Isolated intact porcine thyroid follicles free of contaminating single cells were embedded in "Matrigel", which is a gel-forming basement membrane preparation containing mainly collagen type IV, laminin, heparan sulfate proteoglycans and entactin. Follicles were treated with different growth factors: thyrotropin (TSH), insulin-like growth factor I (IGF-I), epidermal growth factor (EGF) or transforming growth factor beta. Cell proliferation was quantified by counting cell numbers. Morphological studies were done by photodocumentation and analysis of histology by light and electron microscopy. The thyrocytes had the physiological polarity with follicular cell arrangement, microvilli at the apical membrane, desmosomes and tight junctions. The lumen contained colloid. Iodide organification (10.2 +/- 2.1 vs 26.1 +/- 5.8 pmol/10(6) cells; TSH 0.1 mU/ml) and release of thyroid hormones (thyroxine, 1754 +/- 207 vs 2890 +/- 460 pg/10(6) cells; triiodothyronine, 164 +/- 22 vs 412 +/- 106 pg/10(6) cells; TSH, 1mU/ml) were significantly stimulated by TSH. There was no basal growth rate in serum-free medium but proliferation was slightly stimulated with TSH (1 mU/ml; 149 +/- 19%) and in the same order of magnitude with IGF-I (10 ng/ml; 159 +/- 23%) but without follicle neoformation. In contrast, BGF (1.0-5.0 ng/ml) induced thyrocyte proliferation dose dependently three- to sixfold. With BGF up to 2 ng/ml, buds of new follicles formed surrounding pre-existing follicles. With BGF higher than 3 ng/ml, typical papillary structures developed. Transforming growth factor beta inhibited this dedifferentiated growth. A migration of single cells into the gel was never observed. Thus, three-dimensional culture of isolated thyroid follicles in "Matrigel" provides a tool for investigating the regulation of follicular growth and neoformation close to the in vivo situation.
Insulin-like growth factor I (IGF-I) has been shown to be released from thyrocytes in vitro. We investigated IGF-I mRNA expression during treatment with thyrotropin (TSH), forskolin and potassium iodide (KI) in intact porcine thyroid follicles ex vivo. Porcine thyroid follicles were prepared by collagenase digestion and cultured in the presence of TSH, forskolin or KI. After different incubation times, mRNA was isolated and examined by Northern hybridization with a porcine IGF-I cDNA probe of 405 bp in length. In untreated follicles no IGF-I mRNA was found, whereas in follicles stimulated with TSH an IGF-I mRNA of 7.0 kb was detected after 24 h, which persisted for another 24 h. Forskolin treatment mimicked the TSH effect, indicating that IGF-I mRNA expression may be stimulated by the adenylate cyclase pathway. Preincubation of the porcine follicles with KI decreased dose dependently the TSH-induced IGF-I mRNA expression, with complete inhibition at 10 mumol/l KI. These results suggest that TSH acts via the cAMP pathway to enhance IGF-I mRNA expression, which then may lead to an autocrine IGF-I stimulation. The IGF-I mRNA expression is under negative control of iodide.
Thyroid volume, urinary iodine excretion as well as personal nutritional knowledge and individual iodine prophylaxis were determined during a health education program on iodine deficiency and prophylaxis in 1992. Participants were 472 male and 568 female (mean age 27.7 years) students and employees of five universities in the southern part of Germany. The study aimed to clarify the relationship between personal knowledge on iodine, individual iodine prophylaxis and parameters of iodine deficiency (thyroid volume, iodine excretion) in a well known iodine deficient area. Mean thyroid volume (mean +/- SD) was 19.7 +/- 8.3 ml in males and 15.8 +/- 7.1 ml in females. 25.5% of females and 19.9% of males showed thyroid volume above the upper normal values. Total mean urinary iodine excretion was 70.7 +/- 42 micrograms I/g creatinine reflecting WHO-grade-I iodine deficiency. 80.8% of total subjects used iodized salt and 43.2% stated to consume salt-water fish to meet their iodine requirement. The female non-users had significantly lower iodine excretion (no iodized salt, no salt-water fish: 61.4 +/- 31.3 vs. +iodized salt, +salt-water fish: 83.9 +/- 47.6 micrograms I/g creatinine; p < 0.05), however, thyroid volume was identical in these groups. The area of residence over the last 10 years did not significantly influence the thyroid volume. The goiter incidence increased with age. Although our study population was highly educated (81.8% students) and the subjects were provided with educational brochures immediately prior to the study, knowledge about iodine content of food was poor. We conclude that despite a high degree of voluntary iodine prophylaxis and educational programs the iodine intake is insufficient. The use of iodized salt in households, cafeterias, and also in food manufacturing must be increased for sufficient iodine prophylaxis.
It has been proposed from in vivo studies that thyroid angiogenesis during thyroid enlargement may be due to paracrine mitogenic factors released by epithelial thyroid cells. To study this paracrine growth regulating communication between thyroid cells and endothelial cells in vitro, culture medium from isolated porcine thyroid follicles was investigated for a growth promoting effect on porcine aortal endothelial cells. Serum-free conditioned medium (CM) from thyroid follicles in suspension culture contains a dose-related mitogenic activity which stimulates endothelial cell growth up to 197%. Stimulation of the thyroid follicles with TSH (1 mU/ml) significantly reduced the mitogenic activity for endothelial cells in CM to 131%. Thyroid hormones had no influence on mitogenic activity in CM. When follicles were treated with iodide (20 microM) during CM production, no proliferation of endothelial cells was observed by this CM. In contrast, CM from epidermal growth factor-treated thyroid follicles significantly enhanced the mitogenic activity for endothelial cells up to 235%. The mitogenic activity was precipitable by saturated ammonium sulfate, showed high affinity to heparin by chromatography on heparin-sepharose, and was abolished after treatment of CM with trypsin. On gel electrophoresis the heparin-binding fraction showed a double band with a mol wt of 15 and 15.5 k. These data show a paracrine mitogenic activity on endothelial cells released by thyroid follicles which is regulated by TSH, epidermal growth factor, and iodide in parallel with the direct effect of these substances on thyroid cell growth. The data suggest that the mitogenic factor is a polypeptide, which belongs to the heparin-binding growth factors.
Interleukin-1 beta (IL-1 beta) is known to be involved in autoimmune thyroiditis. Since the results of different in vitro-studies on the effect of IL-1 on thyrocytes are controversial, our aim was to investigate the existence of specific binding sites for IL-1 beta and its influence on specific functions and growth of isolated porcine thyroid follicles ex vivo with a preserved iodide metabolism. For binding studies isolated thyroid follicles were incubated with 125I-IL-1 beta (213.5 nCi/ml) and with increasing concentrations of unlabelled IL-1 beta (0.06-11.5 nmol/l) for 24 h at 4 degrees C. The dissociation constant Kd was 0.85 x 10(-10) mol/l and about 800 binding sites per cell were calculated. IL-1 beta (10 U/ml) decreased basal and TSH-stimulated iodide uptake and organification after an incubation time of 45 min to 6 h without any influence on cAMP-formation. In addition, after 40 h of incubation IL-1 beta dose-dependently increased T3-secretion, followed by a decrease during simultaneous TSH-stimulation, whereas there was no effect on T4-secretion. In contrast to these functional effects IL-1 beta showed no influence on the growth of thyroid follicles, so that the cytokine cannot be made responsible for goiter growth in thyroid diseases by directly influencing thyroxytes.
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