Treatment Of Thyrocytes Research Articles

IL-14 and IL-16 are expressed in the thyroid of patients with either Graves' disease or Hashimoto's thyroiditis.

Cytokines have an important role in orchestrating the pathophysiology in autoimmune thyroid disease. The aim of the current study was to analyse the expression of interleukin (IL)-14 and IL-16 in the thyroid tissue of patients with Graves' disease (GD), Hashimoto's thyroiditis (HT) or multinodular goitre (MNG) and in that of normal individuals, in patients' intrathyroidal CD4(+) and CD8(+) T cells, and in patient and normal cultured thyroid follicular cells. The expression of IL-14 and IL-16 mRNA and protein was investigated using reverse transcription-polymerase chain reaction (RT-PCR) amplification, and Western blotting and ELISAs, respectively. IL-14 mRNA expression was detected in thyroid tissue from 8/9 GD, 3/4 HT and 3/13 MNG patients and 1/6 normal individuals, and IL-16 mRNA expression in thyroid tissue from 9/9 GD, 4/4 HT and 9/13 MNG patients and 4/6 normal individuals. IL-14 mRNA expression was detected in intrathyroidal CD4(+) and CD8(+) T cells from 2/2 GD and 2/2 HT patients, while IL-16 mRNA was detected in samples from 1/2 HT patients but not in those from either patient with GD. IL-14 and IL-16 mRNA expression was found in thyroid follicular cells derived from 2/2 patient with GD and 1/1 normal individual. IL-14 protein was detected in thyroid tissue from 6/6 GD, 1/1 HT and 0/6 MNG patients and 0/6 normal individuals, and IL-16 protein in thyroid tissue from 6/6 GD, 1/1 HT and 1/6 MNG patients and 0/6 normal individuals. Expression of IL-14 protein was stimulated in thyroid follicular cells derived from two patients with GD and one normal individual by peripheral blood mononuclear cell (PBMC)-conditioned medium. Treatment of thyrocytes from two patients with GD and one normal individual with PBMC-conditioned medium and tumour necrosis factor (TNF)-α stimulated IL-16 protein expression. In normal thyrocytes, IL-16 protein synthesis was induced also by IL-1β, IL-17A, IL-4 and transforming growth factor (TGF)-β. The data provide evidence that the intrathyroidal production of IL-14 and IL-16 is associated with the pathogenesis of autoimmune thyroid disease. Thyroid follicular cells display the ability to express IL-14 and IL-16 mRNA and can be stimulated to express IL-16 protein, by a panel of cytokines, and IL-14 protein, by as yet unidentified factors.

Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema.

The expression of two autoimmune thyroid diseases. GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-gamma) or IL-1beta for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1beta or IFN-gamma caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1beta or IFN-gamma was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1beta or IFN-gamma, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1beta or IFN-gamma. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1beta or IFN-gamma. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.

Subcellular Distribution, Secretion, and Posttranslational Modifications of Clusterin in Thyrocytes

A prominent secretory glycoprotein was detected in the culture medium of porcine thyrocytes which was identified as clusterin by microsequencing. Treatment of thyrocytes with thyroid stimulating hormone revealed a tight regulation of both synthesis and secretion of clusterin, with a distinct fraction of clusterin being always associated with the cells. At least three N-bound glycans were found on each subunit of clusterin, receiving most of the incorporated [32P]phosphate-label. Binding of clusterin to the immobilized cation-independent mannose 6-phosphate (M6P) receptor indicated that part of the phosphate label was contained in M6P moieties. Immunolabeling of cultured thyrocytes and of thyrocytes in situ showed clusterin on the apical cell surfaces where it colocalized with gp330/megalin, which is known to serve as a binding site for clusterin. The association with the apical plasma membrane, which, in thyrocytes, carries the iodinating system, was confirmed by biosynthetic iodination, an as yet unknown posttranslational modification of clusterin. On the basolateral plasma membranes clusterin was found within distinct, bipartite patches, suggesting that it is a constituent of cell-adhesion complexes and that it participates in cell–cell and cell–matrix interactions.

Thyroid-stimulating hormone inhibits Fas antigen-mediated apoptosis of human thyrocytes in vitro.

The mechanisms of TSH-induced growth stimulation of thyrocytes in vivo have yet to be elucidated. We examined the antiapoptotic effect of TSH toward Fas antigen-mediated apoptosis of thyrocytes. Fas antigen was expressed on approximately 40% of unstimulated thyrocytes, and the expression was significantly inhibited by the addition of TSH in a dose-dependent manner. Treatment of thyrocytes with 8-bromo-cAMP mimicked the effect of TSH, suggesting that the inhibitory effect of TSH on Fas antigen expression was mediated by activating protein kinase A. In contrast, treatment of thyrocytes with either interleukin-1 beta (IL-1 beta) or interferon- gamma (IFN gamma) markedly increased Fas antigen expression on thyrocytes, and these effects were inhibited in the presence of TSH. The expression of the protooncogene product Bcl-2 did not change after the addition of TSH, 8-bromo-cAMP, IL-1 beta, IFN gamma, or a combination of TSH and IL-1 beta or IFN gamma. When thyrocytes stimulated with either IL-1 beta or IFN gamma were treated with anti-Fas IgM mAb, the cells were committed to apoptosis, whereas this apoptotic process was significantly inhibited by the addition of TSH. These results indicate that the Fas antigen is functionally expressed on the surface of thyrocytes, and TSH inhibits Fas antigen-mediated apoptosis of thyrocytes through the inhibitory effect of Fas antigen expression, resulting in the promotion of growth of the thyroid gland.

