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Lars E Ericson and Mikael Nilsson

Ericson LE, Nilsson M. Effects of insulin-like growth factor I on growth, epithelial barrier and iodide transport in polarized pig thyrocyte monolayers. Eur J Endocrinol 1996;135:118–27. ISSN 0804–4643

The effects of insulin-like growth factor I (IGF-I) on mitogenesis, epithelial barrier function and transepithelial iodide transport were studied in confluent, polarized monolayers of pig thyrocytes cultured on filter in Transwell bicameral chambers. The growth rate in controls cultured in 1% fetal calf serum was low. Insulin-like growth factor I stimulated dose-dependently the incorporation of [3H]thymidine, maximally at 100 ng/ml, which corresponded to an increase of DNA content by 60% after 6 days. Thyrotropin (1 mU/ml) alone did not stimulate cell multiplication but inhibited partially the stimulatory effect of IGF-I. Insulin-like growth factor I(100 ng/ml) increased within 10 min the transepithelial potential difference, which remained elevated for several days, but did not significantly change the transepithelial resistance. When added together, IGF-I reinforced the effects of TSH on potential difference (increase) and resistance (decrease). A preserved epithelial barrier in IGF-I-treated cultures was confirmed by observing a normal immunolocalization of the tight junction protein ZO-1 and an unchanged ultrastructure of the junctional complex. Insulin-like growth factor I increased the transepithelial flux of 125I in the basal-to-apical, but not in the opposite, direction. Stimulation of iodide transport by IGF-I was modest after 2 days and pronounced after 6 days. In comparison, TSH-stimulated iodide transport was higher after 2 days but lower after 6 days. Both TSH and IGF-I were strongly synergistic, after 6 days amounting to a 90-fold increase over the control basoapical 125I transfer. The simultaneous accumulation of 125I in the cell layer was increased two- to fourfold by IGF-I and/or TSH. In conclusion, IGF-I is able to induce growth in preformed monolayers of pig thyrocytes cultured on permeable filter. During these conditions, the mitogenic effect of IGF-I is partially inhibited by TSH, which has no growth-promoting action on its own. The transepithelial transport of iodide and bulk electrolytes is altered by IGF-I without affecting the epithelial barrier function. Specifically, IGF-I up-regulates the activity of the basolateral iodide pump and increases the iodide permeability of the apical plasma membrane. The action of IGF-I on iodide transport is independent of, although synergistic with, that of TSH. The findings support the notion that IGF-I may be an important regulator of thyroid growth and differentiated functions.

Lars E Ericson, Institute of Anatomy and Cell Biology, Göteborg University, Medicinaregatan 3, S-413 90 Göteborg, Sweden

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Kerstin Westermark, Mikael Nilsson and F. Anders Karlsson

Abstract.

Interleukin 1, a product primarily of activated macrophages, was found to exert both a stimulatory and an inhibitory effect on porcine thyroid follicles in suspension cultures. A stimulatory effect on [125I] iodide incorporation into protein (iodination) was seen in cultures exposed to human recombinant interleukin 1α (20 μg/l) for 1 h and (0.1 or 10 μg/l) for 18 h, whereas an inhibitory effect on iodination of interleukin 1 (10 μg/l) was registered after pre-incubation for 42 h and significant upon stimulation with TSH (200 mU/l). Cyclic AMP levels were stimulated in cells exposed to interleukin 1, however, significantly only after 42 h of pre-incubation, whereas TSH-stimulated cAMP response was inhibited by interleukin 1 already after 18 h of pre-incubation. The lumen of interleukin 1 incubated follicles was highly dilated and surrounded by a flattened epithelium. There were, however, no morphological signs of follicle disintegration or cellular cytotoxicity in the cultures exposed to interleukin 1. The data suggest that interleukin 1 might have a modulating role on thyroid function in autoimmune thyroid diseases associated with activation of intrathyroidal macrophages.

