A bioassay for thyroid stimulating immunoglobulins of patients with Graves' disease using porcine thyroid monolayer cells

A bioassay for thyroid stimulating immunoglobulins (TSI) of patients with Graves' disease was developed by porcine thyroid monolayer cells. Thyroid cells were prepared by dispersion using collagenase and trypsin. Aliquots of the cell suspension (2 X 10(6) cells/1.5 ml/dish) in Ham's F-12 medium (pH 7.2) containing 10% calf serum and 1.5 mM Hepes were seeded and cultured in air at 36 C. On day 6 of culture, cells were incubated with test samples (IgG or bTSH) in 1 ml of serum-free, 0.5 mM IMX-included fresh medium for an additional time, and cAMP in the cells was measured by radioimmunoassay. Intracellular cAMP was increased within 5 minutes after the addition of bTSH and the maximal increase was observed after 30 min. Responses of cAMP were in a dose-related manner up to 10 mU/ml of bTSH. With the addition of IgG from untreated Graves' patients, dose-related increases in cAMP were also observed up to 10 mg/ml IgG and the maximal response was seen at 2 hours incubation. Thyroid stimulating activity in IgG's from normal subjects and patients with Graves' disease was tested with a dose of 10 mg/ml and 2 hours incubation and the activity was expressed as a percent of the control (incubated in the same experiment without IgG). One hundred forty one of 145 untreated patients showed higher activity (228 +/- 51.8%, mean +/- SD; 127-393%, range) than normal subjects (103 +/- 13.3%, mean +/- SD, n = 24; 80-129%, range). Sequential changes in TSI activity in 27 patients after initiating thionamide drugs were studied for 24 months. Initially all 27 patients showed positive TSI and 6 months later 15 remained positive. At 6 months after that, 10 of 23, 4 of 16, and 2 of 6 followed patients showed positive TSI. These results indicate that this bioassay is clinically useful for detecting TSI.     

Release of an endothelial cell growth factor from cultured porcine thyroid follicles

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.     

Correlation between thyroid stimulating immunoglobulins and thyrotropin binding inhibitory immunoglobulins levels in patients with Graves' disease

INTRODUCTION: The II generation method using human recombination thyrotropin receptors for measurement of thyrotropin binding inhibitory immunoglobulins (TBII) is characterized by increased sensitivity and specificity in comparison with I generation method. AIM OF STUDY was to determine, whether TBII levels measured with II generation assay reflect thyroid stimulation and whether measurement of thyroid stimulating antibodies (TSI) could be replaced by TBII determinations. Specific aim was to evaluate, whether correlation between TSI and TBII levels is stable during antithyroid therapy. MATERIAL AND METHODS: 41 patients with the newly diagnosed Graves' disease were included in the study. TSI (cAMP levels in CHO cell line) and TBII (II generation assay) levels were determined before treatment and after 1, 3, 6, 9 and 12 months of thiamazol therapy. Moreover, thyroid blocking antibodies were determined after 12 months of treatment. RESULTS: 32 patients (82.05%) had positive basic TSI level and 35 patients (89.74%) had positive basic TBII level. After 12 months of therapy negative level of TSI was observed in 67.57% of patients and negative level of TBII was founded in 45.85% of patients. Correlation between TSI and TBII levels was positive during treatment course except time after 9 months of therapy. CONCLUSIONS: TBII level is adequate parameter to assess thyroid stimulation intensity. Positive correlation between TSI and TBII levels is present during almost whole treatment course. TBII seems to be reliable parameter in disease activity monitoring and response to therapy.     

Induction of apoptosis in porcine thyroid follicles by transforming growth factor beta1 and epidermal growth factor.

