated PBMC supernatants from GD was significantly different from that of normal controls (p less than 0.05, ANOVA). TMA-stimulated GD PBMC supernatants increased thyrocyte HLA-DR index [TMA 1 ng/ml, SI 143 +/- 82 (mean +/- SD), p less than 0.05], but normal PBMC supernatants did not. However there was no significant difference in response in terms of the thyrocyte HLA-DR expression induced by TMA-stimulated PBMC supernatants between AMA seronegative vs seropositive GD. These results suggest the possibility of some dissociation of the activities of T lymphocytes and B lymphocytes in patients with GD in response to thyroid microsomal antigen with or without anti-thyroid microsomal antibodies.

In vitro production of interferon-gamma by peripheral blood from patients with Graves' disease, Hashimoto's thyroiditis and rheumatoid arthritis.

The production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells (PBMC), CD4 cells, or CD8 cells in response to interleukin-2 (IL-2) stimulation has been studied; the samples were obtained from 12 healthy control subjects, 19 patients with Graves' disease (10 hyperthyroid and nine euthyroid), 13 patients with Hashimoto's thyroiditis (four hypothyroid and nine euthyroid), and 15 patients with rheumatoid arthritis (11 active and four inactive). A dose of IL-2 (25 U/ml) was utilized to induce IFN-gamma by PBMC from all four groups. The incremental increase in IFN-gamma values (with IL-2 stimulation minus without stimulation) was significantly less in PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis than that in PBMC from control subjects. The values from PBMC in patients with Graves' disease in a euthyroid state were below normal but greater than those from patients with Graves' disease in a hyperthyroid state. The incremental increase in IFN-gamma values from Graves' disease PBMC correlated with the serum TSH values (r = 0.622, P less than 0.01), but not with thyroid autoantibodies (anti-thyroid microsomal antibodies, anti-thyroid microsomal antibodies, nor TSH-binding inhibitory immunoglobulin activities). The incremental increase in IFN-gamma from PBMC from both control subjects and Graves' disease was correlated with that from CD4 cells (r = 0.711, P less than 0.01), but not with that from CD8 cells. The production of IFN-gamma in response to IL-2 from PBMC in Graves' disease correlated inversely with thyroid function, appearing to reflect the very effect of hyperthyroidism in this process. The precise explanation of these phenomena remains unclear. The decreased response of IFN-gamma to IL-2 stimulation by PBMC from patients with Graves' disease, Hashimoto's thyroiditis, and rheumatoid arthritis seems to be a non-specific phenomenon occurring in both organ specific autoimmune disease and systemic autoimmune disease. It may be due to a down-regulation in autoimmune disease of CD4 cells in response to IL-2, a decreased level of IL-2 cellular receptors or a decreased receptor affinity, associated increased soluble IL-2 receptors, or a defect of the intra-CD4 cellular IL-2 signal to produce or release IFN-gamma in the conditions studied.