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Mikael Nilsson, Ulla Björkman, Ragnar Ekholm and Lars E Ericson

The intracellular regulation of thyrotropin-stimulated iodide efflux was studied in polarized porcine thyrocytes grown as a continuous, tight monolayer in bicameral culture chambers. From a previous study using this system we know that thyrotropin rapidly increases iodide efflux in the apical but not basal direction of the polarized epithelium. [125I]-iodide efflux in apical direction was stimulated by thyrotropin in a concentration-dependent manner (1–10 U/I), whereas efflux in basal direction was unchanged at any thyrotropin dose. Thyrotropin-induced elevation of intracellular cAMP showed a corresponding concentration dependence. The selective stimulation of apical efflux by thyrotropin was evident also when re-uptake of iodide released in basal direction was blocked by perchlorate. The effect of thyrotropin on apical efflux was mimicked by 8-bromo-cAMP and forskolin, whereas agents known to activate the Ca2+/phosphatidylinositol cascade (epidermal growth factor) and protein kinase C (phorbol ester) or increase cytosolic [Ca2+] (A23187) were inactive. We conclude that the selective stimulation by thyrotropin of apical iodide efflux, corresponding to efflux in luminal direction in intact follicles, occurs via cAMP-regulated iodide channels present in the apical domain of the plasma membrane.

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Mikael Nilsson, Johanna Husmark, Bengt Nilsson, Lars-Erik Tisell and Lars E Ericson

Nilsson M, Husmark J, Nilsson B, Tisell L-E, Ericson LE. Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function. Eur J Endocrinol 1996;135:469–80. ISSN 0804–4643

Epithelial properties of thyrocytes are difficult to maintain in conventional cell culture systems. We used bicameral chambers (Transwell™) in attempts to establish a functional epithelium of thyrocytes of human origin. Thyroid follicle segments were isolated by collagenase digestion of paradenomatous tissue obtained at surgery for follicular adenoma and of tissue from glands with Graves' disease. After careful separation from connective tissue and single cells by centrifugation, the follicles were plated at high density on the collagen-coated filter of the chambers and cultured in Eagle's essential medium (EMEM) containing 10% fetal calf serum (FCS) or Coon's modified Hams medium enriched with five or six factors (5H, 6H); the latter media contained 5% FCS without (5H) or with (6H) thyrotropin (TSH). The follicles were converted into a confluent cell layer, which had similar DNA content irrespective of type of medium, after 4–6 days. Cells grown in EMEM or 5H established a transepithelial electrical resistance (R) of 200–500Ω·cm2 and was impermeable to [3H]inulin, indicating the formation of epithelial junctions. Addition of 6H to confluent cells initially cultured in EMEM or 5H caused a further increase of R, maximally to 1500 Ω·cm2, along with a rise of the transepithelial potential difference; 6H promoted the monolayer formation of cells, increased the number of apical microvilli and reinforced the junctional distribution of actin, cadherin and ZO-1; 6H also enhanced the polarized secretion of [3H]leucine-labeled thyroglobulin into the apical medium. Cells from Graves' thyroid tissue established an epithelium on the filter with similar characteristics to that of normal thyrocytes; some platings contained in addition large numbers of HLA-DR positive cells with a dendritic shape. HLA-DR expression was generally absent in EMEM- or 5H-grown thyrocytes, but appeared in limited areas of the cell layer after 6H and was expressed by all epithelial cells after interferon-gamma stimulation for 48 h. We conclude that human thyrocytes form a tight and polarized epithelium when cultured on permeable filters. The polarized structure and function of the cells are positively regulated by TSH. The culture system may be useful in studies addressing the role of the epithelial phenotype (cell polarity and tight barrier) in normal thyroid function as well as in pathological processes in the thyroid, such as autoimmunity, cell transformation and tumor progression.

Mikael Nilsson, Institute of Anatomy and Cell Biology, Göteborg University, Medicinaregatan 3, S-413 90 Göteborg, Sweden