Thyroid-stimulating hormone inhibits Fas antigen-mediated apoptosis of human thyrocytes in vitro

The mechanisms of TSH-induced growth stimulation of thyrocytes in vivo have yet to be elucidated. We examined the antiapoptotic effect of TSH toward Fas antigen-mediated apoptosis of thyrocytes. Fas antigen was expressed on approximately 40% of unstimulated thyrocytes, and the expression was significantly inhibited by the addition of TSH in a dose-dependent manner. Treatment of thyrocytes with 8-bromo-cAMP mimicked the effect of TSH, suggesting that the inhibitory effect of TSH on Fas antigen expression was mediated by activating protein kinase A. In contrast, treatment of thyrocytes with either interleukin-1 beta (IL-1 beta) or interferon- gamma (IFN gamma) markedly increased Fas antigen expression on thyrocytes, and these effects were inhibited in the presence of TSH. The expression of the protooncogene product Bcl-2 did not change after the addition of TSH, 8-bromo-cAMP, IL-1 beta, IFN gamma, or a combination of TSH and IL-1 beta or IFN gamma. When thyrocytes stimulated with either IL-1 beta or IFN gamma were treated with anti-Fas IgM mAb, the cells were committed to apoptosis, whereas this apoptotic process was significantly inhibited by the addition of TSH. These results indicate that the Fas antigen is functionally expressed on the surface of thyrocytes, and TSH inhibits Fas antigen-mediated apoptosis of thyrocytes through the inhibitory effect of Fas antigen expression, resulting in the promotion of growth of the thyroid gland.

Endothelin binding to receptors and endothelin production by human thyroid follicular cells: effects of transforming growth factor-beta and thyrotropin.

Measurable endothelin (ET) was found in serum-free medium of cultured primary thyroid cells derived from human thyroid tissues after 3 days incubation at levels ranging from undetectable to 35 fmol/100,000 cells. Out of 12 thyroid glands studied, 2 responded to transforming growth factor-beta (TGF-beta) treatment with increased ET secretion into medium. Detailed study of cells derived from one of these thyroids showed TGF-beta at 1 ng/ml stimulated ET secretion from 13.7 to 33.3 fmol/100,000 cells after 3 days incubation. Although TSH alone did not significantly modulate ET release into medium, TSH enhanced the stimulatory effect of TGF-beta. A combination of TSH at 1 mU/ml and TGF-beta at 1 ng/ml stimulated ET secretion to 63.8 fmol/100,000 cells after 3 days incubation. Immunostaining studies demonstrated the presence of intracellular immunoreactive ET, largely localized in perinuclear regions, which was greatly stimulated by TSH but not by TGF-beta. These observations suggest that TSH may stimulate only ET synthesis, whereas TGF-beta may stimulate both synthesis and secretion. Binding of [125I]ET-1 to receptor on thyroid cells was dose-dependently stimulated by TGF-beta (0-10 ng/ml) pretreatment for 3 days. Scatchard analysis of competitive binding data from TGF-beta (1 ng/ml)-treated cells indicated that increased binding was the result of increased receptor number and not increased receptor affinity. TSH (0-10 mU/ml), though not as potent as TGF-beta, also dose-dependently stimulated ET binding. Treatment of thyrocytes with 1 mU/ml TSH for 3 days did not significantly affect ET receptor number or binding affinity. These results illuminate aspects of a possible autocrine regulation of ET in the thyroid gland.

Islet cells but not thyrocytes are susceptible to lysis by NK cells

BB rats develop both pancreatic insulitis and lymphocytic thyroiditis, but whereas spontaneous autoimmune diabetes is common, hypothyroidism is rare. Splenic natural killer (NK) cells from acutely diabetic (AD) BB rats and from athymic nude rats are known to be cytotoxic to rat islet cells in vitro. To investigate possible differential tissue susceptibility to lysis by NK cells or their cytokines such as cytolysin (perforin) or NK cytotoxic factor (NKCF), we used an in vitro 51Cr-release assay to measure the cytotoxicity of splenocytes, cytolysin or NKCF against Wistar Furth (WF) and Fischer 344 (F-344) rat islet cells, and FRTL-5 F-344-derived and WRT Wistar-derived rat thyrocytes. The results demonstrated that spleen cells from AD-BB (RT1 u) rats and athymic F-344 nude (RT1 1) rats are cytotoxic to WF (RT1 u) islets and F-344 (RT1 1) islets, but not to FRTL-5 (RT1 1) or WRT (class IRT1 1) thyrocytes. WF and F-344 rat spleen cells were not cytotoxic to any of these cells. Thyrocytes are known to express class II molecules on their surface in chronic thyroiditis. We found that treatment of thyrocytes with interferon-gamma (IFN-γ) induced class II expression but did not increase the cytotoxicity of splenocytes against these cells. Cytolysin and NKCF were both cytotoxic to islets in a dose dependent manner, but FRTL-5 thyrocytes were resistant to killing by these cytokines. These findings suggest that islet cells and thyrocytes in vitro are differentially susceptible to lysis by NK cells.