For thyroid cells in culture DNA fragmentation and morphological changes related to apoptosis were first described in dog thyroid cells after deprivation of serum, epidermal growth factor or thyrotropin. With intact porcine thyroid follicles in three-dimensional culture, the effect of deprivation of growth factors and of incubation with transforming growth factor beta1 (TGF-beta1), epidermal growth factor (EGF), thyrotropin (TSH) or insulin-like growth factor I (IGF-I) on the incidence of apoptosis was studied. Thyroid follicles were embedded in growth factor-depleted Matrigel and cultured in serum-free medium with or without growth factors for 7 days followed by incubation for 4, 24 and 72 h with TGF-beta1 (2 or 5 ng/mL). The percentage of apoptotic cells was determined by direct counting in electron-microscopy. Approximately 1% of apoptotic bodies could be detected in unstimulated follicles. This was unchanged in the presence of TSH (1 mU/mL) or IGF (10 ng/mL) but significantly increased up to 3.99 +/- 1.24% with 2 ng/mL of EGF. After incubation with TGF-beta apoptosis increased dose-dependently to 4.05 +/- 0.67% with 2 ng/mL TGF-beta1 and 5.16 +/- 1.75% with 5 ng/mL TGF-beta1. The incidence of necrotic cells remained constant at about 1 to 2%. Preincubation of follicles with 2 ng/mL of EGF followed by incubation with 5 ng/mL TGF-beta1 increased the rate of apoptic bodies up to 13.19 +/- 1.9%. We conclude that growth factor depletion in thyroid follicles in three-dimensional culture does not lead to apoptosis. TGF-beta1, however, induces apoptosis even in quiescent thyroid follicular cells and is significantly more pronounced in growing thyroid cells. EGF, which is a dedifferentiating growth factor for thyroid cells, also induces apoptosis. As EGF enhances TGF-beta1 mRNA and protein in thyroid follicular cells, the induction of apoptosis by EGF might also be due to TGF-beta1.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

Relationship among thyrotropin (TSH), thyroid stimulating immunoglobulins, and results of triiodothyronine (T3) suppression test in patients with Graves' disease

To investigate the relationship between TSH and abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease in whom normal thyroid hormone levels in the serum were maintained by antithyroid drug therapy and in patients with euthyroid Graves' disease, determinations were made of the TSH concentration, action of thyroid stimulating immunoglobulins (TSAb and TBII), and T3 suppression. Out of thirty-three patients with hyperthyroid Graves' disease, twelve patients with subnormal TSH levels were all non-suppressible according to the T3 suppression test results and the detectability of TSAb and/or TBII was as high as 75%. In three out of five patients with euthyroid Graves' disease, the serum TSH level was subnormal. All three showed non-suppressibility in the T3 suppression test and positive action of either TSAb or TBII. One of them became clinically thyrotoxic when the TSAb activity was further increased and TBII became positive, and was therefore diagnosed as having hyperthyroid Graves' disease. The present findings suggest that there are still abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease who have low TSH, even if their thyroid hormone concentrations remain normal. Moreover, it is likely that some of the patients with euthyroid Graves' disease are actually in a state of subclinical hyperthyroidism because of the presence of abnormal thyroid stimulator(s).     

Similar triiodothyronine releasing activity of thyroid stimulating antibodies in human and porcine thyroid slices

In an approach to addressing species specificity of thyroid stimulating antibodies (TSAb) stimulation of T3 release by Graves' sera was comparatively studied in human and porcine thyroid slices. A high sensitivity and specificity was found for the T3 bioassay independently on the use of human or porcine thyroid. Moreover, activity indices of the individual sera in both tissues were significantly correlated to each other and to circulating hormone levels in untreated disease. In conclusion, we suppose a lack of functionally relevant differences between target antigens, brought about probably by the TSH receptor itself and other membrane components, in human and porcine thyroid. Thus, for clinically applicable T3 releasing bioassay porcine thyroid may be alternatively used. In addition, this bioassay renders the advantage of reflecting the activity of disease.     

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Bovine thyrotropin inhibits DNA synthesis inversely with stimulation of cyclic AMP production in cultured porcine thyroid follicles

The effects of bovine thyrotropin (TSH) on DNA synthesis and cyclic AMP production were studied in porcine thyroid follicles using suspension culture. During the early 72 hours incubation, the time-dependent uptake of [3H]thymidine by the follicles was observed. In the presence of 10 mU/ml TSH, the uptake of [3H] thymidine was significantly depressed at 72 hours incubation. TSH inhibition of [3H] thymidine incorporation was related to its concentration and the 50% inhibition was observed by using 1.0 mU/ml TSH. Under the same conditions, cyclic AMP production was stimulated by TSH and the stimulation was observed to be related to TSH concentration. In these experiments, the incubation time was 30 min. Dibutyryl cyclic AMP, an analogue of cyclic AMP, inhibited the [3H] thymidine uptake at 72 hours incubation. From these results, it is suggested that TSH inhibits DNA synthesis, and that the inhibition may be mediated by cyclic AMP that is produced by TSH stimulation.     

Demonstration of growth in porcine thyroid cell culture

Eagle's minimum essential medium supplemented with 20 per cent newborn calf serum (N.C.S.) allows porcine thyroid cell survival but not cell growth in vitro. In NCTC 109 medium supplemented with 20 per cent N.C.S. these cells actively grow and may be serially propagated. Cell population doubling time expressed as DNA doubling value is 3.5 days at 37 degrees C in 95 per cent air-5 per cent CO2. Thyrotropin does not affect porcine thyroid cell multiplication in vitro but stimulates the plating efficiency in primary cultures to about 130 per cent of controls. Cell selection was obtained by replacing media with Earle's balanced salt solution. This operation provoked death of nearly all cells by day 18 but subsequent addition of growth medium resulted in proliferation of epithelial cell clones. From generation 2 to generation 8, cells produce thyroglobulin but they do not actively trap iodide nor form follicles when thyrotropin is added to the media. Cell selection, demonstration of growth, as well as freeze-storage techniques described in this paper permit selection and storage of porcine thyroid cells and the potential constitution of cell collections.     

Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles

Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council     

Measurement of thyroid stimulating hormone (TSH) in human urine

Using a highly sensitive and specific immunoradiometric assay kit for human TSH, we measured TSH concentrations in unprocessed urines in normal subjects, in patients with primary hypothyroidism, and patients with renal disease. In five of ten normal subjects TSH was detectable in urine samples (less than 20-69 microU/day). In five patients with hypothyroidism, the urinary TSH excretion was increased. In seven out of ten patients with nephrotic syndrome, eight out of nine patients with chronic renal failure and two patients with tubular dysfunction, the urinary TSH excretion was increased. The urinary TSH excretion correlated significantly with both urinary protein excretion and urinary beta 2-microglobulin excretion. These results suggest that the renal handling of TSH involves both glomerular filtration and tubular re-absorption, and that urinary TSH excretion is increased when serum TSH is increased and either glomerular or tubular function is impaired.     

Cytoplasmic pH in the action of epidermal growth factor (EGF) in cultured porcine thyroid cells

We demonstrate measurement of cytoplasmic pH (pHi), using 2',7'-bis(2-carboxyethyl)-5 (and 6-) carboxyfluorescein (BCECF), and internalized fluorescent pHi indicator, in thyroid cells. Using cultured porcine thyroid cells, we studied the effects of epidermal growth factor (EGF) on pHi and [3H] thymidine incorporation; 10 nM EGF alkalinizes thyroid cells and stimulates thymidine incorporation. The results indicate that Na+/H+ exchange or cell alkalinization may function as a transmembrane signal transducer in the action of EGF in the thyroid cells.     

Effects of inorganic iodide, epidermal growth factor and phorbol ester on hormone synthesis by porcine thyroid follicles cultured in suspension.

Porcine thyroid follicles cultured in suspension for 96 h synthesized and secreted thyroid hormones in the presence of thyrotropin (TSH). The secretion of newly synthesized hormones was assessed by determining the contents of thyroxine (T4) and triiodothyronine (T3) in the media and by paperchromatographic analysis of 125I-labelled hormones in the media where the follicles were cultured in the presence and absence of inhibitors of hormone synthesis. The hormone synthesis and secretion was modified by exogenously added NaI (0.1-100 microM). The maximal response was obtained at 1 microM. Thyroid peroxidase (TPO) activity in the cultured follicles with TSH for 96 h was dose-dependently inhibited by NaI. One hundred microM of NaI completely inhibited TSH-induced TPO activity. Moreover, both epidermal growth factor (EGF: 10(-9) and 10(-8) M) and phorbol 12-myristate 13-acetate (PMA: 10(-8) and 10(-7) M) inhibited de novo hormone synthesis. An induction of TPO activity by TSH was also inhibited by either agent. These data provide direct evidences that thyroid hormone synthesis is regulated by NaI as well as TSH at least in part via regulation of TPO activity and also that both EGF and PMA are inhibitory on thyroid hormone formation.     

Chromosome regional mapping for the human thyroid stimulating hormone beta subunit (TSHB) gene

The human thyroid stimulating hormone beta subunit (TSHB) gene, located on chromosome 1, was studied to determine its subregional location by in situ hybridization and Southern blot analysis of human x mouse hybrid cells. The results allowed localization of TSHB to the proximal portion of 1p22, which is in the region of localization of the linkage group including amylase (AMY), nerve growth factor beta subunit (NGFB), and NRAS, which are conserved in humans and rodents.     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.